Screening And Functional Analisis Of Botrytis Cinerea Mutants Defective In Cell Wall Integrity And The Related Genes

Botrytis cinerea appeared in a wide range of plants causing considerable economic losses to plant production all over the world.Therefore,it is important to understand its pathogenesis and function of related genes.In this research,cell wall-defective mutant of B.cinerea were screened,and the related genes were analized.In this study,the haploid strain B05.10 of B.cinerea was used as the experimental strain.By using the Agrobacterium tumefaciens-mediated transformation,249 T-DNA inserted mutants of B.cinerea were obtained.In order to search the cell wall-defective mutant,T-DNA insertion mutants of B.cinerea were screened by using Calcofluor White,and 7 sensitive mutants(D-9,D-59,D-122,D-125,D-153,D-241 D-286)and 1 resistant mutant(D-2)were obtained.By testing the hygromycin resistace gene in the T-DNA,it was confirmed that T-DNA has inserted into the genome of above eight mutants.To find out the T-DNA insertion site in the mutant,the flanking sequence of the T-DNA insertion site was amplified using TAIL-PCR,sequenced,and BLAST searched.The result showed that only three mutants(D-2,D-9,D-59)has the value to be further studied.In the RT-PCR,the expression of gene BCLG-00770.1 of mutant D-59 T-DNA insertion sites was not detected,indicating that this gene was inactivated by T-DNA insertion.Southern blot showed the one copy of T-DNA in the mutant D-59.In order to study the functions of the BCLG-00770.1 gene,the gene complementation strain was constructed.By comparing and analyzing the phenotypes of wild strain,mutant D-59 and gene complementation strain,it was showed that BCLG-00770.1 is associated with sporulation,spore germination,chitin synthesis,and the infection capacity of B.cinerea.Because BCLG-00770.1 is still an unknown gene,a large number of further studies are needed to reveal the function and importance of this gene in B.cinerea.