飞蝗几丁质脱乙酰基酶基因1和2的功能分化机理

Chitin deacetylase (CDA) is an important enzyme during chitin metabolism that is essential for insect growth and development. In our previous study, the protein localizations of LmCDA1 and LmCDA2 from Locusta migratoria were found obviously different, and it is speculated that these two genes may have functional differentiation. In this project we will focus on the functional differentiation of LmCDA1 and LmCDA2 and perform the research as follows: 1) precise and fine localization of LmCDA1 and LmCDA2 using immunohistochemistry and colloidal gold technology to determinate the action sites of these two proteins; 2) comparison of the effects of LmCDA1 and LmCDA2 on the chitin content and degree of deacetylation using histochemical stain and quantitative analysis; 3) analysis of their effects on chitin ultrastructure, chitin organization, crystalline structure and cuticular mechanical property using scanning electron microscopy, transmission electron microscopy, X-ray diffraction and nanoindentation technique, respectively, so as to have better understanding of the functional differentiation of these two genes; 4) using site-directed mutagenesis to mutate some different amino acids at active sites of LmCDA1 and LmCDA2, and thus to compare their chitin deacetylase activities. The results from this project will help to reveal the mechanism of functional differentiation between LmCDA1 and LmCDA2, and provide scientific bases for designing new type of pesticide based on chitin deacetylase, and controlling regulate the growth and development of migratory locust by using RNAi technology and eventually, to achieve their effective control.

几丁质脱乙酰基酶（CDA）是几丁质代谢过程中的关键酶，对昆虫生长发育至关重要。课题组前期研究发现飞蝗LmCDA1和LmCDA2的蛋白定位存在明显差异，推测两者功能出现分化。本项目拟围绕LmCDA1和LmCDA2的功能分化开展如下研究：1）采用免疫组化和胶体金标记技术对LmCDA1和LmCDA2进行精细定位，确定其作用位点的差异；2）采用组织化学染色和定量分析比较两个基因对几丁质含量和几丁质脱乙酰度的影响；3）运用扫描和透射电镜、X-射线衍射和纳米压痕技术分析它们对几丁质超微结构、几丁质排布、晶体结构和表皮机械特性的影响，深入阐明其功能分化；4）采用点突变技术对LmCDA1和LmCDA2活性区域的差异氨基酸突变，比较CDA活性的变化。研究结果将揭示飞蝗CDA1和CDA2的功能分化机理，为基于CDA设计新型杀虫剂，应用RNAi技术调控飞蝗的生长发育，实现飞蝗的有效控制提供科学依据。

昆虫表皮覆盖整个虫体，对保护虫体免受机械损伤、物理或化学伤害、病原菌入侵、维持器官的特定位置和保持正常活动是必不可少的。几丁质是昆虫表皮的重要组分，昆虫蜕皮和发育依赖于几丁质代谢和组装的精细调控。几丁质脱乙酰基酶 CDAs 是一种金属蛋白，属于糖酯酶 4 家族的一员，能催化几丁质链上的乙酰氨基水解成壳聚糖。几丁质脱乙酰基酶（CDA）是几丁质代谢过程中的关键酶，对昆虫生长发育至关重要。LmCDA1和LmCDA2的沉默会导致飞蝗蜕皮困难死亡，前期研究发现它们的蛋白定位存在明显差异，推测两者功能出现分化。本项目围绕LmCDA1和LmCDA2的功能分化开展研究，研究结果表明：1）利用LmCDA1和LmCDA2的抗体进行免疫组化，发现LmCDA1定位在细胞质和表皮下半部分，而LmCDA2定位在表皮顶端，说明其作用位点存在差异；2）注射dsLmCDA1后，飞蝗表皮的几丁质含量和脱乙酰度均显著降低，而注射dsLmCDA2后几丁质含量和壳聚糖均未发生明显变化；3）运用透射电镜观察发现，注射dsLmCDA1后表皮片层结构消失不明显，但表皮显著变薄。注射dsLmCDA2后，几丁质片层结构消失，但表皮显著变厚。4）LmCDA1和 LmCDA2基因在飞蝗前后肠表皮中具有与体壁相似的定位。5）LmCDA1和LmCDA2的沉默除了影响表皮结构，还增加了飞蝗表皮渗透性、减少了表皮脂质的沉积，表皮结构和成分的变化促使飞蝗对真菌和杀虫剂的敏感性增加。通过该研究系统阐明了LmCDA1和LmCDA2在细胞定位、几丁质含量、脱乙酰度、几丁质片层结构、表皮脂质沉积等方面均存在差异性，揭示了其功能分化机理。研究结果为基于CDA设计新型杀虫剂，应用RNAi技术调控飞蝗的生长发育，实现飞蝗的有效控制提供科学依据。