AChE参与褐飞虱若虫中肠细胞凋亡进程的研究

The brown planthopper (BPH) (Nilaparvata lugens Hemiptera: Delphacidae) is one of the most destructive pests of rice (Oryza sativa L.). The BPH midgut is the most important interface during the interaction of BPH with rice. Midgut apoptosis occurs in BPH nymphs during normal development. Host rice resistance can induce midgut apoptosis. One of the non-canonical functions of acetylcholinesterase (AChE) is involved in apoptosis. In this study, in situ analysis of acetylcholinesterase catalytic activity will be carried out firstly by biochemical staining. AChE will be located in apoptotic cells by immunohistochemistry. Secondly, AChE expression level will be investigated and the number of the apoptotic cells will be determined in the midgut of BPH nymphs ingested resistant rice or not, or fed with amiRNA-AChE, amiRNA-IAP, respectively, or not. Thirdly, molecular types of AChE involved in apoptotic progress and the distribution characteristics of AChE in subcellular level will be studied. Finally, yeast two-hybrid assay will be employed to screen putative proteins interacted with AChE during apoptosis. Relevance of the proteins to apoptosis will be studied, and then construction of the signal pathway will be attempted. This study will help in intensifying the knowledge of novel functions of AChE involved in apoptotic progress of BPH midgut, and in the process of interaction of BPH with rice. In addition, the data will supply theoretical reference for breeding new resistant rice varieties and provide clues for exploration of novel ways to govern this pest.

褐飞虱是水稻的重要害虫之一。褐飞虱若虫中肠组织是其与水稻互作的重要界面，中肠组织存在发育过程的细胞凋亡现象，且抗性水稻能诱导凋亡加剧。乙酰胆碱酯酶(AChE)的非经典功能之一是参与细胞凋亡进程。本课题首先通过原位催化研究AChE在I型褐飞虱4龄若虫中肠凋亡细胞中的催化活性；并通过免疫组织化学法在中肠细胞中定位AChE；其次研究取食抗性水稻前后，饲喂amiRNA-AChE和amiRNA-IAP前后，中肠AChE表达变化，并检测凋亡细胞数量变化；进一步研究参与凋亡进程的AChE分子类型及其亚细胞分布规律；最后利用酵母双杂交寻找与AChE互作的蛋白，通过RNAi的方法验证该蛋白与中肠细胞凋亡的关联性，尝试构建AChE参与的凋亡信号通路。研究结果将拓展人们对AChE参与褐飞虱中肠细胞凋亡进程，及其参与褐飞虱与水稻互作的新功能的认识，为培育抗性水稻新品种提供参考，为寻找新的治理方案提供线索。

褐飞虱是水稻的重要害虫之一。AChE是害虫体内农药作用的重要靶标，也是调控细胞凋亡的重要因子。为了解AChE在调控褐飞虱中肠细胞凋亡，及其在褐飞虱与水稻互作中的作用，开展了以下研究：对生物型I和II褐飞虱的AChE进行测序，分析表明两型AChE中均出现了碱基的多态性替换，且因起始密码领先导致I型AChE序列更长。构建了凋亡抑制基因IAP-2的原核表达载体Pet23a-IAP2，并在大肠杆菌BL21中实现了异源表达。制备了多克隆抗体，进行了抗体的特异性分析并确定了效价。克隆了细胞色素P450基因CYP6CW1和家蝇NADPH细胞色素P450还原酶(NCPR)基因，通过杆状病毒表达系统实现了在Sf9细胞中的共表达，获得了CYP6CW1重组酶。证明了CYP6CW1能代谢苯巴比妥，为鉴定该酶的天然底物奠定了基础。从4龄Ⅰ型若虫中肠组织分离了原代细胞，用不同抗性的水稻汁液处理第一代继代细胞，检测了凋亡的细胞，对AChE进行亚细胞定位和检测其表达水平的变化。结果表明，感虫水稻TN1、抗性水稻B5和TKM-6汁液处理细胞的凋亡率分别为8％、65%和85％。凋亡的细胞中都存在AChE的积累，且主要分布于细胞质；AChE在凋亡后期集中在细胞核附近。与对照细胞相比，TKM-6，B5和TN1处理的细胞中AChE的表达量分别升高了29.9, 18.4和8倍。用H2O2和花椒毒素分别处理细胞24小时后，检测凋亡的细胞。结果表明：花椒毒素处理的细胞有少量的凋亡信号，而H2O2处理的细胞出现较多的凋亡信号。花椒毒素处理组细胞中AChE的表达量增加了1.44倍，而H2O2处理的细胞中AChE上调了3.36倍。取食AChE-miRNA的若虫体内AChE表达水平下调了1.68倍，中肠细胞凋亡率下降为1.95%。凋亡过程中染色质浓缩并边沿化，凋亡小体开始形成。这些研究结果不仅证实了水稻品种抗性水平与其诱导的细胞凋亡率呈正相关，而且揭示了氧化胁迫同样诱导细胞凋亡，且凋亡的细胞质中都有AChE的积累。这些发现为揭示AChE在褐飞虱与水稻的互作中的功能、促进抗性水稻新品种的选育及开发新的褐飞虱防治措施提供了一定的参考信息。