RNAi法分析广东虫草热激蛋白40 CgDnaJ05基因功能

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2 361 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。

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

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 2 361 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.