大丽轮枝菌致病缺陷型T-DNA插入突变体的筛选与鉴定

Identification of Pathogenicity Defective Mutants from a T-DNA Insertion Mutant Library of Verticillium dahliae

【目的】从大丽轮枝菌T-DNA突变体库中筛选鉴定致病缺陷型突变体并分析致病相关基因。【方法】将落叶型大丽轮枝菌菌株Vd991的294个T-DNA插入突变体在寄主棉花上进行致病力测定。利用Southern杂交和hiTAIL-PCR(High-efficiency thermal asymmetric interlaced PCR)技术分别对筛选获得的9个致病力显著降低的突变体进行拷贝数和侧翼序列分析。【结果】与接种野生型Vd991的棉花相比,接种突变体的棉花的病情指数极显著降低。Southern杂交表明其中1个突变体中T-DNA为双拷贝插入,其余8个突变体均为单拷贝插入。生物学特性分析发现T-DNA的插入导致这些突变体在生长速率和产孢量等方面与野生型Vd991相比均有不同程度的降低。Blast比对分析明确了这些突变体中T-DNA的插入位置和在基因组上的分布,并从野生型菌株Vd991中成功克隆得到了这些可能影响大丽轮枝菌致病力的相关基因。【结论】筛选和鉴定T-DNA插入突变是在全基因组范围内快速获得致病相关基因信息的1种有效方法,极大的促进了大丽轮枝菌的致病分子机制等研究,为进一步培育抗病棉花品种奠定了基础。

[Objective]Our research aimed to identify pathogenicity defective mutants from a T-DNA insertion mutant library of Verticillium dahliae strain Vd991 and to analyze pathogenicity-related genes.[Method]In total,294 T-DNA insertion mutants of V.dahliae were tested for their virulence using a cotton infection assay.Southern blot assays were performed to identify the T-DNA insertion copy number of each pathogenicity defective mutant.DNA sequences flanking the T-DNA insertional sites of each mutant were analyzed by high-efficiency thermal asymmetric interlaced PCR.[Result]Based on the virulence assay,the disease indices of cotton plants inoculated with each mutant decreased very significantly in comparison with the index of those inoculated with Vd991.The Southern blot assay revealed that only one mutant contained two T-DNA insertions,while the remaining eight mutants harbored a single T-DNA insert.An analysis of biological characteristics found that the growth and conidial production of these mutants were impaired by the T-DNA insertions compared with the wild type Vd991.The T-DNAs'insertion position and distribution in each mutant were identified by comparison with genome sequences of strain VdLs.17.Furthermore,the pathogenicity-related genes were cloned from strain Vd991.[Conclusion]The screening and identification of T-DNA insertion mutants is an effective method to identify the pathogenicity-related genes of V.dahliae on a genome-wide scale.This laid a foundation for the further breeding of disease-resistant cotton varieties and will promote the study of the pathogenic molecular mechanisms of V.dahliae.