【作者】 安静杰；

【导师】 吴孔明； 雷朝亮；

【作者基本信息】 华中农业大学 ， 农业昆虫与害虫防治， 2014， 博士

【摘要】 从细菌苏云金芽孢杆菌(Bacillus thuringiensis)中获得的杀虫蛋白成功的导入到棉株内获得的抗虫棉,可以有效的抑制靶标害虫的发生和危害,减少了对化学杀虫剂的使用和依赖,并取得了显著的经济效益和生态效益。我国自1997年引进转Bt-CrylAc抗虫棉后,成功的控制了棉铃虫Helicoverpa armigera的为害。但是随着Bt棉花的常年种植,其表达的Cry1Ac毒素对棉铃虫造成了强大的选择压力,棉铃虫对该毒素的抗性演化将严重威胁Bt棉花的使用寿命。本文将单雌系生测法和DNA分子诊断法相结合,对我国华北棉区的棉铃虫种群进行了系统的监测,评估其对转Bt基因棉的抗性发展水平。同时,本研究以胰蛋白酶基因沉默而导致抗性的品系(LF20.0和LF120.0)为研究对象,以室内饲养的敏感品系(96s和LF)为对照,将4龄幼虫饲喂不同蛋白浓度的饲料,比较各品系营养指标和中肠蛋白酶活性的差异,探索蛋白酶改变后对其适应性的影响。主要结论如下：1在2011-2013年的棉花生长季节,从山东省夏津县和河北省安次县分别收集了1,694和933个棉铃虫单雌家系,在正常饲料和1.0μg/mL的Cry1Ac饲料上进行生物测定。夏津棉铃虫种群的相对平均发育级别(RADR)分别0.59,0.58和0.62,安次种群的相对平均发育级别分别为0.60,0.53和0.62。与2002年的监测结果相比,2011-2013年夏津和安次地区棉铃虫种群的RADR分别上升了1.53-1.63和1.77-2.07倍,而抗性基因频率仍然较低。研究结果表明：与往年相比我国华北地区棉铃虫种群对Cry1Ac的抗性有一定的升高趋势,但并未达到高水平抗性。2根据已经报道的与棉铃虫抗性相关的钙粘蛋白(Cadherin)和氨肽酶N(Aminopeptidase N, APN)突变基因位点,设计特异引物,利用PCR诊断技术对田间收集的棉铃虫样品进行分子检测。2009-2013年共有2,391个样品进行了检测,但并未检测到已报道的相同突变基因。3将胰蛋白酶改变而导致抗性的棉铃虫抗性品系(LF20.0和LF120.0)和敏感品系(96s和LF)饲喂蛋白含量分别为0%、5%、10%、20%和30%的饲料,发现敏感品系取食蛋白浓度0%-10%的饲料,幼虫体重和蛹重随着蛋白含量的增加而增加,当饲料中蛋白浓度在10%-30%时,反而随饲料中蛋白浓度的增加而降低。但是抗性品系并不遵循这一规律,在任一浓度的饲料上,敏感品系的幼虫质量均显著高于抗性品系,并且在这5种饲料上抗性品系均不能化蛹。表明抗性品系对蛋白的消化吸收能力显著低于敏感品系。4将抗性品系(LF20.0和LF120.0)和敏感品系(96s和LF)用不同蛋白浓度的饲料处理,测定幼虫取食后总蛋白酶、类胰蛋白酶和类胰凝乳蛋白酶的活性。结果显示：LF120.0品系取食蛋白浓度为0%和5%的饲料2d时,总蛋白酶活性显著高于其它品系,随着取食时间的延长而降低到与敏感品系无显著差异。当取食蛋白浓度为10%和20%的饲料6d后,敏感品系的类胰蛋白酶和类胰凝乳蛋白酶的活性均显著高于抗性品系。尽管高抗品系LF120.0的类胰蛋白酶活性在取食蛋白浓度为10%的饲料2d后表现最大值,显著高于其它品系,但随着取食时间的延长显著降低,直至低于敏感品系。结果表明,抗性品系在改变蛋白酶产生抗性的同时,中肠相关消化酶活性显著降低影响了对蛋白质的水解作用。棉铃虫抗性监测的数据与以前的研究结果相比,田间种群对Cry1Ac的抗性水平有一定的升高趋势,但未达到高水平的抗性。尽管PCR诊断未检测到已经报道的抗性突变基因,并不排除田间存在其它突变基因的可能。棉铃虫中肠内蛋白酶的改变导致的抗性产生后,消化酶活性显著降低影响了对蛋白的消化吸收,进而影响了抗性品系的适应性。本研究评估了当前棉铃虫自然种群对转Bt基因棉的抗性演化规律,为抗性治理策略的制定和实施提供依据,同时增强了对抗性棉铃虫蛋白质营养代谢的了解,对延缓抗性的产生提供重要的线索。更多还原

【Abstract】 Transgenic cotton that expresses a gene derived from the bacterium Bacillus thuringiensis (Bt) has been widely adopted because of the economic advantages, reducing the use of conventional insecticides and regional suppression populations of a target pest. In China, Bt cotton was approved in1997for commercial use to control cotton bollworm, Helicoverpa armigera, and has steadily been planted by Chinese cotton growers. The widespread planting of transgenic Bt cotton imposes strong selection pressure on cotton bollworm in the field. Resistance to the Bt toxin Cry1Ac in H. armigera is considered to be a major threat for the long-term effectiveness of Bt cotton. In this study, we evaluated resistance to Bt Cry1Ac toxin by the bioassays of isofemale lines and diagnostic PCR in H. armigera populations in North China. Compared to the susceptible strain (96s and LF), loss of function of a trypsin protease in the resistant populations (LF20.0and LF120.0) was linked to high levels of Cry1Ac resistance, the4th instar larvae were restricted to diets varying in concentration of protein. Compare the differences of nutrition indicators and gut proteases activity in the various strains, to investigate the influence of protease changed on the adaptability in H. armigera.The main research results are as follows:1A total of1,694lines from Xiajin and933lines from Anci were screened for growth rate on normal artificial diet and the diet containing1.0μg of CrylA(c) toxin per ml. In2011-2013, the mean relative average development rates of H. armigera larvae in the Xiajin population was0.62,0.59,0.59,0.58and0.62, respectively; and the Anci population was0.54,0.58,0.60,0.53and0.62, respectively. Compared to previous results in2002, the RADR of H. armigera during2011-2013, with ratios of1.53-1.63and1.77-2.07in their respective Xiajin and Anci populations, however, the frequency of resistance alleles of H. armigera was remained low. The result suggested that resistance to Cry1Ac has increased in H. armigera populations in North China, but have not reached a high level of resistance.2According to the mutant allele of APN or cadherin conferring resistance to Bt toxin Cry1Ac, A DNA-based PCR method was developed to screen the mutation alleles from field populations of H. armigera collected from the main cotton planting areas of China in2009-2013, however, no mutation allele was detected from2,246moths.3We used four strains of H. armigera:susceptible strains (96s and LF) and Cry1Ac-resistant strains (LF20.0and LF120.0), the4th instar larvae were restricted to diets varying in concentration of protein (0%、5%、10%、20%and30%). As the protein content of the diet increased, the bigger mass of larval and pupal were observed when bollworm were reared on diet that protein concentration was0%-10%. When the concentration of protein in the diet was10%-30%, the mass of larval and pupal decreased with increasing concentration of protein. But the resistant strains didn’t follow this rule, The larvae mass of susceptible strains were significantly higher than that of resistant strains fed on any protein concentration of diets, and the resistant strains couldn’t pupate in the5kinds of diet. The results indicated that the ability of protein digestion and absorption of resistant strains was significantly lower than the susceptible strains.4The activities of midgut total protease, trypsin-like enzyme and chymotrypsin-like enzyme were estimated after larvae of resistant strains (LF20.0and LF120.0) and susceptible strains (96s and LF) fed on artificial diets containing different concentrations of protein. LF120.0strain feed on lower protein diets (0%and5%) after2days showed significantly higher total protease activity than other strains. However, there was no significant difference between the resistant strains and susceptible strains with the extension of feeding time. Larvae reared on high protein diets (10%and20%) after6d, Trypsin-like activity and chymotrypsin-like activity of the resistant strains were significantly higher than the susceptible strains. The maximum in trypsin-like activity of LF120.0strain feed on artificial diets contains10%concentrations of protein after2d, is significantly higher than other strains, however, as the feeding time was significantly reduced, until it is lower than the sensitive strains. The results showed that the resistant strains at the same time of altered protease lead to resistance to Cry1Ac, reduces the activities of midgut digesting enzyme, and hydrolysis of protein was affected in Cry1Ac-resistant strains.Compared with previous studies, resistance to Cry1Ac has increased in H. armigera populations in northern China, but not reached a high level of resistance. Although PCR diagnosis has not detected resistance mutations which have been reported, does’t excluded the possibility that there were other resistance alleles occured in field population. A laboratory-reared resistant strain that altered protease resulted in resistance to Cry1Ac, the digestion and absorption of protein was affected by digestive enzyme activity decreased significantly, and the adaptability of resistant strains was affected. The present study evaluated the level of Bt toxin susceptibility of cotton bollworm larvae from field populations, provide the basis for resistance management strategy formulation and implementation. Meanwhile, enhances the understanding of protein nutrition metabolism of Cry1Ac-resistant H. armigera, and providing leads that could be delay resistance to Bt cotton.更多还原