小麦抗叶锈近等基因系TcLr38的cDNA-AFLP分析

cDNA-AFLP Analysis of Near-isogenic Line TcLr38 Resistance to Wheat Leaf Rust

【目的】分析经叶锈菌诱导小麦抗叶锈近等基因系TcLr38的基因表达情况,寻找与抗病基因表达相关的片段。【方法】应用cDNA-AFLP技术从mRNA表达水平研究TcLr38和感病对照Thatcher与小麦叶锈菌05-22-65(THTS)互作时基因表达的差异。【结果】76对引物组合检测到约3800条条带,平均每一对选择性引物可以获得50条条带,其中28对能够在小麦近等基因系TcLr38和感病对照Thatcher之间扩增出特异性条带。多态性条带划分为8种类型,其中3种类型可能与抗病基因相关。最终得到95条小麦抗叶锈近等基因系TcLr38的特异性表达的转录衍生片段(TDF),其中19条差异TDFs只在接种叶锈菌的TcLr38中出现,为上调表达,而8条差异TDFs为下调表达。对可能与抗病相关的特异TDFs中的21条片段进行序列测定与分析,经BLASTx比较,17个TDFs所推导的蛋白质序列在数据库中找到其所对应的同源序列,15个TDFs为已知功能的基因。【结论】通过对功能已知的基因分析,推测决定蛋白激酶C、ATP结合蛋白、类受体激酶、信号传导组氨酸激酶(HPK)、肽酶家族M23、Dnak抑制蛋白DksA、甲硫氨酸tRNA合成酶、RanGTP酶激活蛋白1、烯酰辅酶A水合酶的TDFs可能与抗病或防御过程相关。

[Objective] The objective of the research is to analyze the gene expression profile of wheat near-isogenic line TcLr38 induced by Puccinia triticina and to detect the fragments related to disease-resistant gene expression. [Method] The study was carried out to detect the differential expression of genes in TcLr38 and Thatcher inoculated with avirlent P. triticina strain 05-22-65 （THTS） by complementary DNA amplified fragments length polymorphism （cDNA-AFLP） analysis. [ Result] Seventy-six pairs of primers were used to amplify products for cDNA-AFLP analysis. About 3800 bands were detected and the average number of bands per pair of primers amplified was 50. Twenty-eight of 76 primer combinations for cDNA-AFLP analysis amplified specific bands. Polymorphic bands between TcLr38 and Thatcher were grouped into eight different types, and three types of them were supposed to correspond to the disease-resistant reactions. Ninety-five transcripts derived fragments （TDFs） were selected for their differential expression from wheat near-isogenic line TcLr38 and Thatcher. And nineteen TDFs were upregulated and only expressed in the inoculated TcLr38, whereas 8 TDFs were suppressed. Twenty-one TDFs were sequenced and analyzed. BLASTx analysis showed that 17 TDFs had the homologous sequences with the sequence in GenBank, and fifteen were homologous with the known function genes. [Conclusion] The TDFs confer protein kinase C, ATP binding protein, receptor-like kinase,signal transduction histidine kinase, putative dnaK suppressor protein DksA, Methionine-tRNA synthetase, Peptidase M23, Ran GTPase activating protein 1, and Enoyl-Coenzyme A hydratase were supposed to correspond to the expression of the disease-resistant reactions.