Cloning And Analysis Of DREB Transcription Factor Gene In Salt Tolerance Wild Soybean

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.