Differential Expression Of Adult Plant Resistance Gene Lr35 To Wheat Leaf Rust

Wheat Leaf rust resistance gene Lr35 from an Triticum speltoides was transfered to the breed wheat Marquis through backcrossing with diploid T. monococcum. The gene was mapped on chromosome 2B. It closely links with the wheat stem rust resistance gene Sr39. Its resistance first appears at the growth stage of two leaves, and completely expresses at six leaves. Lr35 is highly effective against Puccinia recondita f.sp. tirtici, but not extensively used in the production yet. It is important to study the Lr35 expression for understanding the resistant mechanism, the gene cloning and its use in production.Complementary DNA amplified fragments length polymorphism (cDNA-AFLP) analysis was carried to investigate differential expression of the gene at different leaf-growing stages of TcLr35 with Thatcher as a check. The main results were as follows:1 .The optimized cDNA-AFLP selection amplification reaction system was established for wheat. The reaction system had a total volume of 20uL, containing 5uL template cDNA, 2uL 10×PCR buffer, 4mM dNTP, 0.6U Taq DNA polymerase, 20mM primer. The amplification performed as 2 min at 94℃ for wheat cDNA thermal predenaturalization; as 30s at 94℃ for denaturalizing, as 30s at 65℃ for annealling(dropped -0.7℃ at every cycle), as 60s at 72℃ for extension for 12 cycles; as 30s at 94℃ for denaturalizing, as 30s at 56℃ for annealling, as 30s at 72℃ for extension for 23 cycles and a final extension at 72℃ for 120s after the last cycle.2. Only six out of ninety six pairs of primers screened for cDNA-AFLP analysis amplified polymorphic bands among the treatments inoculated with wheat leaf rust fungus 98-12-1. The six primer pairs are E-TC/M-CAT, E-TG/M-CTC, E-TG/M-CAG, E-TG/N-CAC, E-TG/M-CTG,E-TC/M-CAA. Sixty eight differential expression bands were gained in this research.3. Two differential expression patterns wereobtained. The first one is the increasing of the quantity of differential expression with elongation of inoculation time. The primer combination E-TC/M-CAT amplified polymorphic bands at 260bp, 195bp, 190bp, which phenomena was not found with Thatcher. The second one is that the special differential expression bands appeared. The primers are E-TG/M-CTC,E-TG/M-CAG,E-TG/N-CAC, E-TG/M-CTG and E-TC/M-CAA. Especially primer combination E-TG/M-CTG amplifieda polymorphic band which was found at different leaf-growing stages of Lr35 from two leafs to six leaves and absent in Thatcher, the seedling period of Lr35, Lr35 without inoculation with the pathogen. The polymorphic bands was named E-TG/M-CTG-195. The differential expression band began to express at 60h after inoculation at growth stage of two leaves, at 48h at three leaves and four leaves, at 36h at five leaves and six leaves.4. Sixty eight differential expression bands were recoverd from the Gels and then amplified. Thirty three bands were successfully amplified, the efficiency reached to 49 percent. Through reverse Northern blotting testing, seven differential fragments were obtained. Out of the seven bands, the fragment obtained from primer combination E-TG/M-CTG proved to be a special gene fragment of Lr35.