| Powdery mildew caused by Blumeria graminis f.sp. tritici is one of the most serious diseases which influence the wheat production in China. Breeding for resistant cultivars has been proved the most effective way to control this disease. Pml6 near isogenic line (NIL) developed by Key Laboratory of Crop Germplasm and Biotechnology, possessing powdery mildew resistance gene Pm16, which is effective against all the current biotypes of Blumeria graminis in China. Cloning of Pm16 gene and the related genes for powdery mildew resistance is of significance for understanding disease- resistance mechanism and disease resistance breeding. In this study, bioinformatics method and RT-PCR technique were used to analyze the sequence characteristics and expression profile of the interested genes besides sequencing strategy.After inoculation with No.15 biotype of Blumeria graminis, the leaves from NIL Pml6-were harvested at 1, 3, 6, 9, 12, 16, 20 and 24 hours, respectively. A cDNA library was constructed using the above leaves. The titer of primary library was 1.04 l06pfu/ml.The recombinant percent accounted for 96%. The primary library was directly introduced into the E.coli BM25.8 without amplification. The transformation efficiency exceeded over 90%. The size of insert fragments basically ranged from 0.4 kb to 2.0 kb, with average of about 0.9 kb.216 sequences with high quality were obtained. These sequences were sent to GenBank for homologue search using Blastn and Blastx programs and were further classified based on their functions. The results are as follows: 102 sequences showed high homologue to 13 classes of genes with known function; 42 sequences were matched to the genes whose protein function were unclassified; 58 sequences showed homologoue to ESTs; 14 sequences hit no any sequence in GenBank.Among the 102 sequences which hit the genes with known function, 33 sequences showed homologue to 30 putative genes related to disease resistance, which are involved in the whole course of disease resistance response. These genes included ^? genes whose protein product could recognize pathogen; small GTP-binding protein-like genes; ion channel component; protein kinase participating in signal transduction; transcript factors regulating gene expression; protein induced by hypersensitive; proteins involved in defense response; protein indispensable for basal defense and non-host resistance, protein related to cell self protection in disease-resistance response and host factor interacting with pathogen.Among the 33 sequences which hit putative genes related to disease resistance, 9 sequences were chosen for analyzing sequence characteristics using bioinformatics software. Primers were designed based on their sequences. RT-PCR was performed to study the expression profile in different organs and different sampling time point after inoculation with powdery mildew. The wheat gene TaCDPK with high similarity to calcium-dependent protein kinase genes was 1172 bp length and contained an intact open reading frame with 876 bp length which encoded 291 amino acids. The protein encoded by the TaCDPK gene included STYKc domain and four EF-hands. Compared with other CDPKs, theprotein was short of 13 amino acid residues. Wheat TaXa21 gene was 1120 bp length and contained an intact open reading frame with 378 bp length which encoded 125 amino acids.The expression of the above two genes was strongly influnced by powdery mildew. The two genes were involved in wheat powdery mildew defense response and there existed significant difference between resistant and susceptible plants in expression model and level. The transcript accumulation of the other seven genes showed no changes at all the sampling time point. Organ expression profile indicated that expression level of TaCDPK gene, TaXa21 gene, TaABC gene and TaMYB gene varied with different organs.Based on the obtained sequence of putative glycine-rich protein gene in this study, a pair of primers was designed to amplify genome DNA corresponding to 5 different materials. A fragment with abou...
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