烟草硝酸盐转运蛋白基因NtNRT2.4的克隆及表达分析
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- 英文论文题名：Cloning and characterization of NRT2.4 gene from Nicotiana tabacum
- 论文作者：黄化刚 ; 申燕 ; 王卫峰 ; 连文力 ; 陈雪 ; 翟欣 ; 喻奇伟 ; 杨振智 ; 贾宏昉
- 英文论文作者：HUANG huagang ; SHEN yan ; WANG weifeng ; LIAN wenli ; CHEN xue ; ZHAI xin ; YU qiwei ; YANG chenzhi ; JIA hongfang ; Key laboratory of Tobacco Cultivation ; College of Tobacco Science ; Henan Agricultural University ; Bijie Branch of Guizhou tobacco Company ; Guangxi Zhuang Autonomous Region Tobacco Company of CNTC
- 年：2016
- 作者机构：河南农业大学烟草学院烟草行业栽培重点实验室;贵州省烟草公司毕节市公司;中国烟草总公司广西壮族自治区公司;
- 论文关键词：烟草 ; NtNRT2.4 ; 克隆 ; 表达分析
- 英文论文关键词：Nicotiana tabacum ; NtNRT2.4 ; cloning ; expression analysis
- 会议召开时间：2016-12-01
- 会议录名称：中国烟草学会2016年度优秀论文汇编——烟草农业主题
- 语种：中文
- 分类号：S572
- 学会代码：ZGYG
- 学会名称：中国烟草学会
- 页数：11
- 文件大小：1343k
- 原文格式：D
摘要
植物NRT2基因家族(高亲和硝酸盐转运蛋白基因)在植物吸收和转运硝酸盐过程中发挥重要作用。本研究根据NRT2基因家族的保守序列设计兼并引物,扩增出一条821bp序列,以此序列为信息探针,通过电子克隆和RT-PCR的方法从烟草中克隆获得一个包含1593bp开放阅读框、编码530个氨基酸的c DNA序列,命名为NtNRT2.4。生物信息学分析表明,该基因编码的蛋白质具有NRT家族的共有结构特征,与烟草已发现的三个本家族基因同源性达到77.54%,与拟南芥NRT2家族基因同源性达到81.63%,其启动子中包含多个与硝酸盐、光照及根系特异表达相关的元件;组织特异表达分析结果显示,烟草NtNRT2.4基因的组织表达差异较大,在根部的表达量较高,在叶片和茎部的表达量低;不同氮素形态、光照和蔗糖处理下的基因表达分析结果表明,硝态氮、光照和蔗糖处理诱导NtNRT2.4表达,而铵态氮抑制其表达。
NRT2 plays a critical role in nitrate absorption and transport in plants. In the study, one 821 bp sequence of NRT2 was screened from c DNA library of tobacco. Based on the sequence, Nt NRT2.4 was isolated from tobacco(Nicotiana tabacum L.) by in silico cloning and RT-PCR. Nt NRT2.4 includes an open reading frame(ORF) of 1593 bp and encodes 530 deduced amino acid residues(AAR). Phylogenetic analysis suggested Nt NRT2.4 was high homologous to its paralogous genes from tobacco(77.54%) and orthologous genes from Arabidopsis(81.63%), which were also induced by nitrate stresses. Promoter analysis suggested Nt NRT2.4 had a lot of motif involved in N and light metabolism. Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR(q RT-PCR) was used to determine the expression patterns of Nt NRT2.4 in tobacco. The results revealed that Nt NRT2.4 expressed most strongly in tobacco roots and marginally in leaves, stems. Expression patterns under light and sugar responses suggested that Nt NRT2.4 was involved in response to light and sugar treatment, with significant different responsive profiles.
NRT2 plays a critical role in nitrate absorption and transport in plants. In the study, one 821 bp sequence of NRT2 was screened from c DNA library of tobacco. Based on the sequence, Nt NRT2.4 was isolated from tobacco(Nicotiana tabacum L.) by in silico cloning and RT-PCR. Nt NRT2.4 includes an open reading frame(ORF) of 1593 bp and encodes 530 deduced amino acid residues(AAR). Phylogenetic analysis suggested Nt NRT2.4 was high homologous to its paralogous genes from tobacco(77.54%) and orthologous genes from Arabidopsis(81.63%), which were also induced by nitrate stresses. Promoter analysis suggested Nt NRT2.4 had a lot of motif involved in N and light metabolism. Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR(q RT-PCR) was used to determine the expression patterns of Nt NRT2.4 in tobacco. The results revealed that Nt NRT2.4 expressed most strongly in tobacco roots and marginally in leaves, stems. Expression patterns under light and sugar responses suggested that Nt NRT2.4 was involved in response to light and sugar treatment, with significant different responsive profiles.
引文
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[4]Williams LE,Miller AJ.Transporters responsible for the uptake and partitioning of nitrogenous solutes[J].Annu Rev Plant Physiol Plant Mol Biol,2001,52(1):659-688.
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[7]Vidmar JJ,Zhuo D,Siddiqi MY,et al.Isolation and characterization of Hv NRT2.3 and Hv NRT2.4,c DNAs encoding high-affinity nitrate transporters from roots of barley[J].Plant Physiology,2000,122(3):783-792.
[8]Quesada A,Krapp A,Trueman LJ,et al.PCR-identification of a Nicotiana plumbaginifolia c DNA homologous to the high-affinity nitrate transporters of the crn A family[J].Plant Molecular Biology,1997,34(2):265-274.
[9]Amarasinghe BH,De Bruxelles GL,Braddon M,et al.Regulation of Gm NRT2 expression and nitrate transport activity in roots of soybean(Glycine max)[J].Planta,1998,206(1):44-52.
[10]Quaggiotti S,Ruperti B,Borsa P,et al.Expression of a putative high-affinity NO3-transporter and of an H+-ATPase in relation to whole plant nitrate transport physiology in two maize genotypes differently responsive to low nitrogen availability[J].Journal of Experiment Botany,2003,54:1023-1031.
[11]赵学强,李玉京,刘建中,等.小麦NO3-转运蛋白基因Ta NRT2.3的克隆和表达分析[J].植物学报,2004,46(3):347-354.
[12]张洪映,贾宏昉,张松涛,等.烟草非生物胁迫应答基因Nt Sn RK2.1的克隆与表达分析[J].中国烟草学报,2014,20(4):94-99
[13]Orsel M,Krapp A,Daniel-Vedele F.Analysis of the NRT2 nitrate transporter family in Arabidopsis.Structure and gene expression[J].Plant Physiology,2002(2):886-896.
[14]许金,谢小东,冯爽,等.烟草硝酸盐转运蛋白基因的克隆及表达分析[J].烟草科技,2013,7:68-71
[15]Cai CH,Wang JY,Zhu YG,et al.Gene structure and expression of high-affinity nitrate transport system in rice roots[J].Journal of Integrative Plant Biology,2008,50(2):443-451.
[16]Yan M,Fan XR,Feng HM,et al.Rice Os NAR2.1 interacts with Os NRT2.1,Os NRT2.2 and Os NRT2.3a nitrate transporters to provide uptake over high and low concentration ranges[J].Plant Cell and Environment,2011,34(8):1360-1372.
[17]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[J].Plant Journal,1999,18(5):509-519.
[18]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[J].Plant Journal,2010,63(2):269-282.
[2]Wang YY,Hsu PK,Tsay YF.Uptake,allocation and signaling of nitrate[J].Trends in plant science,2012,17(8):458-467.
[3]Forde BG.Nitrate transporters in plants:structure,function and regulation[J].Biochimica biophysica acta,2000,1465(12):219-235.
[4]Williams LE,Miller AJ.Transporters responsible for the uptake and partitioning of nitrogenous solutes[J].Annu Rev Plant Physiol Plant Mol Biol,2001,52(1):659-688.
[5]Campble WH.Nitrate reductase structure,function and regulation:bridging the gap between biochemistry and physiology[J].Annual Review of Plant Physiology,1999,50(1):277-303.
[6]Trueman LJ,Richardson A,Forde B G.Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans[J].Gene,1996,175(1-2):223-231.
[7]Vidmar JJ,Zhuo D,Siddiqi MY,et al.Isolation and characterization of Hv NRT2.3 and Hv NRT2.4,c DNAs encoding high-affinity nitrate transporters from roots of barley[J].Plant Physiology,2000,122(3):783-792.
[8]Quesada A,Krapp A,Trueman LJ,et al.PCR-identification of a Nicotiana plumbaginifolia c DNA homologous to the high-affinity nitrate transporters of the crn A family[J].Plant Molecular Biology,1997,34(2):265-274.
[9]Amarasinghe BH,De Bruxelles GL,Braddon M,et al.Regulation of Gm NRT2 expression and nitrate transport activity in roots of soybean(Glycine max)[J].Planta,1998,206(1):44-52.
[10]Quaggiotti S,Ruperti B,Borsa P,et al.Expression of a putative high-affinity NO3-transporter and of an H+-ATPase in relation to whole plant nitrate transport physiology in two maize genotypes differently responsive to low nitrogen availability[J].Journal of Experiment Botany,2003,54:1023-1031.
[11]赵学强,李玉京,刘建中,等.小麦NO3-转运蛋白基因Ta NRT2.3的克隆和表达分析[J].植物学报,2004,46(3):347-354.
[12]张洪映,贾宏昉,张松涛,等.烟草非生物胁迫应答基因Nt Sn RK2.1的克隆与表达分析[J].中国烟草学报,2014,20(4):94-99
[13]Orsel M,Krapp A,Daniel-Vedele F.Analysis of the NRT2 nitrate transporter family in Arabidopsis.Structure and gene expression[J].Plant Physiology,2002(2):886-896.
[14]许金,谢小东,冯爽,等.烟草硝酸盐转运蛋白基因的克隆及表达分析[J].烟草科技,2013,7:68-71
[15]Cai CH,Wang JY,Zhu YG,et al.Gene structure and expression of high-affinity nitrate transport system in rice roots[J].Journal of Integrative Plant Biology,2008,50(2):443-451.
[16]Yan M,Fan XR,Feng HM,et al.Rice Os NAR2.1 interacts with Os NRT2.1,Os NRT2.2 and Os NRT2.3a nitrate transporters to provide uptake over high and low concentration ranges[J].Plant Cell and Environment,2011,34(8):1360-1372.
[17]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[J].Plant Journal,1999,18(5):509-519.
[18]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[J].Plant Journal,2010,63(2):269-282.
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