The Establishment And Application Of The RNA Interference System Of Coprinus Chinensis

Coprinopsis cinerea is an ideal object for studies of the developmental processes in basidiomycetes,mainly because it can be completed in the laboratory within 2 weeks.Although it has been sequenced in 2003,but there are large numbers of predicted genes with unknown function.However,the gene disruption by homologous recombination is very difficult,because the probability of homologous recombination is extremely low in C.cinereus.To overcome the limitations of homologous integration we have developed post-transcriptional gene silencing(PTGS)approaches for C.cinereus.It is RNA interference.We construct the RNAi systerm in C.cinereus in our lab and apply it to the study of gene function successfully.In this experiment,We construct the expression plasmid to express the dsRNA of the target gene in C.cinerea.It is composed of gpdll promoter in Agaricus bisporus,TrpC terminator in Aspergillus nidulans,loop sequence,antisense and sense.The dsRNA expression plasmid has been transformed into C.cinerea based on the transformation system of C.cinerea,which has been established by the member in our lab early.Target gene has been silenced to varying degrees by using RT-PCR to detect the expression of silenced gene in positive transformants.The silencing efficiency is between twenty percent and ninety percent.The differentiation of silencing efficiency may be caused of copy number of target gene inserted in host strain's genome.The development process of fruiting body undergoes remarkable morphogenesis:the elongation growth of stipe,the expansion of pileus,the autolysis of pileus.In the previous study,three glucanases(ENG,EXG,BGLU1)were purified from the pileus of C.cinerea.In addition,?-glucosidase(BGLU2)has been purified from the wall of stipe.In a series of studies being conducted,we proposed that they may act on the autolysis of pileus.In order to study the function of the four glucanases further,we applied the RNAi system to silence the four glucanases.The expression of the four glucanases were repressed by the RNAi system,respectively.But no distinct phenotype was observed.The up-regulation of the partial homologous gene of the glucanases has been detected by RT-PCR.The reason of no distinct phenotype is the high expression of the homologous gene of the four glucanases,respectively.We propose the autolysis of pileus is caused of a series of glucanases synergisticly.