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周鹏,顾琼楠,黄俊斌,郑露.希金斯刺盘孢T-DNA插入突变体表型筛选及其特性分析[J].华中农业大学学报,2019,38(4):
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
投稿时间:2019-01-22  
DOI:
中文关键词:  希金斯刺盘孢  农杆菌介导转化  突变体  致病性  插入位点
英文关键词:Colletotrichum higginsianum  Agrobacterium tumefaciens mediated transformation  mutant  pathogenicity  insertion site
基金项目:国家自然科学基金项目(31101399)
作者单位E-mail
周鹏 湖北省烟草公司襄阳市公司襄阳 441100 972513466@qq.com 
顾琼楠 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  
黄俊斌 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  
郑露 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 luzheng@mail.hzau.edu.cn 
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中文摘要:
      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2 000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。
英文摘要:
      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).
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