希金斯刺盘孢T-DNA插入突变体表型筛选及其特性分析  

Screening and characterization of T-DNA insertion mutants of Colletotrichum higginsianum

作　　者：周鹏 顾琼楠 黄俊斌[2] 郑露[2] ZHOU Peng;GU Qiongnan;HUANG Junbin;ZHENG Lu(Xiangyang Municipal Tobacco Company,Hubei Province,Xiangyang 441100,China;College of Plant Science and Technology,Huazhong Agricultural University/Key Lab of Plant Pathology,Hubei Province,Wuhan 430070,China;Institute of Plant Protection and Soil Science,Hubei Academy of Agricultural Sciences/Key Laboratory of Integrated Pest Management on Crops in Central China,Ministry of Agriculture and Rural Affairs/Hubei Key Laboratory of Crop Diseases,Insect Pests and Weeds Control,Wuhan 430064,China)

机构地区：[1]湖北省烟草公司襄阳市公司,襄阳441100 [2]华中农业大学植物科学技术学院/湖北省作物病害监测与安全控制重点实验室,武汉430070 [3]湖北省农业科学院植保土肥研究所/农业农村部华中作物有害生物综合治理重点实验室/农作物重大病虫草害防控湖北省重点实验室,武汉430064

出　　处：《华中农业大学学报》2019年第4期33-39,共7页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(31101399)

摘　　要：以希金斯刺盘孢菌株Ch-1为供试野生型菌株,通过农杆菌介导转化方法获得含2000个转化子的T-DNA插入突变体库,筛选菌落生长异常或致病缺陷突变体,分析致病缺陷突变体的T-DNA插入拷贝数和位点。结果显示,筛选到14株菌落异常突变体,包括2株生长缓慢突变体Ch-1-E393和Ch-1-C135、12株菌落形态异常突变体(包括色素异常、菌落扇变或菌丝坍塌现象),另外筛选到1株致病缺陷突变体Ch-1-G090。致病性测定结果表明,致病缺陷突变体Ch-1-G090接种拟南芥后,叶片发病明显减弱,且未产生水渍状病斑。显微观察发现该突变体分生孢子在叶片上仅能产生少量初生菌丝,不能正常形成次生菌丝。Southern杂交显示,致病缺陷突变体Ch-1-G090为T-DNA双拷贝插入;通过Inverse-PCR法获得插入T-DNA的侧翼序列,明确该突变体T-DNA插入位点分别为假定蛋白(Ch063_10682)和RNA加工蛋白FCF1(CH063_10671)编码区。In this study,insertional mutagenesis by Agrobacterium tumefaciens mediated transformation (ATMT) was used to build a T-DNA insertion library of Colletotrichum higginsianum containing a collection of 2 000 insertion mutants. From the library,development and pathogenicity-deficient mutants were screened and T-DNA insertion copies and sites were analyzed. As a result,we isolated 2 growth- deficient mutants Ch-1-E393 and Ch-1-C135 with significant reduced growth on PDA medium,12 mutants with abnormal colonies and one pathogenicity-deficient mutant Ch-1-G090. Pathogenicity assays showed that after inoculation of the pathogenic defective mutant Ch-1-G090 in Arabidopsis ,the incidence of leaf diseases was significantly reduced. Under microscope,few primary hyphae were found in the leaves and no secondary hyphae was observed in Ch-1-G090. Southern blot analysis indicated that the mutant Ch-1-G090 harbored two T-DNA insertions. Border flanking sequences of T-DNAs from these mutants were recovered by Inverse-PCR. Sequence analyses revealed that the two T-DNA insertion sites in the mutant Ch-1-G090 were coding genes for a hypothetical protein (Ch063_10682) and an RNA processing enzyme (CH063_10671).

关 键 词：希金斯刺盘孢 农杆菌介导转化 突变体 致病性 插入位点 

分 类 号：S436[农业科学—农业昆虫与害虫防治]