深黄被孢霉高产菌株差异表达未知基因UG1 RNAi表达质粒的构建

Construction of RNAi expression vector targeting unknown differentially expressed UG1 gene of high-yield strain of Monierella isabellina

目的构建深黄被孢霉高产菌株差异表达未知基因UG1 RNAi表达质粒。方法以红色荧光蛋白DsReD作为报告基因,构建DsReD RNAi表达质粒pREDi;制备原生质体,通过PEG/CaCl2原生质体转化法将质粒pREDi转化至稳定表达DsReD基因的深黄被孢霉高产菌株中进行表达;在此基础上构建深黄被孢霉高产菌株差异表达未知基因UG1 RNAi表达质粒pUG1i,并对转染了质粒pREDi、pUG1i的受体菌进行Northern blot分析。结果质粒pREDi和pUG1i经酶切和测序鉴定表明构建正确;转化了表达质粒pREDi的菌株呈无色;转染质粒pUG1i的受体菌UG1和DsReD在基因表达水平上受到了抑制。结论成功构建了深黄被孢霉高产菌株差异表达未知基因UG1 RNAi表达质粒,为下一步通过RNAi共沉默DsReD和目的基因,以及进一步深入研究深黄被孢霉未知基因UG1对菌株合成ARA是否有影响和基因功能的分析奠定了基础。

Objective To construct a RNAi expression vector targeting unknown differentially expressed UG1 gene of high-yield strain of Monierella isabellina. Methods DsReD RNAi expression vector pREDi was constructed using red fluorescent protein gene DsReD as a report gene, and transformed to M. isabellina stably expressing DsReD gene by PEG/ CaC12-mediated protoplast transformation, based on which the RNAi expression vector pUGli targeting differentially expressed UG1 gene of high-yield M. isabellina strain was constructed, and the M. isabellina transfected with pREDi and pUGli were analyzed by Northern blot. Results Restriction analysis and sequencing proved that plasmids pREDi and pUGli were constructed correctly. The M. isabellina strain transformed with pREDi was colorless. However, the expressions of UG1 and DsReD in recipient M. isabellina transfected with plasmid pUGli were inhibited at gene level. Conclusion RNAi expression vector targeting unknown differentially expressed UG1 gene of high-yield strain of M. isabellina was constructed successfully, which laid a foundation of co-silencing DsReD and target gene by RNAi and further study on the effect of UG1 gene on synthesis of ARA and analysis of gene function.