棉花黄萎病菌T-DNA插入突变体功能基因及生物学性状分析

Phenotypic Analysis and Functional Identification of T-DNA Insertional Mutants in Verticillium dahliae

【目的】分析棉花黄萎病致病菌相关基因的功能揭示其中致病机制。【方法】通过ATMT法构建了大丽轮枝菌(Verticillium dahliae)650个T-DNA插入突变体。利用hiTAIL-PCR技术获得17株T-DNA插入体侧翼基因序列,通过blast比对分析表明,其中16个基因序列编码酶或蛋白。【结果】突变体的生物学性状与野生型菌株Vd991相比,出现黄色菌丝型2株,中间菌丝型6株;部分突变体在生长速率(7株)、产孢量(14株)和粗毒素产量(4株)上都发生了具有统计学意义的变化(p<0.05);致病力测定表明,9株突变体的致病力较野生型有所降低,在p<0.05水平具有统计学意义。【结论】该研究为大丽轮枝菌致病基因的筛选和鉴定提供了理论基础。

[Purposes] Verticillium wilt is indispensable for revealing the pathogenicity mechanisms through functional analysis of pathogenicity related genes. [Methods] Agrobacterium turnefaciens-mediated transformation （ATMT） method, 650 T-DNA randomly insertional mutants were constructed. The left-flanking sequence of the insertional sites of 17 mutant strains were cloned using hiTAIL-PCR and were identified using Blastn. [Findings]2 strains are yellow mycelium type, and 6 strains are intermediate filament type, which were different from the phenotype of WT V991. Additionally, 7 mutants, 14 mutants and 4 mutants were significantly different from the WT on the growth rate, spore yield and crude toxin content individually （p~〈0.05）. Compared with the pathogenicity of the wild type strains Vd991, that of nine mutant strains were significantly reduced （p 〈0.05）. [Conclusions] The results provide theoretical fundament for the screening and identification of pathogenicity related genes.