The Cloning And Analysis Of Resistant Gene To Phytophthora Capsici

Phytophthora capsici is a fatal disease of pepper. It occurs widespread in pepper planting areas over the world, and having become the main barrier that affects pepper production in China. Phytophthora capsici L. can survive long term in soil and the temperature and humility that suit for pepper production are also suitable for the growth and regeneration of Phytophthora capsici L. So Phytophthora capsici often epidemics outbreak. Breeding resistant varieties is an effective way to avoid or reduce the harm of Phytophthora capsici. Many scholars believe that there is no immune variety that against Phytophthora capsici and the inheritance of resistance to this disease is very complex. Therefore, cloning, marking and molecular locating of Phytophthora capsici resistance related genes are key steps of Molecular Marker Assisted Breeding in selecting pepper resistant varieties.The materials of this research were pepper varieties which are resistant or susceptible to Phytophthora capsici. They were supplied by pepper study team in Collage of Horticulture, Northwest A & F University. Among them, A1, A2, A5 and A15 are resistant varieties and B2 and 2096 are susceptible varieties. Extracted DNA from pepper leaves using development SDS methods. Many RGAs (Resistance gene analogs) were gained by PCR amplification using degenerate primers which designed by the conserved domains in R genes in GenBank. Analyzed these RGAs, the main 4 results were as follows.1. Designed degenerate primers YB5 and YB8 by P-loop and Kinase-2a which are two substructures of the NBS class conserved domains in TMV resistance gene N in tobacco (U15605), rust resistance gene L6 in Flax(U27081)and tomato clove pseudomonas resistance gene RPS2 in Arabidopsis thaliana(U12860). Gained 17 RGAs from 6 different pepper varieties that are resistant and susceptible to Phytophthora capsici using degenerate primer PCR. 17 RGAs were submitted to GenBank and got accession number from EF194114 to EF194130. BLAST analysis was used for 14 read-through RGAs. The result showed that the similarity among these RGAs and resistance genes or resistance gene analogs of tomato, pepper, potato and tobacco are 81%-97%. The amino acid multiple sequence alignments among these RGAs and NBS structure of Gap2 , I2C-1 , Xa1 , L6 , RPP5 , N , RPS2 , Mi-1.2 and Bs showed that they all contains P-loop (GVGKTT) and Kinase-2a( LLVLDDVW ) which are only in NBS class R genes.2. Designed degenerate primers YB17 and YB18 by LRR class conserved domains in leaf mold resistance gene Cf-2(U42445)and Cf-9(U15936)in tomato. Gained 27 RGAs from 6 different pepper varieties that are resistant and susceptible to Phytophthora capsici using degenerate primer PCR. 27 RGAs were submitted to GenBank and got accession number from EF064260 to EF064286. Among them, the longest ORF is more than 130 AA. in 18 RGAs, 80-130 AA. in 7 RGAs and less than 80 AA. in 2 RGAs. The result of BLAST analysis showed that the similarity between 21 RGAs and leaf mold resistance gene of tomato is 80%-96%. The result of multiple sequence alignments showed that there is high similarity between amino acid sequence deduced from nucleotide sequences of A5-1, A5-5 and amino acid sequence of Cf-2, and in some domains the sequences are same.3. Designed primers YB25 and YB26 by the sequence of blight resistance gene in potato (GenBank accession number is AY303171) using Primer software. Gained 10 RGAs from 6 different pepper varieties that are resistant and susceptible to Phytophthora capsici. The result of BLAST analysis showed that the similarity among these RGAs and blight resistance protein genes of potato are 82%-90%. The result of nucleotide multiple sequence alignments showed that the similarity among 10 RGAs are 82%-100%. The result of amino acid multiple sequence alignments among these RGAs and blight resistance genes SH10, RPI, RGA1, RGA2, RGA3, RGA4, SH2 and T118 showed that the similarity of amino acid sequences deduced from nucleotide sequences of these RGAs are 71%-100%, the similarity among these RGAs and R genes are 70%-90%. The similarity of amino acid sequences between A1-2 and RGA4, A5-1 and RGA4, B2-4 and RGA4 are 89%, 89% and 90% respectively.4. RGAs that are associated with resistance to Phytophthora capsici were gained from both resistant and susceptible pepper varieties.