【作者】 李莹；

【导师】 李杰；

【作者基本信息】 东北农业大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 低温、干旱、盐碱等逆境条件严重影响作物的产量和质量,是全球农业生产亟待解决的重大问题。通过基因工程手段改良植物的抗逆性是提高作物产量的有效途径之一。而如何利用抗逆基因资源,从大量的渗透胁迫响应基因中寻找到能够综合性提高作物抗逆性的基因,是植物渗透胁迫基因工程的重要前提。转录因子在植物逆境信号传递过程中发挥着关键作用,一个转录因子可以同时调控多个下游基因的表达,从而提高植物的抗逆性。耐盐野生大豆具有丰富的抗逆基因资源,是基因克隆的理想材料。本研究以耐盐野生大豆为试材,通过电子克隆、同源克隆、酵母单杂交方法克隆DREB(Dehydration ResponsiveElement-Binding protein)类转录因子基因;利用生物信息学手段预测基因的结构、并对获得的全长DREB转录因子基因做进化分析;应用同酵母单杂交系统对获得的DREB类转录因子基因进行体内结合特异性分析,同时比较其与DRE顺式作用元件结合能力的强弱。为野生大豆中DREB类转录因子基因的功能研究及其在植物抗渗透胁迫基因工程中的应用奠定基础。主要研究内容如下:1.电子克隆方法克隆GsDREB1基因应用电子克隆技术在耐盐野生大豆中克隆了1个DREB类转录因子基因,与GmDREB1基因序列相似性达到99.71%,命名为GsDREB1。经SMART和InterProScan预测表明,GsDREB1基因含有AP2结构域。2.同源克隆方法克隆DREB类转录因子基因应用同源克隆方法在耐盐野生大豆中克隆了5个DREB类转录因子基因,分别命名为GsDREBa、GsDREBb、GsDREBc、GsDREB2、GsDREB3。分别将各条基因进行序列比对,比对结果为:GsDREBc与GmDREBc的相似性为99.33%;GsDREBb与GmDREBb的相似性为97.44%;GsDREBa与GmDREBa与的相似性为93.08%;GsDREB2与GmDREB2的相似性为95.60%;GsDREB3与GmDREB3的相似性为100.00%。SMART和InterProScan预测结果表明这5个基因都含有AP2结构域。3.酵母单杂交方法克隆与DRE顺式作用元件结合的转录因子基因将耐盐野生大豆幼苗进行胁迫处理,构建cDNA融合文库。以DRE元件为“诱饵”,应用酵母单杂交方法克隆DREB类转录因子。从A52号克隆中得到了一条232bp的片段,这个片段不含有起始密码子和终止密码子,经SMART预测此片段含有AP2结构域。5’RACE延伸出了198bp,但这198bp在三种读码方式下都含有终止密码子。4.DREB类转录因子基因体内结合特异性分析经SMART预测,获得6个DREB基因的AP2结构域序列。应用Clastalxl.83软件将6个基因的结构域进行比对,结果表明6个基因结构域序列都具有典型的AP2结构域特征。PCR扩增6个基因保守结构域,并在保守结构域和A52克隆两端引入同源重组位点。应用酵母单杂交系统分析6个基因结构域的体内结合特异性,同时将转化混合物涂布在含有不同浓度3-AT的三缺SD培养基上,比较它们与DRE顺式作用元件结合能力的强弱。结果表明GsDREBa、GsDREBb、GsDREBc、GsDREB1、GsDREB2、GsDREB3都能够与DRE元件发生特异性结合,且与DRE顺式作用元件结合能力相当。但A52克隆的结合力较其它6个基因弱很多。5.DREB类转录因子基因的进化分析应用DNAstar软件对GsDREBa、GsDREBb、GsDREBc、GsDREB1、GsDREB2、GsDREB36个基因进行进化分析。进化树直观的表明GsDREB1与GsDREB2聚为一类;GsDREBa与GsDREBc聚为一类;GsDREBb与GsDREB3聚为一类。6个基因与祖先的远近关系为GsDREBb＞GsDREBa＞GsDREBc＞GsDREB1＞GsDREB2=GsDREB3。更多还原

【Abstract】 Environmental factors that impose osmitic stress,such as salinity,drought and cold,place major limits on plant productivity and quality.It is a big problem that deserves global attention. One of the effective ways to improving crop yields is genetic engineering of plant resistance.The important prerequisite for genetic engineering of plants osmotic stress is that how we can find the osmotic stress response genes that comprehensive increase crop resistance genes.Transcription factors play a key role in the signal transduction process.A transcription factor can control the expression of a number of downstream genes,thereby improving plant resistance.Salt-tolerant wild soybean that contains rich genetic resources of resistance is an ideal material for gene cloning.In this study,DREB(Dehydration Responsive Element Binding protein) transcription factor genes from the salt-tolerant wild soybean were cloned by silico cloning, homology cloning,yeast one-hybrid method.The genetic structures were predicted by bioinformatics tools,and evolutionary analysis of the full-length gene transcription factor DREB were performed.Yeast one-hybrid system was used to analyze their DNA-binding specificity and compare binding capacity of their donmain with DRE cis-acting element.That set the base of the function study on DREB transcription factor of wild Soybean and its application to osmotic stress. The main contents were as follows:1.cloning GsDREB1 gene by silico cloningA DREB transcription factor was cloned in salt-tolerant wild soybean by silico cloning.The similarity of the fragment and GmDREB1 gene sequence is 99.71%,named as GsDREB1. GsDREB1 contained AP2 domain through SMART and InterProScan forecasted.2.cloning DREB transcription factor gene by homology cloningFive DREB transcription factor genes were cloned in salt-tolerant wild soybean by homology cloning method.Those were named as GsDREBa,GsDREBb,GsDREBc,GsDREB2 and GsDREB3.The sequences were aligned and the result was following:the similarity of GsDREBc and GraDREBc is 99.33%;the similarity of GsDREBb and GmDREBb is 97.44%;the similarity of GsDREBa and GmDREBa is 93.08%;the similarity of GsDREB2 and GmDREB2 is 95.60%;the similarity of GsDREB3 and GmDREB3 is 100.00%.Six genes all contained AP2 domain through SMART and InterProScan forecasted. 3.Cloning transcription factor gene that binding DRE cis-acting element by Y1HcDNA fusion library was constructed with treated salt-tolerant wild soybean.Taking DRE cis-acting elements as "bait",DREB transcription factor genes were cloned by yeast one-hybrid. There was a 232bp fragment in A52 clone.The fragment contained AP2 domain by SMART forecast and did not contain start codon termination codon.198bp fragment was extended by 5’ RACE,but there was termination codon in the three reading modes of 198bp fragment.4.Analysis of DNA-binding specificitySix DREB domain sequences were found through SMART forecast,Aligned the six domain sequences with Clastalx1.83 software.The result showed that all of six domain sequences contained the typical characteristics of AP2 domain.Six conservative domains were amplified by PCR,and introduced homologous recombination sites at both sides of the conservative domain and A52 clone.Yeast one-hybrid system was used to analyze the DNA-binding specificity of the six genes,and transformation mixture was spreaded on three missing SD medium that contained different concentrations of 3-AT,then binding capacity of six donmain with DRE cis-acting element was compared.The result showed that GsDREBa,GsDREBb,GsDREBc,GsDREB1, GsDREB2,GsDREB3 can bind specificly with DRE cis-acting element,and their binding capacity to DRE cis-acting element were approximate.But the binding capacity of A52 clone was weaker than the other six genes.5.Analysis evolution of the DREB transcription factor geneDNAstar software was used to analyze the evolution of GsDREBa,GsDREBb,GsDREBc, GsDREB1,GsDREB2,GsDREB3.Phylogenetic tree shows directly that GsDREB1 and GsDREB2 clustered together;GsDREBa and GsDREBc clustered together;GsDREBb and GsDREB3 clustered category.The distance between six genes and their ancestors is GsDREBb＞GsDREBa＞GsDREBc＞GsDREB1＞GsDREB2=GsDREB3.更多还原