Cloning And Analyzing Of Powdery Mildew Resistance Gene Analogues In Hexaploid Wheat

Wheat (Triticum aestivum L., 2n=42, AABBDD) is one of the most important food crops in the world. Powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt), is a prevalent world-wide wheat disease. Cultivation of varieties with natural resistance is the most effective, economic and safe way to control powdery mildew disease. It is a new strategy for disease controlling to clone the resistance genes and transfer it into plants for the improvement of disease resistance.Pm3b is the first powdery mildew resistance gene isolated from hexaploid wheat. Pm3b is a member of a large NBS-LRR class gene family cluster. The isolation of other members of the gene family plays a very important role in the study of the evolution of NBS-LRR class resistance gene and the mechanism of disease resistance. In this study, wheat near-isogenic lines in Chancellor background Ulka/XX194/8*Chancellor (Pm2) and Chancellor were employed and degenerate primers were designed based on the conservation of 5'-terminus and 3'-terminus of Pm3b. Eleven resiatance gene analogues (RGAs) were acquired by PCR-based cloning in this study, and the characteristic of them were analyzed by bioinformatics programs. And meanwhile, we optimized the PCR conditions for long products. The main results were as follows:1. In comparison with the amplification of short fragment, the pH value of long PCR condition should be higher. But the pH value of GC buffer bought with LA Taq from TaKaRa was not high enough and should be adjusted higher properly so as to amplify long DNA fragment effectively from high-complexity templates.2. Temperature played an important role in the stable multiplication of the bacteria clone with long fragments. The bacteria clone with long fragment insertion was prone to lose its insertion completely or partially when multiplying in 37℃, but under the condition of 30℃, the clone would be more stable.3. Eleven RGAs were acquired in this study, and registered in GenBank respectively (accession number: EF157980-EF157990). The 11 RGAs shared high similarity with Pm3 alleles. And among them, three RGAs were pseudogenes and the others were protein encoding genes. Phylogenetic analysis revealed that the eight protein encoding genes formed a monophyletic group, and they were paraphyletic with Pm3 alleles. Given that the primers were designed mostly from Pm3b, we inferred that the 11 RGAs were from the large Pm3 family.4. Most positively selected sites distributed in LRR domain, which suggested that LRR domain bore more positive selection pressure than other domains.5. We preliminarily inferred that the evolution mode of NBS-LRR resistance gene was as follows: after the ancestor gene diverged, the majority of evolution events occurred in LRR domain at first, and then, after a period of time, the proportion of evolution events occurred in other domains would increase; the evolution events in LRR domain occurred in other secondary structures firstly, and then inβ-sheet.