Study On The RNAi In Aspergillus Niger

Filamentous fungi are big group in fungus, and have been used in industry, agriculture, themedicine and basic research in biology. Filamentous fungi have been widely studying becauseof the important role in economy and science, and the studies of functional genes areimportant parts. There are many methods to study functional genes, such as gene knockout,transposon tagging and RNA interference, et al. RNA interference (RNAi) is apost-transcriptional gene-silencing (PTGS) phenomenon in which double stranded RNA(dsRNA) triggers the degradation of homologous mRNA species, thereby reducing geneexpression. RNAi has been successfully used in some filamentous fungus, such as Aspergillusoryzae, Aspergillus fumigatus, Aspergillus nidulans and Aspergillus niger, et al. Thistechnology will be more widely applied to study functional genes of filamentous fungus dueto its high performance, high specificity, very stability and transmissibility and heritage.A basic vector pHGW-RNAi with Gateway cloning system used in RNAi was constructedchiefly. Two ccdB genes with attR1and attR2recombination sites were reversely connected,with the eighth intron of amylase gene of Aspergillus oryzae as a spacer fragment. It alsocarried A. oryzae amyA gene promoter and agdA gene terminator for expression of the hairpinRNA cassette. Target gene fragments were PCR-amplified and constructed by the BP reactionwith vector pDONR221to generate an entry vector. Then, an RNAi expression vector wasformed by the LR reaction between the entry vector and the basic vector. Finally, theexpression vector was transformed into A. niger SH-2by Agrobacterium-mediatedtransformation. The A. niger transformants were analyzed by apparent activity detection,RT-PCR and SDS-PAGE to detect the effect of RNAi.GlaA gene was first chosen as the target gene, and two fragments those were784bp and388bp respectively were PCR-amplified with cDNA as the templates then introduced into theexpression vector. The results indicated that RNAi was effective in A. niger and the shorterone was more effective. After this, the fusion fragment of A. niger amyA gene, glaA gene andprtT gene was generated by overlap extension PCR, then was introduced into the expressionvector to inhibit the three genes simultaneously. The results indicated that we could indeed silence the three genes simultaneously by RNAi. That was, co-suppression of RNAiwas feasible in A. niger. We also found through the experiment that the consequence of RNAiwas inhibition of gene expression but not gene knockout.