Bar基因双元载体的快速构建及其在蛹虫草中的功能验证  

Rapid Construction of Binary Vector of Bar Gene and Its Function Verification in Cordyceps militaris

作　　者：娄海伟 林俊芳[2] 赵仁勇[1] 叶志伟[2] 田双起[1] 王新伟[1] 郭丽琼[2] 赵玉 Lou Haiwei;Lin Junfang;Zhao Renyong;Ye Zhiwei;Tian Shuangqi;Wang Xinwei;Guo Liqiong;Zhao Yu(College of Food Science and Technology,Henan University of Technology,Zhengzhou 450001;College of Food Science,South China Agricultural University,Guangzhou 510642)

机构地区：[1]河南工业大学粮油食品学院,郑州450001 [2]华南农业大学食品学院,广州510642

出　　处：《中国食品学报》2021年第10期15-22,共8页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31572178,31772373,31801918);广东省科技创新战略专项资金(2018B020205003,2018B020205001);河南省重点研发与推广专项(科技攻关)项目(212102110194)。

摘　　要：为获得草铵膦完全抑制蛹虫草生长的最小浓度和一种快速构建Bar基因双元载体的方法,本文首先采用浓度梯度法研究草铵膦对蛹虫草的抑制效果,其次采用双接头PCR(DJ-PCR)构建Bar基因表达盒,然后分别采用T4 DNA连接酶法和同源重组法把Bar基因表达盒插入双元载体pAg1-H3的T-DNA区域,以构建Bar基因双元载体pAg-Bar,并比较T4DNA连接酶法和同源重组法的连接效果,最后通过农杆菌介导转化法来验证载体pAg-Bar的有效性及Bar基因在蛹虫草中的功能。结果表明:草铵膦完全抑制蛹虫草分生孢子生长的最小质量浓度为400μg/mL;采用DJ-PCR方法可快速构建Bar基因表达盒;采用T4 DNA连接酶法和同源重组法均可实现双元载体pAg-Bar的构建,而同源重组法更快速、高效;采用农杆菌介导转化法可把双元载体pAg-Bar中的Bar基因表达盒整合入蛹虫草的基因组,使蛹虫草转化子具有草铵膦抗性。本研究建立的载体构建方法具有快速、高效的特点,适用于抗性基因载体、敲除载体、超表达载体等载体的构建,为蛹虫草基因功能的鉴定提供技术支撑。The aim of this study is to obtain the minimum concentration of glufosinate ammonium that can completely inhibit the growth of Cordyceps militaris and a method for quickly constructing the binary vector of bar gene.The concentration gradient method was used to study the inhibitory effect of glufosinate ammonium on the growth of C.militaris.Double-joint PCR(DJ-PCR)was used to construct the bar gene expression cassette.The T4 DNA ligase method and the homologous recombination method were used to insert the bar gene expression cassette into the T-DNA region of the binary vector pAg1-H3 to construct the binary vector pAg-Bar,and the ligation effects of the above two methods were compared.Agrobacterium tumefaciens-mediated transformation method was used to verify the effectiveness of the vector p Ag-Bar and the function of the bar gene in C.militaris.The results showed that the minimum concentration of glufosinate ammonium that could completely inhibit the growth of C.militaris conidia was 400μg/mL.The bar gene expression cassette could be effectively constructed by the DJ-PCR method.The binary vector pAg-Bar could be successfully constructed by the T4 DNA ligase method and the homologous recombination method.However,the homologous recombination method is simpler and more efficient than the T4 DNA ligase method.The bar gene expression cassette in the binary vector pAg-Bar could be successfully integrated into C.militaris genome by Agrobacterium tumefaciens-mediated transformation method,and the obtained C.militaris transformants were resistant to glufosinate ammonium.The method of constructing vectors established in this study is simple and efficient.It is suitable for constructing vectors with resistance gene,knock-out vectors,and overexpression vectors and so on.These results provide technical support for the identification of C.militaris gene function.

关 键 词：BAR基因 选择标记基因 蛹虫草 草铵膦 农杆菌介导转化 双元载体 同源重组 

分 类 号：S567.3[农业科学—中草药栽培]