Disruption And Function Preliminary Exploration Of Two New 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 provide theoretical basis for explore the interaction between the pathogen and its antagonistic fungi, the pathogen and its host in molecular level, we chose two related putative genes from the gene bank of Fusarium oxysporum f.sp.cubense race4（FOC4） for functional analysis. The knock-out mutans of the two genes were obtained and analyzed the roles in FOC4 preliminarily by their phenotypes differences in this paper.Amino acid sequence analysis showed that the deduced amino acid sequence encoded by FOC4B₀01gene had some conserved regions such as:the GTP binding site, the adenylyl cyclase interaction site,β-γcomplex interaction site of the G protein and the putative transmembrane receptor binding site. And then the amino acid sequence alignment showed that the deduced amino acid sequences encoded by FOC4B₀01 gene have relatively higher similarity to Neurospora crassa,Trichoderma and Magnaporthe grisea,but have relatively lower similarity to another two known gene encoding G protein a subunit in Fusarium oxysporun. So we presumed that the FOC4B₀01 gene is a new gene encoding G protein a subunit in Fusarium oxysporun. The FOC4B₀03 gene was a specific gene of FOC4. Amino acid sequence analysis showed that the product of this gene contains a conserved region approximately 150 residues long within various heterokaryon incompatibility proteins （HET）.To understand the role of the two genes, they were knocked out from the wild type FOC4 by homologous recombination. We transformed the protoplast of FOC4 by using a linear DNA fragments contains the green of fluorescent protein and confirmed the genetic transformation system. We constructed two knockou vector, and obtained the fragments to thransform the FOC4 by using restriction enzymes. The DNA fragments contain homologous sequence of target gene was successfully transferred to FOC4 by using the protoplast-mediated genetic transformation system, and obtained the gene deletion mutants. The mutants morphology analysis results indicated that there were some differences in hyphae morphology and heat resistance in all deletion mutants were increaseed.But no obvious differences in fungal colony morphology, conidium morphology, and growth rate between the two gene deletion mutant and wild type strain. Comparised to the wild type, The FOC4B₀03 disruptants had resistance against the tested Trichoderma to a certain extent in the dual-culture and antagonistic test. This result suggests that the FOC4B₀03 gene plays certain role in the interaction between the FOC4 and other fungi.There was no obvious difference in the pathogenicity between the two gene deletion mutants and the wild type.This test preliminary explored two unknown genes function by gene sequence analysis and gene knocked out technology,and determined the gene knockout system in FOC 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 pathogens.