Disruption And Function Exploration Of Two Cellulase Genes In Fusarium Oxysporum F.sp.Cubense

Fusarium wilt of banana caused by Fusarium oxysporum f.sp.cubense(Foc), is considered to be one of the most destructive diseases in banana production. In order to explore the interaction between the pathogen and its antagonistic fungi, the pathogen and its host in molecular level, two putative related gene Ceg-1andCeg-2have been chose from the gene bank of Fusarium oxysporum f.sp.cubense race4(Foc4) for functional analysis.These two new gene have been knockout by the protoplast-mediated genetic transformation system. The knock-out mutans of the two genes were obtained and their phenotypes were analysed in this paper for realizes the roles in Foc4.Fist of all analyse the amino acid sequence of these two gene in the NCBI.By the amino acid sequence analyse found that these two new gene are members of the cellulase gene family.The conserver sequence of these two genes are similar to the endo glucanase.Cellulase is belonged to the cell wall degrading enzyme.So suggesting that these two gene Cge-1and Ceg-2to participate in the process of the cell-wall degrading between the Fusarium oxysporun attack the banana plant.In order to understand the role of these two genes,single gene knock-out and double gene knock-out have being done in this experiment.Fist, build the knock-out vector by the plasmid pCX62carry the hygromycin gene.Second,obtain the DNA fragment which have the hygromycin gene by PCR.And then using this DNA fragment to transformation.Single gene deletion mutants and double gene deletion mutants were got.We have got some positive transformants by the PCR method.After Southern blotting and RT-PCR verification,we have got the single gene knock-out mutants and double gene knock-out mutant.In this study,we choose three mutants,contain single gene knock-out and double gene knock-out.The mycelium of the mutants were difference with wild type.But the spore form,colonies form and the growth rate have no difference with the wild type.In the virulence testing,we found that,the mutants have no difference with wild type in the leaves in vitro,the cauloid and roots pathogenicity testing.This test preliminary explore two unknown genes function by gene sequence analysis and gene knocked out technology,and determine the gene knockout system in Foe at the same time. These results would help to know the detailed function of this two genes in the following researchs. These results would also provides technical support for explore the other gene in the gene bank of this pathogen.