Construction Of A T-NDA Insertion Mutant Library And Preliminary Characterization Of The Pathogenicity-related Genes In Botrytis Cinerea

Botrytis cinerea is the causal agent for plant grey mould disease. It can infect more than200crop hosts worldwide and cause serious economic losses. In order to design novel strategies for control of grey mould disease, it is quite necessary to better understand molecular mechanisms during pathogenesis of B. cinerea.In this thesis, more than1000transformants of B. cinerea strain RoseBC-3have been obtained by Agrobacterium tumefaciens-mediated transformation （ATMT）, several characters and the T-DNA flanking sequences of some transformants have been analyzed. Following results have been achieved:（1） Transformants of B. cinerea have been obtained by ATMT and the cultural conditions for ATMT were optimized. Results showed that the optimum conditions were: hygromycin B at50μg/ml, the conidial concentration of B. cinerea at1×105conidia/ml and the co-culturing temperature at22℃. Under the optimum conditions, the yield reached100transformants per petri dish （9cm diameter）.（2） A mutant library of B.cinerea containing1000transformants was obtained. The hygromycin B-resistant trait in selected transformants was detected to be mitotically stable after several subcultures. Integration of the external gene in the genome of B. cinerea was confirmed by PCR and Southern blot. Results showed that all the tested transformants were T-DNA insertion mutants. Among those transformants,8mutants were identified by Southern blot. Results showed that all the mutants had the blot signal and7of them contained only one copy at the random site with the the percentage of single-copy insertion accounting for87.5%. Only one transformant contained double copies accounting for12.5%. Based on the cultural characteristics, the T-DNA insertion mutants were divided into6classes, namely conidia-deficient mutants, growth-decreasing mutants, aerial hyphae-deficient mutants, colony coloured mutants, pathogenicity-decreasing mutants and pathogenicity-increasing mutants.（3） Sequences flanking the T-DNA were amplified by high-efficiency thermal asymmetric interlaced PCR （hi-tail PCR） and inverse PCR. Results showed that14out of 16sequences belonged to the genome of B. cinerea, whereas the remaining2sequences belonged to the vector. The ratio of the T-DNA insertion sites in the coding regions and the noncoding regions appreaed equal.（4） Mutants AT15, AT239and AT426of B. cinerea were compared with the wild type strain RoseBC-3for colony morphology, growth rate, conidial production and germination, activity of amylase, production of acid and the formation of infection structure. The results showed that all the mutants were able to produce amylase and acids. AT239is a pathogenicity-decreasing mutant with normal mycelial growth and formation of infection cushions. However, it failed to produce conidia. The T-DNA was inserted in600bp upsteam of the conserved hypothetical protein BC1G₀3701gene. AT15is a pathogenicity-increasing mutant with normal mycelial growth. However, It formed infection cushions earlier and more than the wild type strain RoseBC-3. The T-DNA was inserted in the17bp upsteam of the predicted protein BC1G₀0493gene.This study generated numerous mutants with altered pathogenicity, compared to the wild type strain RoseBC-3. The mutants will be useful for elucidation of the pathogenesis mechanisms responsible for infection of plant tissues by B. cinerea.