摘要
光照是高等真菌子实体发育必不可少的环境因子之一,真菌通过光受体感受光信号,WC-1蛋白既有对光信号应答能力,又可以作为转录因子,激活下游基因表达。为了对真菌光感应系统作进一步研究,本研究利用杏鲍菇Ple-tm菌株转录组数据,采用RT-PCR方法克隆获得了杏鲍菇wc-1基因,构建原核表达载体p ET-28a(+)-wc-1并转化大肠杆菌BL21,诱导表达。结果显示:wc-1基因(GenBank登录号:MG705181)长为2614 bp,含有一个2205 bp的开放阅读框(open reading frame,OFR),编码734个氨基酸,理论分子量(molecular weight,Mw)为81 k D,等电点(isoelectric point,PI)为6.29。同源性比对发现,与平菇中蓝光受体在氨基酸水平上相似性达82%以上,显示其较高的保守;成功构建含有wc-1基因的原核表达载体p ET-28a(+)-wc-1,体外诱导表达出融合蛋白,该重组蛋白在18℃、0.1 mmol/L IPTG、12 h时表达量最大,且以包涵体的形式存在。本研究结果为深入研究杏鲍菇子实体形成机理及蓝光受体蛋白的结构奠定了基础。
Light is one of the indispensable environmental factors for the development of higher fungal fruiting bodies. In this study, the transcriptome data of Pleurotus eryngii "Ple-tm" was used to clone and obtain the wc-1 gene of Pleurotus eryngii by RT-PCR method. The prokaryotic expression vector p ET-28 a(+)-wc-1 was constructed and transformed into E. coli BL21 for induction of expression, and then it was analyzed by SDS-PAGE. The results showed that the wc-1 gene(Gen Bank accession number: MG705181) contains a 2 205 bp open reading frame(OFR) encoding 734 amino acids with a theoretical molecular weight(Mw) of 81 kD and an isoelectric point(Isoelectric point, PI) is 6.29. The homology comparison showed that the similarity of the blue light receptor in the Pleurotus ostreatus was more than 82% at the amino acid level, indicating its high conservation. The prokaryotic expression vector pET-28 a(+)-wc-1 was successfully constructed, and the fusion protein was induced to express in vitro. The recombinant protein was expressed in the form of inclusion bodies at 18℃, 0.1 mmol/L IPTG and 12 h.This study laid a foundation for further study on the mechanism of Pleurotus eryngii fruiting body and the structure of blue receptor protein.
作者
闫苗
刘云超
李帆
辛思洁
陈利丁
艾柳英
孙淑静
Yan Miao;Liu Yunchao;Li Fan;Xin Sijie;Chen Liding;Ai Liuying;Sun Shujing(College of Life Sciences,Fujian Agriculture and Forestry University,Fuzhou,350002;Fujian Agricultural and Forestry University(Gutian)Institute of Fungi,Ningde,352200)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2019年第12期5444-5449,共6页
Genomics and Applied Biology
基金
福建省自然科学基金杰出青年项目(2014J06010)
校科技创新专项基金(CXZX2017505)
福建省科技厅区域发展项目(2019N3018)共同资助
关键词
杏鲍菇
蓝光受体
基因克隆
原核表达
Pleurotus eryngii
Blue light receptor
Gene cloning
Prokaryotic expression
