RNAi法分析香菇邻氨基苯甲酸合酶基因功能  

Functional analyses of anthranilate synthase gene Letrp E in Lentinula edodes by RNAi mediated gene knockdown

作　　者：马超君[1] 王刚正 周莎莎 罗义 龚钰华[1] 边银丙[1] MA Chao-Jun;WANG Gang-Zheng;ZHOU Sha-Sha;LUO Yi;GONG Yu-Hua;BIAN Yin-Bing(Institute of Applied Mycology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, Chin)

机构地区：[1]华中农业大学植物科学技术学院应用真菌研究所,湖北武汉430070

出　　处：《菌物学报》2018年第5期576-583,共8页Mycosystema

基　　金：国家自然科学基金(34116162);湖北省技术创新专项重大项目(2016ABA100)~~

摘　　要：邻氨基苯甲酸合酶(Trp E)是生物体内一种重要的逆境诱导蛋白,其在逆境条件下的响应保证了生物体生存相关产物的正常代谢。本文以香菇Lentinula edodes栽培菌株S606为试验材料,以Le Trp E功能结构保守区域395bp的反向互补片段为干扰片段,构建Letrp E基因双向启动子RNAi载体;采用根癌农杆菌介导转化法侵染香菇菌丝,通过PCR方法检测DNA插入片段,获得11个阳性转化子;实时荧光定量PCR分析结果表明,2个转化子Letrp E基因表达量较野生型菌株下调了2–3倍,确认其为Letrp E基因RNAi转化子;菌丝体在40℃高温处理24h后,检查到吲哚-3-乙酸合成途径中基因Le Tam-1在野生菌株S606表达量上调,而在2个RNAi转化子中表达量下调;RNAi转化子菌丝体在25℃下不能恢复生长,而野生型菌丝体可以恢复生长。研究表明,香菇Letrp E基因的功能与耐热性有关。Anthranilate synthase Trp E, an important stress-induced protein in organisms, maintains the balance of metabolites related to organism survival as respond to stresses. In this study, Lentinula edodes strain S606 was treated as the tested strain and RNAi fragment. 395 bp antisense fragment from the conserved domain of Letrp E was promoted dually. The vector was transferred to L. edodes mycelia via Agrobacterium tumefaciens-mediated transformation method. Amplifying insert fragment, 11 positive transformants were gained. q RT-PCR result showed that the relative expression levels of two tranformants had approximate two and three-fold downregulation compared with those of wild type（WT） S606, indicating that they were RNAi transformants. After 24 h 40°C heat stress, expression levels of Le Tam-1, one protein relating to indole-3-acetic acid biosynthesis, were upregulation in WT S606 but downregulation in RNAi transformants; RNAi transformant mycelia could not regrow while WT could regrow. The results demonstrated that Letrp E function was related to L. edodes thermotolerance.

关 键 词：香菇 高温胁迫 耐热性 邻氨基苯甲酸合酶 转化子 

分 类 号：S646.12[农业科学—蔬菜学]