层出镰刀菌T-DNA插入突变体库的构建与分析  

Construction and analysis of the T-DNA insertional mutant library for Fusarium proliferatum

作　　者：刘丽媛[1] 朱立华[1] 刘力伟[1] 王亚南[1] 胡同乐[1] 曹克强[1] 王树桐[1] 

机构地区：[1]河北农业大学植物保护学院,河北保定071001

出　　处：《河北农业大学学报》2017年第1期71-75,共5页Journal of Hebei Agricultural University

基　　金：国家重点研发计划(2016YFD0201100);河北省自然科学基金(c2016204140);国家苹果产业技术体系(CARS-28)

摘　　要：苹果再植病害(Apple replant disease,ARD)是世界苹果主产区广泛发生的病害。前期研究表明,层出镰刀菌是造成苹果再植病害的重要致病菌之一,但其分子致病机制尚缺乏研究报道。本研究的目的是建立农杆菌介导的层出镰刀菌的遗传转化体系,并获得大规模的遗传稳定的层出镰刀菌ATMT突变体库,为研究该菌的分子致病机理奠定基础。对影响农杆菌介导转化效率的主要因子进行单因子条件测验,得到其最优转化体系为:抑制H10菌丝和孢子生长的潮霉素的浓度为100μg/mL,层出镰刀菌的分生孢子浓度为107个/mL,农杆菌OD600值为0.3,AS浓度为200μg/mL,共培养时间为48h,共培养温度为26℃。利用这一体系构建了3000个转化子的突变体库,并对随机选择的200个突变体进行潮霉素抗性基因的PCR检测,并经过在PDA培养基上5代培养,验证了T-DNA插入片段的稳定性。Apple replant disease（ARD）is common to all major apple growing regions of the world.Fusarium proliferatum was proved to be one of the causal agents in our previous study,while the molecular mechanism of its pathogenicity is still unclear.This study aimed to establish the Agrobacterium tumefaciens-mediated transformation of F.proliferatum H10,and obtain a large-scale and genetic stable F.proliferatum ATMT mutant library,providing new insights for the molecular mechanisms of pathogenicity as well as efficient strategy of disease control in F.proliferatum.Single factor test was conducted to improve the transformation efficiency,respectively.The results showed that the optimal ATMT conditions were as follows：the medium containing 100μg/mL hygromycin B could inhibit hyphae growth and spores germination completely;the optimal conidial concentration was 107 conidia/mL;Agrobacerium OD600 was 0.3;AS concentration was 200μg/mL;cultivation time was 48h;and cultivation temperature was 26℃.A mutant library containing 3000 mutants was constructed under the optimized conditions,and 200 randomized selected mutants were identified with PCR test.Mutants were fully stable after five rounds successive culture,indicating that foreign T-DNA segments have been inserted into the genome.

关 键 词：层出镰刀菌 农杆菌介导转化(ATMT) 苹果再植病害 突变体 

分 类 号：S432.44[农业科学—植物病理学]