棉铃虫受体蛋白在Cry1Ac、Cry2Ab交互抗性中的作用

发布时间：2017-12-27 00:34

  本文关键词：棉铃虫受体蛋白在Cry1Ac、Cry2Ab交互抗性中的作用　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

  更多相关文章： Cry1Ac Cry2Ab 棉铃虫 受体蛋白 交互抗性

【摘要】：自1996年开始,转Bt基因棉花的投入使用,标志着一种新的依赖植物自身产生杀虫蛋白的防治策略被应用到保护作物、防治害虫上来。Bt作物的经济效益和环境效益使其种植面积在过去的20年迅速增加,与此同时害虫的抗性问题也日益严重。为了延缓害虫对Bt的抗性,目前很多国家都开始种植转双个或多个Bt基因的棉花(pyramids策略)来取代第一代转单价的Bt棉花。理想的“pyramids”策略认为害虫对其中一个毒素产生了抗性会被另外的毒素杀死。目前,这种策略指导下的转Cry1Ac和Cry2Ab的棉花(Bollgard II)已经在澳大利亚,印度和美国广泛种植,但是目前中国还只是种植一代转Cry1Ac的棉花。中国一代转Bt棉花的大面积种植,田间害虫的抗性水平也在显著的增加。尽管二代双价棉花比一代转Bt棉花要耐受一些,然Cry1Ac和Cry2Ab的交互抗性也是制约该双价棉花大面积应用的一个重要因素。我们的前期研究结果已经表明棉铃虫(Helicoverpa armigera)对Cry1Ac和Cry2Ab存在一定程度的交互抗性。同时,Cry1A类毒素的作用机理和抗性机制复杂多变,Cry2Ab的作用机制和抗性机制鲜为人知,这些问题的日益激化和突出,迫切的需要我们研究Cry1Ac和Cry2Ab交互抗性的机制,这将为二代转基因棉花的引入提供理论依据,为明确毒素的机理和昆虫的抗性机制提供思路。本文首先从基因组学和蛋白质组学的角度分析抗感棉铃虫(H.armigera)中的抗性差异基因。其次,为了快速有效的研究这些抗性差异基因是否参与Cry1Ac和Cry2Ab的作用机制中,本研究利用美洲棉铃虫(Helicoverpa zea)中肠细胞系,脂肪体细胞系和Sf9细胞系,建立细胞毒理测定方法。通过Ligand blot和Western blot分析毒素与受体的结合、细胞内真核表达目的基因、dsRNA细胞内干扰和siRNA活体干扰目的基因的实验方法分析比较了抗性差异基因在Cry1Ac和Cry2Ab作用机制中的作用,并对受体蛋白在Cry1Ac的作用机制中的互作进行了分析,其主要结果如下:1:通过基因水平分析LF敏感和LF抗性品系(LF5、LF10、LF20、LF30、LF60和LF120)之间的基因差异,以及蛋白水平分析LF、LF5、LF10、96S、96+2Ab、96-2Ab、96+1Ac和96-1Ac中可能调控棉铃虫对Cry1Ac和Cry2Ab的抗性差异蛋白,发现cadherin、APN1、ALP2、ABCC2、V-ATPase a、V-ATPase c和ATP synthase都有可能参与棉铃虫对Cry1Ac和Cry2Ab的抗性。2:建立细胞毒理实验的方法体系:细胞培养基是毒理实验的最佳缓冲液;4.5 h和5.5h分别是观察Cry1Ac和Cry2Ab毒理的最佳时间。并发现棉铃虫Cry1Ac和Cry2Ab的受体存在较大差异。3:在细胞中表达受体或干扰受体,以及幼虫活体干扰实验表明:美洲棉铃虫的Cadherin、APN1、ALP2、ABCC2和V-ATPa是Cry1Ac的功能受体、而Cadherin、APN1、ALP2和V-ATPa不是Cry2Ab的功能受体。4:对Cry1Ac和Cry2Ab的交互抗性机制的系统研究,发现ABCC2可能是导致Cry1Ac和Cry2Ab产生交互抗性的主要原因。5:对Cry1Ac的主要受体Cadherin、APN1和ALP2之间的互作关系对Cry1Ac毒力的影响进行了分析,没有发现显著的依赖关系和作用次序,参与Cry1Ac作用模型的反应机制至少有4个。
[Abstract]:Since 1996, the use of transgenic Bt cotton has marked a new strategy based on the insecticidal protein produced by plants, which is applied to protect crops and prevent pests. The economic and environmental benefits of Bt crops have made the planting area increase rapidly in the past 20 years, while the problem of resistance to pests is becoming more and more serious. In order to delay the resistance of pests to Bt, many countries have begun to plant two or more Bt genes of cotton (pyramids strategy) to replace the first generation of Bt cotton. The ideal "pyramids" strategy believes that the insect's resistance to one of the toxins will be killed by another toxin. At present, Cry1Ac and Cry2Ab cotton (Bollgard II) under the guidance of this strategy has been widely planted in Australia, India and the United States, but at present, China is only planting a new generation of cotton that transfers to Cry1Ac. In the large area of Bt cotton planting in China, the resistance level of field pests is also increasing significantly. Although the two generation of bivalent cotton is more tolerant than the first generation of Bt cotton, the interaction resistance between Cry1Ac and Cry2Ab is also an important factor restricting the wide application of the bivalent cotton. The results of our previous studies have shown that Helicoverpa armigera has a certain degree of cross resistance to Cry1Ac and Cry2Ab. At the same time, the mechanism and the mechanism of resistance to Cry1A toxin complex mechanism and resistance mechanism of Cry2Ab is littleunderstood. These problems became increasingly acute and prominent, the urgent need we study Cry1Ac and Cry2Ab interaction mechanism of resistance, which will provide a theoretical basis for the introduction of the two generation of transgenic cotton, resistance mechanism to provide ideas to clear the toxins and insects. This paper first analyzes the resistance differentially genes in H.armigera from the perspective of genomics and proteomics. Secondly, in order to quickly and effectively study whether these resistance differentially genes are involved in the mechanism of Cry1Ac and Cry2Ab, we established the cytotoxicity assay by using the Helicoverpa cell line, the Helicoverpa cell line, the fat body cell line and the Sf9 cell line. By Ligand blot and Western blot analysis, combined with the toxin receptor intracellular eukaryotic expression analysis experimental method of interference and siRNA interference in vivo target gene, dsRNA cells, compared the effect differences of resistance genes in the Cry1Ac and Cry2Ab mechanism, and the interaction of receptor proteins in the mechanism of Cry1Ac in are analyzed, the main results are as follows: 1: by gene level analysis of LF sensitive and LF resistant strains (LF5, LF10, LF20, LF30, LF60 and LF120) gene and protein level differences between the analysis of possible differences in regulation resistance of cotton bollworm to Cry1Ac and Cry2Ab, LF5, LF10, LF protein, 96S 96+2Ab 96+1Ac, 96-2Ab, and 96-1Ac, APN1, ALP2, cadherin, ABCC2, V-ATPase a, V-ATPase C and ATP synthase are likely to be involved in Cotton Bollworm Resistance to Cry1Ac and Cry2Ab. 2: set up a method system of cell toxicology experiment: cell culture medium is the best buffer for toxicological experiment; 4.5 h and 5.5h are the best time to observe Cry1Ac and Cry2Ab toxicology respectively. It was found that the receptors of Cry1Ac and Cry2Ab of cotton bollworm were significantly different. 3: expresses receptors or interfering receptors in cells, and larva interference experiments show that Cadherin, APN1, ALP2, ABCC2 and V-ATPa are functional receptors of Cry1Ac, while Cadherin, APN1, ALP2 and V-ATPa are not functional receptors of APN1. 4:'s systematic study on the interaction resistance mechanism of Cry1Ac and Cry2Ab has found that ABCC2 may be the main cause of the interaction resistance of Cry1Ac and Cry2Ab. 5: analyzed the interaction between Cry1Ac's main receptors Cadherin, APN1 and ALP2 on the virulence of Cry1Ac, and found no significant dependencies and order of action. There were at least 4 mechanisms involved in Cry1Ac action models.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S433.4
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本文编号：1339548