本文選題：腰帶長體繭蜂 + 亞洲玉米螟　； 參考：《中國農(nóng)業(yè)大學》2016年博士論文

【摘要】：轉基因作物在進行商業(yè)化種植之前必須要經(jīng)過嚴格的安全性評價,本研究系統(tǒng)地評價了轉crylAc基因玉米對靶標害蟲的寄生性天敵腰帶長體繭蜂的影響。迄今為止,尚未見到對轉Bt基因作物/Bt蛋白-亞洲玉米螟-腰帶長體繭蜂之間關系的研究。本文首先建立一個系統(tǒng)的評價轉基因作物或殺蟲蛋白對寄生蜂風險的方法。結合生理生化、分子生物學以及蛋白質(zhì)組學等方法研究腰帶長體繭蜂寄生和Cry1Ac蛋白對敏感和抗性亞洲玉米螟幼蟲營養(yǎng)代謝、酶活性代謝以及免疫反應的影響,以進一步揭示寄生蜂與寄主之間的相互調(diào)控機制,明確寄生對于敏感和抗性亞洲玉米螟品系之間的內(nèi)在差異,為提高寄生性天敵在生物防治中的作用和開發(fā)延緩抗性和抗性治理新途徑提供理論依據(jù)和方法指導。主要研究結果如下：1.發(fā)展了一套評價轉基因殺蟲蛋白對腰帶長體繭蜂直接影響和間接影響的試驗體系,并利用該體系評價了CrylAc蛋白對腰帶長體繭蜂的影響,結果表明采用敏感亞洲玉米螟作為寄主,飼喂Cry1Ac蛋白后寄主死亡率明顯升高,且寄生蜂的寄生率和繭塊重量等參數(shù)明顯降低,而采用抗性亞洲玉米螟作為寄主,飼喂Cry1Ac蛋白后,對寄生蜂各項生命表參數(shù)均沒有產(chǎn)生不利影響。說明CrylAc蛋白對寄生蜂的影響是由寄主質(zhì)量下降引起的,而CrylAc對腰帶長體繭蜂不會產(chǎn)生不利影響。并用Cry1Ac蛋白直接飼喂腰帶長體繭蜂成蜂,以PA作為陽性對照,結果顯示取食不同濃度Cry1Ac蛋白對腰帶長體繭蜂成蜂各項生命表參數(shù)均沒有影響,而取食了PA的腰帶長體繭蜂壽命和出蜂量顯著下降。本實驗結果進一步說明CrylAc蛋白對腰帶長體繭蜂沒有不利影響。2.采用蛋白質(zhì)組學方法研究了寄生蜂、Bt和寄主三者之間的相互關系。選用敏感和抗性亞洲玉米螟作為寄主,研究腰帶長體繭蜂寄生和CrylAc蛋白對寄主幼蟲血淋巴蛋白質(zhì)組學的影響。首先利用iTRAQ技術對寄主血淋巴蛋白進行定量分析,結果鑒定到有定量信息的蛋白質(zhì)286個,然后分別對各比較組樣品進行差異表達蛋白分析,并對差異表達蛋白進行GO功能注釋和KEGG分析。然后對iTRAQ結果中篩選到的三個重要差異表達蛋白(貯藏蛋白、酚氧化酶和溶菌酶)進行MRM驗證,并對在各處理組中差異表達都有顯著變化的貯藏蛋白進行基因克隆和qPCR驗證。結果表明寄生蜂和Cry1Ac蛋白單獨或者聯(lián)合作用都會對敏感和亞洲玉米螟體內(nèi)的蛋白產(chǎn)生表達差異。3.對腰帶長體繭蜂寄生和Cry1Ac蛋白對敏感和抗性亞洲玉米螟幼蟲血淋巴內(nèi)的營養(yǎng)調(diào)控進行探討,測定了不同處理后寄主幼蟲血淋巴內(nèi)蛋白質(zhì)、氨基酸、糖類和脂類等的變化。結果表明：敏感和抗性亞洲玉米螟寄生后血淋巴內(nèi)蛋白質(zhì)含量、脂滴濃度、海藻糖含量和總還原糖含量顯著變化,而總氨基酸含量和海藻糖酶活性沒有顯著變化；CrylAc蛋白處理敏感亞洲玉米螟后,蛋白含量、脂滴濃度和總還原糖含量顯著變化,而其他物質(zhì)含量沒有變化。而寄生或未寄生抗性亞洲玉米螟飼喂CrylAc蛋白后,各物質(zhì)含量均沒有顯著變化。這些營養(yǎng)物質(zhì)的變化可能是導致寄主質(zhì)量下降的內(nèi)在原因。4.測定了腰帶長體繭蜂寄生和CrylAc蛋白作用對敏感和抗性亞洲玉米螟血淋巴內(nèi)解毒酶、保護酶和中腸蛋白酶三大酶系活性的影響,結果表明敏感和抗性亞洲玉米螟寄生后ACHE活性降低,GST活性顯著升高,SOD酶活性先升高后降低,而其他酶類活性變化不大；Cry1Ac作用于敏感和抗性亞洲玉米螟后,ACHE顯著升高,敏感亞洲玉米螟GST酶降低,SOD酶活性先升高后降低,類胰蛋白酶活性明顯上升,而抗性亞洲玉米螟GST升高,其他酶活性無顯著變化。以上結果說明這三類不同酶活性的變化也可能與寄主質(zhì)量下降有關。5.以敏感和抗性亞洲玉米螟為寄主,初步探討了腰帶長體繭蜂寄生和Cry1Ac蛋白對寄主敏感和抗性亞洲玉米螟幼蟲血淋巴內(nèi)細胞免疫和體液免疫方面的影響,結果表明寄生和Crry1Ac蛋白處理2d后,敏感和抗性亞洲玉米螟血淋巴內(nèi)血細胞的會出現(xiàn)一系列病態(tài)變化。敏感亞洲玉米螟寄生前后飼喂Cry1Ac蛋白后,血細胞總數(shù)會呈現(xiàn)不同的變化趨勢；敏感亞洲玉米螟飼喂Cry1Ac蛋白后血細胞對Sephadex A-25微珠的包囊能力顯著下降且細胞凋亡率顯著高于其他處理；腰帶長體繭蜂寄生對敏感和抗性亞洲玉米螟血細胞內(nèi)PPO活性和黑化反應均產(chǎn)生明顯抑制作用,而寄生后溶菌酶活性升高。這些免疫反應的差異也許是引起寄主質(zhì)量下降的另一個內(nèi)在機理。
[Abstract]:The effects of transgenic crylAc maize on the parasitic natural enemies of the target pests were systematically evaluated before the transgenic crops were carried out for commercial cultivation. So far, the relationship between the /Bt protein of the Bt gene crop and the Asiatic corn borer, the Asiatic corn borer, the long body cocoon wasp has not yet been seen. In this paper, we first set up a systematic method to evaluate the risk of transgenic crops or insecticidal proteins to parasitoids. In combination with physiological, biochemical, molecular and proteomics methods, the parasitic and Cry1Ac protein of the waistband wasp was studied for the sensitive and resistant Asiatic corn borer's nutrition metabolism, enzyme activity metabolism and immune response. In order to further reveal the mutual regulation mechanism between parasitic wasps and host, the internal differences between parasitic and resistant Asian corn borer lines are clearly defined, and the theoretical basis and method guidance for improving the role of parasitic natural enemies in biological control and the development of new ways of developing resistance and resistance control are proposed. As follows: 1. an experimental system was developed to evaluate the direct and indirect effects of transgenic insecticidal proteins on the long body cocoon of the waistband. The system was used to evaluate the effect of CrylAc protein on the waistband long body cocoon bee. The results showed that the sensitive Asian corn borer was used as host, and the host mortality was significantly increased and parasitized after feeding Cry1Ac protein. The parasitic rate of the bee and the weight of the cocoon block were obviously reduced, while the resistant Asian corn borer was used as the host, and the Cry1Ac protein had no adverse effects on the life table parameters of the parasitic wasps. The effect of the CrylAc protein on parasitic wasps was caused by the drop of host mass, and the CrylAc was not harmful to the long body callus wasp. Cry1Ac protein was used to directly feed the long body cocoon wasp with PA as the positive control. The results showed that the different concentrations of Cry1Ac protein had no effect on the life table parameters of the long body cocoon wasp of the waistband, while the life span and the amount of the wasp feeding on the PA were significantly decreased. The results of the experiment further explained the CrylAc egg. .2. had no adverse effects on the waistband long body wasp. The proteomics method was used to study the relationship between parasitic wasps, Bt and the host three. The sensitivity and resistance of Asiatic corn borer were selected as host, and the effects of the parasitic and CrylAc protein on the haemolymph white matter of host larvae were studied. First, the iTRAQ technique was used. The host hemolymph protein was quantitatively analyzed, and 286 proteins with quantitative information were identified, and then the differential expression proteins were analyzed for the samples of the comparative groups, and the differential expression proteins were annotated with GO function annotation and KEGG analysis. Then, three important differential proteins (storage proteins, phenol oxidation) were selected from the iTRAQ results. The enzyme and lysozyme were verified by MRM, and the storage proteins with significant changes in the different treatments were cloned by gene cloning and qPCR validation. The results showed that both the parasitic wasp and Cry1Ac protein alone or combined with the protein production in the Asiatic corn borer and the Asiatic corn borer were different.3. for the parasitic and Cry1Ac of the waistband long body cocoon bee. Protein regulation of the hemolymph of sensitive and resistant Asian corn borer (Ostrina borer) larvae was discussed. The changes of protein, amino acids, carbohydrates and lipids in host larvae were measured after different treatments. The results showed that the protein content, lipid droplet concentration, trehalose content and total of the hemolymph of the sensitive and resistant Asian corn borer were detected. The content of total amino acids and the activity of alginase were not significantly changed, and the content of protein, the concentration of lipid droplets and the total reducing sugar content of the CrylAc protein changed significantly, but the content of other substances was not changed, while the parasitic or non parasitic Asian corn borer was fed with CrylAc protein. There was no significant change in the quality of the substance. The changes in these nutrients may be the intrinsic cause of the drop in the host quality.4. determined the effects of the parasitic and CrylAc proteins of the waistband on the hemolymph of the sensitive and resistant Asian corn borer, the protective enzyme and the three major protease activity of the midgut protease, which results in sensitivity and resistance. The activity of ACHE in the Asiatic corn borer decreased, the activity of GST increased significantly, the activity of SOD enzyme increased first and then decreased, but the activity of other enzymes changed little. After the sensitivity and resistance of Asiatic corn borer, Cry1Ac was significantly increased, the GST enzyme in the sensitive Asian corn borer was reduced, the activity of SOD enzyme increased and then decreased, and the activity of trypsin like trypsin like increased significantly. The resistance of Asiatic corn borer GST increased and the activity of other enzymes did not change significantly. The above results showed that the changes in the activity of these three kinds of enzymes may also be related to the drop of host mass of.5.. The sensitivity and resistance of the Asiatic corn borer, the parasitism of the waistband and the Cry1Ac egg white on the host sensitivity and resistance to the Asian corn borer larvae were preliminarily discussed. The effects of cellular immunity and humoral immunity in hemolymph showed that a series of pathological changes occurred in the hemolymph of the sensitive and resistant Asian corn borer (Ostrina borer) after the parasitic and Crry1Ac protein was treated with 2D. After feeding the Cry1Ac protein of the sensitive Asian corn borer before and after the parasitism, the total number of blood cells in the Asiatic corn borer would present a different trend of change; After feeding Cry1Ac protein of Ostrina borer, the cysts of Sephadex A-25 microspheres decreased significantly and the apoptosis rate was significantly higher than that of other treatments. The parasitism of the waistband parasitic wasp on the PPO activity and the blackening reaction in the blood cells of the sensitive and resistant Asiatic corn borer were obviously inhibited, and the activity of the parasitic lysozyme increased. The difference in immune response may be another intrinsic mechanism leading to a decline in host quality.

【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S476.3

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本文編號：1787152