Spotting The Positions On The Genome For 4 Pathogenicity-related Mutagenic Sites Of Magnaporthe Grisea And Analyzing On A Peg Deficiency Mutant

Magnaporthe grisea (MG) is the pathogenic fungus of rice blast which is one of the most damaging diseases in the world. The research on the sorts and function of the pathogenic genes, the relationship between the pathogenic genes and the physiological race, the heredity of the pathogenic genes and the gene flow within the population is theoretically and practically helpful, not only to the breeding and selection of rice blast resistant varieties, but also to the disease chemical and biological control.In this research, 7 pathogenicity-related mutants were selected from the T-DNA insertional mutant bank formerly established by ATMT (Agrobacterium tumifaciens -mediated transformation) for further study. The genome DNA of these mutants were extracted and the T-DNA insertion were confirmed by PCR reaction. The sequences flanking the T-DNA inserted sites on the genome DNA were amplified from these 7 pathogenicity-related mutants by DW-ACP-PCR technology. The specific fragments were amplified from all the participant mutants. These fragments were recovered and cloned. All these cloned fragments were sequenced. Among these fragments, 5 were successfully sequenced, 2 failed in sequencing. All the 5 sequences of the successfully sequenced fragments were compared with the published genome database of MG and NCBI by BLASTn, and 1 sequence were found to belong to a part of the vector pCAMBIA1300, 4 were found to have 93%~99% identity to the MG genome, and these 4 fragments were considered to be the flanking sequences of the insertional sites of T-DNA. The locations of these 4 sequences inserted in the MG genome were showed in details as followings:In mutant Y34-0144, inserted site of T-DNA was located in the 1883321st bp in supercontig 5.187, chromosome IV, between two hypothetical gene MG12157.5 and MG04131.5. In mutant Y34-0178, inserted site of T-DNA was located in the 33793rd bp in supercontig 5.139, unmapped scaffolds chromosomal, 473 upstream away from the predicted gene MG14318.5. In mutant Y34-0453, inserted site of T-DNA was located in the 403890th bp in supercontig 5.184, Chromosomalâ…¡, 1773 downstream away from the predicted gene MG02065.5. In mutant Y34-0619, inserted site of T-DNA was located in the 403789th bp in supercontig 5.184, Chromosomalâ…¡, 1874 downstream away from the predicted gene MG02065.5.Study on the morphology and microscopical anatomy were carried out to find the the reason for the pathogenic lost of mutant Y34-0453. Compared phenotype of Y34-0453 with the one of the wild strain, it was found that the growth rate of colony, conidium morphology, appressorium morphology, conidium germination rate and appressorium formation rate did not have any significant differences between Y34-0453 and the wild strain. However, Y34-0453 were etiolated in colony color, had less aerial mycelia and less melanin, the conidium production of Y34-0453 was significantly higher than the one of the wild strain. It was interesting that Y34-0453 was able to infect the wounded rice leaf and caused typical rice blast symptoms, while completely lost its pathogenicity on the unwounded leaf from the rice variety susceptible to the wild strain. By examination under microscope in detail, it was discovered that Y34-0453 completely lost its ability to form the penetration peg on the onion epidermis as the wild strain did. Therefore, it was considered that the pathogenicity lost for Y34-0453 was because of the losed ability to form the penetration peg.In order to relate the ability penetration peg formation to the genes, primers were designed to amplify part of T-DNA sequence and its flanking MG sequence of mutant Y34-0453. An anticipative fragment was obtained, T-DNA insertional site in the MG genome was confirmed. The total RNAs were extracted from Y34-0453 and the wild strain. The first cDNAs were synthesized using the total RNAs as templates. Then the first cDNA were used as templates to amplified by RT-PCR with the primers designed according to the exon sequence of the predicted gene MG02065.5 in MG genome, which was downstream, nearby the T-DNA insertional site. The result showed that the fragment amplified from wild strain Y34 coincided with the exon sequence, while no any fragment was amplified in the same way from Y34-0453. It was primarily concluded that the expression of predicted gene MG02065.5 was interrupted by the insertion of T-DNA in mutant Y34-0453, and the ability penetration peg formation of MG was confered by the predicted gene MG02065.5. However, the conclution needs to be confirmed by some more evidences.