The Analysis On The Phenotypes Of Colletotrichum Gloeosporioides Pathogenicity-related Mutants And The T-DNA Flanking Sequence

Rubber plant anthracnose, one of the most serious rubber foliar diseases, mainly caused by the infection of Colletotrichum gloeosporioides (Penz.) Sacc.The Construction of anthracnose ATMT (Agrobacterium tumefaciens-Mediated Transformation) transformants library, identification of the relevant mutant, using molecular biology techniques to isolate and identificate the disease related genes of Colletotrichum gloeosporioides (Penz.) Sacc, is the way to understand its pathogenesis and mechanism of interaction important tool.This is not only important in plant pathology, but also significant for the production of disease control and further explore new ways to provide clues for the breeding of durable resistant varieties, at the same time, it provide theoretical basis for the developing sustainable management strategies Colletotrichum gloeosporioides (Penz.).In the study, we mutated Colletotrichum gloeosporioides RC-178 with T-DNA insertion Agrobacterium tumefaciens-mediated transformation (ATMT), using the promoter trapping vector pCAHPH which has been constructed by our lab. We increased the number of the T-DNA inserted mutants to 1,685.15 transformants which reduced greatly in pathogenicitv were selected for detection. Observation of the biological properties of these mutants and found that two color abnormalities, three colonies grow slowly, eight sporulation was significantly reduced, two conidia morphology abnormalities, two attached to abnormal cell morphology,3 could not form appressoria, in the onion skin infection nail on the formation rate decreased significantly.These 15 mutants were tested with PCR for their insertional sequence, the results showed that all the genomes of these mutants contained the sequence of hygromycin phosphotransferase (hph) gene, while the wild type genomic DNA amplification of less than, that means mutant phenotype is as the result of exogenous DNA insertion. TAIL-PCR method using these 15 mutant genomic DNA were amplified by specific fragment, and these DNA sequences were cloned and sequenced to obtain three sequences, these sequences in the NCBI Blast search on the site.The results are as follows:A 1275bp fragment was cloned from Mutant T-0900-1 to sites of T-DNA insertion of the right sector, in which there are 134bp (950bp-1143bp)and Neurospora as a conservative assumption that the highest protein homology, while the 113bp (966bp-1143bp) and the Magnaporthe grisea in the 70-15 conserved hypothetical protein (MGG₀3680) also has high homology. T-0003-3 of T-DNA insertion sites cloned 688bp fragment of the right sector, Flanking sequence 1bp-240bp and 240bp-301bp of sequence and Neosartorya fischeri, Aspergillus fumigatus eukaryotic translation initiation factor 3 are highly homologous.712bp fragment of T-1103-2 was cloned into the left border is not found with BLAST analysis of homologous gene sequences, we speculate that the gene is damaged in the fungi in the biological function of genes has not been determined.