Cry1Ac蛋白对腰带长体茧蜂的安全性评价及两者对亚洲玉米螟生理机制的研究

发布时间：2018-04-22 12:08

本文选题：腰带长体茧蜂 + 亚洲玉米螟　；参考：《中国农业大学》2016年博士论文

【摘要】：转基因作物在进行商业化种植之前必须要经过严格的安全性评价,本研究系统地评价了转crylAc基因玉米对靶标害虫的寄生性天敌腰带长体茧蜂的影响。迄今为止,尚未见到对转Bt基因作物/Bt蛋白-亚洲玉米螟-腰带长体茧蜂之间关系的研究。本文首先建立一个系统的评价转基因作物或杀虫蛋白对寄生蜂风险的方法。结合生理生化、分子生物学以及蛋白质组学等方法研究腰带长体茧蜂寄生和Cry1Ac蛋白对敏感和抗性亚洲玉米螟幼虫营养代谢、酶活性代谢以及免疫反应的影响,以进一步揭示寄生蜂与寄主之间的相互调控机制,明确寄生对于敏感和抗性亚洲玉米螟品系之间的内在差异,为提高寄生性天敌在生物防治中的作用和开发延缓抗性和抗性治理新途径提供理论依据和方法指导。主要研究结果如下：1.发展了一套评价转基因杀虫蛋白对腰带长体茧蜂直接影响和间接影响的试验体系,并利用该体系评价了CrylAc蛋白对腰带长体茧蜂的影响,结果表明采用敏感亚洲玉米螟作为寄主,饲喂Cry1Ac蛋白后寄主死亡率明显升高,且寄生蜂的寄生率和茧块重量等参数明显降低,而采用抗性亚洲玉米螟作为寄主,饲喂Cry1Ac蛋白后,对寄生蜂各项生命表参数均没有产生不利影响。说明CrylAc蛋白对寄生蜂的影响是由寄主质量下降引起的,而CrylAc对腰带长体茧蜂不会产生不利影响。并用Cry1Ac蛋白直接饲喂腰带长体茧蜂成蜂,以PA作为阳性对照,结果显示取食不同浓度Cry1Ac蛋白对腰带长体茧蜂成蜂各项生命表参数均没有影响,而取食了PA的腰带长体茧蜂寿命和出蜂量显著下降。本实验结果进一步说明CrylAc蛋白对腰带长体茧蜂没有不利影响。2.采用蛋白质组学方法研究了寄生蜂、Bt和寄主三者之间的相互关系。选用敏感和抗性亚洲玉米螟作为寄主,研究腰带长体茧蜂寄生和CrylAc蛋白对寄主幼虫血淋巴蛋白质组学的影响。首先利用iTRAQ技术对寄主血淋巴蛋白进行定量分析,结果鉴定到有定量信息的蛋白质286个,然后分别对各比较组样品进行差异表达蛋白分析,并对差异表达蛋白进行GO功能注释和KEGG分析。然后对iTRAQ结果中筛选到的三个重要差异表达蛋白(贮藏蛋白、酚氧化酶和溶菌酶)进行MRM验证,并对在各处理组中差异表达都有显著变化的贮藏蛋白进行基因克隆和qPCR验证。结果表明寄生蜂和Cry1Ac蛋白单独或者联合作用都会对敏感和亚洲玉米螟体内的蛋白产生表达差异。3.对腰带长体茧蜂寄生和Cry1Ac蛋白对敏感和抗性亚洲玉米螟幼虫血淋巴内的营养调控进行探讨,测定了不同处理后寄主幼虫血淋巴内蛋白质、氨基酸、糖类和脂类等的变化。结果表明：敏感和抗性亚洲玉米螟寄生后血淋巴内蛋白质含量、脂滴浓度、海藻糖含量和总还原糖含量显著变化,而总氨基酸含量和海藻糖酶活性没有显著变化；CrylAc蛋白处理敏感亚洲玉米螟后,蛋白含量、脂滴浓度和总还原糖含量显著变化,而其他物质含量没有变化。而寄生或未寄生抗性亚洲玉米螟饲喂CrylAc蛋白后,各物质含量均没有显著变化。这些营养物质的变化可能是导致寄主质量下降的内在原因。4.测定了腰带长体茧蜂寄生和CrylAc蛋白作用对敏感和抗性亚洲玉米螟血淋巴内解毒酶、保护酶和中肠蛋白酶三大酶系活性的影响,结果表明敏感和抗性亚洲玉米螟寄生后ACHE活性降低,GST活性显著升高,SOD酶活性先升高后降低,而其他酶类活性变化不大；Cry1Ac作用于敏感和抗性亚洲玉米螟后,ACHE显著升高,敏感亚洲玉米螟GST酶降低,SOD酶活性先升高后降低,类胰蛋白酶活性明显上升,而抗性亚洲玉米螟GST升高,其他酶活性无显著变化。以上结果说明这三类不同酶活性的变化也可能与寄主质量下降有关。5.以敏感和抗性亚洲玉米螟为寄主,初步探讨了腰带长体茧蜂寄生和Cry1Ac蛋白对寄主敏感和抗性亚洲玉米螟幼虫血淋巴内细胞免疫和体液免疫方面的影响,结果表明寄生和Crry1Ac蛋白处理2d后,敏感和抗性亚洲玉米螟血淋巴内血细胞的会出现一系列病态变化。敏感亚洲玉米螟寄生前后饲喂Cry1Ac蛋白后,血细胞总数会呈现不同的变化趋势；敏感亚洲玉米螟饲喂Cry1Ac蛋白后血细胞对Sephadex A-25微珠的包囊能力显著下降且细胞凋亡率显著高于其他处理；腰带长体茧蜂寄生对敏感和抗性亚洲玉米螟血细胞内PPO活性和黑化反应均产生明显抑制作用,而寄生后溶菌酶活性升高。这些免疫反应的差异也许是引起寄主质量下降的另一个内在机理。
[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.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S476.3

【参考文献】