本文關(guān)鍵詞：棉鈴蟲(chóng)受體蛋白在Cry1Ac、Cry2Ab交互抗性中的作用　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： Cry1Ac Cry2Ab 棉鈴蟲(chóng) 受體蛋白 交互抗性

【摘要】：自1996年開(kāi)始,轉(zhuǎn)Bt基因棉花的投入使用,標(biāo)志著一種新的依賴植物自身產(chǎn)生殺蟲(chóng)蛋白的防治策略被應(yīng)用到保護(hù)作物、防治害蟲(chóng)上來(lái)。Bt作物的經(jīng)濟(jì)效益和環(huán)境效益使其種植面積在過(guò)去的20年迅速增加,與此同時(shí)害蟲(chóng)的抗性問(wèn)題也日益嚴(yán)重。為了延緩害蟲(chóng)對(duì)Bt的抗性,目前很多國(guó)家都開(kāi)始種植轉(zhuǎn)雙個(gè)或多個(gè)Bt基因的棉花(pyramids策略)來(lái)取代第一代轉(zhuǎn)單價(jià)的Bt棉花。理想的“pyramids”策略認(rèn)為害蟲(chóng)對(duì)其中一個(gè)毒素產(chǎn)生了抗性會(huì)被另外的毒素殺死。目前,這種策略指導(dǎo)下的轉(zhuǎn)Cry1Ac和Cry2Ab的棉花(Bollgard II)已經(jīng)在澳大利亞,印度和美國(guó)廣泛種植,但是目前中國(guó)還只是種植一代轉(zhuǎn)Cry1Ac的棉花。中國(guó)一代轉(zhuǎn)Bt棉花的大面積種植,田間害蟲(chóng)的抗性水平也在顯著的增加。盡管二代雙價(jià)棉花比一代轉(zhuǎn)Bt棉花要耐受一些,然Cry1Ac和Cry2Ab的交互抗性也是制約該雙價(jià)棉花大面積應(yīng)用的一個(gè)重要因素。我們的前期研究結(jié)果已經(jīng)表明棉鈴蟲(chóng)(Helicoverpa armigera)對(duì)Cry1Ac和Cry2Ab存在一定程度的交互抗性。同時(shí),Cry1A類毒素的作用機(jī)理和抗性機(jī)制復(fù)雜多變,Cry2Ab的作用機(jī)制和抗性機(jī)制鮮為人知,這些問(wèn)題的日益激化和突出,迫切的需要我們研究Cry1Ac和Cry2Ab交互抗性的機(jī)制,這將為二代轉(zhuǎn)基因棉花的引入提供理論依據(jù),為明確毒素的機(jī)理和昆蟲(chóng)的抗性機(jī)制提供思路。本文首先從基因組學(xué)和蛋白質(zhì)組學(xué)的角度分析抗感棉鈴蟲(chóng)(H.armigera)中的抗性差異基因。其次,為了快速有效的研究這些抗性差異基因是否參與Cry1Ac和Cry2Ab的作用機(jī)制中,本研究利用美洲棉鈴蟲(chóng)(Helicoverpa zea)中腸細(xì)胞系,脂肪體細(xì)胞系和Sf9細(xì)胞系,建立細(xì)胞毒理測(cè)定方法。通過(guò)Ligand blot和Western blot分析毒素與受體的結(jié)合、細(xì)胞內(nèi)真核表達(dá)目的基因、dsRNA細(xì)胞內(nèi)干擾和siRNA活體干擾目的基因的實(shí)驗(yàn)方法分析比較了抗性差異基因在Cry1Ac和Cry2Ab作用機(jī)制中的作用,并對(duì)受體蛋白在Cry1Ac的作用機(jī)制中的互作進(jìn)行了分析,其主要結(jié)果如下:1:通過(guò)基因水平分析LF敏感和LF抗性品系(LF5、LF10、LF20、LF30、LF60和LF120)之間的基因差異,以及蛋白水平分析LF、LF5、LF10、96S、96+2Ab、96-2Ab、96+1Ac和96-1Ac中可能調(diào)控棉鈴蟲(chóng)對(duì)Cry1Ac和Cry2Ab的抗性差異蛋白,發(fā)現(xiàn)cadherin、APN1、ALP2、ABCC2、V-ATPase a、V-ATPase c和ATP synthase都有可能參與棉鈴蟲(chóng)對(duì)Cry1Ac和Cry2Ab的抗性。2:建立細(xì)胞毒理實(shí)驗(yàn)的方法體系:細(xì)胞培養(yǎng)基是毒理實(shí)驗(yàn)的最佳緩沖液;4.5 h和5.5h分別是觀察Cry1Ac和Cry2Ab毒理的最佳時(shí)間。并發(fā)現(xiàn)棉鈴蟲(chóng)Cry1Ac和Cry2Ab的受體存在較大差異。3:在細(xì)胞中表達(dá)受體或干擾受體,以及幼蟲(chóng)活體干擾實(shí)驗(yàn)表明:美洲棉鈴蟲(chóng)的Cadherin、APN1、ALP2、ABCC2和V-ATPa是Cry1Ac的功能受體、而Cadherin、APN1、ALP2和V-ATPa不是Cry2Ab的功能受體。4:對(duì)Cry1Ac和Cry2Ab的交互抗性機(jī)制的系統(tǒng)研究,發(fā)現(xiàn)ABCC2可能是導(dǎo)致Cry1Ac和Cry2Ab產(chǎn)生交互抗性的主要原因。5:對(duì)Cry1Ac的主要受體Cadherin、APN1和ALP2之間的互作關(guān)系對(duì)Cry1Ac毒力的影響進(jìn)行了分析,沒(méi)有發(fā)現(xiàn)顯著的依賴關(guān)系和作用次序,參與Cry1Ac作用模型的反應(yīng)機(jī)制至少有4個(gè)。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S433.4
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本文編號(hào)：1339548