RNAi法分析香菇邻氨基苯甲酸合酶基因功能

邻氨基苯甲酸合酶（TrpE）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LeTrpE功能结构保守区域395bp的反向互补片段为干扰片段,构建LetrpE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LetrpE基因表达量较野生型菌株下调了2-3倍,确认其为LetrpE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LeTam-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LetrpE基因的功能与耐热性有关。

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

Anthranilate synthase TrpE, 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. 395bp antisense fragment from the conserved domain of LetrpE 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. qRT-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 24h 40°C heat stress, expression levels of LeTam-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 LetrpE function was related to L. edodes thermotolerance.