菊花CmNRTs基因的克隆及功能鉴定
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摘要
菊花(Chrysanthemum grandiflorum (Ramat.) Kitam.)是我国十大传统名花和世界四大切花之一,观赏和经济价值极高,但切花菊设施周年生产过程中,普遍存在氮肥过度施用、氮肥利用率低和氮素损失严重等问题,严重影响切花产量与品质,并导致水系统富营养化及土壤次生盐渍化。目前,有关菊花氮素营养研究还仅限于氮素水平／形态对其生长影响。菊花硝态氮运输分子机制研究可以为氮素高效利用,提高切花菊产量与品质提供理论基础。本研究包括：从16个南农系列切花品种中筛选出氮高效吸收切花菊品种‘南农雪峰’；克隆了CmNRT2家族7个基因,命名为CmNRT2.1-2.7,并克隆了CmNRT1.1基因和CmNAR2.1基因；通过TAIL-PCR结合Anchored-PCR方法克隆了CmNRT2.1、CmNRT2.4、CmNRT1.1和CmNAR2.1基因的启动子；利用GeNorm软件分析菊花中稳定的内参基因,发现psaA基因在生物胁迫和非生物胁迫下都相对稳定；通过qRT-PCR方法研究了菊花CmNRT基因的表达特性；通过泛素分裂化系统酵母双杂交和双分子荧光互补实验证实CmNAR2.1和CmNRT2.1、CmNRT2.4存在互作关系；超表达CmNAR2.1、CmNRT2.1和CmNRT2.4基因,对拟南芥生理指标及根系形态的分析；构建RNAi干扰载体转化切花菊‘神马’,获得转基因株系,主要研究结果如下：
1.以本实验室自主选育的16个南农系列切花菊品种为试验材料,利用石英砂为基质进行砂培,营养液为改良的休伊特营养液(大量)加Arnon营养液(微量),通过对植株浇灌低氮水平营养液(16mg/L)和正常氮水平营养液(320mg/L),3周之后测定植株干重,叶绿素含量和全氮量,通过比较相对干物质重,相对含氮量和相对叶绿素含量,初步确定‘南农小丽’、‘南农雪峰’和‘南农玉珠’为氮利用效率相对较高的品种,‘南农霞光’、‘南农宫粉’、‘南农花脸’为氮利用效率较低的品种。仍然以相对干物质重,相对含氮量和相对叶绿素含量为评判标准,进一步进行营养液培养复筛,比较初筛的6个品种,最终确定1个氮高效利用品种‘南农雪峰’和1个氮低效利用品种‘南农宫粉’。
2.以‘南农雪峰’为研究材料,利用兼并引物RT-PCR和RACE-PCR方法分离出7个CmNRT2基因家族成员,命名为CmNRT2.1-2.7。同时扩增了CmNRT2基因家族成员的基因组DNA序列。利用DNAman软件及MatGAT2.01程序对家族成员氨基酸序列进行相似性比较及进化分析,发现CmNRT2.5和CmNRT2.7与其它5个成员氨基酸差异性较大。利用相同方法克隆获得2个CmNRT类基因的cDNA全长,经NCBI数据库Blast比对命名为CmNRT1.1和CmNAR2.1.CmNRTl.l基因编码587个氨基酸,CmNAR2.1基因编码199个氨基酸,分别对两个基因做了氨基酸序列同源性比对和聚类分析,发现CmNRT1.1蛋白与双子叶植物大豆(GmNRTl.1-like)进化关系上最接近,CmNAR2.1蛋白与双子叶植物拟南芥(AtNAR2.1、AtNAR2.2),百脉根(LjNAR2.1)和黄瓜(CsNAR2)的等聚为一个亚群。这些表明,我们分离的CmNRTs基因为硝酸盐转运蛋白基因。
3.利用TAIL-PCR结合Anchored PCR方法从菊花基因组中克隆获得了CmNRTl.1、 CmNRT2.1、CmNRT2.4和CmNAR2.1(CmNRT3.1)基因上游启动子序列,长度分别为1096bp、1658bp、1413bp和1683bp,并对其结构和功能元件进行预测分析,发现4个CmNRTs基因的上游启动子序列中除含有典型的真核生物核心启动子结构外,还包含硝酸盐诱导的调控元件、氮代谢调控元件以及多个胁迫响应和激素应答相关的调控元件,同时启动子间还包含许多相同的调控元件,表明基因可能受到共同调控。
4.利用geNorm软件评估了菊花中8个候选内参基因在生物胁迫和非生物胁迫下的稳定性,八个候选内参基因分别为：tubulin (alpha-2,4tubulin)、actin、 EFl a (elongation factor1α)、UBC (ubiquitin C)、GAPDH (glyceraldehyde-3-phosphate dehydrogenase)、 psaA (photosynthesis-related plastid gene representing photosystem Ⅰ)、 PP2Acs (catalytic subunit of protein phosphatase2A)和PGK(phosphoglycerate kinase).广泛使用的EF1α基因在蚜虫胁迫下表现稳定,但是涝胁迫下不稳定。PP2Acs基因为在热胁迫下和涝害胁迫下最稳定的内参基因之一,但在蚜虫胁迫下最不稳定。在三种胁迫下,常用的actin基因都不是最稳定的。psaA基因在生物胁迫和非生物胁迫下都相对稳定。
5.利用荧光定量PCR方法对CmNRT2.1、CmNRT2.4和CmNAR2.1基因的表达特性进行了分析。结果表明,CmNRT2.1、CmNRT2.4和CmNAR2.1均受硝酸盐诱导型表达,其中CmNAR2.1、CmNRT2.4为组成型诱导型表达基因。铵盐作为植物体另一种氮源,可以诱导CmNRT2.1和CmNRT2.4的表达,但会抑制CmNAR2.1的表达。谷氨酰胺作为另一种还原性氮,能抑制硝酸盐对CmNRT2.1、CmNRT2.4和CmNAR2.1的诱导表达。
6.构建了酵母表达载体pBT3-C-CmNAR2.1和pPR3-N-CmNRT2.1、pPR3-N-CmNRT2.4,利用分裂泛素化系统证明了CmNAR2.1与CmNRT2.1、CmNRT2.4互作。为验证酵母实验结果,进行了BiFC (bimolecular fluorescence complementation双分子荧光互补)实验,结果表明CmNAR2.1与CmNRT2.1、CmNRT2.4之间确实存在互作关系。在分析CmNAR2.1和OsNAR2.1、AtNAR2.1、HvNAR2.3氨基酸序列的基础上,将CmNAR2.1进行氨基酸分段,初步证明了153－199氨基酸是CmNAR2.1和CmNRT2.4互作的关键区域,这些初步证明菊花中协同作用蛋白CmNAR2.1可以辅助高亲和运输蛋白CmNAR2.1和CmNRT2.4运输硝酸盐。
7.构建了植物正义表达载体pCAMBIA1301-220-CmNAR2.1和pCAMBIA1301-220-CmNRT2.1、pCAMBIA1301-220-CmNRT2.4载体,利用农杆菌介导法转化野生型拟南芥(Col-0),通过潮霉素抗性、GUS染色、RT-PCR、qRT-PCR筛选鉴定阳性苗,每个基因选取两个转基因表达量高的株系(T3代)进一步分析。结果表明,转CmNRT2.1和CmNRT2.4基因拟南芥地上部鲜重,地下部鲜重以及地上部鲜重与地下部鲜重比值均高于野生型拟南芥；两个CmNRT2.1转基因株系的地上部,地下部硝酸盐含量均高于野生型；但是35S:CmNRT2.4-3株系的地上部,地下部硝酸盐含量高于野生型,35S:CmNRT2.4-7却低于野生型。转CmNAR2.1株系在固体培养基生长8-14天,主根较野生型更长,侧根发生明显优于野生型。我们认为CmNRT2.1和CmNRT2.4具硝酸盐转运功能,CmNAR2.1可能调控根系发育。
Chrysanthemum morifolium is one often most famous flowers in China and one of the top four cut flowers in the world with high ornamental and practical value. Cut chrysanthemum is one of our major export flowers with year-round production in protected cultivation. However, excessive application, low utilization rate and serious loss of nitrogen fertilizer are common which seriously affect the yield and quality of cut flowers. Moreover, it leads to water eutrophication and soil secondary salinization. The study of chrysanthemum nitrogen was restricted to physiological level. Studying molecular mechanisms of nitrate transporter in chrysanthemum can lay the foundation to improve nitrogen use efficiency and improve the yield and quality of cut chrysanthemum. Several aspects are included in our study.'Nannongxuefeng' was selected as high nitrogen efficiency characterization from variety from sixteen cut chrysanthemum varieties; then seven CmNRT2genes named CmNRT2.1-2.7with CmNRTl.l and CmNAR2.1were cloned from 'Nannongxuefeng'; on the basis of the genomic DNA, the promoter regions of CmNRT2.1, CmNRT2.4, CmNRT1.1and CmNAR2.1were cloned by TAIL-PCR and Anchored-PCR; by using GeNorm software, we analyzed the stability of the reference gene in chrysanthemum. Expression characterization of CmNRTwas analyzed by qRT-PCR; the protein interaction between CmNAR2.1with CmNRT2.1, CmNRT2.4was dentified by split-ubiquitin system and bimolecular fluorescence complementation system; physiological indexes and root morphology were analyzed in transgenic Arabidopsis overexpressing CmNAR2.1, CmNRT2.1and CmNRT2.4; transgenic chrysanthemum lines were obtained by transforming interference vectors. The main results were as follows:
1. Sixteen 'Nannong' cut chrysanthemum were used in our study. After cultivating by water culture, seedlings were transferred to the sand culture. Nutrient solution was improved hewitt solution (macro) plus Arnon solution (micro). Two groups of plants were treated by low nitrogen level (16mg/L) and normal nitrogen level (320mg/L). After three weeks, dry weight, chlorophyll content and total N content were determined. As a result, high nitrogen efficiency varieties 'Nannongxiaoli','Nannongxuefeng','Nannongyuzhu' and low nitrogen efficiency varieties 'Nannongxiaguang','Nannonggongfen','Nannonghualian' were selected by comparing relative dry weight, relative chlorophyll content and relative total N content. The next step was the selection of one high nitrogen efficiency variety and one low nitrogen efficiency variety from the six varieties which were took out in the primary screening. And the screening index was the same as in the primary screening.
2. Seven CmNRT2genes named CmNRT2.1-CmNRT2.7were obtained by degenerate primer RT-PCR and RACE-PCR from 'Nannongxuefeng', respectively. After that, CmNRT2genome DNA sequece were cloned. CmNRT2.5and CmNRT2.7have large differences with other five family numbers using DNAman software and MatGAT2.01program. We also cloned CmNRT1.1and CmNAR2.1. The putative protein of CmNRT1.1and CmNAR2.1separately contained587and199amino acids. The phylogenetic analysis suggested that CmNRT1.1and GmNRT1.1-like, CmNAR2.1had the closest relationship with AtNAR2.1, AtNAR2.2, LjNAR2.1and CsNAR2in the evolutionary. These showed that CmNRTs were nitrate transporter genes.
3. The promoter regions of CmNRT1.1, CmNRT2.1, CmNRT2.4and CmNAR2.1(CmNRT3.1) were cloned by TAIL-PCR and Anchored PCR. The length was1096bp,1658bp,1413bp and1683bp, respectively. Besides typical eukaryotic core promoter structure, NO3-cis-elements, nitrogen metabolism cis-elements and many stress response motifs and hormone response motifs were found based on the analysis of the predicted motifs. We also analyzed the similarities and differences of four gene promoter regulatory elements. Promoters also contained many of the same regulatory elements, indicating that the gene may be under common control.
4. The stability of expression of eight potential reference genes, namely tubulin (alpha-2,4tubulin),actin,EF1α (elongation factor1α),UBC (ubiquitin C),GAPDH (glyceraldehyde-3-phosphate dehydrogenase),psaA (photosynthesis-related plastid gene representing photosystem Ⅰ), PP2Acs (catalytic subunit of protein phosphatase2A) and PGK (phosphoglycerate kinase), was assessed in chrysanthemum plants subjected to biotic and abiotic stress using geNorm software. The widely used reference gene EF1α performed well for aphid infested plants but poorly for waterlogged ones. The catalytic subunit of protein phosphatase2A (PP2Acs) was the best performing during heat and waterlogging stress, but the worst during aphid infestation. The commonly used reference gene actin was generally the least stable of the set. psaA was relatively stable in the biotic and abiotic stress.
5. The expression characterization of CmNRT2.1, CmNRT2.4and CmNAR2.1was analyzed by qRT-PCR, respectively. The three genes were induced by nitrate and CmNRT2.4and CmNAR2.1were constitutively inducible genes. NH4+can induce the expression of CmNRT2.1and CmNRT2.4but repress the expression of CmNAR2.1. As another reduced nitrogen, glutamine would repress the inducible effect which nitrate played.
6. The interaction between CmNAR2.1and CmNRT2.1, CmNRT2.4was identified by transforming vectors pBT3-C-CmNAR2.1and pPR3-N-CmNRT2.1, pPR3-N-CmNRT2.4to the yeast NMY51. By BiFC (bimolecular fluorescence complementation) method, split-ubiquitin system result was verified. Based on the conserved sequence between CmNAR2.1and OsNAR2.1, AtNAR2.1, HvNAR2.3, the CmNAR2.1amino acids sequence was deduced, we preliminary proved that153-199AA was the key region in CmNAR2.1interacts with CmNRT2.4through deletion analysis in yeast. These preliminarily proofed that CmNAR2.1could assist with high affinity transport proteins CmNAR2.1and CmNRT2.4to transport nitrate.
7. The vectors pCAMBIA1301-220-CmNAR2.1, pCAMBIA1301-220-CmNRT2.1and pCAMBIA1301-220-CmNRT2.4was successfully constructed and then transformed into A. thaliana (Col-0) by the floral dip method, respectively. Transgenic lines were selected by hygromycin resistance, histochemical GUS assay, RT-PCR and qRT-PCR. We chosed two high expression lines (T3) to carry out downstream experiments from every CmNRT gene transform lines. Shoot fresh weight, root fresh weight and S/R ratio in CmNRT2.1and CmNRT2.4transgenic lines were higher than that in wild type. The shoot and root nitrate contents in CmNRT2.1transgenic lines and35S:CmNRT2.4-3transgenic lines were higher than in wild type. However, the shoot and root nitrate contents in35S:CmNRT2.4-3transgenic lines were lower than that in wild type. The length of main roots in CmNAR2.1transgenic lines were longer than that in wild lines and the number of lateral root in CmNAR2.1transgenic lines were more than that in wild lines. We thought that CmNRT2.1 and CmNRT2.4had the function of nitrate transport, however, CmNAR2.1may regulate root development.
1.以本实验室自主选育的16个南农系列切花菊品种为试验材料,利用石英砂为基质进行砂培,营养液为改良的休伊特营养液(大量)加Arnon营养液(微量),通过对植株浇灌低氮水平营养液(16mg/L)和正常氮水平营养液(320mg/L),3周之后测定植株干重,叶绿素含量和全氮量,通过比较相对干物质重,相对含氮量和相对叶绿素含量,初步确定‘南农小丽’、‘南农雪峰’和‘南农玉珠’为氮利用效率相对较高的品种,‘南农霞光’、‘南农宫粉’、‘南农花脸’为氮利用效率较低的品种。仍然以相对干物质重,相对含氮量和相对叶绿素含量为评判标准,进一步进行营养液培养复筛,比较初筛的6个品种,最终确定1个氮高效利用品种‘南农雪峰’和1个氮低效利用品种‘南农宫粉’。
2.以‘南农雪峰’为研究材料,利用兼并引物RT-PCR和RACE-PCR方法分离出7个CmNRT2基因家族成员,命名为CmNRT2.1-2.7。同时扩增了CmNRT2基因家族成员的基因组DNA序列。利用DNAman软件及MatGAT2.01程序对家族成员氨基酸序列进行相似性比较及进化分析,发现CmNRT2.5和CmNRT2.7与其它5个成员氨基酸差异性较大。利用相同方法克隆获得2个CmNRT类基因的cDNA全长,经NCBI数据库Blast比对命名为CmNRT1.1和CmNAR2.1.CmNRTl.l基因编码587个氨基酸,CmNAR2.1基因编码199个氨基酸,分别对两个基因做了氨基酸序列同源性比对和聚类分析,发现CmNRT1.1蛋白与双子叶植物大豆(GmNRTl.1-like)进化关系上最接近,CmNAR2.1蛋白与双子叶植物拟南芥(AtNAR2.1、AtNAR2.2),百脉根(LjNAR2.1)和黄瓜(CsNAR2)的等聚为一个亚群。这些表明,我们分离的CmNRTs基因为硝酸盐转运蛋白基因。
3.利用TAIL-PCR结合Anchored PCR方法从菊花基因组中克隆获得了CmNRTl.1、 CmNRT2.1、CmNRT2.4和CmNAR2.1(CmNRT3.1)基因上游启动子序列,长度分别为1096bp、1658bp、1413bp和1683bp,并对其结构和功能元件进行预测分析,发现4个CmNRTs基因的上游启动子序列中除含有典型的真核生物核心启动子结构外,还包含硝酸盐诱导的调控元件、氮代谢调控元件以及多个胁迫响应和激素应答相关的调控元件,同时启动子间还包含许多相同的调控元件,表明基因可能受到共同调控。
4.利用geNorm软件评估了菊花中8个候选内参基因在生物胁迫和非生物胁迫下的稳定性,八个候选内参基因分别为：tubulin (alpha-2,4tubulin)、actin、 EFl a (elongation factor1α)、UBC (ubiquitin C)、GAPDH (glyceraldehyde-3-phosphate dehydrogenase)、 psaA (photosynthesis-related plastid gene representing photosystem Ⅰ)、 PP2Acs (catalytic subunit of protein phosphatase2A)和PGK(phosphoglycerate kinase).广泛使用的EF1α基因在蚜虫胁迫下表现稳定,但是涝胁迫下不稳定。PP2Acs基因为在热胁迫下和涝害胁迫下最稳定的内参基因之一,但在蚜虫胁迫下最不稳定。在三种胁迫下,常用的actin基因都不是最稳定的。psaA基因在生物胁迫和非生物胁迫下都相对稳定。
5.利用荧光定量PCR方法对CmNRT2.1、CmNRT2.4和CmNAR2.1基因的表达特性进行了分析。结果表明,CmNRT2.1、CmNRT2.4和CmNAR2.1均受硝酸盐诱导型表达,其中CmNAR2.1、CmNRT2.4为组成型诱导型表达基因。铵盐作为植物体另一种氮源,可以诱导CmNRT2.1和CmNRT2.4的表达,但会抑制CmNAR2.1的表达。谷氨酰胺作为另一种还原性氮,能抑制硝酸盐对CmNRT2.1、CmNRT2.4和CmNAR2.1的诱导表达。
6.构建了酵母表达载体pBT3-C-CmNAR2.1和pPR3-N-CmNRT2.1、pPR3-N-CmNRT2.4,利用分裂泛素化系统证明了CmNAR2.1与CmNRT2.1、CmNRT2.4互作。为验证酵母实验结果,进行了BiFC (bimolecular fluorescence complementation双分子荧光互补)实验,结果表明CmNAR2.1与CmNRT2.1、CmNRT2.4之间确实存在互作关系。在分析CmNAR2.1和OsNAR2.1、AtNAR2.1、HvNAR2.3氨基酸序列的基础上,将CmNAR2.1进行氨基酸分段,初步证明了153－199氨基酸是CmNAR2.1和CmNRT2.4互作的关键区域,这些初步证明菊花中协同作用蛋白CmNAR2.1可以辅助高亲和运输蛋白CmNAR2.1和CmNRT2.4运输硝酸盐。
7.构建了植物正义表达载体pCAMBIA1301-220-CmNAR2.1和pCAMBIA1301-220-CmNRT2.1、pCAMBIA1301-220-CmNRT2.4载体,利用农杆菌介导法转化野生型拟南芥(Col-0),通过潮霉素抗性、GUS染色、RT-PCR、qRT-PCR筛选鉴定阳性苗,每个基因选取两个转基因表达量高的株系(T3代)进一步分析。结果表明,转CmNRT2.1和CmNRT2.4基因拟南芥地上部鲜重,地下部鲜重以及地上部鲜重与地下部鲜重比值均高于野生型拟南芥；两个CmNRT2.1转基因株系的地上部,地下部硝酸盐含量均高于野生型；但是35S:CmNRT2.4-3株系的地上部,地下部硝酸盐含量高于野生型,35S:CmNRT2.4-7却低于野生型。转CmNAR2.1株系在固体培养基生长8-14天,主根较野生型更长,侧根发生明显优于野生型。我们认为CmNRT2.1和CmNRT2.4具硝酸盐转运功能,CmNAR2.1可能调控根系发育。
Chrysanthemum morifolium is one often most famous flowers in China and one of the top four cut flowers in the world with high ornamental and practical value. Cut chrysanthemum is one of our major export flowers with year-round production in protected cultivation. However, excessive application, low utilization rate and serious loss of nitrogen fertilizer are common which seriously affect the yield and quality of cut flowers. Moreover, it leads to water eutrophication and soil secondary salinization. The study of chrysanthemum nitrogen was restricted to physiological level. Studying molecular mechanisms of nitrate transporter in chrysanthemum can lay the foundation to improve nitrogen use efficiency and improve the yield and quality of cut chrysanthemum. Several aspects are included in our study.'Nannongxuefeng' was selected as high nitrogen efficiency characterization from variety from sixteen cut chrysanthemum varieties; then seven CmNRT2genes named CmNRT2.1-2.7with CmNRTl.l and CmNAR2.1were cloned from 'Nannongxuefeng'; on the basis of the genomic DNA, the promoter regions of CmNRT2.1, CmNRT2.4, CmNRT1.1and CmNAR2.1were cloned by TAIL-PCR and Anchored-PCR; by using GeNorm software, we analyzed the stability of the reference gene in chrysanthemum. Expression characterization of CmNRTwas analyzed by qRT-PCR; the protein interaction between CmNAR2.1with CmNRT2.1, CmNRT2.4was dentified by split-ubiquitin system and bimolecular fluorescence complementation system; physiological indexes and root morphology were analyzed in transgenic Arabidopsis overexpressing CmNAR2.1, CmNRT2.1and CmNRT2.4; transgenic chrysanthemum lines were obtained by transforming interference vectors. The main results were as follows:
1. Sixteen 'Nannong' cut chrysanthemum were used in our study. After cultivating by water culture, seedlings were transferred to the sand culture. Nutrient solution was improved hewitt solution (macro) plus Arnon solution (micro). Two groups of plants were treated by low nitrogen level (16mg/L) and normal nitrogen level (320mg/L). After three weeks, dry weight, chlorophyll content and total N content were determined. As a result, high nitrogen efficiency varieties 'Nannongxiaoli','Nannongxuefeng','Nannongyuzhu' and low nitrogen efficiency varieties 'Nannongxiaguang','Nannonggongfen','Nannonghualian' were selected by comparing relative dry weight, relative chlorophyll content and relative total N content. The next step was the selection of one high nitrogen efficiency variety and one low nitrogen efficiency variety from the six varieties which were took out in the primary screening. And the screening index was the same as in the primary screening.
2. Seven CmNRT2genes named CmNRT2.1-CmNRT2.7were obtained by degenerate primer RT-PCR and RACE-PCR from 'Nannongxuefeng', respectively. After that, CmNRT2genome DNA sequece were cloned. CmNRT2.5and CmNRT2.7have large differences with other five family numbers using DNAman software and MatGAT2.01program. We also cloned CmNRT1.1and CmNAR2.1. The putative protein of CmNRT1.1and CmNAR2.1separately contained587and199amino acids. The phylogenetic analysis suggested that CmNRT1.1and GmNRT1.1-like, CmNAR2.1had the closest relationship with AtNAR2.1, AtNAR2.2, LjNAR2.1and CsNAR2in the evolutionary. These showed that CmNRTs were nitrate transporter genes.
3. The promoter regions of CmNRT1.1, CmNRT2.1, CmNRT2.4and CmNAR2.1(CmNRT3.1) were cloned by TAIL-PCR and Anchored PCR. The length was1096bp,1658bp,1413bp and1683bp, respectively. Besides typical eukaryotic core promoter structure, NO3-cis-elements, nitrogen metabolism cis-elements and many stress response motifs and hormone response motifs were found based on the analysis of the predicted motifs. We also analyzed the similarities and differences of four gene promoter regulatory elements. Promoters also contained many of the same regulatory elements, indicating that the gene may be under common control.
4. The stability of expression of eight potential reference genes, namely tubulin (alpha-2,4tubulin),actin,EF1α (elongation factor1α),UBC (ubiquitin C),GAPDH (glyceraldehyde-3-phosphate dehydrogenase),psaA (photosynthesis-related plastid gene representing photosystem Ⅰ), PP2Acs (catalytic subunit of protein phosphatase2A) and PGK (phosphoglycerate kinase), was assessed in chrysanthemum plants subjected to biotic and abiotic stress using geNorm software. The widely used reference gene EF1α performed well for aphid infested plants but poorly for waterlogged ones. The catalytic subunit of protein phosphatase2A (PP2Acs) was the best performing during heat and waterlogging stress, but the worst during aphid infestation. The commonly used reference gene actin was generally the least stable of the set. psaA was relatively stable in the biotic and abiotic stress.
5. The expression characterization of CmNRT2.1, CmNRT2.4and CmNAR2.1was analyzed by qRT-PCR, respectively. The three genes were induced by nitrate and CmNRT2.4and CmNAR2.1were constitutively inducible genes. NH4+can induce the expression of CmNRT2.1and CmNRT2.4but repress the expression of CmNAR2.1. As another reduced nitrogen, glutamine would repress the inducible effect which nitrate played.
6. The interaction between CmNAR2.1and CmNRT2.1, CmNRT2.4was identified by transforming vectors pBT3-C-CmNAR2.1and pPR3-N-CmNRT2.1, pPR3-N-CmNRT2.4to the yeast NMY51. By BiFC (bimolecular fluorescence complementation) method, split-ubiquitin system result was verified. Based on the conserved sequence between CmNAR2.1and OsNAR2.1, AtNAR2.1, HvNAR2.3, the CmNAR2.1amino acids sequence was deduced, we preliminary proved that153-199AA was the key region in CmNAR2.1interacts with CmNRT2.4through deletion analysis in yeast. These preliminarily proofed that CmNAR2.1could assist with high affinity transport proteins CmNAR2.1and CmNRT2.4to transport nitrate.
7. The vectors pCAMBIA1301-220-CmNAR2.1, pCAMBIA1301-220-CmNRT2.1and pCAMBIA1301-220-CmNRT2.4was successfully constructed and then transformed into A. thaliana (Col-0) by the floral dip method, respectively. Transgenic lines were selected by hygromycin resistance, histochemical GUS assay, RT-PCR and qRT-PCR. We chosed two high expression lines (T3) to carry out downstream experiments from every CmNRT gene transform lines. Shoot fresh weight, root fresh weight and S/R ratio in CmNRT2.1and CmNRT2.4transgenic lines were higher than that in wild type. The shoot and root nitrate contents in CmNRT2.1transgenic lines and35S:CmNRT2.4-3transgenic lines were higher than in wild type. However, the shoot and root nitrate contents in35S:CmNRT2.4-3transgenic lines were lower than that in wild type. The length of main roots in CmNAR2.1transgenic lines were longer than that in wild lines and the number of lateral root in CmNAR2.1transgenic lines were more than that in wild lines. We thought that CmNRT2.1 and CmNRT2.4had the function of nitrate transport, however, CmNAR2.1may regulate root development.
引文
Alboresi, A., Gestin, C., LEYDECKER, M.T., et al. Nitrate, a signal relieving seed dormancy in Arabidopsis. Plant Cell Environ,2005,28:500-512.
Almagro, A., Lin, S.H., Tsay, Y.F. Characterization of theE Arabidopsis nitrate transporter NRT 1.6 reveals a role of nitrate in early embryo development. Plant Cell,2008,20:3289-3299.
Andersen, C.L., Jensen, J.L.,Orntoft, T.F. Normalization of real-time quantitative reverse transcription-PCR data:a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res,2004,64:5245.
Anderson, L.E., Carol, A.A. Enzyme co-localization in the pea leaf cytosol:3-P-glycerate kinase, glyceraldehyde-3-P dehydrogenase, triose-P isomerase and aldolase. Plant Sci,2005,169:620-628.
Araki, R., Hasegawa, H. Expression of rice (Oryza sativa L.) genes involved in high-affinity nitrate transport during the period of nitrate induction. Breeding Sci,2006,56:295-302.
Armengaud, P., Breitling, R., Amtmann, A. The potassium-dependent transcriptome of Arabidopsis reveals a prominent role of jasmonic acid in nutrient signaling. Plant Pysiol,2004,136:2556-2576.
Aronheim, A., Zandi, E., Hennemann, H., et al. Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol Cell Biol,1997,17:3094-3102.
Artico, S., Nardeli, S.M., Brilhante, O., et al. Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data. BMC Plant Biol,2010,10:49.
Baxter, I., Hosmani, P.S., Rus, A., et al. Root suberin forms an extracellular barrier that affects water relations and mineral nutrition in Arabidopsis. PLoS Genet,2009,5:e1000492.
Bernier, G., Havelange, A., Houssa, C., et al. Physiological signals that induce flowering. Plant Cell, 1993,5:1147-1155.
Bezier, A., Lambert, B., Baillieul, F. Study of defense-related gene expression in grapevine leaves and berries infected with Botrytis cinerea. Eur J Plant Pathol,2002,108:111-120.
Bi, Y.M., Zhang, Y, Signorelli, T., et al. Genetic analysis of Arabidopsis GATA transcription factor gene family reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity. Plant J,2005,44:680-692.
Broder, Y.C., Katz, S., Aronheim, A. The ras recruitment system, a novel approach to the study of protein-protein interactions. Curr Biol,1998,8:1121-1130.
Brunner, A., Yakovlev, I., Strauss, S. Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol,2004,4:14.
Bustin, S. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol,2000,25:169-193.
Bustin, S. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR):trends and problems. J Mol Endocrinol,2002,29:23-39.
Cai, C., Wang, J.Y., Zhu, Y.G, et al. Gene Structure and Expression of the High-affinity Nitrate Transport System in Rice Roots. J Integr Plant Biol,2008,50:443-451.
Cai, C., Zhao, X.Q., Zhu, Y.G, et al. Regulation of the high-affinity nitrate transport system in wheat roots by exogenous abscisic acid and glutamine. J Integr Plant Biol,2007,49:1719-1725.
Cai, W., Ma, J., Chi, W., et al. Cooperation of LPA3 and LPA2 is essential for photosystem II assembly in Arabidopsis. Plant Physiol,2010,154:109-120.
Camanes, G, Pastor, V., Cerezo, M., et al. A deletion in NRT2.1 attenuates Pseudomonas syringae-induced hormonal perturbation, resulting in primed plant defenses. Plant Pysiol,2012,158: 1054-1066.
Canning, R.E., Kramer, P.J. Salt absorption and accumulation in various regions of roots. Am J Bot, 1958:378-382.
Castaings, L., Marchive, C., Meyer, C., et al. Nitrogen signalling in Arabidopsis:how to obtain insights into a complex signalling network. J Exp Bot,2011,62:1391-1397.
Cerezo, M., Tillard, P., Filleur, S., et al. Major Alterations of the Regulation of Root NO3 Uptake Are Associated with the Mutation of Nrt2.1 and Nrt2.2 Genes in Arabidopsis. Plant Physiol,2001,127: 262-271.
Chen, A., Gu, M., Sun, S., et al. Identification of two conserved cis-acting elements, MYCS and P1BS, involved in the regulation of mycorrhiza-activated phosphate transporters in eudicot species. New Phytol,2011,189:1157-1169.
Chen, S., Miao, H., Chen, F., et al. Analysis of Expressed Sequence Tags (ESTs) Collected from the Inflorescence of Chrysanthemum. Plant Mol Biol Rep,2009,27:503-510.
Chen, Y, Chen, S., Chen, F., et al. Functional Characterization of a Chrysanthemum dichrum Stress-Related Promoter. Mol Biotechnol,2011:1-9.
Chen, Y.F., Etheridge, N., Schaller, G.E. Ethylene signal transduction. Ann Bot-London,2005,95: 901-915.
Chiu, C.C., Lin, C.S., Hsia, A.P., et al. Mutation of a nitrate transporter, AtNRTl:4, results in a reduced petiole nitrate content and altered leaf development. Plant Cell Physiol,2004,45:1139-1148.
Chopin, F., Orsel, M., Dorbe, M.F., et al. The Arabidopsis ATNRT2.7 nitrate transporter controls nitrate content in seeds. Plant Cell,2007,19:1590-1602.
Chopin, F., Wirth, J., Dorbe, M.F., et al. The Arabidopsis nitrate transporter AtNRT2.1 is targeted to the root plasma membrane. Plant Physiol Biochem,2007,45:630-635.
Citovsky, V., Lee, L.Y., Vyas, S., et al. Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta. J Mol Biol,2006,362:1120-1131.
Czechowski, T., Bari, R., Stitt, M., et al. Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors:unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J, 2004,38:366-379.
Deng, Y., Chen, S., Lu, A., et al. Production and characterisation of the intergeneric hybrids between Dendranthema morifolium and Artemisia vulgaris exhibiting enhanced resistance to chrysanthemum aphid (Macrosiphoniellasanbourni). Planta,2010,231:693-703.
Diaz, I., Martinez, M., Isabel-LaMoneda, I., et al. The DOF protein, SAD, interacts with GAMYB in plant nuclei and activates transcription of endosperm-specific genes during barley seed development. Plant J,2005,42:652-662.
Dorbe, M.F., Caboche, M., Daniel-Vedele, F. The tomato nia gene complements a Nicotiana plumbaginifolia nitrate reductase-deficient mutant and is properly regulated. Plant Mol Biol,1992, 18:363-375.
Druege, U., Zerche, S., Kadner, R., et al. Relation between nitrogen status, carbohydrate distribution and subsequent rooting of chrysanthemum cuttings as affected by pre-harvest nitrogen supply and cold-storage. Ann Bot-London,2000,85:687-701.
Enstone, D.E., Peterson, C.A., Ma, F. Root endodermis and exodermis:structure, function, and responses to the environment. J Plant Growth Regul,2002,21:335-351.
Fan, R.C., Peng, C.C., Xu, Y.H., et al. Apple sucrose transporter SUT1 and sorbitol transporter SOT6 interact with cytochrome b5 to regulate their affinity for substrate sugars. Plant Physiol,2009,150: 1880-1901.
Fan, S.C., Lin, C.S., Hsu, P.K., et al. The Arabidopsis nitrate transporter NRT 1.7, expressed in phloem, is responsible for source-to-sink remobilization of nitrate. Plant Cell,2009,21:2750-2761.
Fan, X., Shen, Q., Ma, Z., et al. A comparison of nitrate transport in four different rice (Oryza sativa L.) cultivars. Sci China Seri C,2005,48:897-911.
Feng, H., Yan, M., Fan, X., et al. Spatial expression and regulation of rice high-affinity nitrate transporters by nitrogen and carbon status. J Exp Bot,2011,62:2319-2332.
Fernandez, P., Di Rienzo, J.A., Moschen, S., et al. Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence transcript analysis. Plant Cell Rep, 2010:1-12.
Filleur, S., Dorbe, M.F., Cerezo, M., et al. An Arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake. FEBS Lett,2001,489:220-224.
Forde, B.G. Nitrate transporters in plants:structure, function and regulation. BBA-Biomembranes,2000, 1465:219-235.
Fraisier, V., Dorbe, M.F., Daniel-Vedele, F. Identification and expression analyses of two genes encoding putative low-affinity nitrate transporters from Nicotiana plumbaginifolia. Plant Mol Biol,2001,45: 181-190.
Fraisier, V., Gojon, A., Tillard, P., et al. Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia:evidence for post-transcriptional regulation by a reduced nitrogen source. Plant J,2000,23:489-496.
Frohman, M.A., Dush, M.K., Martin, GR. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. PNAS,1988,85:8998-9002.
Gansel, X., Mu os, S., Tillard, P., et al. Differential regulation of the NO3 and NH4 transporter genes AtNrt2.1 and AtAmtl.1 in Arabidopsis:relation with long-distance and local controls by N status of the plant. Plant J,2001,26:143-155.
Gao, H., Song, A., Zhu, X., et al. The heterologous expression in Arabidopsis of a chrysanthemum Cys2/His2 zinc finger protein gene confers salinity and drought tolerance. Planta.2011:1-15.
Gasic, K., Korban, S.S. Expression of Arabidopsis phytochelatin synthase in Indian mustard(Brassica juncea) plants enhances tolerance for Cd and Zn. Planta.2007,225:1277-1285.
Gasic, K., Korban, S.S. Transgenic Indian mustard (Brassica juncea) plants expressing an Arabidopsis phytochelatin synthase (AtPCS1) exhibit enhanced As and Cd tolerance. Plant Mol Biol,2007,64: 361-369.
Girin, T., Lejay, L., Wirth, J., et al. Identification of a 150 bp cis-acting element of the AtNRT2.1 promoter involved in the regulation of gene expression by the N and C status of the plant. Plant Cell Environ,2007,30:1366-1380.
Gojon, A., Nacry, P., Davidian, J.C. Root uptake regulation:a central process for NPS homeostasis in plants. Curr Opin Plant Biol,2009,12:328-338.
Gordon, A.J., Lea, P.J., Rosenberg, C., et al. Nodule formation and function. Plant nitrogen. Berlin: Springer-Verlag.2001:101-146.
Guillaumot, D., Lelu-Walter, M.A., Germot, A., et al. Expression patterns of LmAP2Ll and LmAP2L2 encoding two-APETALA2 domain proteins during somatic embryogenesis and germination of hybrid larch (Larix × marschlinsii). J Plant Physiol,2008,165:1003-1010.
Guo, F.Q., Wang, R., Chen, M., et al. The Arabidopsis dual-affinity nitrate transporter gene AtNRTl.l (CHL1) is activated and functions in nascent organ development during vegetative and reproductive growth. Plant Cell,2001,13:1761-1777.
Guo, F.Q., Young, J., Crawford, N.M. The nitrate transporter AtNRTl.1q (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis. Plant Cell,2003,15:107-117.
Guo, J., Dai, X., Xu, W., et al. Overexpressing GSH1 and AsPCS1 simultaneously increases the tolerance and accumulation of cadmium and arsenic in Arabidopsis thaliana. Chemosphere.2008,72: 1020-1026.
Guo, J., Wang, S., Wang, J., et al. Dissection of the relationship between RACK1 and heterotrimeric G-proteins in Arabidopsis. Plant Cell Physiol,2009,50:1681-1694.
Han, Y., Cao, H., Jiang, J., et al. Rice ROOT ARCHITECTURE ASSOCIATED1 binds the proteasome subunit RPT4 and is degraded in a D-box and proteasome-dependent manner. Plant Physiol,2008, 148:843-855.
He, Q., Qiao, D., Zhang, Q., et al. Cloning and expression study of a putative high-affinity nitrate transporter gene from Dunaliella salina. J. Appl Phycol,2004,16:395-400.
Hershko, A. The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle&ast. Cell Death Differ,2005,12:1191-1197.
Hershko, A., Ciechanover, A. The ubiquitin system for protein degradation. Annu Rev Biochem,1992, 61:761-807.
Ho, C.H., Lin, S.H., Hu, H.C., et al. CHL1 functions as a nitrate sensor in plants. Cell.2009,138: 1184-1194.
Ho, C.H., Tsay, Y.F. Nitrate, ammonium, and potassium sensing and signaling. Curr Opin Plant Biol, 2010,13:604-610.
Hu, C.D., Chinenov, Y., Kerppola, T.K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell,2002,9:789-798.
Hu, C.D., Kerppola, T.K. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol,2003,21:539-545.
Huang, N.C, Chiang, C.S., Crawford, N.M., et al. CHL1 encodes a component of the low-affinity nitrate uptake system in Arabidopsis and shows cell type-specific expression in roots. Plant Cell,1996,8: 2183-2191.
Huang, N.C, Liu, K.H., Lo, H.J., et al. Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake. Plant Cell,1999,11: 1381-1392.
Huis, R., Hawkins, S., Neutelings, G. Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biol,2010,10:71.
Hwang, C.F., Lin, Y., D'Souza, T., et al. Sequences necessary for nitrate-dependent transcription of Arabidopsis nitrate reductase genes. Plant Physiol,1997,113:853-862.
Hynes, T.R., Yost, E., Mervine, S., et al. Multicolor BiFC analysis of competition among G protein p and y subunit interactions. Methods,2008,45:207-213.
Immink, R.G.H., Angenent, G.C. Transcription factors do it together:the hows and whys of studying protein-protein interactions. Trends Plant Sci,2002,7:531-534.
Iwabuchi, K., Li, B., Bartel, P., et al. Use of the two-hybrid system to identify the domain of p53 involved in oligomerization. Oncogene,1993,8:1693-1896.
Jain, M., Nijhawan, A., Tyagi, A., et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Bioph Res Co,2006,345:646-651.
Jian, B., Liu, B., Bi, Y, et al. Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol,2008,9:59.
Johnsson, N., Varshavsky, A. Split ubiquitin as a sensor of protein interactions in vivo. PNAS,1994,91: 10340-10344.
Johnsson, N., Varshavsky, A. Ubiquitin-assisted dissection of protein transport across membranes. EMBOJ,1994,13:2686-2698.
JongMyung, C., ChangYong, S., HaeJoon, C., et al. Effect of liming fertilization on changes of nutrient concentrations in rice-hull based media, crop growth and nutrient uptake of chrysanthemum. Journal of the Korean Society for Horticultural Science SCI.2001,42.
Kang, S.G., Price, J., Lin, P.C., et al. The Arabidopsis bZIP1 transcription factor is involved in sugar signaling, protein networking, and DNA binding. Mol plant,2010,3:361-373.
Katayama, H., Kawamura, Y, Tanaka, T., et al. Production and characterization of transgenic rice plants carrying a high-affinity nitrate transporter gene (OsNRT2.1). Breeding Sci,2009,59:237-243.
Kerppola, T.K. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protoc,2006,1:1278-1286.
Kiba, T., Feria-Bourrellier, A.B., Lafouge, F., et al. The Arabidopsis Nitrate Transporter NRT2.4 Plays a Double Role in Roots and Shoots of Nitrogen-Starved Plants. Plant Cell,2012,24:245-258.
Kiba, T., Kudo, T., Kojima, M., et al. Hormonal control of nitrogen acquisition:roles of auxin, abscisic acid, and cytokinin. J Exp Bot,2011,62:1399-1409.
Kikuchi, K., Ueguchi-Tanaka, M., Yoshida, K., et al. Molecular analysis of the NAC gene family in rice. Mol Gen Genet,2000,262:1047-1051.
Kodama, Y., Wada, M. Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis. Plant Mol Biol,2009,70:211-217.
Koltermann, M., Moroni, A., Gazzarini, S., et al. Cloning, functional expression and expression studies of the nitrate transporter gene from Chlorella sorokiniana (strain 211-8k). Plant Mol Biol,2003,52: 855-864.
Konishi, M., Yanagisawa, S. Identification of a nitrate-responsive cis-element in the Arabidopsis NIR1 promoter defines the presence of multiple cis-regulatory elements for nitrogen response. Plant J, 2010,63:269-282.
Kotur, Z., Mackenzie, N., Ramesh, S., et al. Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2.1. New Phytol,2012, doi: 10.1111/j.1469-8137.2012.04094.x.
Krapp, A., Fraisier, V., Scheible, W.R., et al. Expression studies of Nrt2:1Np, a putative high-affinity nitrate transporter:evidence for its role in nitrate uptake. Plant J,1998,14:723-731.
Krishnaswamy, S., Verma, S., Rahman, M.H., et al. Functional characterization of four APETALA2-family genes (RAP2.6, RAP2.6L, DREB19 and DREB26) in Arabidopsis. Plant Mol Biol,2011,75:107-127.
Krouk, G, Crawford, N.M., Coruzzi, G.M., et al. Nitrate signaling:adaptation to fluctuating environments. Curr Opin Plant Biol,2010,13:265-272.
Krouk, G, Tillard, P., Gojon, A. Regulation of the high-affinity NO3-uptake system by NRT1.1-mediated NO3- demand signaling in Arabidopsis. Plant Physiol,2006,142:1075-1086.
L vdal, T. and Lillo, C. Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Anal Biochem,2009,387:238-242.
Langer, K., Ache, P., Geiger, D., et al. Poplar potassium transporters capable of controlling K+ homeostasis and K+-dependent xylogenesis. Plant J,2002,32:997-1009.
Laugier, E., Bouguyon, E., Mauries, A., et al. Regulation of High-Affinity Nitrate Uptake in Roots of Arabidopsis Depends Predominantly on Posttranscriptional Control of the NRT2.1/NAR2.1 Transport System. Plant Physiol,2012,158:1067-1078.
Lee, S., Moon, J.S., Ko, T.S., et al. Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress. Plant Physiol,2003,131:656-663.
Lehmann, M. Anything else but GAGA:a nonhistone protein complex reshapes chromatin structure. Trends Genet,2004,20:15-22.
Lejay, L., Tillard, P., Lepetit, M., et al. Molecular and functional regulation of two NO3- uptake systems by N-and C-status of Arabidopsis plants. Plant J,1999,18:509-519.
Lejay, L., Wirth, J., Pervent, M., et al. Oxidative pentose phosphate pathway-dependent sugar sensing as a mechanism for regulation of root ion transporters by photosynthesis. Plant Physiol,2008,146: 2036-2053.
Li, E., Wang, S., Liu, Y., et al. OVATE FAMILY PROTEIN4 (OFP4) interaction with KNAT7 regulates secondary cell wall formation in Arabidopsis thaliana. Plant J,2011,67:328-341.
Li, J.Y., Fu, Y.L., Pike, S.M., et al. The Arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance. Plant Cell,2010,22:1633-1646.
Li, W., Wang, Y., Okamoto, M., et al. Dissection of the AtNRT2.1:AtNRT2.2 inducible high-affinity nitrate transporter gene cluster. Plant Physiol,2007,143:425-433.
Lin, C.M., Koh, S., Stacey, G., et al. Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRTl, from rice. Plant Physiol,2000,122:379-388.
Lin, S.H., Kuo, H.F., Canivenc, G., et al. Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport. Plant Cell,2008,20:2514-2528.
Lin, Y., Lai, Z. Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree. Plant Sci,2010,178:359-365.
Lindsey, K., Wei, W, Clarke, M.C., et al. Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants. Transgenic Res,1993,2:33-47.
Little, D.Y., Rao, H., Oliva, S., et al. The putative high-affinity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues. PNAS,2005,102:13693-13698.
Liu, K.H., Huang, C.Y., Tsay, Y.F. CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake. Plant Cell,1999,11:865-874.
Liu, K.H., Tsay, Y.F. Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation. EMBO J,2003,22:1005-1013.
Liu, L., Zhu, K., Yang, Y., et al. Molecular cloning, expression profiling and trans-activation property studies of a DREB2-like gene from chrysanthemum (Dendranthema vestitum). J Plant Res,2008, 121:215-226.
Liu, Y.G., Mitsukawa, N., Oosumi, T., et al. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J,1995,8:457-463.
Liu, Y.G., Whittier, R.F. Thermal asymmetric interlaced PCR:automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics,1995,25: 674-681.
Liu, Z., Gu, C., Chen, F., et al. Heterologous Expression of a Nelumbo nucifera Phytochelatin Synthase Gene Enhances Cadmium Tolerance in Arabidopsis thaliana. Appl Biochem. Biotechnol,2012: 1-13.
Livak, K.J., Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT Method. Methods,2001,25:402-408.
Luo, H., Chen, S., Jiang, J., et al. The expression of floral organ identity genes in contrasting water lily cultivars. Plant Cell Rep,2011:1-10.
Luo, H., Chen, S., Wan, H., et al. Candidate reference genes for gene expression studies in water lily. Anal Biochem,2010,404:100-102.
Ma, Z., Zhang, H., Zheng, J., et al. Interaction between M-Like Protein and Macrophage Thioredoxin Facilitates Antiphagocytosis for Streptococcus equi ssp. zooepidemicus. PloS one,2012,7:e32099.
Malagoli, P., Laine, P., Le Deunff, E., et al. Modeling nitrogen uptake in oilseed rape cv Capitol during a growth cycle using influx kinetics of root nitrate transport systems and field experimental data. Plant Physiol,2004,134:388-400.
Martin, R.C., Hollenbeck, V.G., Dombrowski, J.E. Evaluation of Reference Genes for Quantitative RT-PCR in Lolium perenne. Crop Sci,2008,48:1881-1887.
Masclaux-Daubresse, C., Daniel-Vedele, F., Dechorgnat, J., et al. Nitrogen uptake, assimilation and remobilization in plants:challenges for sustainable and productive agriculture. Ann Bot-London, 2010,105:1141-1157.
Matsumura, H., Xie, Y., Shirakata, S., et al. Crystal structures of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylases. Structure.2002,10:1721-1730.
Mazars, G.R., Moyret, C., Jeanteur, P., et al. Direct sequencing by thermal asymmetric PCR. Nucleic Acids Res,1991,19:4783.
McNulty, S.E., Toscano, W. Transcriptional regulation of glyceraldehyde-3-phosphate dehydrogenase by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem Bioph Res Co,1995,212:165-171.
Miao, H., Jiang, B., Chen, S., et al. Isolation of a gibberellin 20-oxidase cDNA from and characterization of its expression in chrysanthemum. Plant breeding.2010,128:1-8.
Miller, A.J., Shen, Q., Xu, G. Freeways in the plant:transporters for N, P and S and their regulation. Curr Opin Plant Biol,2009,12:284-290.
Morere-Le Paven, M.C., Viau, L., Hamon, A., et al. Characterization of a dual-affinity nitrate transporter MtNRT1.3 in the model legume Medicago truncatula. J Exp Bot,2011,62:5595-5605.
Nasholm, T., Kielland, K., Ganeteg, U. Uptake of organic nitrogen by plants. New Phytol,2009,182: 31-48.
Nandanavanam, R. Investigations on the contribution of AtNRT2.6 gene to nitrate transport in Arabidopsis thaliana.2011.
Nazoa, P., Vidmar, J.J., Tranbarger, T.J., et al. Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana:responses to nitrate, amino acids and developmental stage. Plant Mol Biol, 2003,52:689-703.
Nicot, N., Hausman, J., Hoffmann, L., et al. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot,2005,56:2907-2914.
Nicot, N., Hausman, J.F., Hoffmann, L., et al. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot,2005,56:2907.
Okamoto, M., Kumar, A., Li, W., et al. High-affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1. Plant Physiol,2006,140:1036-1046.
Okamoto, M., Vidmar, J.J., Glass, A.D.M. Regulation of NRT1 and NRT2 gene families of Arabidopsis thaliana:responses to nitrate provision. Plant Cell Physiol,2003,44:304-317.
Orsel, M., Chopin, F., Leleu, O., et al. Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interaction. Plant Physiol,2006,142: 1304-1317.
Orsel, M., Eulenburg, K., Krapp, A., et al. Disruption of the nitrate transporter genes AtNRT2.1 and AtNRT2.2 restricts growth at low external nitrate concentration. Planta,2004,219:714-721.
Orsel, M., Krapp, A., Daniel-Vedele, F. Analysis of the NRT2 nitrate transporter family in Arabidopsis. Structure and gene expression. Plant Physiol,2002,129:886.
Perez, R., Tupac-Yupanqui, I., Dunner, S. Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue. BMC Mol Biol,2008,9:79.
Pandey, S., Assmann, S.M. The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein a subunit GPA1 and regulates abscisic acid signaling. Plant Cell,2004,16: 1616-1632.
Paolacci, A., Tanzarella, O., Porceddu, E., et al. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Biol,2009,10:11.
Plett, D., Toubia, J., Garnett, T., et al. Dichotomy in the NRT gene families of dicots and grass species. PloS one.2010,5:e15289.
Quesada, A., Galvan, A., Fernandez, E. Identification of nitrate transporter genes in Chlamydomonas reinhardtii. Plant J,1994,5:407-419.
Quesada, A., Hidalgo, J., Fernandez, E. Three Nrt2 genes are differentially regulated in Chlamydomonas reinhardtii. Mol Gen Genet,1998,258:373-377.
Raab, T.K., Terry, N. Carbon, nitrogen, and nutrient interactions in Beta vulgaris L. as influenced by nitrogen source, NO3- versus NH4+. Plant Physiol,1995,107:575-585.
Ramakers, C., Ruijter, J., Deprez, R., et al. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett,2003,339:62-66.
Rastogi, R., Bate, N.J., Sivasankar, S., et al. Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants. Plant Mol Biol,1997,34:465-476.
Reid, K., Olsson, N., Schlosser, J., et al. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Mol Biol, 2006,6:27.
Reisenauer, H. Mineral nutrients in soil solution. Environ Biol,1966:507-508.
Remans, T., Nacry, P., Pervent, M., et al. The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches. PNAS,2006,103: 19206-19211.
Remans, T., Nacry, P., Pervent, M., et al. A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to nitrogen limitation in Arabidopsis. Plant Physiol,2006,140:909-921.
Rexach, J., Llamas, A., Fernandez, E., et al. The activity of the high-affinity nitrate transport system I (NRT2;1, NAR2) is responsible for the efficient signalling of nitrate assimilation genes in Chlamydomonas reinhardtii. Planta,2002,215:606-611.
Richard-Molard, C., Krapp, A., Brun, F., et al. Plant response to nitrate starvation is determined by N storage capacity matched by nitrate uptake capacity in two Arabidopsis genotypes. J Exp Bot,2008, 59:779-791.
Rinehart, J.A., Petersen, M.W., John, M.E. Tissue-specific and developmental regulation of cotton gene FbL2A (Demonstration of promoter activity in transgenic plants). Plant Physiol,1996,112: 1331-1341.
Rioux, S., Neyt, C., Di Paolo, E., et al. Transcriptional regulation, occurrence and putative role of the Pht family of Streptococcus pneumoniae. Microbiology,2011,157:336-348.
Salamov, A., Solovyev, V. The Gene-Finder computer tools for analysis of human and model organisms genome sequences. Ismb,1997,5:294-302.
Schallau, A., Kakhovskaya, I., Tewes, A., et al. Phylogenetic footprints in fern spore-and seed-specific gene promoters. Plant J,2008,53:414-424.
Shan, H., Chen, S., Jiang, J., et al. Heterologous Expression of the Chrysanthemum R2R3-MYB Transcription Factor CmMYB2 Enhances Drought and Salinity Tolerance, Increases Hypersensitivity to ABA and Delays Flowering in Arabidopsis thaliana. Mol Biotechnol,2011: 1-14.
Sharma, M.K., Kumar, R., Solanke, A.U., et al. Identification, phylogeny, and transcript profiling of ERF family genes during development and abiotic stress treatments in tomato. Mol Gen Genet,2010: 1-21.
Shchennikova, A.V., Shulga, O.A., Immink, R., et al. Identification and characterization of four chrysanthemum MADS-box genes, belonging to the APETALA1/FRUITFULL and SEPALLATA3 subfamilies. Plant Physiol,2004,134:1632-1641.
Siebrecht, S., Mack, G., Tischner, R. Function and contribution of the root tip in the induction of NO3-uptake along the barley root axis. J Exp Bot,1995,46:1669-1676.
Silveira, E.D., Alves-Ferreira, M., Guimaraes, L.A., et al. Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha. BMC Mol Biol,2009,9:84.
Solovyev, V. Statistical approaches in eukaryotic gene prediction. Handbook of statistical genetics.2001.
Solovyev, V.V. and Shahmuradov, I. PromH:promoters identification using orthologous genomic sequences. Nucleic Acids Res,2003,31:3540-3545.
Sturzenbaum, S., Kille, P. Control genes in quantitative molecular biological techniques:the variability of invariance. Comp Biochem Physiol B:Biochem Mol Biol,2001,130:281-289.
Stagljar, I., Korostensky, C., Johnsson, N., et al. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. PNAS,1998,95:5187-5192.
Strahm, B.D., Harrison, R.B. Nitrate sorption in a variable-charge forest soil of the Pacific Northwest. Soil Sci,2006,171:313-321.
Suzuki, T., Higgins, P., Crawford, D. Control selection for RNA quantitation. Biotechniques,2000,29: 332-337.
Takahashi-Terada, A., Kotera, M., Ohshima, K., et al. Maize Phosphoenolpyruvate Carboxylase. J Biol Chem,2005,280:11798-11806.
Terauchi, R., Kahl, G. Rapid isolation of promoter sequences by TAIL-PCR:The 5'-flanking regions of Pal and Pgi genes from yams (Dioscorea). Mol Gen Genet,2000,263:554-560.
Thaminy, S., Auerbach, D., Arnoldo, A., et al. Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system. Genome Res,2003,13:1744-1753.
Thomas, C., Meyer, D., Wolff, M., et al. Molecular characterization and spatial expression of the sunflower ABP1 gene. Plant Mol Biol,2003,52:1025-1036.
Thornton, B. Inhibition of nitrate influx by glutamine in Lolium perenne depends upon the contribution of the HATS to the total influx. J Exp Bot,2004,55:761-769.
Tjaden, G, Edwards, J.W., Coruzzi, G.M. Cis elements and trans-acting factors affecting regulation of a nonphotosynthetic light-regulated gene for chloroplast glutamine synthetase. Plant Physiol,1995, 108:1109-1117.
Todd, C.D., Zeng, P., Huete, A.M.R., et al. Transcripts of MYB-like genes respond to phosphorous and nitrogen deprivation in Arabidopsis. Planta.2004,219:1003-1009.
Tong, Y., Zhou, J.J., Li, Z., et al. A two-component high-affinity nitrate uptake system in barley. Plant J, 2005,41:442-450.
Tong, Z., Gao, Z., Wang, F., et al. Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol Biol,2009,10:71.
Tong, Z., Hong, B., Yang, Y., et al. Overexpression of two chrysanthemum DgDREBl group genes causing delayed flowering or dwarfism in Arabidopsis. Plant Mol Biol,2009,71:115-129.
Topping, J.F., Lindsey, K. Promoter trap markers differentiate structural and positional components of polar development in Arabidopsis. Plant Cell,1997,9:1713-1725.
Trevisan, S., Borsa, P., Botton, A., et al. Expression of two maize putative nitrate transporters in response to nitrate and sugar availability. Plant Biology,2008,10:462-475.
Tsay, Y.F., Chiu, C.C., Tsai, C.B., et al. Nitrate transporters and peptide transporters. FEBS Lett,2007, 581:2290-2300.
Tsay, Y.F., Schroeder, J.I., Feldmann, K.A., et al. The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter. Cell,1993,72:705-713.
Tzfira, T., Citovsky, V. Partners-in-infection:host proteins involved in the transformation of plant cells by Agrobacterium. Trends Cell Biol,2002,12:121-129.
Vandesompele, J., De Preter, K., Pattyn, F., et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol,2002,3.
Vidmar, J.J., Zhuo, D., Siddiqi, M.Y., et al. Isolation and Characterization of HvNRT2.3 and HvNRT2.4, cDNAs Encoding High-Affinity Nitrate Transporters from Roots of Barley. Plant Physiol,2000, 122:783-792.
Vidmar, J.J., Zhuo, D., Siddiqi, M.Y., et al. Regulation of high-affinity nitrate transporter genes and high-affinity nitrate influx by nitrogen pools in roots of barley. Plant Physiol,2000,123:307-318.
Von Arnim, A. Subcellular Localization of GUS-and GFP-Tagged Proteins in Onion Epidermal Cells. Cold Spring Harbor Protocols,2007,2007:pdb. prot4689.
Voss, A.K., Thomas, T., Gruss, P. Compensation for a gene trap mutation in the murine microtubule-associated protein 4 locus by alternative polyadenylation and alternative splicing. Dev Dynam,1998,212:258-266.
Wan, H., Zhao, Z., Qian, C, et al. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem,2010,399: 257-261.
Wang, R., Liu, D., Crawford, N.M. The Arabidopsis CHL1 protein plays a major role in high-affinity nitrate uptake. PNAS,1998,95:15134-15139.
Wang, Y.Y., Tsay, Y.F. Arabidopsis Nitrate Transporter NRT1.9 Is Important in Phloem Nitrate Transport. Plant Cell,2011,23:1945-1957.
Wang, Z., Ye, S., Li, J., et al. Fusion primer and nested integrated PCR (FPNI-PCR):a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning. BMC Biotech, 2011,11:109.
Watanabe, M., Mochida, K., Kato, T., et al. Comparative genomics and reverse genetics analysis reveal indispensable functions of the serine acetyltransferase gene family in Arabidopsis. Plant Cell,2008, 20:2484-2496.
Weis, K. Regulating access to the genome:nucleocytoplasmic transport throughout the cell cycle. Cell. 2003,112:441-451.
Wirth, J., Chopin, F., Santoni, V., et al. Regulation of root nitrate uptake at the NRT2.1 protein level in Arabidopsis thaliana. J Biol Chem,2007,282:23541-23552.
Xu, M., Zhang, B., Su, X., et al. Reference gene selection for quantitative real-time polymerase chain reaction in Populus. Anal Biochem,2011,408:337-339.
Xu, Z., Zhou, Y, Meng, C., et al. Characterization of promoter expression patterns of OsNrt2.1, a nitrate transporter gene of rice (Oryza sativa L.). Frontiers of Agriculture in China.2009,3:402-412.
Yan, J., He, C, Zhang, H. The BAG-family proteins in Arabidopsis thaliana. Plant Sci,2003,165:1-7.
Yan, M., Fan, X., Feng, H., et al. Rice OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a nitrate transporters to provide uptake over high and low concentration ranges. Plant Cell Environ, 2011,1360-1372.
Yanagisawa, S. Dofl and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize. Plant J,2000,21:281-288.
Yanagisawa, S., Schmidt, R.J. Diversity and similarity among recognition sequences of Dof transcription factors. Plant J,1999,17:209-214.
Yang, Y, Wu, J., Zhu, K., et al. Identification and characterization of two chrysanthemum (Dendronthemax moriforlium) DREB genes, belonging to the AP2/EREBP family. Mol Biol Rep, 2009,36:71-81.
Yin, D., Chen, S., Chen, F., et al. Morphological and physiological responses of two chrysanthemum cultivars differing in their tolerance to waterlogging. Environ Exp Bot.,2009,67:87-93.
Yin, L.P., Li, P., Wen, B., et al. Characterization and expression of a high-affinity nitrate system transporter gene (TaNRT2.1) from wheat roots, and its evolutionary relationship to other NTR2 genes. Plant Sci,2007,172:621-631.
Yong, Z., Kotur, Z., Glass, A.D.M. Characterization of an intact two-component high-affinity nitrate transporter from Arabidopsis roots. Plant J,2010,63:739-748.
Zhang, G, Chen, M., Chen, X., et al. Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean (Glycine max L.). J Exp Bot,2008,59:4095-4107.
Zhang, H., Jennings, A., Barlow, P.W., et al. Dual pathways for regulation of root branching by nitrate. PNAS,1999,96:6529-6534.
Zhang, H., Rong, H., Pilbeam, D. Signalling mechanisms underlying the morphological responses of the root system to nitrogen in Arabidopsis thaliana. J Exp Bot,2007,58:2329-2338.
Zheng, C., Oba, S., Matsui, S., et al. Effects of calcium and magnesium treatments on growth, nutrient contents, ethylene production, and gibberellin content in Chrysanthemum plants. J Jap Soc Hortic Sci,2005,74:144-149.
Zhou, J.J., Fernandez, E., Galvan, A., et al. A high affinity nitrate transport system from Chlamydomonas requires two gene products. FEBS Lett,2000,466:225-227.
Zhou, J.J., Theodoulou, F.L., Muldin, I., et al. Cloning and functional characterization of a Brassica napus transporter that is able to transport nitrate and histidine. J Biol Chem,1998,273: 12017-12023.
Zhuang, J., Cai, B., Peng, R.H., et al. Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa. Biochem Bioph Res Co,2008,371:468-474.
Zhuo, D., Okamoto, M., Vidmar, J.J., et al. Regulation of a putative high-affinity nitrate transporter (Nrt2;1At) in roots of Arabidopsis thaliana. Plant J,1999,17:563-568.
陈柏君,孙超,王勇,等.锚定PCR(Anchored PCR):一种新的染色体步行方法.科学通报,2004,49(15)：1569-1571.
陈军营,孙佩,王德勤,等.一种改良的克隆小麦GLP3基因启动子的TAIL-PCR技术.植物生理学通讯,2007,43(4)：754-758.
春亮.玉米氮高效品种选育及根系形态对低氮反应的遗传分析.中国农业大学博士论文,2004.
崔颖.甜菜M14品系表达基因M14-100, M14-265, M14-G12 cDNA全长克隆与序列分析.黑龙江大学硕士论文,2008.
范晓荣.作物硝态氮跨膜运输的生理和分子机理.南京农业大学博士论文,2005.
方进,李素文.转基因水稻T-DNA侧翼序列的扩增与分析.遗传学报,2001,28(4)：345-351.
高翔,陈磊,云鹏,等.不同基因型小麦侧根生长对硝态氮的响应差异.植物营养与肥料学报,2010,16(4),1013-1019.
管志勇,陈发棣,滕年军,等.5种菊花近缘种属植物的耐盐性比较.中国农业科学,2010,43(4)：787-794.
管志勇,陈素梅,陈发棣,等.32个菊花近缘种属植物耐盐性筛选.中国农业科学,2010,43(19)：4063-4071.
郭亚芬,米国华,陈范骏,等.局部供应硝酸盐诱导玉米侧根生长的基因型差异.植物营养与肥料学报,2005,11(2)：155-159.
洪娟.油菜氮高效种质的筛选及其生理机制的初步研究.华中农业大学硕士论文,2007.
胡尚连,曹颖,黄胜雄,等.慈竹4CL基因的克隆及其生物信息学分析.西北农林科技大学学报：自然科学版,2009,37(8)：204-210.
黄长兵,房伟民,杨勇,等.不同水平和形态氮素供应对盆栽小菊外观品质和光合特性的影响.浙江农业学报,2010,22(1)：45-50.
姜贝贝,房伟民,陈发棣等.氮磷钾配比对切花菊‘神马’生长发育的影响.浙江林学院学报,2009,25(6)：692-697.
蒋志平,鲁剑巍,张文君,等.菊花氮钾肥效及配方筛选研究.中国农学通报,2010,26(10)：182-186.
靳苗苗,杨青川,金洪,等.紫花苜蓿柠檬酸合成酶基因的克隆及对烟草的转化.草地学报,2010,18(4)：550-555.
康健,裴蓓,周丕生,等.不同氮素水平对郁金香磷素与钾素的累积与分配的影响.上海交通大学学报：农业科学版,2006,23(4)：377-382.
李雪妮.棉花氮高效品种筛选及其机理的初步研究.新疆农业大学硕士论文,2007.
李玉中,祝廷成,Redmann, R.三种利用类型羊草草地氮总矿化,硝化和无机氮消耗速率的比较研究.生态学报,2002,22(5)：668-673.
李悦,张涛,齐秀兰.切花菊氮磷钾营养特性的研究进展.安徽农业科学,2005,33(8)：1486-1487.
李秋莉,张毅,尹辉,等.辽宁碱蓬甜菜碱醛脱氢酶基因(BADH)启动子分离及序列分析.生物工程学报,2006,22(1)：77-81.
李芳,沈欣杰,赵凯,等.苹果山梨醇运输基因MdSOT3启动子克隆与功能分析.园艺学报,2011,38(增刊)：2457
刘霞,邓馨.复苏植物牛耳草BhLEA2基因启动子的分离和基因表达模式研究.自然科学进展,2009,19(1)：39-44.
刘晓庆,丁志鑫,刘晶,等.2种PCR方法扩增大豆rbcS启动子5'侧翼序列比较.吉林农业大学学报,2011,33(5)：511-517.
刘云,何书萱.菊花无土栽培技术研究.山西农业科学,1999,27(3)：62-65.
刘振林,曹华雯,夏新莉,等.甘菊BADH基因启动子的克隆与瞬时表达分析.园艺学报,2008,35(12)：1787-1794.
卢艳丽,陆卫平,王继丰,等.不同基因型糯玉米氮素吸收利用效率的研究.植物营养与肥料学报,2006,12(3)：321-326.
马彩艳,刘冬成,阳文龙等.短柄草磷转运蛋白家族基因的克隆及表达分析.分子植物育种,2011,9(4)：482-490.
马传营.中国水仙(Narcissus tazetta L. var. chinensis Roem)花香相关基因DXPS, DXR和PAL的克隆.福建农林大学硕士论文,2010.
马翠,范晓荣,徐国华.武运粳7号超表达OsNRT1.2后对硝酸盐的响应.中国水稻科学,2011,25(4)：349-356.
马赛箭,苏乔,吴凇,等.盐角草磷酸乙醇胺N-甲基转移酶基因(SePEAMT)启动子的克隆及瞬时表达.中国生物工程杂志,2008,28(8)：69-73.
彭少兵,黄见良,钟旭华,等.提高中国稻田氮肥利用率的研究策略.中国农业科学,2002,35(9)：1095-1103.
曲宪成,崔严慧,周正峰,等.团头鲂促性腺激素GtHI β亚基基因5’端启动子区克隆及表达载体构建.水生生物学报,2008,32(4)：558-567.
曲宪成,崔严慧,周正峰,等.团头鲂促性腺激素GtⅡ β亚基基因5’端启动子区克隆及表达载体构建.中国水产科学,2010,17(4)：649-658.
沈瑾.苹果醇酰基转移酶基因(MdAAT2)启动子克隆与功能鉴定.山东农业大学硕士论文,2009.
史正军,樊小林.作物对氮素养分高效吸收的根系形态学研究进展.广西农业生物科学,2003,22(3),225-229.
宋长年,房经贵,王晨,·等.基于EST库的枳APETALA2基因cDNA克隆及其表达分析.园艺学报,2009,36(6)：799-806.
孙凯.切花菊‘神马’氮营养研究.北京林业大学硕士论文,2007.
王小兵,吴平,胡彬,等.硝态氮(N03-)对水稻侧根生长及其氮吸收的影响.植物学报,2002,6,678-683.
王艳,米国华,陈范骏,等.玉米氮素吸收的基因型差异及其与根系形态的相关性.生态学报,2003,23(2),297-302.
魏跃,陈啸寅,王全智,等.应用改良TAIL-PCR克隆黄瓜6PGDH基因上游序列.浙江农业学报,2011,23(5)：900-904.
乌云塔娜,刘学英,李振国.中国梨S-RNase基因启动子的TAIL-PCR克隆及功能预测.南京林业大学学报：自然科学版,2010,34(4)：21-25.
吴金燕.番木瓜eIF4E和eIFiso4E的克隆及其与PRSV-VPg互作研究.海南大学硕士论文,2010.
徐海荣.水稻新型硝酸盐转运蛋白基因OsTNrt2.1的克隆,表达和遗传转化.河北农业大学硕士论文.2007.
许瑛,陈煜,陈发棣,等.菊花耐寒特性分析及其评价指标的确定.中国农业科学,2009,42(3)：974-981.
杨帆,林俊芳,郭安平,等.利用TAIL-PCR技术克隆猴头菇β-glucosidase基因.食品与发酵工业,2005,31(5)：9-14.
杨伟,陈发棣,陈素梅,等.不同菊花品种不同开花阶段舌状花耐热性比较.南京农业大学学报,2011,34(2)：47-53.
杨迎东.氮营养对切花菊‘优香’品质影响研究.中国农业科学院硕士论文,2010.
杨宗灿.利用BiFC技术研究拟南芥GPX3和RRTF1与DOS1体内相互作用.河南大学硕士论文,2011.
尹冬梅,管志勇,陈素梅,等.菊花及其近缘种属植物耐涝评价体系建立及耐涝性鉴定.植物遗传资源学报,2009,10(3)：399-404.
尹西翔.水稻氮高效品种的筛选及其机理的初步探讨.华中农业大学硕士论文,2005.
应革,武威,何朝族.’[AIL-PCR方法快速分离Xcc致病相关基因序列.生物工程学报,2002,18(2)：182-186.
于莹.利用羊草EST文库克隆耐盐碱相关基因.吉林大学博士论文,2011.
赵付江.茄子氮高效基因型筛选及氮效率基因型差异机理的研究.河北农业大学硕士论文,2008.
赵九洲,陈洁敏,陈松笔,等.无土基质与营养液EC值对切花菊生长发育的影响.园艺学报,1999,26(5)：327-330.
赵英,牛建新.利用3'和5'RACE克隆新疆梨树苹果茎痘病毒末端序列.园艺学报,2008,35(4)：493-500.
郑成淑,王文莉,孙宪芝,等.硝酸钙处理对菊花扦插生根及抗氧化酶活性的影响.园艺学报,2008,35(2)：263-268.
郑路,傅玉兰.菊花抗寒性与营养特性的研究.园艺学报,1994,21(2)：185-188.
周忠新.不同氮素水平下施磷对小麦产量和品质的影响及其生理基础.山东农业大学硕士论文,2006.
朱树勇,迟伟,张立新.分裂泛素化酵母双杂交系统研究光合膜蛋白相互作用.生物化学与生物物理进展.2006,33(4)：350-356.
祝丽香,王建华,毕建杰,等.不同氮素用量对杭白菊养分累积,转运及产量的影响.植物营养与肥料学报,2010,16(4)：992-997.
Almagro, A., Lin, S.H., Tsay, Y.F. Characterization of theE Arabidopsis nitrate transporter NRT 1.6 reveals a role of nitrate in early embryo development. Plant Cell,2008,20:3289-3299.
Andersen, C.L., Jensen, J.L.,Orntoft, T.F. Normalization of real-time quantitative reverse transcription-PCR data:a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res,2004,64:5245.
Anderson, L.E., Carol, A.A. Enzyme co-localization in the pea leaf cytosol:3-P-glycerate kinase, glyceraldehyde-3-P dehydrogenase, triose-P isomerase and aldolase. Plant Sci,2005,169:620-628.
Araki, R., Hasegawa, H. Expression of rice (Oryza sativa L.) genes involved in high-affinity nitrate transport during the period of nitrate induction. Breeding Sci,2006,56:295-302.
Armengaud, P., Breitling, R., Amtmann, A. The potassium-dependent transcriptome of Arabidopsis reveals a prominent role of jasmonic acid in nutrient signaling. Plant Pysiol,2004,136:2556-2576.
Aronheim, A., Zandi, E., Hennemann, H., et al. Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol Cell Biol,1997,17:3094-3102.
Artico, S., Nardeli, S.M., Brilhante, O., et al. Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data. BMC Plant Biol,2010,10:49.
Baxter, I., Hosmani, P.S., Rus, A., et al. Root suberin forms an extracellular barrier that affects water relations and mineral nutrition in Arabidopsis. PLoS Genet,2009,5:e1000492.
Bernier, G., Havelange, A., Houssa, C., et al. Physiological signals that induce flowering. Plant Cell, 1993,5:1147-1155.
Bezier, A., Lambert, B., Baillieul, F. Study of defense-related gene expression in grapevine leaves and berries infected with Botrytis cinerea. Eur J Plant Pathol,2002,108:111-120.
Bi, Y.M., Zhang, Y, Signorelli, T., et al. Genetic analysis of Arabidopsis GATA transcription factor gene family reveals a nitrate-inducible member important for chlorophyll synthesis and glucose sensitivity. Plant J,2005,44:680-692.
Broder, Y.C., Katz, S., Aronheim, A. The ras recruitment system, a novel approach to the study of protein-protein interactions. Curr Biol,1998,8:1121-1130.
Brunner, A., Yakovlev, I., Strauss, S. Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol,2004,4:14.
Bustin, S. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol,2000,25:169-193.
Bustin, S. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR):trends and problems. J Mol Endocrinol,2002,29:23-39.
Cai, C., Wang, J.Y., Zhu, Y.G, et al. Gene Structure and Expression of the High-affinity Nitrate Transport System in Rice Roots. J Integr Plant Biol,2008,50:443-451.
Cai, C., Zhao, X.Q., Zhu, Y.G, et al. Regulation of the high-affinity nitrate transport system in wheat roots by exogenous abscisic acid and glutamine. J Integr Plant Biol,2007,49:1719-1725.
Cai, W., Ma, J., Chi, W., et al. Cooperation of LPA3 and LPA2 is essential for photosystem II assembly in Arabidopsis. Plant Physiol,2010,154:109-120.
Camanes, G, Pastor, V., Cerezo, M., et al. A deletion in NRT2.1 attenuates Pseudomonas syringae-induced hormonal perturbation, resulting in primed plant defenses. Plant Pysiol,2012,158: 1054-1066.
Canning, R.E., Kramer, P.J. Salt absorption and accumulation in various regions of roots. Am J Bot, 1958:378-382.
Castaings, L., Marchive, C., Meyer, C., et al. Nitrogen signalling in Arabidopsis:how to obtain insights into a complex signalling network. J Exp Bot,2011,62:1391-1397.
Cerezo, M., Tillard, P., Filleur, S., et al. Major Alterations of the Regulation of Root NO3 Uptake Are Associated with the Mutation of Nrt2.1 and Nrt2.2 Genes in Arabidopsis. Plant Physiol,2001,127: 262-271.
Chen, A., Gu, M., Sun, S., et al. Identification of two conserved cis-acting elements, MYCS and P1BS, involved in the regulation of mycorrhiza-activated phosphate transporters in eudicot species. New Phytol,2011,189:1157-1169.
Chen, S., Miao, H., Chen, F., et al. Analysis of Expressed Sequence Tags (ESTs) Collected from the Inflorescence of Chrysanthemum. Plant Mol Biol Rep,2009,27:503-510.
Chen, Y, Chen, S., Chen, F., et al. Functional Characterization of a Chrysanthemum dichrum Stress-Related Promoter. Mol Biotechnol,2011:1-9.
Chen, Y.F., Etheridge, N., Schaller, G.E. Ethylene signal transduction. Ann Bot-London,2005,95: 901-915.
Chiu, C.C., Lin, C.S., Hsia, A.P., et al. Mutation of a nitrate transporter, AtNRTl:4, results in a reduced petiole nitrate content and altered leaf development. Plant Cell Physiol,2004,45:1139-1148.
Chopin, F., Orsel, M., Dorbe, M.F., et al. The Arabidopsis ATNRT2.7 nitrate transporter controls nitrate content in seeds. Plant Cell,2007,19:1590-1602.
Chopin, F., Wirth, J., Dorbe, M.F., et al. The Arabidopsis nitrate transporter AtNRT2.1 is targeted to the root plasma membrane. Plant Physiol Biochem,2007,45:630-635.
Citovsky, V., Lee, L.Y., Vyas, S., et al. Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta. J Mol Biol,2006,362:1120-1131.
Czechowski, T., Bari, R., Stitt, M., et al. Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors:unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J, 2004,38:366-379.
Deng, Y., Chen, S., Lu, A., et al. Production and characterisation of the intergeneric hybrids between Dendranthema morifolium and Artemisia vulgaris exhibiting enhanced resistance to chrysanthemum aphid (Macrosiphoniellasanbourni). Planta,2010,231:693-703.
Diaz, I., Martinez, M., Isabel-LaMoneda, I., et al. The DOF protein, SAD, interacts with GAMYB in plant nuclei and activates transcription of endosperm-specific genes during barley seed development. Plant J,2005,42:652-662.
Dorbe, M.F., Caboche, M., Daniel-Vedele, F. The tomato nia gene complements a Nicotiana plumbaginifolia nitrate reductase-deficient mutant and is properly regulated. Plant Mol Biol,1992, 18:363-375.
Druege, U., Zerche, S., Kadner, R., et al. Relation between nitrogen status, carbohydrate distribution and subsequent rooting of chrysanthemum cuttings as affected by pre-harvest nitrogen supply and cold-storage. Ann Bot-London,2000,85:687-701.
Enstone, D.E., Peterson, C.A., Ma, F. Root endodermis and exodermis:structure, function, and responses to the environment. J Plant Growth Regul,2002,21:335-351.
Fan, R.C., Peng, C.C., Xu, Y.H., et al. Apple sucrose transporter SUT1 and sorbitol transporter SOT6 interact with cytochrome b5 to regulate their affinity for substrate sugars. Plant Physiol,2009,150: 1880-1901.
Fan, S.C., Lin, C.S., Hsu, P.K., et al. The Arabidopsis nitrate transporter NRT 1.7, expressed in phloem, is responsible for source-to-sink remobilization of nitrate. Plant Cell,2009,21:2750-2761.
Fan, X., Shen, Q., Ma, Z., et al. A comparison of nitrate transport in four different rice (Oryza sativa L.) cultivars. Sci China Seri C,2005,48:897-911.
Feng, H., Yan, M., Fan, X., et al. Spatial expression and regulation of rice high-affinity nitrate transporters by nitrogen and carbon status. J Exp Bot,2011,62:2319-2332.
Fernandez, P., Di Rienzo, J.A., Moschen, S., et al. Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence transcript analysis. Plant Cell Rep, 2010:1-12.
Filleur, S., Dorbe, M.F., Cerezo, M., et al. An Arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake. FEBS Lett,2001,489:220-224.
Forde, B.G. Nitrate transporters in plants:structure, function and regulation. BBA-Biomembranes,2000, 1465:219-235.
Fraisier, V., Dorbe, M.F., Daniel-Vedele, F. Identification and expression analyses of two genes encoding putative low-affinity nitrate transporters from Nicotiana plumbaginifolia. Plant Mol Biol,2001,45: 181-190.
Fraisier, V., Gojon, A., Tillard, P., et al. Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia:evidence for post-transcriptional regulation by a reduced nitrogen source. Plant J,2000,23:489-496.
Frohman, M.A., Dush, M.K., Martin, GR. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. PNAS,1988,85:8998-9002.
Gansel, X., Mu os, S., Tillard, P., et al. Differential regulation of the NO3 and NH4 transporter genes AtNrt2.1 and AtAmtl.1 in Arabidopsis:relation with long-distance and local controls by N status of the plant. Plant J,2001,26:143-155.
Gao, H., Song, A., Zhu, X., et al. The heterologous expression in Arabidopsis of a chrysanthemum Cys2/His2 zinc finger protein gene confers salinity and drought tolerance. Planta.2011:1-15.
Gasic, K., Korban, S.S. Expression of Arabidopsis phytochelatin synthase in Indian mustard(Brassica juncea) plants enhances tolerance for Cd and Zn. Planta.2007,225:1277-1285.
Gasic, K., Korban, S.S. Transgenic Indian mustard (Brassica juncea) plants expressing an Arabidopsis phytochelatin synthase (AtPCS1) exhibit enhanced As and Cd tolerance. Plant Mol Biol,2007,64: 361-369.
Girin, T., Lejay, L., Wirth, J., et al. Identification of a 150 bp cis-acting element of the AtNRT2.1 promoter involved in the regulation of gene expression by the N and C status of the plant. Plant Cell Environ,2007,30:1366-1380.
Gojon, A., Nacry, P., Davidian, J.C. Root uptake regulation:a central process for NPS homeostasis in plants. Curr Opin Plant Biol,2009,12:328-338.
Gordon, A.J., Lea, P.J., Rosenberg, C., et al. Nodule formation and function. Plant nitrogen. Berlin: Springer-Verlag.2001:101-146.
Guillaumot, D., Lelu-Walter, M.A., Germot, A., et al. Expression patterns of LmAP2Ll and LmAP2L2 encoding two-APETALA2 domain proteins during somatic embryogenesis and germination of hybrid larch (Larix × marschlinsii). J Plant Physiol,2008,165:1003-1010.
Guo, F.Q., Wang, R., Chen, M., et al. The Arabidopsis dual-affinity nitrate transporter gene AtNRTl.l (CHL1) is activated and functions in nascent organ development during vegetative and reproductive growth. Plant Cell,2001,13:1761-1777.
Guo, F.Q., Young, J., Crawford, N.M. The nitrate transporter AtNRTl.1q (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis. Plant Cell,2003,15:107-117.
Guo, J., Dai, X., Xu, W., et al. Overexpressing GSH1 and AsPCS1 simultaneously increases the tolerance and accumulation of cadmium and arsenic in Arabidopsis thaliana. Chemosphere.2008,72: 1020-1026.
Guo, J., Wang, S., Wang, J., et al. Dissection of the relationship between RACK1 and heterotrimeric G-proteins in Arabidopsis. Plant Cell Physiol,2009,50:1681-1694.
Han, Y., Cao, H., Jiang, J., et al. Rice ROOT ARCHITECTURE ASSOCIATED1 binds the proteasome subunit RPT4 and is degraded in a D-box and proteasome-dependent manner. Plant Physiol,2008, 148:843-855.
He, Q., Qiao, D., Zhang, Q., et al. Cloning and expression study of a putative high-affinity nitrate transporter gene from Dunaliella salina. J. Appl Phycol,2004,16:395-400.
Hershko, A. The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle&ast. Cell Death Differ,2005,12:1191-1197.
Hershko, A., Ciechanover, A. The ubiquitin system for protein degradation. Annu Rev Biochem,1992, 61:761-807.
Ho, C.H., Lin, S.H., Hu, H.C., et al. CHL1 functions as a nitrate sensor in plants. Cell.2009,138: 1184-1194.
Ho, C.H., Tsay, Y.F. Nitrate, ammonium, and potassium sensing and signaling. Curr Opin Plant Biol, 2010,13:604-610.
Hu, C.D., Chinenov, Y., Kerppola, T.K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell,2002,9:789-798.
Hu, C.D., Kerppola, T.K. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol,2003,21:539-545.
Huang, N.C, Chiang, C.S., Crawford, N.M., et al. CHL1 encodes a component of the low-affinity nitrate uptake system in Arabidopsis and shows cell type-specific expression in roots. Plant Cell,1996,8: 2183-2191.
Huang, N.C, Liu, K.H., Lo, H.J., et al. Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake. Plant Cell,1999,11: 1381-1392.
Huis, R., Hawkins, S., Neutelings, G. Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biol,2010,10:71.
Hwang, C.F., Lin, Y., D'Souza, T., et al. Sequences necessary for nitrate-dependent transcription of Arabidopsis nitrate reductase genes. Plant Physiol,1997,113:853-862.
Hynes, T.R., Yost, E., Mervine, S., et al. Multicolor BiFC analysis of competition among G protein p and y subunit interactions. Methods,2008,45:207-213.
Immink, R.G.H., Angenent, G.C. Transcription factors do it together:the hows and whys of studying protein-protein interactions. Trends Plant Sci,2002,7:531-534.
Iwabuchi, K., Li, B., Bartel, P., et al. Use of the two-hybrid system to identify the domain of p53 involved in oligomerization. Oncogene,1993,8:1693-1896.
Jain, M., Nijhawan, A., Tyagi, A., et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Bioph Res Co,2006,345:646-651.
Jian, B., Liu, B., Bi, Y, et al. Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol,2008,9:59.
Johnsson, N., Varshavsky, A. Split ubiquitin as a sensor of protein interactions in vivo. PNAS,1994,91: 10340-10344.
Johnsson, N., Varshavsky, A. Ubiquitin-assisted dissection of protein transport across membranes. EMBOJ,1994,13:2686-2698.
JongMyung, C., ChangYong, S., HaeJoon, C., et al. Effect of liming fertilization on changes of nutrient concentrations in rice-hull based media, crop growth and nutrient uptake of chrysanthemum. Journal of the Korean Society for Horticultural Science SCI.2001,42.
Kang, S.G., Price, J., Lin, P.C., et al. The Arabidopsis bZIP1 transcription factor is involved in sugar signaling, protein networking, and DNA binding. Mol plant,2010,3:361-373.
Katayama, H., Kawamura, Y, Tanaka, T., et al. Production and characterization of transgenic rice plants carrying a high-affinity nitrate transporter gene (OsNRT2.1). Breeding Sci,2009,59:237-243.
Kerppola, T.K. Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells. Nat Protoc,2006,1:1278-1286.
Kiba, T., Feria-Bourrellier, A.B., Lafouge, F., et al. The Arabidopsis Nitrate Transporter NRT2.4 Plays a Double Role in Roots and Shoots of Nitrogen-Starved Plants. Plant Cell,2012,24:245-258.
Kiba, T., Kudo, T., Kojima, M., et al. Hormonal control of nitrogen acquisition:roles of auxin, abscisic acid, and cytokinin. J Exp Bot,2011,62:1399-1409.
Kikuchi, K., Ueguchi-Tanaka, M., Yoshida, K., et al. Molecular analysis of the NAC gene family in rice. Mol Gen Genet,2000,262:1047-1051.
Kodama, Y., Wada, M. Simultaneous visualization of two protein complexes in a single plant cell using multicolor fluorescence complementation analysis. Plant Mol Biol,2009,70:211-217.
Koltermann, M., Moroni, A., Gazzarini, S., et al. Cloning, functional expression and expression studies of the nitrate transporter gene from Chlorella sorokiniana (strain 211-8k). Plant Mol Biol,2003,52: 855-864.
Konishi, M., Yanagisawa, S. Identification of a nitrate-responsive cis-element in the Arabidopsis NIR1 promoter defines the presence of multiple cis-regulatory elements for nitrogen response. Plant J, 2010,63:269-282.
Kotur, Z., Mackenzie, N., Ramesh, S., et al. Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2.1. New Phytol,2012, doi: 10.1111/j.1469-8137.2012.04094.x.
Krapp, A., Fraisier, V., Scheible, W.R., et al. Expression studies of Nrt2:1Np, a putative high-affinity nitrate transporter:evidence for its role in nitrate uptake. Plant J,1998,14:723-731.
Krishnaswamy, S., Verma, S., Rahman, M.H., et al. Functional characterization of four APETALA2-family genes (RAP2.6, RAP2.6L, DREB19 and DREB26) in Arabidopsis. Plant Mol Biol,2011,75:107-127.
Krouk, G, Crawford, N.M., Coruzzi, G.M., et al. Nitrate signaling:adaptation to fluctuating environments. Curr Opin Plant Biol,2010,13:265-272.
Krouk, G, Tillard, P., Gojon, A. Regulation of the high-affinity NO3-uptake system by NRT1.1-mediated NO3- demand signaling in Arabidopsis. Plant Physiol,2006,142:1075-1086.
L vdal, T. and Lillo, C. Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Anal Biochem,2009,387:238-242.
Langer, K., Ache, P., Geiger, D., et al. Poplar potassium transporters capable of controlling K+ homeostasis and K+-dependent xylogenesis. Plant J,2002,32:997-1009.
Laugier, E., Bouguyon, E., Mauries, A., et al. Regulation of High-Affinity Nitrate Uptake in Roots of Arabidopsis Depends Predominantly on Posttranscriptional Control of the NRT2.1/NAR2.1 Transport System. Plant Physiol,2012,158:1067-1078.
Lee, S., Moon, J.S., Ko, T.S., et al. Overexpression of Arabidopsis phytochelatin synthase paradoxically leads to hypersensitivity to cadmium stress. Plant Physiol,2003,131:656-663.
Lehmann, M. Anything else but GAGA:a nonhistone protein complex reshapes chromatin structure. Trends Genet,2004,20:15-22.
Lejay, L., Tillard, P., Lepetit, M., et al. Molecular and functional regulation of two NO3- uptake systems by N-and C-status of Arabidopsis plants. Plant J,1999,18:509-519.
Lejay, L., Wirth, J., Pervent, M., et al. Oxidative pentose phosphate pathway-dependent sugar sensing as a mechanism for regulation of root ion transporters by photosynthesis. Plant Physiol,2008,146: 2036-2053.
Li, E., Wang, S., Liu, Y., et al. OVATE FAMILY PROTEIN4 (OFP4) interaction with KNAT7 regulates secondary cell wall formation in Arabidopsis thaliana. Plant J,2011,67:328-341.
Li, J.Y., Fu, Y.L., Pike, S.M., et al. The Arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance. Plant Cell,2010,22:1633-1646.
Li, W., Wang, Y., Okamoto, M., et al. Dissection of the AtNRT2.1:AtNRT2.2 inducible high-affinity nitrate transporter gene cluster. Plant Physiol,2007,143:425-433.
Lin, C.M., Koh, S., Stacey, G., et al. Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRTl, from rice. Plant Physiol,2000,122:379-388.
Lin, S.H., Kuo, H.F., Canivenc, G., et al. Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport. Plant Cell,2008,20:2514-2528.
Lin, Y., Lai, Z. Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree. Plant Sci,2010,178:359-365.
Lindsey, K., Wei, W, Clarke, M.C., et al. Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants. Transgenic Res,1993,2:33-47.
Little, D.Y., Rao, H., Oliva, S., et al. The putative high-affinity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues. PNAS,2005,102:13693-13698.
Liu, K.H., Huang, C.Y., Tsay, Y.F. CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake. Plant Cell,1999,11:865-874.
Liu, K.H., Tsay, Y.F. Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation. EMBO J,2003,22:1005-1013.
Liu, L., Zhu, K., Yang, Y., et al. Molecular cloning, expression profiling and trans-activation property studies of a DREB2-like gene from chrysanthemum (Dendranthema vestitum). J Plant Res,2008, 121:215-226.
Liu, Y.G., Mitsukawa, N., Oosumi, T., et al. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J,1995,8:457-463.
Liu, Y.G., Whittier, R.F. Thermal asymmetric interlaced PCR:automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics,1995,25: 674-681.
Liu, Z., Gu, C., Chen, F., et al. Heterologous Expression of a Nelumbo nucifera Phytochelatin Synthase Gene Enhances Cadmium Tolerance in Arabidopsis thaliana. Appl Biochem. Biotechnol,2012: 1-13.
Livak, K.J., Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT Method. Methods,2001,25:402-408.
Luo, H., Chen, S., Jiang, J., et al. The expression of floral organ identity genes in contrasting water lily cultivars. Plant Cell Rep,2011:1-10.
Luo, H., Chen, S., Wan, H., et al. Candidate reference genes for gene expression studies in water lily. Anal Biochem,2010,404:100-102.
Ma, Z., Zhang, H., Zheng, J., et al. Interaction between M-Like Protein and Macrophage Thioredoxin Facilitates Antiphagocytosis for Streptococcus equi ssp. zooepidemicus. PloS one,2012,7:e32099.
Malagoli, P., Laine, P., Le Deunff, E., et al. Modeling nitrogen uptake in oilseed rape cv Capitol during a growth cycle using influx kinetics of root nitrate transport systems and field experimental data. Plant Physiol,2004,134:388-400.
Martin, R.C., Hollenbeck, V.G., Dombrowski, J.E. Evaluation of Reference Genes for Quantitative RT-PCR in Lolium perenne. Crop Sci,2008,48:1881-1887.
Masclaux-Daubresse, C., Daniel-Vedele, F., Dechorgnat, J., et al. Nitrogen uptake, assimilation and remobilization in plants:challenges for sustainable and productive agriculture. Ann Bot-London, 2010,105:1141-1157.
Matsumura, H., Xie, Y., Shirakata, S., et al. Crystal structures of C4 form maize and quaternary complex of E. coli phosphoenolpyruvate carboxylases. Structure.2002,10:1721-1730.
Mazars, G.R., Moyret, C., Jeanteur, P., et al. Direct sequencing by thermal asymmetric PCR. Nucleic Acids Res,1991,19:4783.
McNulty, S.E., Toscano, W. Transcriptional regulation of glyceraldehyde-3-phosphate dehydrogenase by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem Bioph Res Co,1995,212:165-171.
Miao, H., Jiang, B., Chen, S., et al. Isolation of a gibberellin 20-oxidase cDNA from and characterization of its expression in chrysanthemum. Plant breeding.2010,128:1-8.
Miller, A.J., Shen, Q., Xu, G. Freeways in the plant:transporters for N, P and S and their regulation. Curr Opin Plant Biol,2009,12:284-290.
Morere-Le Paven, M.C., Viau, L., Hamon, A., et al. Characterization of a dual-affinity nitrate transporter MtNRT1.3 in the model legume Medicago truncatula. J Exp Bot,2011,62:5595-5605.
Nasholm, T., Kielland, K., Ganeteg, U. Uptake of organic nitrogen by plants. New Phytol,2009,182: 31-48.
Nandanavanam, R. Investigations on the contribution of AtNRT2.6 gene to nitrate transport in Arabidopsis thaliana.2011.
Nazoa, P., Vidmar, J.J., Tranbarger, T.J., et al. Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana:responses to nitrate, amino acids and developmental stage. Plant Mol Biol, 2003,52:689-703.
Nicot, N., Hausman, J., Hoffmann, L., et al. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot,2005,56:2907-2914.
Nicot, N., Hausman, J.F., Hoffmann, L., et al. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot,2005,56:2907.
Okamoto, M., Kumar, A., Li, W., et al. High-affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1. Plant Physiol,2006,140:1036-1046.
Okamoto, M., Vidmar, J.J., Glass, A.D.M. Regulation of NRT1 and NRT2 gene families of Arabidopsis thaliana:responses to nitrate provision. Plant Cell Physiol,2003,44:304-317.
Orsel, M., Chopin, F., Leleu, O., et al. Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interaction. Plant Physiol,2006,142: 1304-1317.
Orsel, M., Eulenburg, K., Krapp, A., et al. Disruption of the nitrate transporter genes AtNRT2.1 and AtNRT2.2 restricts growth at low external nitrate concentration. Planta,2004,219:714-721.
Orsel, M., Krapp, A., Daniel-Vedele, F. Analysis of the NRT2 nitrate transporter family in Arabidopsis. Structure and gene expression. Plant Physiol,2002,129:886.
Perez, R., Tupac-Yupanqui, I., Dunner, S. Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue. BMC Mol Biol,2008,9:79.
Pandey, S., Assmann, S.M. The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein a subunit GPA1 and regulates abscisic acid signaling. Plant Cell,2004,16: 1616-1632.
Paolacci, A., Tanzarella, O., Porceddu, E., et al. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Mol Biol,2009,10:11.
Plett, D., Toubia, J., Garnett, T., et al. Dichotomy in the NRT gene families of dicots and grass species. PloS one.2010,5:e15289.
Quesada, A., Galvan, A., Fernandez, E. Identification of nitrate transporter genes in Chlamydomonas reinhardtii. Plant J,1994,5:407-419.
Quesada, A., Hidalgo, J., Fernandez, E. Three Nrt2 genes are differentially regulated in Chlamydomonas reinhardtii. Mol Gen Genet,1998,258:373-377.
Raab, T.K., Terry, N. Carbon, nitrogen, and nutrient interactions in Beta vulgaris L. as influenced by nitrogen source, NO3- versus NH4+. Plant Physiol,1995,107:575-585.
Ramakers, C., Ruijter, J., Deprez, R., et al. Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett,2003,339:62-66.
Rastogi, R., Bate, N.J., Sivasankar, S., et al. Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants. Plant Mol Biol,1997,34:465-476.
Reid, K., Olsson, N., Schlosser, J., et al. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Mol Biol, 2006,6:27.
Reisenauer, H. Mineral nutrients in soil solution. Environ Biol,1966:507-508.
Remans, T., Nacry, P., Pervent, M., et al. The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches. PNAS,2006,103: 19206-19211.
Remans, T., Nacry, P., Pervent, M., et al. A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to nitrogen limitation in Arabidopsis. Plant Physiol,2006,140:909-921.
Rexach, J., Llamas, A., Fernandez, E., et al. The activity of the high-affinity nitrate transport system I (NRT2;1, NAR2) is responsible for the efficient signalling of nitrate assimilation genes in Chlamydomonas reinhardtii. Planta,2002,215:606-611.
Richard-Molard, C., Krapp, A., Brun, F., et al. Plant response to nitrate starvation is determined by N storage capacity matched by nitrate uptake capacity in two Arabidopsis genotypes. J Exp Bot,2008, 59:779-791.
Rinehart, J.A., Petersen, M.W., John, M.E. Tissue-specific and developmental regulation of cotton gene FbL2A (Demonstration of promoter activity in transgenic plants). Plant Physiol,1996,112: 1331-1341.
Rioux, S., Neyt, C., Di Paolo, E., et al. Transcriptional regulation, occurrence and putative role of the Pht family of Streptococcus pneumoniae. Microbiology,2011,157:336-348.
Salamov, A., Solovyev, V. The Gene-Finder computer tools for analysis of human and model organisms genome sequences. Ismb,1997,5:294-302.
Schallau, A., Kakhovskaya, I., Tewes, A., et al. Phylogenetic footprints in fern spore-and seed-specific gene promoters. Plant J,2008,53:414-424.
Shan, H., Chen, S., Jiang, J., et al. Heterologous Expression of the Chrysanthemum R2R3-MYB Transcription Factor CmMYB2 Enhances Drought and Salinity Tolerance, Increases Hypersensitivity to ABA and Delays Flowering in Arabidopsis thaliana. Mol Biotechnol,2011: 1-14.
Sharma, M.K., Kumar, R., Solanke, A.U., et al. Identification, phylogeny, and transcript profiling of ERF family genes during development and abiotic stress treatments in tomato. Mol Gen Genet,2010: 1-21.
Shchennikova, A.V., Shulga, O.A., Immink, R., et al. Identification and characterization of four chrysanthemum MADS-box genes, belonging to the APETALA1/FRUITFULL and SEPALLATA3 subfamilies. Plant Physiol,2004,134:1632-1641.
Siebrecht, S., Mack, G., Tischner, R. Function and contribution of the root tip in the induction of NO3-uptake along the barley root axis. J Exp Bot,1995,46:1669-1676.
Silveira, E.D., Alves-Ferreira, M., Guimaraes, L.A., et al. Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha. BMC Mol Biol,2009,9:84.
Solovyev, V. Statistical approaches in eukaryotic gene prediction. Handbook of statistical genetics.2001.
Solovyev, V.V. and Shahmuradov, I. PromH:promoters identification using orthologous genomic sequences. Nucleic Acids Res,2003,31:3540-3545.
Sturzenbaum, S., Kille, P. Control genes in quantitative molecular biological techniques:the variability of invariance. Comp Biochem Physiol B:Biochem Mol Biol,2001,130:281-289.
Stagljar, I., Korostensky, C., Johnsson, N., et al. A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. PNAS,1998,95:5187-5192.
Strahm, B.D., Harrison, R.B. Nitrate sorption in a variable-charge forest soil of the Pacific Northwest. Soil Sci,2006,171:313-321.
Suzuki, T., Higgins, P., Crawford, D. Control selection for RNA quantitation. Biotechniques,2000,29: 332-337.
Takahashi-Terada, A., Kotera, M., Ohshima, K., et al. Maize Phosphoenolpyruvate Carboxylase. J Biol Chem,2005,280:11798-11806.
Terauchi, R., Kahl, G. Rapid isolation of promoter sequences by TAIL-PCR:The 5'-flanking regions of Pal and Pgi genes from yams (Dioscorea). Mol Gen Genet,2000,263:554-560.
Thaminy, S., Auerbach, D., Arnoldo, A., et al. Identification of novel ErbB3-interacting factors using the split-ubiquitin membrane yeast two-hybrid system. Genome Res,2003,13:1744-1753.
Thomas, C., Meyer, D., Wolff, M., et al. Molecular characterization and spatial expression of the sunflower ABP1 gene. Plant Mol Biol,2003,52:1025-1036.
Thornton, B. Inhibition of nitrate influx by glutamine in Lolium perenne depends upon the contribution of the HATS to the total influx. J Exp Bot,2004,55:761-769.
Tjaden, G, Edwards, J.W., Coruzzi, G.M. Cis elements and trans-acting factors affecting regulation of a nonphotosynthetic light-regulated gene for chloroplast glutamine synthetase. Plant Physiol,1995, 108:1109-1117.
Todd, C.D., Zeng, P., Huete, A.M.R., et al. Transcripts of MYB-like genes respond to phosphorous and nitrogen deprivation in Arabidopsis. Planta.2004,219:1003-1009.
Tong, Y., Zhou, J.J., Li, Z., et al. A two-component high-affinity nitrate uptake system in barley. Plant J, 2005,41:442-450.
Tong, Z., Gao, Z., Wang, F., et al. Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Mol Biol,2009,10:71.
Tong, Z., Hong, B., Yang, Y., et al. Overexpression of two chrysanthemum DgDREBl group genes causing delayed flowering or dwarfism in Arabidopsis. Plant Mol Biol,2009,71:115-129.
Topping, J.F., Lindsey, K. Promoter trap markers differentiate structural and positional components of polar development in Arabidopsis. Plant Cell,1997,9:1713-1725.
Trevisan, S., Borsa, P., Botton, A., et al. Expression of two maize putative nitrate transporters in response to nitrate and sugar availability. Plant Biology,2008,10:462-475.
Tsay, Y.F., Chiu, C.C., Tsai, C.B., et al. Nitrate transporters and peptide transporters. FEBS Lett,2007, 581:2290-2300.
Tsay, Y.F., Schroeder, J.I., Feldmann, K.A., et al. The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter. Cell,1993,72:705-713.
Tzfira, T., Citovsky, V. Partners-in-infection:host proteins involved in the transformation of plant cells by Agrobacterium. Trends Cell Biol,2002,12:121-129.
Vandesompele, J., De Preter, K., Pattyn, F., et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol,2002,3.
Vidmar, J.J., Zhuo, D., Siddiqi, M.Y., et al. Isolation and Characterization of HvNRT2.3 and HvNRT2.4, cDNAs Encoding High-Affinity Nitrate Transporters from Roots of Barley. Plant Physiol,2000, 122:783-792.
Vidmar, J.J., Zhuo, D., Siddiqi, M.Y., et al. Regulation of high-affinity nitrate transporter genes and high-affinity nitrate influx by nitrogen pools in roots of barley. Plant Physiol,2000,123:307-318.
Von Arnim, A. Subcellular Localization of GUS-and GFP-Tagged Proteins in Onion Epidermal Cells. Cold Spring Harbor Protocols,2007,2007:pdb. prot4689.
Voss, A.K., Thomas, T., Gruss, P. Compensation for a gene trap mutation in the murine microtubule-associated protein 4 locus by alternative polyadenylation and alternative splicing. Dev Dynam,1998,212:258-266.
Wan, H., Zhao, Z., Qian, C, et al. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem,2010,399: 257-261.
Wang, R., Liu, D., Crawford, N.M. The Arabidopsis CHL1 protein plays a major role in high-affinity nitrate uptake. PNAS,1998,95:15134-15139.
Wang, Y.Y., Tsay, Y.F. Arabidopsis Nitrate Transporter NRT1.9 Is Important in Phloem Nitrate Transport. Plant Cell,2011,23:1945-1957.
Wang, Z., Ye, S., Li, J., et al. Fusion primer and nested integrated PCR (FPNI-PCR):a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning. BMC Biotech, 2011,11:109.
Watanabe, M., Mochida, K., Kato, T., et al. Comparative genomics and reverse genetics analysis reveal indispensable functions of the serine acetyltransferase gene family in Arabidopsis. Plant Cell,2008, 20:2484-2496.
Weis, K. Regulating access to the genome:nucleocytoplasmic transport throughout the cell cycle. Cell. 2003,112:441-451.
Wirth, J., Chopin, F., Santoni, V., et al. Regulation of root nitrate uptake at the NRT2.1 protein level in Arabidopsis thaliana. J Biol Chem,2007,282:23541-23552.
Xu, M., Zhang, B., Su, X., et al. Reference gene selection for quantitative real-time polymerase chain reaction in Populus. Anal Biochem,2011,408:337-339.
Xu, Z., Zhou, Y, Meng, C., et al. Characterization of promoter expression patterns of OsNrt2.1, a nitrate transporter gene of rice (Oryza sativa L.). Frontiers of Agriculture in China.2009,3:402-412.
Yan, J., He, C, Zhang, H. The BAG-family proteins in Arabidopsis thaliana. Plant Sci,2003,165:1-7.
Yan, M., Fan, X., Feng, H., et al. Rice OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a nitrate transporters to provide uptake over high and low concentration ranges. Plant Cell Environ, 2011,1360-1372.
Yanagisawa, S. Dofl and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize. Plant J,2000,21:281-288.
Yanagisawa, S., Schmidt, R.J. Diversity and similarity among recognition sequences of Dof transcription factors. Plant J,1999,17:209-214.
Yang, Y, Wu, J., Zhu, K., et al. Identification and characterization of two chrysanthemum (Dendronthemax moriforlium) DREB genes, belonging to the AP2/EREBP family. Mol Biol Rep, 2009,36:71-81.
Yin, D., Chen, S., Chen, F., et al. Morphological and physiological responses of two chrysanthemum cultivars differing in their tolerance to waterlogging. Environ Exp Bot.,2009,67:87-93.
Yin, L.P., Li, P., Wen, B., et al. Characterization and expression of a high-affinity nitrate system transporter gene (TaNRT2.1) from wheat roots, and its evolutionary relationship to other NTR2 genes. Plant Sci,2007,172:621-631.
Yong, Z., Kotur, Z., Glass, A.D.M. Characterization of an intact two-component high-affinity nitrate transporter from Arabidopsis roots. Plant J,2010,63:739-748.
Zhang, G, Chen, M., Chen, X., et al. Phylogeny, gene structures, and expression patterns of the ERF gene family in soybean (Glycine max L.). J Exp Bot,2008,59:4095-4107.
Zhang, H., Jennings, A., Barlow, P.W., et al. Dual pathways for regulation of root branching by nitrate. PNAS,1999,96:6529-6534.
Zhang, H., Rong, H., Pilbeam, D. Signalling mechanisms underlying the morphological responses of the root system to nitrogen in Arabidopsis thaliana. J Exp Bot,2007,58:2329-2338.
Zheng, C., Oba, S., Matsui, S., et al. Effects of calcium and magnesium treatments on growth, nutrient contents, ethylene production, and gibberellin content in Chrysanthemum plants. J Jap Soc Hortic Sci,2005,74:144-149.
Zhou, J.J., Fernandez, E., Galvan, A., et al. A high affinity nitrate transport system from Chlamydomonas requires two gene products. FEBS Lett,2000,466:225-227.
Zhou, J.J., Theodoulou, F.L., Muldin, I., et al. Cloning and functional characterization of a Brassica napus transporter that is able to transport nitrate and histidine. J Biol Chem,1998,273: 12017-12023.
Zhuang, J., Cai, B., Peng, R.H., et al. Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa. Biochem Bioph Res Co,2008,371:468-474.
Zhuo, D., Okamoto, M., Vidmar, J.J., et al. Regulation of a putative high-affinity nitrate transporter (Nrt2;1At) in roots of Arabidopsis thaliana. Plant J,1999,17:563-568.
陈柏君,孙超,王勇,等.锚定PCR(Anchored PCR):一种新的染色体步行方法.科学通报,2004,49(15)：1569-1571.
陈军营,孙佩,王德勤,等.一种改良的克隆小麦GLP3基因启动子的TAIL-PCR技术.植物生理学通讯,2007,43(4)：754-758.
春亮.玉米氮高效品种选育及根系形态对低氮反应的遗传分析.中国农业大学博士论文,2004.
崔颖.甜菜M14品系表达基因M14-100, M14-265, M14-G12 cDNA全长克隆与序列分析.黑龙江大学硕士论文,2008.
范晓荣.作物硝态氮跨膜运输的生理和分子机理.南京农业大学博士论文,2005.
方进,李素文.转基因水稻T-DNA侧翼序列的扩增与分析.遗传学报,2001,28(4)：345-351.
高翔,陈磊,云鹏,等.不同基因型小麦侧根生长对硝态氮的响应差异.植物营养与肥料学报,2010,16(4),1013-1019.
管志勇,陈发棣,滕年军,等.5种菊花近缘种属植物的耐盐性比较.中国农业科学,2010,43(4)：787-794.
管志勇,陈素梅,陈发棣,等.32个菊花近缘种属植物耐盐性筛选.中国农业科学,2010,43(19)：4063-4071.
郭亚芬,米国华,陈范骏,等.局部供应硝酸盐诱导玉米侧根生长的基因型差异.植物营养与肥料学报,2005,11(2)：155-159.
洪娟.油菜氮高效种质的筛选及其生理机制的初步研究.华中农业大学硕士论文,2007.
胡尚连,曹颖,黄胜雄,等.慈竹4CL基因的克隆及其生物信息学分析.西北农林科技大学学报：自然科学版,2009,37(8)：204-210.
黄长兵,房伟民,杨勇,等.不同水平和形态氮素供应对盆栽小菊外观品质和光合特性的影响.浙江农业学报,2010,22(1)：45-50.
姜贝贝,房伟民,陈发棣等.氮磷钾配比对切花菊‘神马’生长发育的影响.浙江林学院学报,2009,25(6)：692-697.
蒋志平,鲁剑巍,张文君,等.菊花氮钾肥效及配方筛选研究.中国农学通报,2010,26(10)：182-186.
靳苗苗,杨青川,金洪,等.紫花苜蓿柠檬酸合成酶基因的克隆及对烟草的转化.草地学报,2010,18(4)：550-555.
康健,裴蓓,周丕生,等.不同氮素水平对郁金香磷素与钾素的累积与分配的影响.上海交通大学学报：农业科学版,2006,23(4)：377-382.
李雪妮.棉花氮高效品种筛选及其机理的初步研究.新疆农业大学硕士论文,2007.
李玉中,祝廷成,Redmann, R.三种利用类型羊草草地氮总矿化,硝化和无机氮消耗速率的比较研究.生态学报,2002,22(5)：668-673.
李悦,张涛,齐秀兰.切花菊氮磷钾营养特性的研究进展.安徽农业科学,2005,33(8)：1486-1487.
李秋莉,张毅,尹辉,等.辽宁碱蓬甜菜碱醛脱氢酶基因(BADH)启动子分离及序列分析.生物工程学报,2006,22(1)：77-81.
李芳,沈欣杰,赵凯,等.苹果山梨醇运输基因MdSOT3启动子克隆与功能分析.园艺学报,2011,38(增刊)：2457
刘霞,邓馨.复苏植物牛耳草BhLEA2基因启动子的分离和基因表达模式研究.自然科学进展,2009,19(1)：39-44.
刘晓庆,丁志鑫,刘晶,等.2种PCR方法扩增大豆rbcS启动子5'侧翼序列比较.吉林农业大学学报,2011,33(5)：511-517.
刘云,何书萱.菊花无土栽培技术研究.山西农业科学,1999,27(3)：62-65.
刘振林,曹华雯,夏新莉,等.甘菊BADH基因启动子的克隆与瞬时表达分析.园艺学报,2008,35(12)：1787-1794.
卢艳丽,陆卫平,王继丰,等.不同基因型糯玉米氮素吸收利用效率的研究.植物营养与肥料学报,2006,12(3)：321-326.
马彩艳,刘冬成,阳文龙等.短柄草磷转运蛋白家族基因的克隆及表达分析.分子植物育种,2011,9(4)：482-490.
马传营.中国水仙(Narcissus tazetta L. var. chinensis Roem)花香相关基因DXPS, DXR和PAL的克隆.福建农林大学硕士论文,2010.
马翠,范晓荣,徐国华.武运粳7号超表达OsNRT1.2后对硝酸盐的响应.中国水稻科学,2011,25(4)：349-356.
马赛箭,苏乔,吴凇,等.盐角草磷酸乙醇胺N-甲基转移酶基因(SePEAMT)启动子的克隆及瞬时表达.中国生物工程杂志,2008,28(8)：69-73.
彭少兵,黄见良,钟旭华,等.提高中国稻田氮肥利用率的研究策略.中国农业科学,2002,35(9)：1095-1103.
曲宪成,崔严慧,周正峰,等.团头鲂促性腺激素GtHI β亚基基因5’端启动子区克隆及表达载体构建.水生生物学报,2008,32(4)：558-567.
曲宪成,崔严慧,周正峰,等.团头鲂促性腺激素GtⅡ β亚基基因5’端启动子区克隆及表达载体构建.中国水产科学,2010,17(4)：649-658.
沈瑾.苹果醇酰基转移酶基因(MdAAT2)启动子克隆与功能鉴定.山东农业大学硕士论文,2009.
史正军,樊小林.作物对氮素养分高效吸收的根系形态学研究进展.广西农业生物科学,2003,22(3),225-229.
宋长年,房经贵,王晨,·等.基于EST库的枳APETALA2基因cDNA克隆及其表达分析.园艺学报,2009,36(6)：799-806.
孙凯.切花菊‘神马’氮营养研究.北京林业大学硕士论文,2007.
王小兵,吴平,胡彬,等.硝态氮(N03-)对水稻侧根生长及其氮吸收的影响.植物学报,2002,6,678-683.
王艳,米国华,陈范骏,等.玉米氮素吸收的基因型差异及其与根系形态的相关性.生态学报,2003,23(2),297-302.
魏跃,陈啸寅,王全智,等.应用改良TAIL-PCR克隆黄瓜6PGDH基因上游序列.浙江农业学报,2011,23(5)：900-904.
乌云塔娜,刘学英,李振国.中国梨S-RNase基因启动子的TAIL-PCR克隆及功能预测.南京林业大学学报：自然科学版,2010,34(4)：21-25.
吴金燕.番木瓜eIF4E和eIFiso4E的克隆及其与PRSV-VPg互作研究.海南大学硕士论文,2010.
徐海荣.水稻新型硝酸盐转运蛋白基因OsTNrt2.1的克隆,表达和遗传转化.河北农业大学硕士论文.2007.
许瑛,陈煜,陈发棣,等.菊花耐寒特性分析及其评价指标的确定.中国农业科学,2009,42(3)：974-981.
杨帆,林俊芳,郭安平,等.利用TAIL-PCR技术克隆猴头菇β-glucosidase基因.食品与发酵工业,2005,31(5)：9-14.
杨伟,陈发棣,陈素梅,等.不同菊花品种不同开花阶段舌状花耐热性比较.南京农业大学学报,2011,34(2)：47-53.
杨迎东.氮营养对切花菊‘优香’品质影响研究.中国农业科学院硕士论文,2010.
杨宗灿.利用BiFC技术研究拟南芥GPX3和RRTF1与DOS1体内相互作用.河南大学硕士论文,2011.
尹冬梅,管志勇,陈素梅,等.菊花及其近缘种属植物耐涝评价体系建立及耐涝性鉴定.植物遗传资源学报,2009,10(3)：399-404.
尹西翔.水稻氮高效品种的筛选及其机理的初步探讨.华中农业大学硕士论文,2005.
应革,武威,何朝族.’[AIL-PCR方法快速分离Xcc致病相关基因序列.生物工程学报,2002,18(2)：182-186.
于莹.利用羊草EST文库克隆耐盐碱相关基因.吉林大学博士论文,2011.
赵付江.茄子氮高效基因型筛选及氮效率基因型差异机理的研究.河北农业大学硕士论文,2008.
赵九洲,陈洁敏,陈松笔,等.无土基质与营养液EC值对切花菊生长发育的影响.园艺学报,1999,26(5)：327-330.
赵英,牛建新.利用3'和5'RACE克隆新疆梨树苹果茎痘病毒末端序列.园艺学报,2008,35(4)：493-500.
郑成淑,王文莉,孙宪芝,等.硝酸钙处理对菊花扦插生根及抗氧化酶活性的影响.园艺学报,2008,35(2)：263-268.
郑路,傅玉兰.菊花抗寒性与营养特性的研究.园艺学报,1994,21(2)：185-188.
周忠新.不同氮素水平下施磷对小麦产量和品质的影响及其生理基础.山东农业大学硕士论文,2006.
朱树勇,迟伟,张立新.分裂泛素化酵母双杂交系统研究光合膜蛋白相互作用.生物化学与生物物理进展.2006,33(4)：350-356.
祝丽香,王建华,毕建杰,等.不同氮素用量对杭白菊养分累积,转运及产量的影响.植物营养与肥料学报,2010,16(4)：992-997.