【作者】 徐照龙；

【导师】 马鸿翔；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2013， 博士

【摘要】 我国约有3000万公顷盐碱化和次生盐碱化土地,土地盐渍化已成为我国农业生产发展的主要限制因素之一。研究植物的耐盐机理,筛选和培育适应盐土环境的耐盐植物,对于盐碱土地生态系统的改善及土地资源的可持续发展与利用具有深远的意义。大豆是重要油料作物和经济作物,研究大豆耐盐机理对培育耐盐大豆品种具有重要意义。本研究以栽培大豆和滨海野生大豆为研究对象,分析盐胁迫差异表达基因,并研究了其中3个表达差异显著的基因bZIP110、WRKY49和WRKY111的表达特征、调控机制与生理功能,试图为大豆耐盐性的遗传改良提供参考依据。利用数字表达谱(DGEP)对耐盐的野生大豆品系“野大豆2号”和盐敏感的栽培大豆品种“东农690”进行盐胁迫差异表达基因分析,发现野生大豆中共有1327个基因发生差异表达,826个上调表达,501个下调表达；栽培大豆中共有3627个基因表达发生差异表达,上调表达1709个,下调表达1918个。对几个差异表达基因进行qRT-PCR验证,结果显示与DGEP数据中的表达趋势一致。经过筛选分析,选择转录因子bZIP110、WRKY49和WRKY111作为与大豆耐盐性相关的候选基因。利用大豆转基因复合植株、转基因拟南芥以及转基因烟草鉴定候选基因耐盐性,结果显示bZIP110、WRKY49以及WRKY111基因均具有提高转基因植株耐盐能力的作用。大豆转录因子bZIP110基因全长1542bp,没有内含子,编码区(CDS)长507bp,编码168个氨基酸。亚细胞定位将该基因定位于细胞核中,聚类分析其属于S组群。bZIP110受NaCl诱导,NaCl处理12h后,在栽培大豆和野生大豆中都出现显著响应。组织表达分析发现,bZIP110在根、茎、叶中都有较高表达,只有在R4(盛荚期)叶中的表达量较低。过表达bZIP110能提高转基因拟南芥植株的抗盐胁迫能力,减少叶片内Na+的积累,上调胁迫应答基因MYB2、PADS、UGT71B6、LCL1、DREB2、 NHX1、SOS1和RCI3。其中RCI3比野生型上调达30倍,RCI3编码一个过氧化物酶(POD),可催化过氧化氢(H202)、酚类和胺类化合物分解,具有消除过氧化氢和酚类、胺类毒性的作用,推测这可能是bZIPHO能提高转基因拟南芥耐盐性的主要原因。转基因拟南芥中胁迫应答基因CCA1.LTP3和P5CS没有显著差异,LHY有显著降低。酵母双杂交试验发现bZIP110具有自激活作用,并且能与C组群的bZIP105互作。酵母单杂交试验发现bZIP110与DNA基序ACGT有一定的结合作用,但没有bZIP105与之的结合作用强。综合酵母单杂交和双杂交结果,可以推断bZIP110与bZIP105相互作用形成异源复合二聚体后,bZIP105绑定DNA序列,而bZIP110行使激活下游基因的作用。大豆转录因子WRKY49基因全长3519bp,有4个内含子, CDS长1728bp,编码575个氨基酸,亚细胞定位结果显示其定位于细胞核中,具有两个WRKY结构域和一个“锌指基序”,聚类分析显示属于Ⅰ组群。VRKY49被盐胁迫强烈诱导。200mMNaCl处理后,WRKY49在栽培大豆中响应速度快,但是在野生大豆中表达更稳定。这可能与野生大豆更耐盐有一定关系。组织表达分析发现,WRKY49在所有组织中都有表达,在根和叶中表达量高,在豆荚中的表达量低。拟南芥突变体证明其功能缺失后,盐胁迫条件下种子发芽率比野生型低；幼苗根长也显著比野生型短。过表达WRKY49能提高转基因拟南芥和烟草植株的耐盐性,上调表达LHY、UGT71B6、 DREB2、PAD3、RCI3、LTP3、NHX1和SOS1等8个基因,其中RCI3上调表达60倍以上。CCA1、MYB2、LCL1和P5CS等4个基因的表达水平没有明显变化。但转基因拟南芥叶片中Na+含量与野生型无显著差异,WRKY49可能提高了转基因拟南芥Na+区隔化的能力。大豆转录因子WRKY111基因全长3618bp,3个内含子,CDS长1197bp,编码398个氨基酸,具有1个WRKY结构域,属于IIc组群。WRKY111受盐胁迫强烈诱导。盐胁迫后WRKY111在栽培大豆中响应速度快,而在野生大豆中表达逐步上调,相对栽培大豆中更稳定,这可能跟野生大豆更耐盐有一定的关系。主要在根中表达,在茎、叶和花中表达量低,而在荚中基本不表达。过表达WRKY111能提高转基因拟南芥和烟草植株的耐盐性,上调表达DREB2、MYB2、PAD3、RCI3、LTP3、NHX1和SOS1等7个基因,其中编码POD基因RCI3比野生型高100倍,CCA1、LCL1、UGT71B6和P5CS等4个基因没有明显变化,LHY比对照低。转WRKY111基因拟南芥植株Na+含量显著低于野生型。上调表达过氧化物酶基因RCI3和降低叶片内Na+含量可能是WRKY111提高转基因植株耐盐性的主要原因。更多还原

【Abstract】 There are about30million hectares of salinization land in China. Therefore soil salinization becomes one of the main limiting factors in agricultural production. Considerably, researches on the mechanism of salt tolerance, selection and development of salt tolerant varieties have profound significances for the improvement ecosystem in salinization area and the sustainable application of land resources.In this regard, soybean is an important industrial crop in the world. To research the mechanism of salt tolerance in soybean can be helpful for the genetic improvement for salt tolerance in soybean. In this study, we analyzed differentially expressed genes under salt stress in coastal wild soybean (Glycine soja) and cultivated soybean (Glycine max) by using DGEP (digital gene expression profiling) technology. Three differential expressed genes, bZIP110, WRKY49and WRKY111were selected as the candidates of salt response genes for further study on their physiological function and molecular mechanism.In DGEP data, there were1327genes which responded salt stress in Glycine soja line "Yedade2",826up-regulated,501down-regulated. Our results demonstrated there were3627genes responded salt stress in Glycine max cultivar "Dongnong690",1709up-regulated,1918down-regulated. We got the same result with DGEP data about some respond genes’expression tendency using qRT-PCR. In addition, we chose significant salt respond genes as candidates including bZIP110, WRKY49and WRKY111, to research their function on salt stress using composite plants and transgenic plants. In the result we found that all the candidate genes could improve salt tolerance of composite and transgenic plants.Transcription factor gene bZIP110is1542bp in soybean genome, with no intron, coding168amino acids. Its subcellular localization is in the nucleus and belongs to S group. bZIP110was induced by200mM NaCl stress, responded salt stress distinctly in Glycine soja and Glycine max under200mM NaCl for12h. In the analysis of expression in different tissues, we found it had a high expression level in the root, stem and leaf in Glycine max, while had a low expression level in leaf only in R4(full pod stage). Moreover our results indicated that, bZIP110enhanced salt tolerance in transgenic Arabidopsis plants, the content of Na+was less than in transgenic plants’leaves, and the expression level of stress respond genes MYB2, PAD3, UGT71B, LCL1, DREB2, NHX1, SOS1and a POD (peroxidase) gene RCI3was higher than wild type. POD can remove H2O2(hydrogen peroxide) and the toxic of phonel, keep oxidative balance in plant under abiotic stress, and this may related to the major reason of more salt tolerance of transgenic plant than wild tpye. The expression levels of genes CCA1、LTP3and P5CS were no difference in transgenic plant compared to wild type, but the expression level of LHY was down regulated. Considerably we realized that, bZIP110had an activation function and interactive role with bZIP105belonging to C group in Y2H (yeast two hybrid), and could bind ACGT motif in Y1H (yeast one hybrid). But we must mention that, the capacity of binding ACGT motif was recorded weaker than bZIP105. We also infer that bZIP105binds promoter of a gene and bZIPl10activates it under salt stress in plant.Transcription factor gene WRKY49is3519bp in soybean genome, with4introns, coding575amino acids. Its subcellular localization is in the nucleus. It has two WRKY domains and one zinc-finger motif, belongs to group Ⅰ. WRKY49was induced by200mM NaCl stress, it responded salt stress more swift and violent in Glycine max under200mM NaCl, but the respond was more stable in Glycine soja, this maybe has relations with the reason of Glycine soja more salt tolerant. WRKY49expressed in all tissues,it had a high expression level in root and leaf, low expression level in pod. Over-expression WRKY49could enhance the salt tolerance in transgenic tobacco and Arabidopsis plants. Germination rate of seeds was lower and length of root was shorter in Arabidopsis mutants than wild type under salt stress. The POD gene RCI3was significant up regulated in transgenic Arabidopsis, stress respond genes LHY, UGT71B6, DREB2, PAD3, LTP3, HNX1and SOS1were up regulated too, but the content of Na+in transgenic leaves was no distinct difference compared to wild type. So we conclude that WRKY49have the ability to enhance the compartmentation of Na+in transgenic plant.Transcription factor gene WRKY111is3618bp in soybean genome, with3introns, coding398amino acids. It has one WRKY domains and one zinc-finger motif, belongs to group Ⅱc. WRKY111was induced by200mM NaCl stress, it responded salt stress more swift and violent in Glycine max under200mM NaCl, but the respond was more stable in Glycine soja, this maybe has relations with the reason of Glycine soja more salt tolerant. WRKYlll mainly expressed in root, low expression level in leaf and flower, no expression in pod. Over-expression WRKY111could enhance the salt tolerance in transgenic tobacco and Arabidopsis plants. The expression level of POD gene RCI3was100folds higher in transgenic Arabidopsis than wild type, and also up-regulated other six stress-respond genes DREB2, MYB2, PADS, LTP3, NHX1and SOS1. Four genes CCA1, LCL1, UGT71B6and P5CS expression levels in transgenic plants were no differences with wild type, but the expression level of LHY was down regulated. The content of Na+was less in transgenic plants’leaves than in wild type. Moreover, we confer that the major reason of WRKYlll enhancing salt tolerance in transgenic plants is up-regulating RCI3and limiting the accumulation of Na+in leaves.更多还原