Differential Expression Of Wheat Leaf Rust Resistance Near-isogenic Line TcLr38

Leaf rust is one of the most important diseases of wheat throughout the world. The use of resistant cultivars is the most economical, effective and safest method in leaf rust disease control. The resistance gene Lr38 derived from Agropyron intermedium, expressed high resistant levels for leaf rust disease is a resistance gene conferred by major gene which will have high potential in application. Therefore, it has great significance to reveal the resistance mechanism of wheat leaf rust resistance near-isogenic line TcLr38, further to seek fragments that related to disease-resistant gene expression.The study was carried out to detect the differential expression of genes in TcLr38 inoculated with Puccinia triticina strain 05-22-65 (THTS) by complementary DNA amplified fragments length polymorphism (cDNA-AFLP) analysis. The infection type of THTS to TcLr38 is"0;"while the infection type of THTS to Thatcher is"4". The main results were as follows:1. Two hundreds and twenty-four pairs of primers were used to screen for differential expression analysis between TcLr38 and Thatcher. As a result, 11200 bands were nearly detected totally, and fifty bands were amplified by each primer pair on average. Seventy-six primer pairs could amplify differential expression bands among TcLr38 and Thatcher, and the detection rate of differential expression bands was up to 33.9%. Polymorphic bands were grouped into eight different types, and three types of them were supposed to correspond to disease-resistant gene, Type I, including 19 bands in all, was presnet only in the TcLr38 inoculated with race THTS, was up-regulated. Type II containning with 17 bands, expressed in both TcLr38 and Thatcher were constitutive expression. Type III, including 8 bands was down-regulated.2. Twenty-one differential expression bands were cloned and sequenced. Blastx analysis showed that the seventeen sequences all had the homology sequences, and the amino acids showed similarity with the known sequences from 28%～98%, fifteen sequences were homology with the known function genes. Deduced 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 Ahydratase were supposed to correspond to the expression of the disease-resistant gene. 3. RT-PCR was employed to detect the differential expression of genes in TcLr38 and Thatcher at different time point (0h, 6h, 12h, 16h, 20h, 24h, 36h, 48h, 60h, 72h, 96h) postinoculation with P. triticina strain. RT-PCR analysis revealed that protein kinase C was present in TcLr38 at all the time point, and the highest expression time point was at 24h postinoculation. DksA can be detected only in inoculated TcLr38 and start expression at 6h postinocultation, the highest expression was detected at 24h postinoculation. The results indicated that the different types of expression have different expression patterns in the processing of the interaction between wheat leaf rust resistance gene Lr38 and the avirulent pathogen in leaves. DksA is the induced production only in the TcLr38, was deduced related with resistant event. Protein kinase C can be detected only in TcLr38 at all the time point in the test, but also be induced by P. triticina. The result indicats that Protein kinase C is related with the incompaetible interaction between Lr38 and P. tritici.4. According to the target fragment (351bp) amplified by primer P-AT/M-CAC, 867bp length fragment the sequence were gained, electric clone was carried out according to the fragment, and ganid Os06g0223800 hypothetical protein.