High efficiency and stable RNA interference vector construction for Penicillium species

【Author】

Haizhao Xue;Yibo Hu;Xiaoran Fang;Xin Song;

【Abstract】

RNA interference(RNAi),which can turn down gene expression effectively and specifically,has showed great potential in gene function research and gene modification.In this study,a RNA interference vector pHX-RNAi,which carries a hygromycin resistance cassette and a transcriptional unit for hairpin RNA expression was constructed.A gene amyl5A which encodes the major amylase in a high cellulase producer strain Penicillium oxalicum Al 1 was targeted knocking-down.The results of SDS-PAGE and RT-qPCR showed that both the transcription and secretion levels of Amyl5A in all of the hygromycin-resistant transformants with the silencing plasmid were dramatically down-regulated,they all showed high degree of silencing in expression of amy15A.Then amy15A and cel7A-2 were connected to the PHX-RNAi.The results of enzyme activity assay,SDS-PAGE and RT-qPCR analysis showed that the expression of amy15A and ce17A-2 were severely inhibited,which indicated that multiple genes could be silenced by only one step.Since RNAi mechanism is conservative,the high efficient RNAi vector pHX-RNAi will be universal and can be applied in other filamentous fungi to silence genes.

【Keywords】

RNA interference;;Penicillium oxalicum;;Filamentous fungi;;Amylase;;Amy15A;;Plasmid;;pHX-RNAi

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