Cloning Of Three DUF Family Genes And Their Responses To Abiotic Stress In Wheat

In the process of growth and development,the wheats consistently suffer from varios environmental challenges,including drought,high salinity,high and low temperature,which adversely affect plant growth and induce severe losses in crop production.The EST sequences of water and cold stress-induced upregulated gene from cDNA microarray datas in the early were obtained.In this study,the full-length of TaDUF4220,TaDUF677 and TaDUF2042 genes which are related to abiotic stress were cloned according to these EST sequences.The three genes exhibited different expression levels in different tissues and developmental stages,and their expression patterns under high salt,PEG,high/low temperature and ABA treatment were studied.In order to explore the functions of TaDUF4220 gene,the overexpression vector of TaDUF4220 was builded and transferred into the arabidopsis thaliana.The main research results were as follows:1.Three stress resistance correlated genes were cloned by Reverse transcription-Polymerase Chain Reaction(RT-PCR)technique in the wheat.The sequence analysis showed that the full-length cDNA sequence of TaDUF4220 gene was 2016 bp,containing a 1782 bp open reading frame(ORF),with 32 bp in the 5'-UTR and 202 bp in the 3'-UTR,and predicted consists of 593 amino acids which is 68.275 kD.The genome sequence analysis revealed that TaDUF4220 gene contained two exons and one intron.The full-length cDNA sequence of TaDUF677 gene was 1731 bp,containing a 1230 bp open reading frame(ORF),with 298 bp in the 5'-UTR and 203bp in the 3'-UTR.Its predicted protein consists of 409 amino acids with the molecular weight is 45.044 kD.Sequence analysising showed that the full-length cDNA sequence of TaDUF2042 gene was 2555 bp,containing a 2457 bp open reading frame(ORF),with 84 bp in the 5' UTR and 15 bp in the 3' UTR,and predicted the molecular weight is 89.773kD which consists of 818 amino acids.2.The spatial expression patterns of the cloned genes in different tissues by real time PCR were studied.The results showed that the TaDUF4220 gene was mainly expressed in the tiller leaf,and seldom expressed in young ear.The expression quantity of TaDUF677 was the highest in the bud of 1 d,but the lowest in the tiller leaf and leaf of 10 d.And the expression quantity of TaDUF2042 was the highest in the bud of 1 d,but the lowest in the seed of post-flowering 25 d.The expression quantities of each gene in different tissues was different,and it showed that a certain tissue expression specificity of the three genes was existed in the wheat.3.The expression patterns of cloned genes under the high salt(NaCl),PEG,high temperature,low temperature and ABA stress treatments were analyzed.The results showed that except under the high temperature(42?)treatment,the expression levels of TaDUF4220 gene were all up-regulated in different levels under the conditions of other stress treatments in the leaf of 'jing 84 1';Even though the expression levels of TaDUF4220 gene were all up-regulated under all kinds of stress treatments in the leaf of ' Chinese Spring',the levels of the gene' s up-regulation were lower than that in'jing 841' The expression trends of TaDUF677 gene in the leaf of 'jing 841 ' and'Chinese Spring' were basically identical and all up-regulated,except in 'jing 841'leaf under PEG treeament.But the expression levels in 'Chinese Spring' leaf was higher than that in 'jing 841 ' leaf and the expression levels under different stress treatments were different.Except under the low temperature(4?)treatment,the expression levels of TaDUF2042 gene were all up-regulated under the conditions of other stress treatments in the leaf of 'Chinese Spring',but the expression patterns were opposite in the leaf of 'jing 841 ',with the expression up-regulated under low temperature treatment and the expression down-regulated under other stress treatment.4.The overexpression vector of TaDUF4220 was constructed and transferred into the arabidopsis thaliana via agrobacterium inflorescence infection method.eighteen transgenic arabidopsis strains by transformation and screening was gained.The primary function of transgenic T2 generation plants were identified and it showed that the seed germination rate of T2 generation obviously decreased 34%than that in controls after 3 days by 45?treatnent for 2h;And under different concentrations of ABA and 150 mM Nacl stress treatment in seedling stage,the growth conditions of transgenic Arabidopsis were weaker than that of wild type controls arabidopsis and it revealed that the excessive expression of TaDUF4220 gene may make the resistance of plants restrained.