Functional Analysis Of Sphingolipid Metabolic Pathway Gene Bcsphl1 In Botrytis Cinerea

Botrytis cinerea is an important plant pathogen,which can infect many important economic crops,cause gray mould disease,and lead to huge economic losses.The sclerotia of B.cinerea is a dense dormant structure formed by entanglement of its hyphae,which can resist the bad environment.As an important source of initial infection,sclerotia play a very important role in the infection cycle of gray mould.Sphingolipids are important parts of the structure of biological membranes and perform important functions in the organism.Many intermediate products in the pathway of sphingolipid biosynthesis are important signal molecules between biological cells.Genes related to the metabolism of sphingolipid and the formation of sclerotia may become a new type of targets for the chemical prevention and control of B.cinerea.In this study,by analyzing the transcriptome data of B.cinerea strain B05.10 at different stages of sclerotia development,the sphingolipid biosynthesis pathway genes Bcsphl1 and Bcsphl2 were selected.A preliminary study on the structure of these genes,the structure of the protein encoded by these genes,and the effect of growth and development to B.cinerea of the gene Bcsphl1 in B.cinerea was carried out.The following results were obtained:1.The structure of Bcsphl1 and Bcsphl2 was elucidated.The protein encoded by Bcsphl1 gene contains 360 amino acids,which contains two transmembrane domains and a fatty acid hydroxylase conserved domain.The amino acid sequence has 98.23% homology with the similar to sphingosine hydroxylase protein of B.cinerea strain T4.The protein encoded by Bcsphl2 gene contained 355 amino acids,which contained a transmembrane domain and a fatty acid hydroxylase conserved domain.The amino acid sequence has 98.23% homology with another similar to sphingosine hydroxylase protein of B.cinerea strain T4.Both of these proteins are structurally stable integrated membrane protein,and they are very similar to that of Saccharomyces cerevisiae Sur2p(This protein contains four transmembrane domains and a fatty acid hydroxylase conserved domain).It was speculated that these two genes might be sphingosine hydroxylase genes.2.Bcsphl1 was successfully knocked outWe used the principle of homologous recombination to knock out the Bcsphl1 gene in B.cinerea strain B05.10.And then PCR and Southern blot were performed on these candidate mutants.Finally,4 knockout mutants of this gene were obtained,which are named ?Bcsphl1-32,?Bcsphl1-99,?Bcsphl1-104 and ?Bcsphl1-106.3.Basic biological features of the Bcsphl1 knockout mutants were determinedThe growth rate,colony morphology,sclerotia size and other characteristics of the knockout mutant on the PDA medium are not significantly different from those of the wild-type strain B05.10,but the number of sclerotia is reduced,indicating that the Bcsphl1 doesn't affect the mycelial growth of B.cinerea,but it affect the number of sclerotia formation.To a certain extent,this gene is related to the sclerotia development of B.cinerea.Compared with wild-type strain B05.10,the growth rates of the knockout mutants were significantly increased on MCDA medium with glucose,fructose and sucrose as carbon sources,and the number of sclotinia formation was significantly decreased.On the medium with sucrose as the carbon source,the growth rate of mycelia of the knockout mutants were significantly increased,but the number of sclerotia formation was not significantly different.This further suggests that this gene is related to the sclerotia development of B.cinerea,and may have some influence on the ability of B.cinerea to utilize different carbon sources.4.The effect of Bcsphl1 knockout mutant on stress and pathogenicity was determinedThere was no significant difference between the growth rate of Bcsphl1 knockout mutant and that of wild-type strain B05.10 on different concentrations of sorbitol,Na Cl,SDS and different p H of PDA medium.This indicated that the gene had no effect on the ability of B.cinerea to resist the stress of these substances and p H stress.Compared with wild-type strain B05.10,the virulence of Bcsphl1 knockout mutant to tomato and tobacco leaves was reduced to a certain extent.In tomato and tobacco leaves,the lesion diameter of the mutant was reduced by 3.3%-44.8% and 11.9%-31.3%,respectively.These results indicated that the gene was related to the pathogenicity of B.cinerea.These results provided some clues for revealing the role of sphingolipid synthesis in the growth,development and pathogenicity of B.cinerea.