摘要
转基因棉花的大规模种植对控制棉花害虫的危害发挥了重要作用,但害虫长期处于转基因作物Bt毒蛋白的高压选择下,害虫对Bt作物抗性问题将不容忽视。昆虫对Bt毒素的抗性机制是复杂和多样的,但中肠结合位点的改变是最主要的一种机制。棉铃虫中已知的受体有氨肽酶N和钙粘蛋白。保存在本实验室的高抗Cry1A蛋白的棉铃虫品系经前人研究未发现该品系存在钙粘蛋白基因的缺失、失活,比较抗、感品系棉铃虫的钙粘蛋白、APN序列,只发现少量氨基酸的变异,因此该棉铃虫品系对Cry1A产生抗性很可能与受体蛋白表达水平变化相关或存在其它的受体蛋白。
本文首先比较了两种双向电泳系统以及几种BBMV提取方法对双向电泳的影响,建立了棉铃虫中肠Bt结合蛋白蛋白质组学的研究方法,利用蛋白质组学的方法分离和分析了棉铃虫中肠Cry1A毒素的结合蛋白,比较了抗、感品系棉铃虫中Cry1Ac结合蛋白的变化,探讨该棉铃虫品系对Bt毒蛋白产生抗性的可能机制。并比较了抗、感品系棉铃虫中与不同Bt毒蛋白作用的结合蛋白的差异。在国内首次开展了利用蛋白质组学研究棉铃虫的中肠Bt结合蛋白及其与抗性的关系。主要结果如下:
1、方法的建立:比较ISO-DALT和IPG-DALT两种双向电泳系统,发现这两种系统都可以用于棉铃虫Cry1Ac结合蛋白的分离,ISO-DALT对结合蛋白的分离效果虽比IPG-DALT更好,尤其对高分子量蛋白的分离明显优于IPG-DALT法,但由于上样量少,因此对后续的质谱分析非常不利;IPG-DALT虽较昂贵但有利于进行后续试验,因此本文选用IPG-DALT系统进行双向电泳。比较已报道的这三种BBMV提取方法:Wolfersberger法、English-Readdy法、Abdul-Rauf法,结果显示这三种方法对分离Cry1Ac结合蛋白的种类没有影响,但这三种方法获得的蛋白量不一样结果。总结三种方法的优点,建立了一种新的综合BBMV提取方法,获得了高量的蛋白,通过双向电泳和结合试验证明该方法适合双向电泳Cry1A结合蛋白研究。
2、利用双向电泳结合Western杂交以及生物质谱的方法,研究了棉铃虫中肠Cry1Ac结合蛋白,首次鉴定得到棉铃虫中肠四种新的Cry1Ac结合蛋白:V-ATPase亚基B、热激蛋白、肌动蛋白以及一种未知的新蛋白。肌动蛋白和V-ATPase亚基A已报道为烟芽夜蛾中Cry1Ac结合蛋白,热激蛋白和未知新蛋白为首次报道的昆虫中肠Cry1Ac结合蛋白。
3、用蛋白质组学的方法比较了抗、感棉铃虫中肠结合蛋白的数量和表达量,结果发现结合蛋白种类无差异,但其中一种新的结合蛋白在抗性品系中表达量至少下降了6.49倍。前人的研究说明,碘标记结合试验证明结合位点或受体数目减少是该抗性品系对Cry1A毒蛋白产生抗性的主要原因。结合前人研究和本文结果,本文新结合蛋白表达量严重降低可能是该抗性品系产生抗性的原因之一。
4、初步比较Cry1Ac、Cry1Ab和Cry1Ah作用于敏感品系和抗性品系的结合蛋白。结果发现无论是抗性品系和敏感品系Cry1Ab的结合都是最少的,在敏感品系中,Cry1Ab未结合肌动蛋白和V-ATPase,而Cry1Ac和Cry1Ah对肌动蛋白和V-ATPase结合强,说明新蛋白和热激蛋白对毒素的作用过程来说可能是必需的,肌动蛋白和V-ATPase可能与毒力相关。前人研究Cry1Ab和Cry1Ah对Cry1Ac毒蛋白抗性品系存在一定的交互抗性,各个Bt蛋白在Cry1Ac抗性品系和敏感品系中结合蛋白的差异还是存在于新蛋白的表达量上,新蛋白表达量下降可能与交互抗性产生有关,也可能存在其他的原因。
The wide spread plant of Bt cotton has played an important role in cotton pest control, pests are selected under the pressure of high-dose Bt toxin produced by Bt cotton for a long time, therefore the resistance evolution of pest to Bt cotton should not be ignored. The resistance mechaniam of Bt toxin to insects is complex and variable, but the change of the binding proteins in the midgut is the main one. The known receptors in cotton bollworm (Helicoverpa armigera (Hübner), CBW) are Aminoopeptidase N (APN) and Cadherin-like proteins. According to the test results of predecessors, no disruption and inactivation of cadherin gene but only a few change of amino acids had been found in APN and Cadherin-like proteins of the Cry1Ac-resistant CBW strain preserving in this lab, probably its Cry1Ac-resistant relates to the change of expressional level of receptor protein or there may have some other related receptors proteins.
Firstly we compared two systems of two-dimension electrophresis (2-DE) and three prepared methods of BBMV, then build the proteomic analysis approach for CBW midgut proteins. Secondly we separated and identified Cry1Ac binding protein in CBW midgut by using proteomic approach. Thirdly the binding proteins in susceptible and resistant strain of CBW were compared, so as to find the possible resistant mechanism of Bt toxin. And finally the binding proteins of different Bt in susceptible and resistant CBW strains were also compared. The results were as follows:
1. Methods: The ISO-DALT and IPG-DALT 2-DE system were compared, results showed that these two system could be used to separate the Cry1Ac binding proteins, though the separation results of ISO-DALT were better than IPG-DALT, especially for the larger protein separation, because of the limit protein loading quantity, this system was adverse for the mass spectrum. The IPG-DALT was more expensive, but it was good for the following mass spectrum work, so we chose IPG-DALT for 2-DE. The three different BBMV preparation protocols (ER, ARE and W) did not affect the number or types of CBW Cry1Ac binding proteins detected, However, different methods yielded different protein quantities. We consolidated a BBMV isolation protocol according to advantage of these reported protocols, this protocol could get high protein quantities and also suitable for 2-DE.
2. Bacillus thuringiensis Cry1Ac binding proteins in CBW BBMV were investigated using proteomic analysis, we identified four other Cry1Ac binding proteins in CBW BBMV for the first time: vacuolar ATP synthase subunit B, actin, a heat shock cognate protein (HSP) and a novel protein. Actin and vacuolar ATP synthase subunit A had been reported as Cry1Ac binding proteins in Heliothis virescens. Heat shock cognate and this novel protein was the first report as Cy1Ac binding protein in insect.
3. The types and expression quantities of Cry1Ac binding proteins of susceptible- and resistant-CBW were compared, it showed that the types of binding proteins were the same between susceptible- and resistant-strain, but the novel protein in the resistant-strain were 6.49-fold lower expression than the susceptible-strain. Studies before said that less binding of Bt in the midgut or less number of receptors were the main resistant mechanism for this resistant strain. Combined studies before and this paper, much lower expression of the novel protein might be one of the resistant reasons.
4. Binding proteins of Cry1Ah, Cry1Ab and Cry1Ac in susceptible- and resistant-CBW were compared, the binding of Cry1Ab to binding proteins was weak than the other two toxins whether in susceptible- or resistant-strain. In susceptible-strain, Cry1Ab bound to novel protein and HSP, did not bind to actin and V-ATPase, but Cry1Ac and Cry1Ah bound to these four binding proteins, maybe novel protein and HSP are essential for the toxin action, actin and V-ATPase may be related to toxicity. Predecessors results showed that Cy1Ab and Cry1Ah have cross resistance to Cry1Ac-resistant CBW, the difference of different Bt binding proteins between susceptible- and resistant-CBW were lower expression of novel protein, maybe this was reason for cross resistance or other reasons need further study.
引文
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