小麦渐渗系山融3号盐胁迫应答基因TaHB30和TaCHP的功能鉴定
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摘要
小麦作为世界上分布最广、种植面积最大、加工制品最为丰富的粮食作物之一,其农业地位的重要性不可忽视。然而随着水资源的日益匮乏及土地荒漠化的日趋严重,非生物胁迫如高盐、干旱等对小麦品质的提高及产量的增加形成了较大的负面影响。加之非生物胁迫应答是由多基因控制的复杂性状,涉及到细胞防御、离子平衡、次生代谢以及能量代谢等诸多方面。且小麦作为异源六倍体,同拟南芥等模式植物相比,耐盐机制研究的复杂性相应增加,生长周期也相对延长,因此,即便与水稻、玉米等其他作物相比,小麦逆境分子机理的研究仍然进展缓慢。
由于小麦属于甜土植物,自身耐盐能力不强,而其野生近缘物种则是抗逆改良的重要基因库,可作为基因工程和细胞工程技术培育耐逆新品种的重要基因源。本实验利用不对称体细胞杂交渐渗技术,获得了盐敏感小麦品种济南177(Triticum aestivum2n=42, JN177)与强耐盐性小麦近缘属长穗偃麦草(Thinopyrum ponticum,2n=70)的渐渗系抗盐新品种山融3号(SR3)。山融3号的耐盐指数及各项生理生化指标均明显优于亲本小麦JN177,其耐盐性由主效基因和微效基因共同控制。
在本实验前期对SR3全基因组芯片和蛋白组分析的基础上,我们发现两个受胁迫表达变化的基因,结合SR3盐胁迫cDNA全长文库和RACE手段,获得基因全长。下面分别对两个基因进行功能验证,主要的研究内容及结果包括：
1.小麦耐盐相关基因TaHB30的克隆及初步功能分析
通过分析SR3盐胁迫cDNA全长文库基因,发现了一个受盐胁迫表达显著上调的基因,依据文库编号将其命名为TaHB30。在对其编码的蛋白序列进行Blast分析时,发现该基因含有Cu/Zn超氧化物歧化酶典型的激活位点,并包含有4个Cu2+结合位点和4个Zn2+结合位点,将其归类于Cu/Zn超氧化物歧化酶大类。
200mM NaCl处理条件下,在SR3及其亲本JN177根叶中总体为上调的表达趋势；10mM过氧化氢处理后,根部的变化情况SR3同JN177较为一致,都呈现先下调后上调的波动变化。叶中SR3仍为先下调后上调的趋势,而JN177则是下调表达。在这些处理中,TaHB30在SR3中的表达量均高于JN177。
由于Cu/Zn超氧化物歧化酶在植物细胞中分布比较广泛,为确定TaHB30的功能作用部位,对基因表达产物进行亚细胞定位。通过该蛋白偶联GFP,基因枪法转化洋葱表皮细胞和PEG诱导拟南芥原生质体,GFP荧光信号一致出现在细胞质与细胞核中,表明该基因产物主要在胞质同核中起作用。
构建TaHB30表达载体并分别转化拟南芥和小麦,对拟南芥过表达转基因株系(OE系)在多种逆境胁迫下的表型进行分析。在对照条件下,OE系和对照株系生长状况正常,没有明显差异；在不同浓度的Mannitol处理下,OE系与对照株系差异也不明显。但当NaCl浓度增加到50mM以后,OE系的生长状态明显好于对照株系,主根根长同对照植株相比较长,侧根的生长也比对照株系发达。在添加H2O2的培养基上,OE系的生长状况也明显优于对照株系。随后进行了不同种类激素,包括IAA、JA、ABA与GA3的处理,在对照和转基因株系中都未发现表型的明显差异。初步推测,TaHB30通过增强ROS途径促进OE系的耐盐能力,同相关的植物激素信号通路没有交汇。利用拟南芥突变体验证该基因功能,将TaHB30转化拟南芥突变体,发现TaHB30过表达拟南芥突变体在正常条件下比对照系的根短,而在NaCl和H202的处理下这种根长度的差异消除,推测与该基因调控拟南芥内源ROS的信号途径有关。
RT-PCR和real time PCR结果显示,正常生长条件下,拟南芥过表达株系(OEl,OE2)本底的超氧化物歧化酶(SOD)编码基因比对照株系表达量增高；而其下游多个ROS清除酶编码基因表达上调：谷胱甘肽过氧化物酶(GPX)表达量的增幅最为显著,其他如过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)分别编码的2个基因在OE系的表达量同对照也具有显著差异。值得注意的是：位于ROS途径上游、与H2O2产生密切相关的NADPH氧化酶编码基因RBOH-D, RBOH-F的表达明显提高,并且在同时含有H202和NADPH氧化酶抑制剂的培养基上,OE系的生长状况不再优于对照株系；在正常条件下,TaHB30过表达拟南芥突变体的RBOH-D基因表达变化不显著,而GPX基因的表达量对照系比较明显上调。以上结果表明：小麦Cu/Zn超氧化物歧化酶基因TaHB30通过调控NADPH氧化酶的变化,进入ROS信号途径而在耐盐中起作用。
进一步测定了OE系及空载体对照的ROS相关酶活性,结果表明：OE系的ROS清除酶系统中总超氧化物歧化酶(SOD)和Cu/Zn超氧化物歧化酶的酶活力都较对照提高；过氧化氢酶(CAT)的酶活力约为对照的1.5-2倍；谷胱甘肽过氧化物酶(GPX)的酶活力约为对照的1.3倍。而ROS的产生酶-非特异性NADPH氧化酶(NOX)活性也在过表达拟南芥中明显升高(1.6倍)；DAB染色显示,TaHB30过表达株系中H202含量明显低于对照植株；而NBT染色后,超氧阴离了的含量高于对照植株。
以上结果表明：TaHB30通过调节ROS信号转导系统提高植物耐盐性。2.小麦耐盐相关基因TaCHP的功能分析
通过本实验前期工作,发现了一个盐胁迫应答新基因TaCHP,编码一个CHP-rich(半胱氨酸,组氨酸和脯氨酸丰富)的锌指蛋白家族基因。TaCHP含有能特异性结合磷脂信号分子甘油二酯(DAG)的C1结构域和可能的激酶结构域,含有该结构域的蛋白质大多具有蛋白激酶(定位细胞膜)和/或转录调控因子(定位细胞核)活性。本研究发现,TaCHP定位在细胞膜和细胞核,推测是一个具有蛋白激酶和转录因子双重功能的抗逆相关重要蛋白。体外PKC激酶活性测定表明,TaCHP表达蛋白无激酶活性。转录激活活性分析发现C’端两个C1结构域及TaCHP全长具有转录激活活性,表明该基因可能作为一个转录因子行使功能。
进行TaCHP胁迫表达模式及其定位分析,发现TaCHP主要在三叶期的小麦根部表达,原位杂交结果表明转录产物定位于根尖的皮层和分生组织细胞内。TaCHP表达量在SR3中比亲本JN177高,通过NaCl,干旱,ABA等处理后,二者都呈现下调趋势。但是,SR3总体表达量均维持在比JN177明显高的水平。
将TaCHP转化盐敏感小麦济南17,小麦的耐盐、抗旱、抗氧化能力及相关生理生化指标都明显提高,证明了该基因的抗逆功能。构建了TaCHP过表达(BS、A5)拟南芥株系,进一步研究该基因可能的作用机制。除了耐盐性以外,拟南芥对H202和ABA的耐受性也有所增加。在盐处理下,过表达系中一部分胁迫响应基因(AtCBF3, AtDREB2A, AtABI2和AtABI1)的表达量升高,而ABA合成基因AtAA03, AtABA2和AtMYB15的表达量降低。Real-time PCR显示转基因拟南芥BS、A5的AtRBOHD表达量平均增幅达7倍,其编码的NADPH氧化酶是ROS产生的重要组成部分,而活性氧清除酶CAT和APX编码基因AtCAT2、AtAPX2在拟南芥过表达株系中被诱导,结合转基因拟南芥BS、A5株系中较高的SOD和CAT酶活性,表明ROS的产生及清除能力在过表达株系中都增强。
以上结果表明：TaCHP通过ABA依赖和非依赖信号途径以及ROS信号途径参与植物对非生物胁迫的响应。
Wheat has the most extensive distribution, the largest amount of acreage and the most abundance of processed products among major crops. Its yield and quality were adversely affected by the scarcity of water resources, land desertification and increasingly serious of abiotic stresses such as drought and salt, which promotes us to dissect its rules of stress response with the aim to stress-tolerance cultivar breeding. However, the response upon abiotic stress is governed by multi-genes, and so many physiological evens are involved, including cellular defense, ion transport, secondary metabolism, energy flow and so on. On account of the different chromosome type to Arabidopsis and rice (allohexaploid to diploid), the salt tolerance mechanism of wheat would be more complicated, such research progress in wheat is also much slower than in rice, corn and other crops.
Wheat is a glycophyte with limited salt tolerance, but its wild relatives have been demonstrated to be the pivotal gene pool for salt tolerance improvement breeding via genetic and cell engineering technologies. In our previous work, a new somatic hybrid introgression line Shanrong No.3(SR3) had been generated from hybrids of common wheat Jinan177(Triticum aestivum2n=42, JN177) with Thinopyrum ponticum (2n=70), a salt and drought tolerant grass. The salinity tolerance indices as well as physiological, biochemical and genetic parameters of SR3were significantly better than JN177, suggested that major salt tolerance genes and some minor genes controlled the salt tolerance of SR3.
Based on the transcriptomic and proteomic data of SR3, we found two genes showing obvious stress-responsive patterns, and isolated their full-length sequences via PCR from the SR3salt-stressed cDNA library and RACE cloning, respectively. Then, their functions in stress tolerance were performed. The main research contents and results were summarized as follows:
1. Cloning and preliminary functional analysis of TaHB30gene involved in salt stress
Based on the screening of SR3salt-stressed cDNA library, we found a gene that was up-regulated in response to salinity treatment, which was named TaHB30according to the library number. Its deduced protein sequence possesses a typical active site of Cu/Zn superoxide dismutase, including four Cu2+and four Zn2+binding sites. Therefore, TaHB30was classified into the Cu/Zn superoxide dismutase gene family.
The transcription of TaHB30was gradually induced both in SR3and JN177when treated with200mM NaCl. After exposure to10mM H2O2, the expression of TaHB30was down-regulated at the early phase and then gradually resumed in roots of two cultivars; in leaves, the gene appeared the comparable expression profile in SR3, while it was down-regulated during the whole treatment course. Moreover, SR3accumulated more TaHB30transcripts in SR3than in JN177under the two treatments.
Cu/Zn superoxide dismutase is found to be ubiquitously distributed in plant cells. To determine the action site of TaHB30, a subcellular localization assay was conducted by transiently expressing TaHB30-GFP fusion protein in onion epidermal cells and Arabidopsis protoplasts. The GFP signals traced that TaHB30located in both cytoplasm and nucleus.
TaHB30was transformed into Arabidopsis and wheat for determining its role in abiotic stress tolerance, and transgenic Arabidopis overexpression (OE) lines were used to further analyze. Under the control conditions, OE lines and vector control (VC) line had no developmental and reproductive difference during the whole life cycle. Under more than50mM NaCl treatment, OE lines showed more vigorous growth ability than VC line, and they had longer main roots and more amounts of lateral roots. Moreover, OE lines had higher tolerance capacity to H2O2. By contrast, OE and VC lines had unconspicuous phenotype in medium plate containing different concentrations of mannitol. Besides, TaHB30overexpression did not change the sensitivity to phytohormone such as IAA, JA, ABA, and GA3. These results speculates that TaHB30enhance the plant salt tolerance through the ROS pathway, which seems to have no interplay with the hormone pathways. TaHB30was further transformed into Arabidopsis defective in the homologue of TaHB30. In comparison with VC line, the mutants overexpressing TaHB30had shorter main roots under the control conditions, but similar ones under NaCl or H2O2treatments, which is possibly attributed to the modulating effect of TaHB30in ROS signaling pathway.
Semi-quantitative RT-PCR and real-time quantitative PCR indicated that OE lines had higher abundance of endogenous SOD (superoxide dismutase) transcripts than VC line under the control conditions. The genes encoding ROS scavenging enzymes catalyzing in downstream steps of SOD were also up-regulated, of which the transcription of GPX (glutathione peroxidase) was notably elevated, and APX (ascorbate peroxidase) and CAT (catalase) also had a higher expression levels. It was noteworthy that the expression of AtRBOH-D and AtRBOH-F, encoding two subunits of NADPH oxidase and functioning in H2O2generation, was significantly raised, what's more, OE lines did not show more vigorous growth ability than VC line to H2O2and inhibitor of NADPH oxidase; The expression of AtRBOH-D did not change and GPX had a higher expression levels in the mutants overexpressing TaHB30than VC line under the control conditions. The results above showde that Wheat Cu/Zn superoxide dismutase gene TaHB30played a pivotal role in salt tolerance through regulating NADPH oxidase of ROS signaling pathways.
Following the transcriptional profiles, the activities of these ROS generating and scavenging enzymes were also improved in TaHB30overexpressors. Of them, the activities of total SOD and Cu/Zn SOD were both remarkably increased in OE lines, the CAT and GPX activities of transgenic plants were higher approximately1.5-2and1.3times respectively than those of VC line, and the activity of nonspecific NADPH oxidase (NOX) was also increased by1.6fold. DAB staining showed OE lines had less H2O2content than VC line, while NBT staining exhibited that they had higher amount of superoxide anion.
These results confirmed that wheat Cu/Zn superoxide dismutase enhanced salt tolerance through ROS signaling pathways.
2. Functional analysis of TaCHP gene involved in salt stress
In our previous study, we identified a salt stress response gene TaCHP, which encoded a CHP-rich (for cysteine, histidine, and proline rich) zinc finger protein family gene. TaCHP possesses three divergent C1domains; C1domain was found to specifically bind to phospholipid signaling molecule diacylglycerol (DAG) in animals, and most of proteins with this domain have kinase activity (localize at cytoplasm) and/or act as transcriptional factors (localize in nucleus). Here, TaCHP was found to localize at cytoplasm and in nucleus, suggesting that TaCHP might serve as both a transcription factor and a putative DAG binding protein to confer salt tolerance. The in vitro PKC kinase activity result showed that TaCHP had no kinase activity. Transactivation assay showed that the full-length TaCHP and C-terminal two C1domains had transcriptional activation activity, which indicated that TaCHP might function as a transcription factor.
TaCHP was majorly transcribed in roots of seedlings at the three-leaf stage with a more remarkable level in SR3than in JN177. in situ hybridization further showed that the transcript localized in the cells of the root tip cortex and meristem. Its transcript abundance was reduced by the imposition of salinity or drought stress, as well as by the exogenous supply of ABA, but SR3overall expression maintained at a significantly higher level than JN177.
When JN17, a salinity hypersensitive wheat cultivar, was engineered to overexpress TaCHP, its performances in the face of salinity, drought and the indexes of physiological and biochemical indicators all improved, proving the abiotic stress tolerance of TaCHP. To study the mechanism of its action, TaCHP overexpression Arabidopsis lines(BS, A5) were constructed. In addition to the salt tolerance, the tolerance about H2O2and ABA increased in overexpression Arabidopsis lines. The expression levels of a number of stress reporter genes (AtCBF3, AtDREB2A, AtABI2, and AtABIl) were raised in the transgenic lines in the presence of salinity stress, while ABA synthetic genes AtAAO3, AtABA2and AtMYB15reduced. Real-time PCR displayed that the average increase expression of AtRBOH-D, which encoded a component of NADPH oxidase required for ROS production, was about7times in the overexpression lines, the ROS scavenging enzymes CAT and APX encoding genes AtCAT2and AtAPX2were induced, combining with the improved enzymatic activity results of SOD and CAT, indicating that ROS production and scavenging capacity were increased in BS and A5lines.
These data indicated that TaCHP participates in abiotic stress response through the ABA-dependent, ABA-independent and ROS signaling pathways.
由于小麦属于甜土植物,自身耐盐能力不强,而其野生近缘物种则是抗逆改良的重要基因库,可作为基因工程和细胞工程技术培育耐逆新品种的重要基因源。本实验利用不对称体细胞杂交渐渗技术,获得了盐敏感小麦品种济南177(Triticum aestivum2n=42, JN177)与强耐盐性小麦近缘属长穗偃麦草(Thinopyrum ponticum,2n=70)的渐渗系抗盐新品种山融3号(SR3)。山融3号的耐盐指数及各项生理生化指标均明显优于亲本小麦JN177,其耐盐性由主效基因和微效基因共同控制。
在本实验前期对SR3全基因组芯片和蛋白组分析的基础上,我们发现两个受胁迫表达变化的基因,结合SR3盐胁迫cDNA全长文库和RACE手段,获得基因全长。下面分别对两个基因进行功能验证,主要的研究内容及结果包括：
1.小麦耐盐相关基因TaHB30的克隆及初步功能分析
通过分析SR3盐胁迫cDNA全长文库基因,发现了一个受盐胁迫表达显著上调的基因,依据文库编号将其命名为TaHB30。在对其编码的蛋白序列进行Blast分析时,发现该基因含有Cu/Zn超氧化物歧化酶典型的激活位点,并包含有4个Cu2+结合位点和4个Zn2+结合位点,将其归类于Cu/Zn超氧化物歧化酶大类。
200mM NaCl处理条件下,在SR3及其亲本JN177根叶中总体为上调的表达趋势；10mM过氧化氢处理后,根部的变化情况SR3同JN177较为一致,都呈现先下调后上调的波动变化。叶中SR3仍为先下调后上调的趋势,而JN177则是下调表达。在这些处理中,TaHB30在SR3中的表达量均高于JN177。
由于Cu/Zn超氧化物歧化酶在植物细胞中分布比较广泛,为确定TaHB30的功能作用部位,对基因表达产物进行亚细胞定位。通过该蛋白偶联GFP,基因枪法转化洋葱表皮细胞和PEG诱导拟南芥原生质体,GFP荧光信号一致出现在细胞质与细胞核中,表明该基因产物主要在胞质同核中起作用。
构建TaHB30表达载体并分别转化拟南芥和小麦,对拟南芥过表达转基因株系(OE系)在多种逆境胁迫下的表型进行分析。在对照条件下,OE系和对照株系生长状况正常,没有明显差异；在不同浓度的Mannitol处理下,OE系与对照株系差异也不明显。但当NaCl浓度增加到50mM以后,OE系的生长状态明显好于对照株系,主根根长同对照植株相比较长,侧根的生长也比对照株系发达。在添加H2O2的培养基上,OE系的生长状况也明显优于对照株系。随后进行了不同种类激素,包括IAA、JA、ABA与GA3的处理,在对照和转基因株系中都未发现表型的明显差异。初步推测,TaHB30通过增强ROS途径促进OE系的耐盐能力,同相关的植物激素信号通路没有交汇。利用拟南芥突变体验证该基因功能,将TaHB30转化拟南芥突变体,发现TaHB30过表达拟南芥突变体在正常条件下比对照系的根短,而在NaCl和H202的处理下这种根长度的差异消除,推测与该基因调控拟南芥内源ROS的信号途径有关。
RT-PCR和real time PCR结果显示,正常生长条件下,拟南芥过表达株系(OEl,OE2)本底的超氧化物歧化酶(SOD)编码基因比对照株系表达量增高；而其下游多个ROS清除酶编码基因表达上调：谷胱甘肽过氧化物酶(GPX)表达量的增幅最为显著,其他如过氧化氢酶(CAT)、抗坏血酸过氧化物酶(APX)分别编码的2个基因在OE系的表达量同对照也具有显著差异。值得注意的是：位于ROS途径上游、与H2O2产生密切相关的NADPH氧化酶编码基因RBOH-D, RBOH-F的表达明显提高,并且在同时含有H202和NADPH氧化酶抑制剂的培养基上,OE系的生长状况不再优于对照株系；在正常条件下,TaHB30过表达拟南芥突变体的RBOH-D基因表达变化不显著,而GPX基因的表达量对照系比较明显上调。以上结果表明：小麦Cu/Zn超氧化物歧化酶基因TaHB30通过调控NADPH氧化酶的变化,进入ROS信号途径而在耐盐中起作用。
进一步测定了OE系及空载体对照的ROS相关酶活性,结果表明：OE系的ROS清除酶系统中总超氧化物歧化酶(SOD)和Cu/Zn超氧化物歧化酶的酶活力都较对照提高；过氧化氢酶(CAT)的酶活力约为对照的1.5-2倍；谷胱甘肽过氧化物酶(GPX)的酶活力约为对照的1.3倍。而ROS的产生酶-非特异性NADPH氧化酶(NOX)活性也在过表达拟南芥中明显升高(1.6倍)；DAB染色显示,TaHB30过表达株系中H202含量明显低于对照植株；而NBT染色后,超氧阴离了的含量高于对照植株。
以上结果表明：TaHB30通过调节ROS信号转导系统提高植物耐盐性。2.小麦耐盐相关基因TaCHP的功能分析
通过本实验前期工作,发现了一个盐胁迫应答新基因TaCHP,编码一个CHP-rich(半胱氨酸,组氨酸和脯氨酸丰富)的锌指蛋白家族基因。TaCHP含有能特异性结合磷脂信号分子甘油二酯(DAG)的C1结构域和可能的激酶结构域,含有该结构域的蛋白质大多具有蛋白激酶(定位细胞膜)和/或转录调控因子(定位细胞核)活性。本研究发现,TaCHP定位在细胞膜和细胞核,推测是一个具有蛋白激酶和转录因子双重功能的抗逆相关重要蛋白。体外PKC激酶活性测定表明,TaCHP表达蛋白无激酶活性。转录激活活性分析发现C’端两个C1结构域及TaCHP全长具有转录激活活性,表明该基因可能作为一个转录因子行使功能。
进行TaCHP胁迫表达模式及其定位分析,发现TaCHP主要在三叶期的小麦根部表达,原位杂交结果表明转录产物定位于根尖的皮层和分生组织细胞内。TaCHP表达量在SR3中比亲本JN177高,通过NaCl,干旱,ABA等处理后,二者都呈现下调趋势。但是,SR3总体表达量均维持在比JN177明显高的水平。
将TaCHP转化盐敏感小麦济南17,小麦的耐盐、抗旱、抗氧化能力及相关生理生化指标都明显提高,证明了该基因的抗逆功能。构建了TaCHP过表达(BS、A5)拟南芥株系,进一步研究该基因可能的作用机制。除了耐盐性以外,拟南芥对H202和ABA的耐受性也有所增加。在盐处理下,过表达系中一部分胁迫响应基因(AtCBF3, AtDREB2A, AtABI2和AtABI1)的表达量升高,而ABA合成基因AtAA03, AtABA2和AtMYB15的表达量降低。Real-time PCR显示转基因拟南芥BS、A5的AtRBOHD表达量平均增幅达7倍,其编码的NADPH氧化酶是ROS产生的重要组成部分,而活性氧清除酶CAT和APX编码基因AtCAT2、AtAPX2在拟南芥过表达株系中被诱导,结合转基因拟南芥BS、A5株系中较高的SOD和CAT酶活性,表明ROS的产生及清除能力在过表达株系中都增强。
以上结果表明：TaCHP通过ABA依赖和非依赖信号途径以及ROS信号途径参与植物对非生物胁迫的响应。
Wheat has the most extensive distribution, the largest amount of acreage and the most abundance of processed products among major crops. Its yield and quality were adversely affected by the scarcity of water resources, land desertification and increasingly serious of abiotic stresses such as drought and salt, which promotes us to dissect its rules of stress response with the aim to stress-tolerance cultivar breeding. However, the response upon abiotic stress is governed by multi-genes, and so many physiological evens are involved, including cellular defense, ion transport, secondary metabolism, energy flow and so on. On account of the different chromosome type to Arabidopsis and rice (allohexaploid to diploid), the salt tolerance mechanism of wheat would be more complicated, such research progress in wheat is also much slower than in rice, corn and other crops.
Wheat is a glycophyte with limited salt tolerance, but its wild relatives have been demonstrated to be the pivotal gene pool for salt tolerance improvement breeding via genetic and cell engineering technologies. In our previous work, a new somatic hybrid introgression line Shanrong No.3(SR3) had been generated from hybrids of common wheat Jinan177(Triticum aestivum2n=42, JN177) with Thinopyrum ponticum (2n=70), a salt and drought tolerant grass. The salinity tolerance indices as well as physiological, biochemical and genetic parameters of SR3were significantly better than JN177, suggested that major salt tolerance genes and some minor genes controlled the salt tolerance of SR3.
Based on the transcriptomic and proteomic data of SR3, we found two genes showing obvious stress-responsive patterns, and isolated their full-length sequences via PCR from the SR3salt-stressed cDNA library and RACE cloning, respectively. Then, their functions in stress tolerance were performed. The main research contents and results were summarized as follows:
1. Cloning and preliminary functional analysis of TaHB30gene involved in salt stress
Based on the screening of SR3salt-stressed cDNA library, we found a gene that was up-regulated in response to salinity treatment, which was named TaHB30according to the library number. Its deduced protein sequence possesses a typical active site of Cu/Zn superoxide dismutase, including four Cu2+and four Zn2+binding sites. Therefore, TaHB30was classified into the Cu/Zn superoxide dismutase gene family.
The transcription of TaHB30was gradually induced both in SR3and JN177when treated with200mM NaCl. After exposure to10mM H2O2, the expression of TaHB30was down-regulated at the early phase and then gradually resumed in roots of two cultivars; in leaves, the gene appeared the comparable expression profile in SR3, while it was down-regulated during the whole treatment course. Moreover, SR3accumulated more TaHB30transcripts in SR3than in JN177under the two treatments.
Cu/Zn superoxide dismutase is found to be ubiquitously distributed in plant cells. To determine the action site of TaHB30, a subcellular localization assay was conducted by transiently expressing TaHB30-GFP fusion protein in onion epidermal cells and Arabidopsis protoplasts. The GFP signals traced that TaHB30located in both cytoplasm and nucleus.
TaHB30was transformed into Arabidopsis and wheat for determining its role in abiotic stress tolerance, and transgenic Arabidopis overexpression (OE) lines were used to further analyze. Under the control conditions, OE lines and vector control (VC) line had no developmental and reproductive difference during the whole life cycle. Under more than50mM NaCl treatment, OE lines showed more vigorous growth ability than VC line, and they had longer main roots and more amounts of lateral roots. Moreover, OE lines had higher tolerance capacity to H2O2. By contrast, OE and VC lines had unconspicuous phenotype in medium plate containing different concentrations of mannitol. Besides, TaHB30overexpression did not change the sensitivity to phytohormone such as IAA, JA, ABA, and GA3. These results speculates that TaHB30enhance the plant salt tolerance through the ROS pathway, which seems to have no interplay with the hormone pathways. TaHB30was further transformed into Arabidopsis defective in the homologue of TaHB30. In comparison with VC line, the mutants overexpressing TaHB30had shorter main roots under the control conditions, but similar ones under NaCl or H2O2treatments, which is possibly attributed to the modulating effect of TaHB30in ROS signaling pathway.
Semi-quantitative RT-PCR and real-time quantitative PCR indicated that OE lines had higher abundance of endogenous SOD (superoxide dismutase) transcripts than VC line under the control conditions. The genes encoding ROS scavenging enzymes catalyzing in downstream steps of SOD were also up-regulated, of which the transcription of GPX (glutathione peroxidase) was notably elevated, and APX (ascorbate peroxidase) and CAT (catalase) also had a higher expression levels. It was noteworthy that the expression of AtRBOH-D and AtRBOH-F, encoding two subunits of NADPH oxidase and functioning in H2O2generation, was significantly raised, what's more, OE lines did not show more vigorous growth ability than VC line to H2O2and inhibitor of NADPH oxidase; The expression of AtRBOH-D did not change and GPX had a higher expression levels in the mutants overexpressing TaHB30than VC line under the control conditions. The results above showde that Wheat Cu/Zn superoxide dismutase gene TaHB30played a pivotal role in salt tolerance through regulating NADPH oxidase of ROS signaling pathways.
Following the transcriptional profiles, the activities of these ROS generating and scavenging enzymes were also improved in TaHB30overexpressors. Of them, the activities of total SOD and Cu/Zn SOD were both remarkably increased in OE lines, the CAT and GPX activities of transgenic plants were higher approximately1.5-2and1.3times respectively than those of VC line, and the activity of nonspecific NADPH oxidase (NOX) was also increased by1.6fold. DAB staining showed OE lines had less H2O2content than VC line, while NBT staining exhibited that they had higher amount of superoxide anion.
These results confirmed that wheat Cu/Zn superoxide dismutase enhanced salt tolerance through ROS signaling pathways.
2. Functional analysis of TaCHP gene involved in salt stress
In our previous study, we identified a salt stress response gene TaCHP, which encoded a CHP-rich (for cysteine, histidine, and proline rich) zinc finger protein family gene. TaCHP possesses three divergent C1domains; C1domain was found to specifically bind to phospholipid signaling molecule diacylglycerol (DAG) in animals, and most of proteins with this domain have kinase activity (localize at cytoplasm) and/or act as transcriptional factors (localize in nucleus). Here, TaCHP was found to localize at cytoplasm and in nucleus, suggesting that TaCHP might serve as both a transcription factor and a putative DAG binding protein to confer salt tolerance. The in vitro PKC kinase activity result showed that TaCHP had no kinase activity. Transactivation assay showed that the full-length TaCHP and C-terminal two C1domains had transcriptional activation activity, which indicated that TaCHP might function as a transcription factor.
TaCHP was majorly transcribed in roots of seedlings at the three-leaf stage with a more remarkable level in SR3than in JN177. in situ hybridization further showed that the transcript localized in the cells of the root tip cortex and meristem. Its transcript abundance was reduced by the imposition of salinity or drought stress, as well as by the exogenous supply of ABA, but SR3overall expression maintained at a significantly higher level than JN177.
When JN17, a salinity hypersensitive wheat cultivar, was engineered to overexpress TaCHP, its performances in the face of salinity, drought and the indexes of physiological and biochemical indicators all improved, proving the abiotic stress tolerance of TaCHP. To study the mechanism of its action, TaCHP overexpression Arabidopsis lines(BS, A5) were constructed. In addition to the salt tolerance, the tolerance about H2O2and ABA increased in overexpression Arabidopsis lines. The expression levels of a number of stress reporter genes (AtCBF3, AtDREB2A, AtABI2, and AtABIl) were raised in the transgenic lines in the presence of salinity stress, while ABA synthetic genes AtAAO3, AtABA2and AtMYB15reduced. Real-time PCR displayed that the average increase expression of AtRBOH-D, which encoded a component of NADPH oxidase required for ROS production, was about7times in the overexpression lines, the ROS scavenging enzymes CAT and APX encoding genes AtCAT2and AtAPX2were induced, combining with the improved enzymatic activity results of SOD and CAT, indicating that ROS production and scavenging capacity were increased in BS and A5lines.
These data indicated that TaCHP participates in abiotic stress response through the ABA-dependent, ABA-independent and ROS signaling pathways.
引文
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