Construction Of The RNAi Vector And Agrobacterium Mediated Transformation Of Hydrophobin Gene In Pleurotus Ostreatus

The barriers to the gene functional research of the higher fungi mainly lie in the characteristics of its own multicellular core,which brings a lot of technical difficulties for the study of gene function of the multi nuclear cells.This study used the RNA interference(RNAi)technology try to inhibition of the expression of Pleurotus ostreatus hydrophobic protein(Hydrophobin)gene in the level of transcription.Overcome the disadvantages of using traditional gene knock out technology not easy to obtain the homozygous except,and combined with Po.hyd gene over expression vector and antisense RNA vector and Agrobacterium mediated transformation method to jointly explore the gene function of Po.hyd.Hydrophobin play an important role in process of fungal life such as the gas hyphae formation,spores germination,fruiting body development and so on.But relative studies on hydrophobic protein gene function is few,and there has not found the Pleurotus ostreatus hydrophobic protein gene function reports.This study through gene horizontal to explore Pleurotus ostreatus hydrophobic protein gene function,and in order to lay the foundation for Pleurotus ostreatus hydrophobic protein gene function research and industrial production.First of all,use of PCR cloning technology to get Po.Hyd gene coding region(CDS)sequence which is 330 bp and the endogenous type glyceraldehyde-3-phosphate dehydrogenase gene promoter(GPD)sesequence of Pleurotus ostreatus which is 960 bp.Use the p CAMBIA1300 as the skeleton,the hydrophobic protein expression vector p1300-gh-gOEhyd and the antisense RNA vector p1300-ghgantihyd were constructed.At the same time,use the Golden Gate cloning technology constructs the Pleurotus ostreatus hydrophobic protein interference vector p1302-GGihyd,which was based on the vector p CAMBIA1302,with hyg B resistance gene and green fluorescent protein(GFP)as a marker gene.Finally,by electroporation technology,the binary vector into the Agrobacterium tumefaciens,and under the inducing effect of acetosyringone(as),Agrobacterium tumefaciens will transform the Pleurotus ostreatus young hypha.The transformation was verified by the technology of PCR,the expression of green fluorescent protein,Southern hybridization,and so on.Finally,the expression of the gene was detected by Real-time PCR.The results showed that the received interference vector transformants with hygromycin antibiotic resistance can also detect signal of green fluorescence under the fluorescence microscope,the expression of the Hydrophobin gene accounted for 43 per cent of the wild type,and the interference vector T-DNA fragment with a single copy in the form of integration into a subbase for group in this mushroom.Transformants of over expression vector and antisense RNA vector,with hygromycin resistance gene and genetic stability.