Screening Of The Sporulation-related Mutants Of Magnaporthe Grisea And The T-DNA Flanking Sequence Analysis

The rice blast pathogen (Magnaporthe grisea (Hebert) Barr.) is important both in the rice production economically and the study of plant-microbe interaction. Functional genomic analysis of the sporulation-related genes is significant for the control of the rice blast. In this dissertation, we report the results of the screening of the sporulation-related mutants generated from this pathogenic fungus by T-DNA insertion and the analysis of the T-DNA Flanking Sequence.Magnaporthe grisea strain Y34 was mutated through T-DNA insertion mediated by Agrobacterium tumifaciens strain AGL-1 and the number of the T-DNA tag mutants was increased to 6855. In average, the transformation efficiency was 300 mutants per 1.0x106 conidia of Y34. We randomly selected 1,600 mutants from the all the mutants for analysis on the morphological development and assay on the virulence to rice. By comparison these mutants with the wild type (Y34) on virulence to rice seedlings, we obtained a total of 173 pathogenic mutants which virulence to rice completely lost or significantly decrease. By comparison these mutants with the wild type on morphological development, we screened 33 sporulation-related mutants out of the 173 pathogenic mutants. Of the 33 sporulation-related mutants, 4 were abnormal in conidium morphology, 10 completely lost their ability of sporulation and 19 significantly decreased in this ability.We then randomly selected 13 from the 33 sporulation-related mutants for PCR amplification. The results showed that all the genome DNAs from the 13 mutants contained hph gene, while the genome DNA from wild type didn't, suggesting that the phenotype change was the results of the foreign DNA insertion. We carried out the TAIL-PCR with the genome DNAs from the 13 mutants as templates and using four arbitrary degenerate (AD) primers which were paired with three nested specific primers according to the T-DNA sequences. We obtained 10 specific DNA fragments by this TAIL-PCR. We cloned these fragments and then had them sequenced. Among the 6 fragments successfully sequenced, 3 were the sequences of Magnaporthe grisea genomic DNA, 3 belonged to the sequences of the vector used...