RNAi法分析广东虫草热激蛋白40 CgDnaJ05基因功能  

Function of the heat shock protein 40 gene CgDnaJ05 in Cordyceps guangdongensis analysed by RNAi

作　　者：郝转 张成花 黄秋菊 黄浩 李泰辉 王刚正 邓旺秋 HAO Zhuan;ZHANG Chenghua;HUANG Qiuju;HUANG Hao;LI Taihui;WANG Gangzheng;DENG Wangqiu(Key Laboratory for Ecology and Environment of River Wetlands in Shaanxi Province,College of Environment and Life Science,Weinan Normal University,Weinan 714099,Shaanxi,China;Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application,State Key Laboratory of Applied Microbiology Southern China,Institute of Microbiology,Guangdong Academy of Sciences,Guangzhou 510075,Guangdong,China)

机构地区：[1]渭南师范学院环境与生命科学学院陕西省河流湿地生态与环境重点实验室,陕西渭南714099 [2]广东省科学院微生物研究所华南应用微生物国家重点实验室广东省菌种保藏与应用重点实验室,广东广州510075

出　　处：《菌物学报》2022年第11期1786-1795,共10页Mycosystema

基　　金：国家自然科学基金(31900015);广东省科学院专项基金(2020GDASYL-20200103014);广州市科技计划(202102021051);广东省自然科学基金(2022A1515011083);陕西省教育厅科学研究计划(22JP022)。

摘　　要：DnaJ蛋白(热激蛋白40)在机体生长发育和适应逆境环境过程中起着重要作用。以广东虫草GDGM30035为材料,克隆获得CgDnaJ05基因,CDS为2361 bp,编码786个氨基酸,生物信息学分析结果表明CgDnaJ05蛋白含有DnaJ蛋白结构域,属于碱性、不稳定和亲水性蛋白质。以CgDnaJ05蛋白DnaJ保守结构域240 bp的反向互补片段为干扰片段,构建CgDnaJ05基因的双向启动子沉默载体;通过根癌农杆菌Agrobacterium tumefaciens介导转化法侵染广东虫草菌丝,筛选获得9个相对稳定的CgDnaJ05沉默转化子。qRT-PCR分析表明,转化子CgDnaJ05相对表达量下调至野生型菌株的20%-82%;与野生菌丝相比,转化子菌丝生长速度显著下降,且CgDnaJ05下调越明显,菌丝减缓速度越显著,同时,转化子原基的形成受到显著抑制。研究表明:广东虫草CgDnaJ05基因明显影响广东虫草菌丝生长和原基形成,对广东虫草生长发育具有重要的调控作用。DnaJ,namely heat shock protein 40,plays an important role in regulating growth and development and resisting adversity of organisms.CgDnaJ05 cloned from Cordyceps guangdongensis strain GDGM30035 has coding sequence of 2361 bp,encoding a protein of 786 amino acids.Bio-informatics analysis showed that CgDnaJ05 protein contained J domain and was basic,unstable and hydrophilic.240 bp antisense fragment from the conserved domain of CgDnaJ05 protein was promoted dually,and used for constructing the vector of gene silence.The vector was transferred to C.guangdongesis mycelia via Agrobacterium tumefaciens-mediated transformation method,and 9 relatively stable CgDnaJ05-silenced transformants were obtained.The results of qRT-PCR showed that relative expression levels of CgDnaJ05 in transfortmants decreased to 20%-82%of that in wild-type strain.Compared with the wild-type strain,the mycelial growth rate of CgDnaJ05-silenced strains significantly decreased,and the more significantly CgDnaJ05 expression was down-regulated,the more obviously mycelial growth rate slowed down.Furthermore,gene silence of CgDnaJ05 markedly inhibited the primordial formation.The results indicate that CgDnaJ05 obviously affects mycelial growth and primordial formation of C.guangdongensis,and shows an important role in regulating growth and development of the cordyceps.

关 键 词：广东虫草 CgDnaJ05 菌丝生长 原基形成 

分 类 号：S567.3[农业科学—中草药栽培]