甜菜夜蛾中肠钙粘蛋白的Cry蛋白受体功能分析

发布时间：2018-09-17 17:54

【摘要】：单价转Cry1Ac基因抗虫棉的大面积推广成功的控制了棉铃虫的为害,并显著减轻农药施用量及对环境的污染。但是,研究发现部分鳞翅目害虫对单价转基因棉产生了抗性,同时发现单价转基因棉花对甜菜夜蛾(Spodoptera exigua)等害虫的防治效果不理想。为了延缓靶标害虫对抗虫棉抗性的发展,以及扩大转基因抗虫棉的防治谱,国际上开始使用双价转基因策略,即同一转基因作物中表达两种不同杀虫机理的杀虫蛋白,Cry2A类蛋白是双基因策略中应用的重要蛋白之一。钙粘蛋白(Cadherin)是Cry1类蛋白在鳞翅目害虫中肠的主要受体,但其与Cry2类蛋白的相互作用关系还不甚明确。本文通过检测钙粘蛋白被沉默以后甜菜夜蛾对Cry蛋白(Cry1Ac和Cry2Aa)的敏感性,检验钙粘蛋白多肽片段与两种Cry蛋白的结合能力,以期阐明甜菜夜蛾Cad1b与两种Cry蛋白间的作用关系,此外借助竞争性结合试验,探究了双价转基因植物的潜在风险。主要研究结果如下:1.通过饲喂甜菜夜蛾初孵幼虫ds Se Cad1b,对钙粘蛋白基因进行RNA干扰研究。结果表明:甜菜夜蛾初孵幼虫取食ds Se Cad1b 24h后,钙粘蛋白基因表达量显著降低;钙粘蛋白基因被沉默以后,甜菜夜蛾对Cry1Ac、Cry2Aa蛋白的敏感性下降,死亡率显著降低。2.将体外表达并纯化的钙粘蛋白多肽片段Se Cad1b、Se CR7-8F、Se CR9-MPED分别与生物素标记的Cry1Ac、Cry2Aa结合,发现三段多肽均可以与两种蛋白特异性结合。3.将纯化后对钙粘蛋白多肽片段Se Cad1b、Se CR7-8F、Se CR9-MPED分别与Cry1Ac和Cry2Aa做同源性竞争和异源性竞争点杂交试验。同源竞争时,两种Cry蛋白在三个多肽片段上均存在竞争性结合,而异源竞争时,Cry蛋白在多肽片段上不存在竞争性结合。综上所述,本研究明确了甜菜夜蛾Se Cad1b基因作为Cry1Ac和Cry2Aa蛋白的受体功能,并且Cry1Ac和Cry2Aa在甜菜夜蛾Se Cad1b蛋白上存在不同的结合位点。本研究为阐述甜菜夜蛾对Cry蛋白的抗性机制研究奠定了理论基础,并对双价转基因作物的研制和开发具有重要意义。
[Abstract]:The application of monovalent Cry1Ac transgenic cotton successfully controlled the damage of Helicoverpa armigera and significantly reduced the amount of pesticide applied and the pollution to the environment. However, it was found that some Lepidoptera pests were resistant to monovalent transgenic cotton, and the control effect of monovalent transgenic cotton on insect pests such as Spodoptera exigua (Spodoptera exigua) was not satisfactory. In order to delay the development of target insect resistance to cotton and expand the control spectrum of transgenic cotton, bivalent transgenic strategy has been used in the world. Cry2A protein, which expresses two different insecticidal mechanisms in the same transgenic crop, is one of the important proteins used in the double gene strategy. Cadherin (Cadherin) is the main receptor of Cry1 proteins in the midgut of Lepidoptera pests, but its interaction with Cry2 proteins is not clear. In this paper, the sensitivity of Spodoptera exigua to Cry protein (Cry1Ac and Cry2Aa) after the silencing of cadherin was detected, and the binding ability of cadherin polypeptide fragment to two Cry proteins was tested in order to elucidate the interaction between Cad1b and two Cry proteins of Spodoptera exigua. In addition, the potential risk of bivalent transgenic plants was explored by competitive binding test. The main results are as follows: 1. The RNA interference of cadherin gene was studied by feeding ds Se Cad1b, of newly hatched larvae of Spodoptera exigua. The results showed that after feeding ds Se Cad1b for 24 hours, the expression of cadherin gene decreased significantly, and the sensitivity to Cry1Ac,Cry2Aa protein decreased and mortality decreased significantly after silencing the cadherin gene. The expression and purification of cadherin polypeptide fragment Se Cad1b,Se CR7-8F,Se CR9-MPED were combined with biotin labeled Cry1Ac,Cry2Aa, and it was found that all three segments of peptides could bind to two proteins specifically. 3. After purification, homologous competitive and heterogenous competitive dot hybridization of cadherin polypeptide Se Cad1b,Se CR7-8F,Se CR9-MPED with Cry1Ac and Cry2Aa were performed respectively. In homologous competition, the two Cry proteins were competitively bound on the three polypeptide fragments, but not on the polypeptide fragments in the heterologous competition. In conclusion, the receptor function of Se Cad1b gene as Cry1Ac and Cry2Aa protein in Spodoptera exigua was confirmed, and Cry1Ac and Cry2Aa had different binding sites on Se Cad1b protein of Spodoptera exigua. This study laid a theoretical foundation for the study of resistance mechanism of Spodoptera exigua to Cry protein and was of great significance to the development of bivalent transgenic crops.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433.4

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