尖孢镰刀菌古巴专化型中SIX2和SIX6基因敲除突变体的生物学特性

尖孢镰刀菌古巴专化型(Fusarium oxysporum f. sp. cubense,Foc)依据对寄主易感性分为4个不同的生理小种,其中4号生理小种(Foc4)几乎能危害目前所有栽培品种。为研究其SIX(secreted in xylem)蛋白编码基因SIX2和SIX6在Foc4对寄主差异性选择中的作用,利用PEG介导的原生质体转化法将基于pCT74质粒框架构建的SIX2、SIX6基因敲除质粒分别转入Foc4 B2菌株,分别得到了SIX2和SIX6基因敲除突变体,然后分析敲除突变体与野生型的生物学特性差异。生物学研究结果表明:SIX2、SIX6基因的缺失突变体均呈现菌丝稀疏、生长速率减慢、产孢率降低、菌丝异核率增加,对渗透压、外源氧等外源胁迫更为敏感等特征。致病力分析实验发现ΔFoSIX2和ΔFoSIX6突变体的孢子在香蕉苗的幼嫩根部附着量减少,孢子根部定殖能力降低;ΔFoSIX2菌株基本上丧失了对巴西蕉的致病力,而对粉蕉仍有较强的致病能力;ΔFoSIX6菌株则对粉蕉苗、巴西香蕉苗盆栽致病力均呈极显著下降。依据生物学与致病力测定结果,推测Foc4中SIX6基因决定Foc4对寄主的致病力,而SIX2基因则决定Foc4对寄主的差异性选择能力。

Biological Characteristics of SIX2 and SIX6 Gene Knock-out Mutants of Fusarium oxysporum f. sp. cubense

There are four recognized races of Fusarium oxysporum f. sp. cubense which are separated based on host susceptibility. Race 4 is capable of attacking ‘Cavendish’ （AAA） as well as the other varieties of banana affected by races 1 and 2. In order to study the roles of SIX2 and SIX6 genes encoding SIX （Secreted in xylem） protein in host xylem of Foc4 on host diversity selection, SIX2 and SIX6 gene knockout plasmids constructed from pCT74 plasmid framework were transferred into Foc4 B2 strain by PEG -mediated protoplast transformation respectively,SIX2 and SIX6 gene knockout mutants were obtained respectively, then the biological characteristics between knock-out strains and wild-type strains were analyzed. The results of biological studies showed that, compared with wild -type strain, SIX2 and SIX6 gene knock -out mutants had sparse hyphae, slow growth rate, less spores production, increased rate of mycelial heteronuclear, more sensitive to osmotic stress and other exogenous stress.Pathogenicity analysis found that compared with wild-type strain, the spores in both ΔFoSIX2 and ΔFoSIX6 knockout mutants decreased the adhesion ability to the young roots of banana seedlings and had reduced colonization ability in the roots. ΔFoSIX2 knock-out mutants basically lost the pathogenicity to Musa acuminata AAA Cavendish cv. Bax, while still had a strong pathogenic ability to Musa sp ABB Pisang Awak. The pathogenicity of ΔFoSIX6 knock-out mutants were significantly decreased to M. acuminata AAA Cavendish cv. Bax and Musa sp ABB Pisang Awak. Based on the results of biology and pathogenicity, it was speculated that the SIX6 gene determined thepathogenicity of Foc4 to host, while SIX2 gene determined the ability of Foc4 to select wider hosts.