大丽轮枝菌(Verticillim dahliae)突变体库的构建与分析  

Construction and Preliminary Analysis of Verticillim dahliae Mutant Library

作　　者：潘国强 吴思源 刘璐 郭惠明[1] 程红梅[1] 苏晓峰[1] PAN Guo-qiang;WU Si-yuan;LIU Lu;GUO Hui-ming;CHENG Hong-mei;SU Xiao-feng(Biotechnology Research Institute,Chinese Academy of Agricultural Sciences,Beijing 100081;College of Plant Protection,China Agricultural University,Beijing 100193)

机构地区：[1]中国农业科学院生物技术研究所,北京100081 [2]中国农业大学植物保护学院,北京100193

出　　处：《生物技术通报》2023年第5期112-119,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(32072376)。

摘　　要：为了更加快捷地筛选大丽轮枝菌致病关键基因,利用聚乙二醇介导的原生质体转化法构建病原菌随机插入突变体库,对部分阳性转化子的生长表型指标和致病力进行分析,筛选生长发育与致病缺陷突变体并进行靶标基因定位。结果表明,本研究共获得13030多个阳性转化子,随机挑选5个转化子与V991野生型菌株进行比较分析,发现其中一个突变体在PDA和不同碳源培养基上的菌落直径、产孢量以及致病力均显著降低。侧翼序列测序结果结合Blast比对分析表明,该缺陷突变体中的潮霉素抗性基因表达盒定位于大丽轮枝菌3号染色体1528782 bp处,属于内切葡聚糖酶1基因(VDAG_04017)。综上所述,本研究通过大丽轮枝菌插入突变体库构建、突变体生长表型指标和致病力鉴定及靶标基因定位等一系列分子生物学手段,可初步鉴定病原菌致病相关基因,为从全基因组层面深入研究大丽轮枝菌致病机制奠定了一定基础。In order to rapidly screen the key pathogenic genes of Verticillim dahliae,the random insertion mutant library was constructed by polyethylene glycol-mediated protoplast transformation.The growth phenotype and pathogenicity of partial positive transformants were analyzed to screen the growth,development and pathogenic defect mutants,and the target gene was located.The results showed that more than 13030 positive transformants were obtained in this study,and five positive transformants were randomly selected.The colony diameter and spore production on PDA and different carbon source medium as well as pathogenicity of one mutant significantly decreased compared with V991 wild type strain.The results of flanking sequence sequencing and Blast alignment analysis demonstrated that the hygromycin resistance gene expression cassette in the defective mutant was located at 1528782 bp of chromosome 3 of V.dahliae,belonging to endoglucanase 1 gene(VDAG_04017).Taken together,this study can preliminarily identify pathogenicity-related genes through a series of molecular biological technique,including the construction of insertion mutant library of V.dahliae,identification of the growth phenotypic indices and pathogenicity,and mapping of target genes so on,which lays a foundation for investigating the pathogenic mechanism of V.dahliae at the whole genome level.

关 键 词：大丽轮枝菌 聚乙二醇介导的原生质体转化法 致病相关基因 致病力 侧翼序列分析 

分 类 号：S51[农业科学—作物学]