【作者】 许超；

【导师】 沈志成；

【作者基本信息】 浙江大学 ， 农业昆虫与害虫防治， 2018， 博士

【摘要】 自从1996年第一批遗传改良农作物商业化种植以来,转基因农作物的种植历史已经跨越了整整20个年头。至今,全球范围内转基因农作物的累计种植面积已经超过20亿公顷,各个国家的农民因此而获得的利益累计超过1500亿美元。第一代Bt转基因农作物只含有单个的抗虫基因,经过多年的单一化种植后,许多地方出现了抗性害虫和次要害虫的爆发;在我国,Bt农作物绝大多数是含有单个Cry1Ac蛋白的抗虫棉花,近年来已经陆续出现抗性棉铃虫(Helicoverpa armigera)和棉红铃虫(Pectinophoragossypiella)等的发生。提升害虫治理的策略、发展第二代甚至第三代Bt转基因农作物的工作迫在眉睫。传统上,治理害虫抗性的方法有“高剂量/庇护所”(high-dose/refuge)策略和“基因堆叠”(pyramids)策略。前者注重农业的生产方法,后者则是提高产品自身的性能。基因堆叠的方法可以是将二个或者二个以上表达单个Bt基因的转化体通过杂交聚合在一个品种中,也可以通过在同一个T-DNA构建几个串联的开放阅读框而实现。但是,这样获得的转基因株系中容易出现不同Bt蛋白之间表达量差异大、启动子的同源沉默等情况。鉴于此,本研究首次设计并改良了一种Bt基因融合策略,将两种不同类型的Bt基因整合成一个融合基因,导入到农作物玉米和水稻中,使得转基因农作物能够同时、等量、稳定地获得两种Bt蛋白的抗虫能力;同时分别设计编码两种不同长度连接肽的序列,插入到两个基因的连接处,使得转基因农作物中能够稳定表达完整的、全长的抗虫融合蛋白。本研究选用了一种Cry蛋白和一种Vip3蛋白,将其核苷酸序列融合;使用的Cry蛋白是商业化最成熟的Bt杀虫晶体蛋白之一 Cry1Ab,已经成功在玉米、棉花等农作物上商业化生产;Vip3蛋白是本课题组自主研发的高效抗虫蛋白Vip3da,这是一种Bt营养期杀虫蛋白;这两种蛋白的抗性机理是完全不一样的。本课题组早前在研究过程中发现,这两个基因直接串联转入植物后,转化事件中往往无法检测到全长的融合蛋白。因此,本研究设计了两段连接肽分别用于连接两个基因:一段是由八个非极性氨基酸组成的短肽linker,另一端是来源于玉米几丁质酶两个结构域之间的连接序列(Chitinaselinker,CL)。这两段连接肽都没有生物活性,只起到连接的作用,用于避免Cry1Ab蛋白与Vip3da蛋白的三维结构发生挤压和拮抗、从而导致融合蛋白的降解;含有短肽linker的融合基因命名为CAL,含有Chitinaselinker的融合基因命名为CACL。对原核表达蛋白的生物活性测定结果显示,CAL与CACL蛋白兼具有Cry1Ab蛋白和Vip3da蛋白的活性和杀虫谱,对Cry1Ab无法杀灭的小地老虎和粘虫,CAL/CACL具有良好的杀虫活性。本研究构建了具有抗虫融合基因的T-DNA载体,并以不同的启动子启动抗虫融合基因在玉米和水稻中的表达。研究结果显示,具有编码连接肽序列的融合基因转入玉米和水稻后,转基因植株中可以检测到完整的、全长的抗虫融合蛋白,并且植株具有相应的抗虫活性。最终,筛选出了 SL34和SL21这两个玉米转化株系,以及A1L、SL6和SL14三个水稻转化株系。对这些转化株系进行分子特征的鉴定,并测定了它们的外源蛋白表达量、T-DNA插入位点和拷贝数等信息,并利用棉铃虫、粘虫和小地老虎等主要的鳞翅目害虫测定了它们的生物活性,为后续的商业化应用提供依据。更多还原

【Abstract】 It has been over 20 years since the first commercialization of genetically modified crops in 1996.Today,a cumulative acrage of GM crops has been over 2 billion hectares worldwide.It was estimated that the total benefits of GM crops to the famers in this period was more US$150 billion.The first generation of Bt crops contained only one insecticidal gene.After more than 10 year’s utilzation,insect resistance to Bt have been developled both in lab and farmland globally.Recently in China,cotton bollworm and pink bollworm were found to have gained some level of resistance in Xinjiang where cultivated transgenic cotton expressing Cry 1 Ac were widely planted.Therefore,Bt resistance management strateges should be implemented in the new generation of GM crops to be developed.Traditionally,high-dose/refuge strategy and pyramids strategy are two major methods for the management of insect resistance.Pyramids strategy stacked two or more toxins in a single crop cultivar by hybridization of two or more single Bt gene events or by molecular stacking,which two or more Bt expression cassettes were constructred into into one T-DNA.In these cases,unbalanced expression of different toxins and gene silencing would be potentially a problem.To over come these issues,we designed a Bt fusion strategy of two Bt toxins with different mode of action.Transgenic corn and rice introduced with this fusion gene would express the entire fusion protein stably and simultaneously.In this study,we fused CrylAb and Vip3da to generate fusion proteins with a linker between the two peptides.CrylAb was a widely used toxin in commercial transgenic crops.Vip3da was developed by our group with high activity against several Lepidopteran pests.A short peptide linker consist of 8 non-polarity amino acids was used as linker to connect CrylAb and Vip3DA.A longer linker derived from a section of nucleotide connecting two domains of maize chitinase was also utilized to link crylAb and vip3da.Bioassay revealed that E.coli expressed fusion protein had high activities against fall armyworm,cotton bollworm and black cutworm,which suggested that this novel fusion proteins maintain the specific activity of both Cry 1 Ab and Vip3da.We constructed T-DNA plasmids carrying the crylAb and vip3da fusion gene using different promoters to transform corn and rice.Results revealed that the entire fusion protein could been detected in transgenic corn and rice events,and they all showed expected bioactivities against several Lepidopteran pests.Finally,we picked two transgenic corn lines SL21 and SL34,and three transgenic rice lines AIL,SL6 and SL14 for detailed analysis of insect resistance and for molecular chacterization.Molecular characters such as expression quantities,border sequences and T-DNA copy numbers had been measured and determined.All of them could been candidates for further commercialization and these data would provide references.更多还原