深海真菌Dichotomomyces cejpii胶霉毒素生物合成基因启动子的克隆和功能鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2017-0941

生物技术通报 ›› 2018, Vol. 34 ›› Issue (4): 144-150.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0941

深海真菌Dichotomomyces cejpii胶霉毒素生物合成基因启动子的克隆和功能鉴定

黄自磊^{1, 2, 3}, 章卫民², 叶伟², 李赛妮², 李浩华², 朱牧孜²

1. 中国科学院南海海洋研究所,广州 510301;
2. 广东省微生物研究所省部共建华南应用微生物国家重点实验室广东省菌种保藏与应用重点实验室广东省微生物应用新技术公共实验室,广州 510070;
3. 中国科学院大学,北京 100049

收稿日期:2017-11-03 出版日期:2018-04-20 发布日期:2018-05-04
作者简介:黄自磊,男,硕士研究生,研究方向：海洋微生物功能基因;E-mail：752261407@qq.com
基金资助:
国家自然科学基金项目（31500037）,广州市科技计划重点项目（21607020018）,广东省科技计划项目（2015A030302061,2016A020222022）,广东省海洋经济创新发展区域示范专项项目（GD2012-D01-002）

Cloning and Identification of Gene Promoter for Gliotoxin Biosynthesis from Deep-Sea-Derived Fungus Dichotomomyces cejpii

HUANG Zi-lei^{1, 2, 3}, ZHANG Wei-min², YE Wei², LI Sai-ni², LI Hao-hua², ZHU Mu-zi²

1. South China Sea Institute of Oceanology,Chinese Academy of Sciences,Guangzhou 510301;
2. State Key Laboratory of Applied Microbiology Southern China,Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application,Guangdong Open Laboratory of Applied Microbiology,Guangdong Institute of Microbiology,Guangzhou 510070;
3. University of Chinese Academy of Sciences,Beijing 100049

Received:2017-11-03 Published:2018-04-20 Online:2018-05-04

摘要/Abstract

摘要： 利用染色体步移技术对深海真菌Dichotomomyces cejpii中胶霉毒素的生物合成基因GliG、GliI和GliO启动子进行克隆,并将其核心区域导入pGL3-basic荧光素酶报告基因载体,通过荧光强度分析发现GliG的启动子转录活性最强。进一步将GliG启动子核心区域导入含潮霉素抗性标记的pAN7-1载体,以替换原有启动子pgpdA,并将重组质粒导入酿酒酵母,用潮霉素抗性平板进行筛选,发现GliG启动子能在酿酒酵母中启动潮霉素抗性基因的表达。

关键词: Dichotomomyces cejpii, 胶霉毒素, 启动子, 克隆, 鉴定, 海洋真菌

Abstract: The promoters of gliotoxins biosynthesis genes including GliG,GliI and GliO from deep-sea-derived fungus Dichotomomyces cejpii were cloned by the method of genome walking,and the core regions of these promoters were inserted into the reporter gene vector of luciferase pGL-3-basic,and the transcriptional activities of GliG promoter was the highest according to its fluorescence intensity. Further,the core region of GliG promoter was further inserted into the pAN7-1 vector with hygromycin resistance marker to replace original pgpdA promoter,then the recombinant plasmid was transferred into Saccharomyces cerevisiae by electronic transformation. The positive clones were screened by plate with hygromycin resistance,and the results demonstrated that the GliG promoter initiated the expression of hygromycin resistance gene.

Key words: Dichotomomyces cejpii, gliotoxin, promoter, cloning, identification, marine fungus

黄自磊, 章卫民, 叶伟, 李赛妮, 李浩华, 朱牧孜. 深海真菌Dichotomomyces cejpii胶霉毒素生物合成基因启动子的克隆和功能鉴定[J]. 生物技术通报, 2018, 34(4): 144-150.

HUANG Zi-lei, ZHANG Wei-min, YE Wei, LI Sai-ni, LI Hao-hua, ZHU Mu-zi. Cloning and Identification of Gene Promoter for Gliotoxin Biosynthesis from Deep-Sea-Derived Fungus Dichotomomyces cejpii[J]. Biotechnology Bulletin, 2018, 34(4): 144-150.

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深海真菌Dichotomomyces cejpii胶霉毒素生物合成基因启动子的克隆和功能鉴定

Cloning and Identification of Gene Promoter for Gliotoxin Biosynthesis from Deep-Sea-Derived Fungus Dichotomomyces cejpii

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