Resistance Of Transgenic Silkworm Expressing DsRNA To BmNPV

The nuclearpolyhedrosis,without any effective drug for prevention and cure,is a contaguious virus disease caused by nuclear polyhedrosis virus,and the most harmful disease on sericultural industry.When it breaks out,sericultural farms normally suffer huge losses.It is a new path,breaking through the limitation of traditional breeding procedure,to improve silkworm resistence to BmNPV by efficienly alternating genome of Bombyx mori so as to confer enhanced resistence to BmNPV on silkworm using of RNAi technology and transgenic technology.To approach the goal,the two primary systems are essential.The one is the transgenic system for silkworm including transferring exogenous genes into silkworm and effective screen method,and the two is to select the target genes which can be interfered obviously.Some studyies revealed that the use of RNAi technology could interfere ie-1,lef-1 and gp64,among those the efficiency was highest of silencing ie-1,and lowest of gp64 to inhibit BmNPV.But another problem is that even targeting the same gene the silent efficiency will bc much different when focusing different regions of the gene.Still,there is no successful method to screen transgenic silkworm.To confer resistence to BmNPV on silkworm by silencing relavent genes of BmNPV,a series studies was carried out on the study.First,we compared the silent efficiencies of RNAi targeting different regions of ie-1 and lef-1 genes in BmN cells, and the results showed ie-siRNAs were more effective than that of lef-siRNA at a whole, as well as ie-97-siRNA was the most effective among the synthesized siRNAs.Second, the transgenic system for silkworm was optimized,of which the exogenuous DNA could be transferred into silkworm eggs with high efficiency by sperm-mediated of injection after mated.And the procedure of well-timed proper concentration of G418 to screen transgenic silkworm was adding G418 at a concentration of 20 mg/mL for 1 day when the first instar silkworm completeing moulting followed by adding 15 mg/mL for 1 day one day before 1-moulting silkworm,then the transgenic silkworms could be selected in the rough from the third instar silkworm depending the differences in their size.Third,the constructed dsRNA expression cassette containg homologous region of ie-97-siRNA and the neo expression cassette were cloned into piggyBac transposon to form a transgenic vector.Four,the transgenic vector was transfected into BmN cells, and the stable transformed cells demonstrated resistence to BmNPV at 10^-5 viral concentration.Afterwards the transgenic silkworms were gained and identified after transgerring transgenic vextor and selecting via G418 and green fluorescence had been processed.And the last,the improvement of the resistence to BmNPV on the G2 and G3 generation transgenic silkworm artificially inoculated with BmNPV has been confirmed. The mortality of transgenic silkworm infected with BmNPV was decrease of 10 centesimal points compared with original silkworm,and qPCR demonstrated that after being infected the ie-1 mRNA and lef-1 mRNA from transgenic silkworm were about 230 folds and 580 folds relatively lower than that from original silkworm,respectively.The study might accelerate creating new materal of silkworm resisting BmNPV, and the use value of created transgenic silkworm is practicably desired.