RNAi法分析香菇色氨酸合酶基因的功能

Functional Analyses of Tryptophan Synthase Gene LetrpB in Lentinula edodes by RNAi Method

色氨酸合酶(tryptophan synthase,TrpB)是生物体内一种重要的功能蛋白,在生物抗逆应激反应过程中发挥着重要的调控作用。本研究以香菇(Lentinula edodes)耐热栽培菌株S606为实验材料,采用双向启动子(Leactin和Legpd)启动LetrpB蛋白β亚基功能结构域的反向互补片段(400 bp),构建了LetrpB基因的RNAi载体,利用农杆菌介导法将该载体转入香菇菌丝中,在潮霉素培养基上筛选转化子,再通过PCR检测片段是否插入,获得了稳定的阳性转化子。实时荧光定量PCR结果显示,与野生型菌株相比,转化子的LetrpB基因表达量显著下调,转化子LeALDH、LeamiE等基因的表达量也显著下调,而LeYuccA的表达量显著上调。40℃高温处理24 h后,野生型S606的菌丝体在25℃条件下可以恢复生长,而RNAi转化子的菌丝体则不能恢复生长。研究结果表明,香菇菌株S606的LetrpB基因参与了热胁迫应激反应。

Tryptophan synthase(TrpB)is an important regulatory protein in response to various stresses.In this study,a TrpB gene LetrpB of a heat-resistant Lentinula edodes strain S606 was studied using the RNAi method.To construct the RNAi vector,double promoters Leactin and Legpd were used to promote theβsubunit for its function antisense fragment of LetrpB gene from either direction.The vector was then transferred to L.edodes mycelia using the Agrobacterium tumefaciens-mediated transformation method.Positive RNAi transformants were first screened by hygromycin and then confirmed by PCR analysis.The qRT-PCR results showed that the relative expression level of LetrpB in positive RNAi tranformants was down-regulated by two folds compared to that of S606.Besides,LeALDH and LeamiE were also significantly down-regulated.On the other hand,LeYuccA was significantly up-regulated.Contrary to S606 that restored mycelial growth after 40℃heat stress challenge for 24 h,RNAi transformants did not restore mycelial growth.The results indicated that LeTrpB was involved in heat stress response of L.edodes.