Cloning And Analysis Of NBS-LRR Type Resistance Gene Analogs In Sweet Potato (Ipomoea Batatas)

Clonging resistance gene analog (RGA) is a new method for finding disease resistant genes from plants. The proceedures are as follows, first the polymerase chain reaction (PCR) for amplifing RGAs were carried out by using the degenerate primer that designed according to the conserved domains of the cloned R genes and the plant genomic DNA or cDNAs as template; second the RGAs were sequenced and homologous and phylogenetic relations among the RGAs and the cloned R genes were analyzed; third some of RGAs were selected as the candidate disease resistance genes; and finally a new plant resistant genes were discovered from these candidate. Compare with other methods of clonging genes, this method has the strongpoint of convenience, save labor and time. Up to now, some RGAs from different plants had been isolated and localized at resistant gene locus of chromosomes. Root rot disease of sweet potato caused by Fusarium solani(Mart) Sacc. f.sp. batatas Mcglure is one of the most serious disease in sweet potato production area. Shen jialian, a famous sweet potato breeding expert, breeded a variety, Xushu 18, that can highly resistant against Fusarium solani f.sp. batatas in 1976. But so far, there is no disease resistance-relative gene has been cloned from Ipomoea batatas. In our study, we cloned some RGAs from xu shu 18 by above method. The results are as follows:1. The results of RGAs cloneA specific fragment of 500bp was cloned from genomic DNA of Xushu 18 by PCR with degernate primers (PI: 5'-GGNATGGGNGGNNTNGGNAARACNAC-3' and P2: 5'-NACYTTNAGNGCNAGNGGNAGNCC-3'(R=A/G,N=A/T/G/C,Y=C/T)) designed according to the conserved domains (P-loop (mggvgktt) and "PLAL" (glplal)) in the NBS region of reported R genes. Before tansformed into E.coli DH5 a this specific fragment was recycled and inserted into pMD18-T vector. 158 recombinations were obtained and confirmed through colony PCR identification. Restriction digestion analysis divided these recombinations into 37 groups.2. The results of sequencing and BLAST37 clones representative of each group respectively were sequenced, among which 20 RGAs with complete open reading frames (ORFs) and recognizable NBS domains were found. These 20 RGAs sorted into two distinct types, TIR-NBS-LRR type and non-TIR-...