dsRNA介导的RNA干扰沉默烟曲霉色素形成相关基因arp1的研究

Silencing arp1 Gene in Aspergillus fumigatus by dsRNA Mediated RNA Interference

目的:用dsRNA介导的RNA干扰(RNAi)特异性沉默烟曲霉色素形成相关基因arp1,使arp1基因mRNA水平降低,产生与母代绿色菌株不同的灰色菌株,类似基因敲除的表型。方法:液氮研磨烟曲霉,提取总mRNA,用含T7启动子序列的特异引物进行RT-PCR,扩增出两端含T7启动子序列的arp1基因,以此为模版使用MEGAscriptRNAiKit体外转录合成dsRNA,用合成的dsRNA电转化烟曲霉后,观察其表型变化,对转化菌进行RT-PCR,分析mRNA变化。结果:转化菌的arp1基因mRNA水平降低,产生与母代绿色菌株不同的灰色菌株,类似基因敲除的表型。结论:RNAi在烟曲霉中的应用使快速敲除靶基因产生类似基因敲除的表型,为研究靶基因功能、识别可能的药物作用靶位奠定了良好的基础。

Objective： Silencing arp1 gene in Aspergillus fumigatus which is included in the conidial pigment biosynthesis by dsRNA mediated RNA interference（RNAi） results in reduced in mRNA levels for the gene, with the production of grey conidia which is different to the wild-type strain producing bluish green conidia, which phenotypic consequences are similar to that of gene disruption. Methods： Pour some liquid nitrogen to the mortar and grind aspergillus fumigatus to powder. Extract A.fumigatus total mRNA. The specifical primers added the T7 promoter sequence to the 5＇ end were used in RT-PCR to generate arp1 gene fragment which contain the T7 promoter sequence on the both ends. It was used in the Ambion MEGAscript T7 kit for the transcription reaction to synthesis dsRNA in vitro. Transform A.fumigatus with synthetic dsRNA by electroporation, then observe the change in A.fumigatus phaenotype, perform RT-PCR, analyze change in mRNA level. Results： After electroporation A.fumigatus by dsRNA, arp1 gene mRNA was reduced in the transformants, with the production of grey conidia which was different to the wild-type strain producing bluish green conidia, which phenotypic consequences in transformants were similar to that of gene disruption. Conclusion： Silencing the investigated gene of A.fumigatus by RNAi will allow easier knockout a gene of interest to generate the phenotypic consequences of reducing expression of target gene, it settle a favourable foundation to research gene function and to identify potential target site of drug action.