Cloning And Functional Characterization Of Heat Stress Responsive Gene TaFER?TaOEP16-2 And TaPEPKR2 In Wheat(Triticum Aestivum L.)

As the world warms,the intensity and frequency of extreme high temperature weather increase.Heat stress badly affects wheat yield and quality.As a major crop,the wheat yield has significant contribution to the national food security.The complexity of the wheat genome and practical difficulties of large-scale identification of heat tolerant wheat in the field are against the research of forward genetics.So,we identified heat stress responsive genes and applied them into wheat breeding to develop the heat tolerant cultivars.On the basis of genome-wide analysis of heat stress response profiles using Affymetrix Genechip(?)Wheat Genome Array,three genes were isolated and the function of these genes were analyzed under heat stress conditions.The study will establish the basis for wheat genetic engineering.The main results of this thesis are as follows:1.The complete ORF corresponding to the probe "Ta.681.1.Sl_x_at" encoding a ferritin protein was isolated.Based on the previous reported ferritin gene sequences,this gene was mapped to the chromosome 5B,and named TaFER-5B.Expression pattern analysis revealed that the relative mRNA abundance of TaFER-5B was induced by heat,PEG,H2O2 and Fe-EDDHA.In order to study the biological function of TaFER-5B,TaFER-5B was transformed into the wheat cultivar Jimai5265(JM5265)?Arabidopsis wild type plants and ferritin mutant ferl-2-3-4.Under heat stress conditions,TaFER-5B reduced the ion leakage rate of wheat and Arabidopsis,enhanced the chlorophyll fluorescence parameter Fv/Fm and could compensate the heat sensitivity phenotype of ferl-2-3-4.These results proved that TaFER-5B enhanced the themotolerance of transgenic wheat and Arabidopsis plants.The transgenic plants accumulated less ROS under heat stress conditions and suggested that TaFER-5B may enhance the themotolerance by scavenging ROS.In addition,TaFER-5B could enhance the drought,excess iron and oxidative stress tolerance,which was supported by the longer main root length of transgenic wheat and Arabidopsis plants under stress conditions.2.On the basis of cloning the gene corresponding to the probe "Ta.4874.1.S1_at" and verifing the thermotolerance in transgenic yeast.We demonstrated that this gene encoded an outer envelope protein,and named TaOEP16-2.Three homoeologous sequences of TaOEP16-2 were isolated from hexaploid wheat,which were localized on the chromosomes 5A,5B and 5D,respectively,using the Chinese Spring nulli-tetrasomic lines.These three homoeologues were all induced by heat,PEG and ABA,however,their expression intensity was different.TaOEP16-2-5B,the dominant one under heat stress conditions,was transformed into Arabidopsis.Compared with wild type plants,transgenic Arabidopsis plants overexpressing TaOEP16-2-5B gene exhibited enhanced tolerance to heat and drought stresses,which was supported by improved survival rate,strengthened cell membrane stability and reduced water loss rate.3.On the basis of cloning and verifying the TaPEPKR2 gene expression pattern,we transformed TaPEPKR2 into Arabidopsis and demonstrated that TaPEPKR2 could enhance heat and drought stress tolerance of transgenic plants,which was supported by improved survival rate,strengthened cell membrane stability and reduced water loss rate.In order to further confirm the biological function of TaPEPKR2 in wheat,TaPEPKR2 was transformed into the wheat cultivar Liaochun 10(LC10)and also demonstrated that TaPEPKR2 could enhance heat and drought stress tolerance of transgenic wheat plants.