Wheat Disease Resistance Related Gene Cloning And Powdery Mildew Resistance Mechanism Study

In order to study molecular mechanisms of wheat(Triticum aestivum L.)disease resistance,using RNAs of Arabidopsis thaliana,Triticum aestivum and Triticum turgidum as templates,different primers designed based on sequences of disease resistance related genes were used for PCR amplification,one arabidopsis NPR1 gene(No.ZS863),three wheat TaXa genes(No.ZS2000,ZS2001, ZS860)were isolated.Then we constructed plant expression vectors with these genes to research their functions.To investigate the function of PR genes and salicylic acid (SA)in wheat powdery mildew resistance reaction,the expression patterns of PR-1, PR-2 and PR-5 in seedling leaves of susceptible and resistant wheat lines were analyzed by semi-quantitative RT-PCR after Blumeria graminis f.sp.tritici(Bgt) infection or SA treatment.Results were as follows:1.The arabidopsis NPR1 gene played an important role in activating various plant defensive responses,including expression of the pathogenesis-related genes and systemic acquired resistance.The NPR1 gene was amplified from Arabidopsis thaliana RNA by RT-PCR method.Compared with NPR1 reported before,our clone had one different nucleotide,the nucleic acid sequence homology was 99%,and protein sequence was exactly the same.NPR1 protein structure included two Ankyrin repeats and one BTB/POZ domain.We constructed a plant over-expressin vector of pAH-NPR1,using pAHC25 as basic vector.Then pAH-NPR1 was transferred into susceptible cultivars by pollen-tuber pathway.Molecular detection of the transgenic plants was undergo.2.Near the HMW-glutenin gene locus of wheat(Triticum aestivum),there is a locus(temporarily named TaXa)encoding an LRR-receptor like protein kinase,which is homologous to rice(Oryza sativa)disease resistance protein Xa21.Through RT-PCR approach,three cDNA clones of ZS860(GenBank accession No.EF394367.), ZS2000(GenBank accession No.EF394368)and ZS2001(GenBank accession No. EF394369)were isolated at the orthologous loci of TaXa in T.aestivum and Triticum turgidum respectively.The ancestral TaXa locus probably encoded a 1 028 amino acid long peptide.Nucleotide base pair substitution,deletion and insertion happened during gene evolution leaded to the open reading frame change,and the encoded peptides were shortened.A complete TaXa protein includes a N-terminal conserved sequence,LRR domains,a transmembrane region and a serine/threonine protein kinase domain.The amino acid sequences encoded by TaXa orthologous loci in Triticum and one of Hordeum vulgare were compared in detail.3.Pathogenesis-related protein genes(PRs)of PR-1,PR-2 and PR-5 are the marker genes of plant disease resistance(R)gene-mediated resistance and systemic acquired resistance(SAR).To investigate the function of PR genes and salicylic acid (SA)in wheat powdery mildew resistance reaction,the expression patterns of PR-1, PR-2 and PR-5 in seedling leaves of susceptible and resistant wheat lines were analyzed by semi-quantitative RT-PCR after Bgt infection or SA treatment.The results showed that the transcribing of PR-1,PR-2 or PR-5 was significantly enhanced by Bgt infection in Zhoumai 18.The expressions of PR-1 and PR-5 were activated more quickly and strongly in resistant wheat line than in susceptible wheat line.SA significantly activated the transcriptions of PR-1 and PR-5,but had less effect on PR-2.Both Bgt and SA could significantly upregulate the expressions of PR-1 and PR-5,but the effect of Bgt was stronger than that of SA.Results indicated that PR-1 and PR-5 played an important role in wheat powdery mildew resistance.PR-1 and PR-5 may be considered as the marker genes of SAR in wheat.The powdery mildew resistance of Zhoumai 18 and Chinese Spring was enhanced after SA treatment.This suggested that SA played a role in wheat powdery mildew resistant signal transduction cascades.