【作者】 杨帆；

【导师】 吴益东；

【作者基本信息】 南京农业大学 ， 农业昆虫与害虫防治， 2013， 硕士

【摘要】 棉铃虫是一种多食性农业害虫,在世界范围内广泛发生。转Bt基因作物自1996年开始在全球范围内推广种植,有效控制了靶标害虫的为害,同时也显著减少了化学杀虫剂的使用。我国自1997年开始推广种植表达Cry1Ac毒素蛋白的Bt棉花,到2012年种植面积已达400万公顷。Bt棉花的广泛种植大大降低了棉花及其他寄主作物上棉铃虫的种群密度。鉴于国外已有田间害虫对Bt作物产生抗性的报道及我国北方棉区部分棉铃虫种群对Cry1AC敏感性下降的事实,棉铃虫对Bt棉花的抗性演化将是Bt棉花持续应用的最大威胁。腺苷三磷酸结合盒式转运蛋白(ATP结合盒式蛋白,ABC转运蛋白)是一类膜整合蛋白,其利用水解ATP的能量对溶质中各种生物分子进行跨膜转运,对维持细胞正常的生理功能起重要作用。已有研究表明ABC转运蛋白超家族成员中的ABCC2基因变异能够引起昆虫对Bt产生抗性。本文克隆了棉铃虫HaABcC2基因的全长cDNA序列,研究了该基因在棉铃虫体内的时空表达,并检测了HaABCC2基因在棉铃虫不同品系间的表达。本文结果表明HaABCC2与所检测的4个棉铃虫品系的Cry1Ac抗性无关。1.克隆棉铃虫HaABCC2基因转运蛋白ABCC2负责在昆虫的肠腔及消化道中富集运送重金属离子、次生代谢物到体外,是昆虫体内新发现的潜在的毒素受体。本研究利用Rr-PCR和RACE技术,克隆了棉铃虫ABCC2transporter的全长cDNA序列。HaABCC2基因的全长cDNA序列大小为4113bp,开放阅读框为4017bp,编码1339个氨基酸,并进行同源性分析得出与棉铃虫HaABCC2基因序列与烟芽夜蛾的相似度最高(93%),与其他昆虫类物种的相似度的取值范围是63％-68％.ABCC2transporter的开放阅读框存在单核苷酸多态性位点。2.棉铃虫HaABCC2基因的时空表达采用实时荧光定量PCR方法,将本实验室饲养的敏感品系在时间上(卵、1-2龄幼虫、3~4龄幼虫、5龄幼虫、雄性蛹、雌性蛹、雄性成虫、雌性成虫)、空间上(5龄幼虫的整头虫体、5龄幼虫的前肠、5龄幼虫的中肠、5龄幼虫的后肠、5龄幼虫的脂肪体、五龄幼虫的马氏管、5龄幼虫的外皮)分别做相对定量PCR,比较HaABCC2基因在时间上、空间上的表达情况。结果表明：1、时间上,从卵到5龄幼虫,目标基因表达量逐渐提高并且各组间有明显差异,到雄性蛹等其余四组的目标基因表达量降低到最低且无明显差异。以雌性成虫HaABCC2基因的相对表达量为1,则HaABCC2基因在各个生长发育阶段的相对表达量分别为卵期(5.50)、1-2龄幼虫(9.16)、3-4龄(13.85)、5龄(26.72)、雌性蛹(2.83)、雄性蛹(1.78)、雄性成虫(1.38)；2、空间上,前肠中的HaABCC2基因的相对表达量水平最高,后肠的、中肠的、马氏管的、脂肪体的HaABCC2基因的相对表达量水平依次降低,外皮的HaABCC2基因的相对表达量最低。以相对表达量最低的外皮的HaABCC2基因的相对表达量为1,则各组织部位的HaABCC2基因的相对表达量分别为前肠(977.04)、后肠(883.32)、中肠(528.44)、马氏管(59.44)、脂肪体(2.83)；此外在整头虫体内的HaABCC2基因的相对表达量为164.72。3.棉铃虫HaABCC2基因在敏感品系与抗性品系间表达量的比较采用实时荧光定量PCR方法,比较了棉铃虫HaABCC2基因在本实验内抗性品系棉铃虫(4个品系)与敏感品系(1个品系)的表达情况。结果表明,HaABCC2基因在棉铃虫敏感品系及四个抗性品系的虫体内中均有表达,并互相之间存在一定的差异。HaABCC2基因在CAD品系中表达量较高,与敏感品系SCD的表达量没有明显差异。目的基因在其余三个抗性品系中的表达量与SCD的相比,均有一定程度的下调,其中：HaABCC2基因在CYTO品系中表达量约为SCD的0.67倍,HaABCC2基因在AY423品系中表达量约为SCD的0.68倍,HaABCC2基因在廊坊品系中表达量约为SCD的0.67倍；并且HaABCC2基因在CYTO品系、AY423品系、廊坊品系中的表达量之间没有明显差异。更多还原

【Abstract】 Helicoverpa armigera (Hubner) is one of the most serious insect pests of cotton in China and other countries in the world. In China, transgenic cotton expressing Bacillus thuringiensis (Bt) toxins was commercialized in1997, and in2012the Bt cotton acreage in China is about four million hectares. The Bt cotton has been widely adopted to control some key lepidopteran pests, especially the cotton bollworm Helicoverpa armigera. Bt cotton has indeed helped to control the damage of cotton bollworm effectively, reduced the need for insecticide sprays, and also enhanced biocontrol services. But evolution of resistance to Bt cotton among target pests is a major threat to the continued success of Bt cotton. There is a major potential risk of the development of resistance to the Bt toxin under the strong selection pressure in the field.The transporter ABCC2has been reported to related to resistance to Cry1Ac in several insects. In this study, the full-length cDNA of the ABCC2gene was cloned from H. armigera by using RT-PCR and RACE techniques. The relative expression of ABCC2gene in the susceptible and Cry1Ac-resistant strains was compared, and the spatio-temporal expression of ABCC2gene was also determined. The results of the study showed that the transporter ABCC2plays no role in the formation of Cry1Ac resistance in the four strains tested.1. Cloning and sequence analysis of HaABCC2gene from H. armigeraThe full-length cDNA of the transporter ABCC2gene (HaABCC2) was cloned from H. armigera. Sequence analysis indicated that the full-length of this target gene was4185bp. HaABCC2has the highest cDNA sequence similarity (93%) with the ABCC2of Heliothis virescens. Comparing the full-length cDNA of HaABCC2between the susceptible and resistant strains of H. armigera, we found the amino acid sequences encoded were identical between strains.2. Temporal and spatial expression of HaABCC2geneThe levels of HaABCC2gene expression in different developmental stages and different tissues of H. armigera were measured by qPCR. From egg to larval stage, the levels of HaABCC2gene expression were on the upward trend. Then the expression level dropped dramatically at pupal stage, and continues to decline to the bottom at adult stage eventually. The levels of HaABCC2gene expression in each stage were respectively5.50(eggs),9.16(1st-2nd),13.85(3rd-4th),26.72(5th),2.83/1.78(female pupae/male pupae) and1.38(male adult) times than that in female adult stage. The levels of HaABCC2gene expression were highest in the foregut; lower in the hindgut, midgut, Malpighian tube, fat body and lowest in the cuticle. The levels of HaABCC2gene expression in all above mentioned tissues and the whole body were respectively977.04,883.32,528.44,59.44,2.83and164.72times than that in the cuticle.3. Expression of HaABCC2gene in susceptible and Bt-resistant strains of H. armigeraThe levels of HaABCC2gene expression in susceptible and Cry1Ac-resistant strains of H. armigera are detected by qPCR. The resistant strains of H. armigera were four representative strains that were resistant to Cry1Ac toxin, including AY423, CYTO, CAD and Lang Fang. The levels of HaABCC2gene expression in the above mentioned4resistant strains were respectively0.68,0.67,1.01and0.67times compared with that of the susceptible strain (SCD).更多还原