绿木霉(Trichoderma virens)T23甲基转移酶基因gliN-T对胶毒素合成的调控研究  

Regulation effect of methyltransferase gene gliN-T on the gliotoxin synthesis regulation in Trichoderma virens T23

作　　者：华丽霞[1,2] 孙佩 蒋秋平[1,2] 曾华兰[1,2] 叶鹏盛 何炼[1,2] 曾静 王明娟[1] 张敏 罗飞[1] 杨晓丫 何晓敏[1] 刘勇 HUA Lixia;Sun Pei;JIANG Qiuping;ZENG Hualan;YE Pengsheng;HE Lian;ZENG Jing;WANG Mingjuan;ZHANG Min;LUO Fei;YANG Xiaoya;HE Xiaomin;Liu Yong(Industrial Crops Research Institute,Sichuan Academy of Agricultural Sciences,Chengdu 610300,China;Key Laboratory of Integrated Pest Management on Crops in Southwest of Ministry of Agriculture and Rural affairs,Chengdu 610066,China)

机构地区：[1]四川省农业科学院经济作物育种栽培研究所,四川成都610300 [2]农业农村部西南作物有害生物综合治理重点实验室,四川成都610066

出　　处：《中国农业大学学报》2020年第6期75-81,共7页Journal of China Agricultural University

基　　金：国家自然科学基金项目(31701830);国家重点研发专项(2017YFD0201103);四川省农业科学院领军人才研究基金(2019LJRC036)。

摘　　要：为探明绿木霉(Trichoderma virens)胶毒素合成的分子调控机制,本研究以绿木霉菌T23为研究对象,对农杆菌介导的遗传转化技术(Agrobacterium tumefaciens-mediated transformation,ATMT)进行改良,并利用改良的ATMT技术对绿木霉菌T23胶毒素合成候选基因簇中的甲基转移酶基因gliN-T进行基因敲除,通过薄层色谱法及高效液相色谱法分析基因敲除突变体培养液中的胶毒素含量变化情况,明确gliN-T对胶毒素合成的调控作用。结果表明:在绿木霉菌T23分生孢子液中添加终浓度为15mg/mL的细胞壁裂解酶(Glucanex),30℃处理2~3h后用于农杆菌介导的遗传转化,每106个孢子转化子数从0.25个提高10个;利用改良的ATMT技术,成功获得基因敲除突变体ΔgliN-T。胶毒素检测结果发现:在野生型T23的24h培养液中未产生胶毒素,而在48、72、96h的培养液中均检测到胶毒素;在相同的条件下,ΔgliN-T在4个生长时期均无法产生胶毒素,表明gliN-T的缺失抑制了绿木霉菌T23胶毒素的合成,说明该基因在胶毒素合成途径中扮演着重要角色。To understand the molecular regulation mechanism of gliotoxin synthesis in Trichoderma virens,T.virens strain T23was used as study material,and Agrobacterium tumefaciens-mediated transformation(ATMT)technology was modified to carry out gene disruption experiment.By using the modified ATMT technology,a methyltransferase gene,gliN-T,which belongs to gliotoxin synthesis candidate cluster in T23,was knocked out for further determination of its regulation effect on gliotoxin synthesis.Thin layer chromatography and high-performance liquid chromatography were adopted to analyze the dynamic change of gliotoxin in the culture medium of the gene knockout mutant.The results showed that:By adding cell wall lyase glucanex with a final concentration of 15mg/mL to T23conidia and treated at 30℃for 2~3h,the number of transformants was increased from 0.25to 10per 106 spores,and the knockout mutantΔgliN-T was successfully obtained.Gliotoxin analysis results showed that:No gliotoxin was produced in the 24hculture of wild-type T23,but gliotoxin was then detected in the culture medium after 48,72and 96h;Under the same conditions,gliotoxin wasn’t detected inΔgliN-T at these four growth stages.These results showed that the absence of gliN-Tinhibited the synthesis of gliotoxin in T23,suggesting that gliN-T played an important role in the gliotoxin synthesis regulation.

关 键 词：农杆菌介导转化 绿木霉菌 胶毒素 甲基转移酶 基因敲除 

分 类 号：S182[农业科学—农业基础科学]