蛹虫草hat启动子的克隆及功能分析  

Cloning and Functional Analysis of hat Promoter from Cordyceps militaris

作　　者：张琳[1,2,3] 梅生杰 王莉颖 程爽爽 李长田 李玉 赫荣琳[2,3] ZHANG Lin;MEI Shengjie;WANG Liying;CHENG Shuangshuang;LI Changtian;LI Yu;HE Ronglin(Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi,Changchun 130118,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China;National Technology Innovation Center of Synthetic Biology,Tianjin 300308,China;College of Mycology,Jilin Agricultural University,Changchun 130118,China)

机构地区：[1]食药用菌教育部工程研究中心,长春130118 [2]中国科学院天津工业生物技术研究所,天津300308 [3]国家合成生物技术创新中心,天津300308 [4]吉林农业大学菌物学院,长春130118

出　　处：《菌物研究》2024年第2期166-172,共7页Journal of Fungal Research

基　　金：天津市合成生物技术创新能力提升行动项目(TSBICP-CXRC-006)。

摘　　要：从蛹虫草中克隆出1个组蛋白乙酰转移酶(Histone acetyltransferase)基因的hat启动子,长度为1509 bp,并对该启动子的序列进行详细分析。依据作用元件预测结果显示,hat启动子含有CAAT-box和TATA-box等典型的顺式作用元件,以及参与光响应的顺式作用元件G-box,参与水杨酸响应的顺式作用元件SARE等。将hat启动子连接于报告基因绿色荧光蛋白基因gfp(Green fluorescence gene)的上游,与gfp融合基因表达构建来鉴定启动子的活性。经过农杆菌转化,得到的疑似转化子进行PCR验证、gfp表达水平分析及荧光检测,结果表明:该启动子在蛹虫草菌丝中有较强的驱动GFP表达的能力,在荧光显微镜下可以观察到转化子具有强烈的绿色荧光。开展此研究为食用菌菌种改造提供启动子元件及建立高效的蛹虫草异源基因表达体系奠定基础。In this study,a promoter region of hat gene(Histone acetyltransferase)with a length of 1509 bp was cloned from Cordyceps militaris.Sequences and structure were analyzed by using the Plant CARE database.Results showed that the hat promoter carried those typical cis-acting regulatory elements,such as CAAT-box and TATA-box.It also contained the G-box which was a cis-acting regulatory element involving in the responsibility to light,the SARE which was also a cis-acting element known to involve in the responsibility to salicylic acid and so on.A GFP(Green fluorescent protein)reporter cassette carrying the hat promoter,gfp gene and gpd terminator was successfully constructed for analysis.After introduction of the cassette into C.militaris through the ATMT method,transformants were applied for quality check with PCR and qRT-PCR techniques,and examinations of green fluorescence as well.As a result,a strong green fluorescence was detected in the mycelium of C.militaris.The present study thus provides us with a fundamental basis for the reconstruction of endogenous promoter elements available for transformation experiments in edible fungi and establishment of a highly effective expression system of heterologous genes in C.militaris.

关 键 词：蛹虫草 hat启动子 绿色荧光蛋白 序列分析 

分 类 号：Q949.325[生物学—植物学] Q78