【作者】 薛仁宇；

【导师】 贡成良；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2009， 博士

【摘要】 家蚕血液型脓病是由家蚕核型多角体病毒引起的一种传染性病毒病,是养蚕业中危害最大的病害,至今尚无有効的防治药物,一旦发生往往会造成巨大的损失。利用RNAi对病毒基因的干扰作用,通过转基因的方法赋予家蚕对BmNPV的抗性,是突破传统育种方法,定向改造家蚕性状的一种新途径。要实现这一目标,必需有二个基础,一是外源基因高效导入及筛选的家蚕转基因系统,二是合适的能通过RNAi有效抑制病毒复制的靶基因。已有的研究表明利用RNAi技术通过干扰ie-1、lef-1和gp64基因均可以有效抑制BmNPV的增殖,其中以ie-1作为RNAi的靶基因抑制BmNPV复制的效率最高,以gp64作为靶基因抑制BmNPV复制的效率最差。由于选择干扰的靶基因不同,抑制病毒复制的效率相差甚远,即使对于同一基因,针对不同区域的siRNA其效率也有很大差异;尽管荧光蛋白报告基因方便了转基因蚕筛选,但至今似乎仍没有一个快速有效的理想方法。为了能通过RNAi和转基因有机结合,提高家蚕对BmNPV的抗性,本研究首先在体外,比较了不同siRNA在家蚕培养细胞中抑制病毒复制的效率,针对ie-1、lef-1基因不同区域,研究siRNA抑制BmNPV复制效果,结果显示ie-siRNA对BmNPV抑制的效率要显著高于lef-siRNA,其中以ie-1-97-siRNA（5′-GCCCAACGACUAUUUGAAUdTdT-3′）效果最好;其次开展了家蚕转基因体系的优化研究,结果显示外源基因导入家蚕卵中的效率以先交后注的精子介导法为高,用G418对转基因蚕筛选的合适浓度和时机应选择在1眠前一天添食15 mg/mL的G418一天,接着在2龄起蚕再用20 mg/mL的G418筛选一天,从3龄开始可以通过蚕的大小实现对转基因蚕的初步筛选;第三将包含ie-97-siRNA对应区域的ie-1 dsRNA表达元件以及neo表达元件一起克隆进piggyBac转座子载体形成pigA3-IE-Neo的转基因载体;第四将该载体转染细胞后获得的稳定转化细胞对10^-5滴度的BmNPV有一定的抗性,通过精子介导法将转基因载体导入家蚕卵、利用G418和绿色荧光双重筛选获得了转基因家蚕,并通过PCR和点杂交对转基因家蚕进行了鉴定。最后通过人工感染BmNPV对G2、G3代转基因家蚕的抗性进行了鉴定,结果显示不同系统的转基因家蚕对病毒的抗性存在显著差异,转基因蚕比正常蚕的平均死亡率均低10%左右,有些系统可以达到20%-60%,而在被感染的转基因蚕中ie-1 mRNA、lef-1 mRNA相对水平较被感染的正常蚕分别低230和850倍左右。本研究促进了抗BmNPV的家蚕新素材创造,获得的转基因家蚕具有潜在的应用价值。更多还原

【Abstract】 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.更多还原