Construction Of Botrytis Cinerea Mutant Library By T-DNA Insertion And Molecular Cloning And Characterization Of Pathogenesis-Related Genes

Botrytis cinerea is an important plant pathogenic fungus.Dissection of gene functions at the whole genome level will speed up our understanding about the fungal biology of B.cinerea and its pathogenisis molecular mechanism.In this study,the technique of the Agrobacterium tumefaciens-mediated transformation(ATMT)by T-DNA insertion of B.cinerea was established.A library of 465 transformants was constructed with a high transformation efficiency of over 80 transformants per 1×106 conidia.Genetic stability test of the marker hygromycin B-resistant gene(hph)was performed and stable transformants were obtained and subcultured through single-spore isolates.After screening through pathogenicity assays on tomato leaves,some mutants with reduced virulence were obtained for further study.The integration of the hph gene was characterized by PCR analysis with specific primers.We also amplified the DNA sequence flanked the integration sites of the T-DNA using high-efficiency thermal asymmetric interlaced PCR(TAIL-PCR).43 fragments were cloned and 27 of them were sequenced and analyzed successfully.Sequence comparisons revealed that in 9 transformants,the transfer-DNA were integrated into the B.cinerea transcript.Integration pattern of T-DNA tags in fungal genome were further analyzed and the result showed that Left border(LB)insert into fungal genome with high probability,the right borders of the T-DNA were frequently truncated,and by contrast the left borders were less prone to degradation and appeared to have been inserted in a relatively integrated manner.The truncted LB sequence show similarity to 59-90%range when compared to the complete sequence.The base excision position of RB may be conserved,occurred between the 5’-TTTG-and subsequent base-A-3’.Extra sequence also occurred in T-DNA integration sites.Comparison between mutant strains and wild strains indicated that some biological phenotypes were changed in some of the mutants,which include changes in colony color,sclerotia production,conidium morphology and sporulation.Among those transformants,T-DNA insertion in some mutants were analysised by Southern blot.The results showed that 3 mutants(7,H-96,H-79)contained only one copy at random site.The gene inserted in 7 mutant T-DNA encodes unknown protein according to analysis of flanking sequences.H-96 encodes a protein with similar to DUF221 family protein,H-79 encodes a protein similar to protein phosphatase 2A regulatory B subunit.Because of the insertion of T-DNA in the H-79 mutant,the phenotype of this mutant was different from the wild type strain.Its phenotypes include:slow growth rate,reduced sporulation capacity,sparse mycelium and decreased pathogenicity on tomato,this gene dedicated as BcPP2A.By using RT-PCR,the gene expression was analysised and the loss of gene expression indicates that this gene was inactivated.We then cloned BcPP2A genomic sequence(promoter-coding region-termination sequence)and constructed binary vector which has been transformed into the A.tumefaciens for gene complementary and function confirmation.We chose rapid amplification of cDNA ends(RACE)technology for cloning of full-longth cDNA.In addition,the gene expression vectorwas constructed for genetic complementation and phenotype characterization of mutant 7.