目录
摘要III
关键词III
AbstractIV
引言
引言1
1 文献综述2
1.1 棉铃虫的危害 2
1.2 昆虫细胞色素P450简介 2
1.3 昆虫细胞色素P450的进化 3
1.4 昆虫细胞色素P450介导的化学防御4
1.4.1 昆虫细胞色素P450介导的植物次生性化合物解毒代谢3
1.4.2 昆虫细胞色素P450介导的杀虫剂解毒代谢3
1.5 细胞色素P450基因功能主要研究方法5
1.5.1 体外功能研究方法5
1.5.2 体内功能研究方法5
1.6 本研究的目的与意义6
2 材料与方法8
2.1 供试昆虫8
2.2 主要试剂和仪器8
2.3 基因克隆、系统发育分析及载体构建8
2.3.1 引物设计9
2.3.2 基因克隆9
2.3.3 系统发育分析9
2.3.4 载体构建9
2.3.5 Bacmid的制备和提取10
2.4 细胞色素P450的体外表达11
2.4.1 昆虫细胞培养11
2.4.2 Bacmid的转染与杆状病毒的制备11
2.4.3 细胞色素P450在High Five细胞中的表达11
2.4.4 细胞微粒体的纯化12
2.4.5 还原型CO差光谱检测12
2.4.6 模式活性底物的测定12
3 结果与分析14
3.1 棉铃虫CYP6AB亚家族P450基因物理图谱及系统发育分析14
3.2 棉铃虫CYP6AB亚家族P450基因克隆14
3.3 表达载体及Bacmid构建16
3.4 细胞转染及杆状病毒制备 *51今日免费论文网|www.51jrft.com +Q: ^351916072^ 
16
3.5 棉铃虫CYP6AB亚家族P450基因在Sf9细胞中的体外表达16
3.6 模式底物活性测定 17
4 讨论18
4.1 鳞翅目昆虫CYP6AB亚家族P450分化时间较早18
4.2 成功克隆4个棉铃虫CYP6AB亚家族P450并进行功能表达18
4.3 棉铃虫CYP6AB10具有MOBFCOD活性18
参考文献19
致谢21
棉铃虫CYP6AB亚家族P450基因的克隆及功能表达
摘要
棉铃虫是有一种世界性分布的多食性害虫，因其寄主植物多、繁殖迁飞能力强，它已成为一种全球性的重要害虫。由于过量地使用杀虫剂，棉铃虫对大多数杀虫剂都产生了高倍的抗药性，造成了巨大的经济损失。之前的研究发现，世界多地棉铃虫种群的杀虫剂抗性是由细胞色素P450介导的解毒代谢能力增强导致的，且其体内的CYP6、CYP9以及CYP337等家族的二十多个P450目前已被证实参与对杀虫剂以及植物次生性化合物的解毒代谢。然而棉铃虫基因组内有114个P450基因，多数成员的表达量曾被报道可以被外源化合物诱导上调，但其具体功能仍然未知。本研究以棉铃虫体内发生扩张且可以被杀虫剂和植物毒素诱导的CYP6AB亚家族作为研究对象，克隆了该亚家族全部4个成员并成功进行了体外功能表达，测定了其对多种模式底物的代谢活性。同时通过对多个鳞翅目昆虫体内CYP6AB P450基因的系统发育分析，揭示了该亚家族的进化路径。本研究为棉铃虫CYP6AB亚家族P450的功能以及进化研究奠定重要基础，并将为棉铃虫杀虫剂抗性演化、化学防御以及寄主选择提供更多的直接证据。
CLONING AND FUNCTIONAL CHARACTERIZATION OF P450 GENES FROM THE CYP6AB SUBFAMILY OF HELICOVERPA ARMIGERA
ABSTRACT
The cotton bollworm, Helicoverpa armigera (Hübner), is a polyphagous pest distributed all over the world. Because of its large number of host plants, high fecundity and migration capacities, it has become an important global pest. Due to the overuse of insecticides, the cotton bollworm developed high resistance to most kinds of insecticide, causing huge economic losses. Previous studies have found that the insecticide resistance of the cotton bollworm population in many places of the world is caused by P450mediated involvement of detoxification, and more than two dozen P450s in the CYP6, CYP9 and CYP337 families in cotton bollworm are found to be involved in the detoxification of pesticides and plant secondary compounds. However, there are 114 P450 genes in the cotton bollworm genome, and the expression levels of many members have been reported that can be upregulated by xenobiotics, but their specific functions are still unknown. In this research, we focused on the amplified CYP6AB subfamily in the cotton bollworm which could be induced by insecticides and phytotoxins. All 4 members were cloned and successfully expressed in vitro. The activities to various model substrates were tested. Phylogenetic analysis of the CYP6AB P450 genes from multiple Lepidoptera was conducted and revealed the evolutionary path of this subfamily. This study lays an important foundation for the function and evolution of the cotton bollworm CYP6AB subfamily, and will provide more direct evidences for research of insecticide resistance mechanism, chemical defense, and host shift of the cotton bollworm.

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