Gene Disruption And Characterization Of Bcjen1 In Botrytis Cinerea

A BLAST search of the Botrytis cinerea B05.10 genome database,using Saccharomyces cerevisiae ScJEN1 protein as query sequence,allowed the identification of a putative JEN1 homologues.The putative protein is similar to carboxylic acid transporter in Botryris cinerea,the study therefore named the gene as Bcjen1?BC1G₀3160.1?.In this study,the function of BcJen1 was analyzed in Botryris cinerea B05.10 strain.Bioinformatical methods was used to predict the physical-chemical characteristics,hydropathy and hydrophobicity,signal peptide,subcellular localization,transmembrane region,structure and function of Bc JEN1,construct protein phylogenetic tree and analyze the key conserved sequence region.The results showed that BcJEN1 was a transmembrane protein located in the cell membrane and may be involved in the lactate or carboxylic acid transport.By constructing the knockout binary vector pCAMBIA-Bar-jen1 by means of Gateway system,through Agrobacterium tumefaciens mediated transformation,three stabilized mutants were obtained.By PCR and RT-PCR analysis,the results showed that Bcjen1-7 and Bcjen1-9 were the real knockout mutants,and Bcjen1-8 was an ectopic transformant.In order to study the Bcjen1,the Bcjen1 mutants were characterized via a series of experiments compared with the wild-type B.cinerea.The results showed that the growth rate of Bcjen1 knockout mutants had no significant difference with the wild type strain on PDA medium,after 14 days,the wild-type could form sclerotia,but knockout mutants almost had no sclerotium formation,in addition,the sporulation of the knockout mutants reduced significantly,the number of spores of wild type strain was about 2.5 times than that of knockout mutants,the results indicated that Bcjen1 had an effect on the formation of spores and sclerotia.Compared with the wild-type,knockout mutants grew slowly on minimal medium containing lactate as sole carbon source,the rate of radial growth was reduced by about 17.1%.However the growth rate was almost no difference with the wild type on minimal medium agar plates supplemented with pyruvate,acetate,malate,fumarate and succinate as the sole source of carbon.The results showed BcJEN1 might be involved in lactate transport and unabled to play a positive role in transport of pyruvate,acetate and malate,succinate and fumarate.On tomato leaves,the pathogenicity of Bcjen1 knockout mutants were similar to the wild type strain,the result showed that Bcjen1 had no effect on the pathogenicity of Botrytis cinerea.These results primarily indicated that Bcjen1 had an effect on growth and pathogenicity of B.cinerea,and might play a role in the transport of lactate,and affect the formation of spores and sclerotia.