Agrobacterium-mediated Transformation Of Botrytis Cinerea And Analysis Of Flanking Sequences

Botrytis cinerea is not only an important plant pathogenic fungus threatening economical crop, but also a important model fungus in studying plant-microbe interaction. Dissection of gene functions at the whole genome level will speed up our understanding in the fungal biology of B.cinerea and its pathogenisis molecular mechanism, provide theoretical evidence for breeding plant with durable resistance and the disease integrated mangament. The technique of the Agrobacterium tumefaciens-mediated transformation(ATMT) has become a useful tool for studying the interaction between fungi and their hosts by its special advantage.In this thesis, a transformation system of B.cinerea mediated by ATMT had been established and several characters of some transformants have been analyzed. The results are as follows:(1) In order to obtain transformants of B.cinerea, the conditions of the Agrobacterium- mediated transformation were optimized. The result showed that 50μg/mL was the most suitable concentration of hygromycin B for screening transformants and 200μg/mL cefotaxime and carbenicillin had the best effective to inhibit Agrobacterium growth. Conidia of B. cinerea and active Agrobacterium co-smeared on glass paper was the best way of culture. 48 h was the fittest co-cultivation time. Using this optimized transformation system, about 120-150 transformants of B.cinerea were obtained from 105 spores.(2) In this study, more than 400 transformants were obtained and hygromycin resistance gene were mitotically stable after several subcultures. Parts of transformants were screened by PCR amplification and Southern blot. The primers were designed by the sequence of hph gene which we have already known. The results showed that all the transformants we screened contained the target hph fragment. Among those transformants, 6 mutants were identified by Southern blot, which have been screened by PCR. The results showed that all the 6 mutants had the blot signal and five of them contained only one copy at random site.(3) Sequence analysis of 28 fragments amplified by thermal asymmetric interlaced PCR(TAIL-PCR) showed that 19 of them were B.cinerea genomic DNA, and 9 fragments contained vector backbone sequences. Comparison between mutant strains and wild strains indicated that some biological characters were changed in some of the mutants, and the characters included colony color, sclerotia production, conidium morphology, and sporulation. Pathogenicity results showed that only a singal mutant lost virulence.In summary, T-DNA was successfully inserted into genome of the B.cinerea by ATMT technique; a large scale of T-DNA insertional mutants were generated; and several genes related to important biological functions were found to be tagged by T-DNA in this study. This is a good start for functional genomics of the fungus and it will have an important impact on science and future application.