白蜡虫泌蜡关键基因鉴定及其分子功能研究

Wax secretion of the scale insects is a special survival strategy derived from the evolution of adaptation to a sedentary life as plant parasites. The specialized characteristic of wax secretion of the white wax scale insect (Ericerus pela) is the typical representative of the scale insects. Revealing the molecular mechanism of white wax secretion will deepen our understanding of the universal generating mechanism of the special survival advantage of scale insects, and will also push the E. pela breeding to molecular breeding level. The fatty acyl-CoA reductase (FAR) and the wax synthase (WS) are the key enzymes involved in wax biosynthesis. We have identified two far candidate genes and one ws candidate gene of E. pela. RNAi of far genes resulted in the significant reduction of wax secretion, which showed they are potential key genes involved in the white wax biosynthesis. Further identification and function research will disclose the wax secretion molecular mechanism in E. pela to a great extent, and obtain the key genes responsible for wax secretion in scale insects for the first time. In this project, we plan to reveal the molecular functions of the three candidate genes in E. plea through the molecular characteristics, biochemical characteristics, and physiological functions research. Firstly, the far and ws genes location in the cuticular wax gland will be analyzed and their activity will be validated in vivo. Then FAR and WS will be biochemical characterized after heterologous expressed in insect cells, and the biochemical mechanism of FAR and WS involved in white wax biosynthesis will be disclosed. Finally, the key role of FAR and WS in wax secretion will be determined by RNAi. These studies will disclose the mechanism and effect of FAR and WS on wax secretion in E. pela.

泌蜡是介壳虫为适应固定寄生在植物上而进化产生的特殊生存策略，白蜡虫特化的泌蜡性状是介壳虫的典型代表，揭示白蜡虫泌蜡的分子机理，将从深层次了解介壳虫特殊生存优势进化产生的普遍机制，也有助于推动白蜡虫育种工作进入分子育种层面。FAR和WS是生物蜡酯合成途径的关键酶，我们前期筛选到了白蜡虫2 个far和1 个ws基因，far基因RNA干涉导致白蜡虫泌蜡量显著降低，表明它们是参与泌蜡的重要候选基因，进一步的鉴定和功能研究将在很大程度上揭示白蜡虫泌蜡的分子机理，并首次获得介壳虫泌蜡的关键基因。为此，本项目拟通过分子特性、生化特性和生理功能等研究解析3个基因在白蜡虫中的分子功能：首先分析候选基因在表皮蜡腺中的分布和体内活性，然后体外表达分析其生化特性，阐述它们参与白蜡合成的生化机理，最后通过RNA干涉分析它们对白蜡虫泌蜡的关键作用，从而阐述它们在白蜡虫泌蜡中的功能和作用机理。

白蜡虫是我国传统的资源昆虫，分泌的白蜡具有重要的医药价值，但是白蜡虫泌蜡的分子机理并不清楚，我们前期鉴定到了参与白蜡虫泌蜡的3个候选基因，但是这3个基因缺乏深入的功能鉴定和作用机理研究。本项目在前期获得的3个重要候选基因的基础上，从分子特性、生化特性和生理功能3方面对候选基因进行系统研究，分析它们的组织表达特点以及生化特性，通过RNA干涉的方法研究其生理功能。我们采用荧光定量PCR分析了3个关键酶基因在不同发育时间和不同组织的基因表达动态，表明3个基因在白蜡虫泌蜡高峰期表达量最高，在雄虫中表皮中表达量最高。制备了单克隆抗体，分析了蛋白的表达情况，表明蛋白表达主要在白蜡虫雄虫泌蜡高峰期，与白蜡虫泌蜡特点一致。分别采用bac-bac杆状病毒表达系统以及构建细胞系的方法，在sf9细胞系和家蚕细胞中对关键酶基因分别进行了蛋白表达，建立了稳定表达目的基因的细胞系。加入不同碳链长度的酰基辅酶A，发现2个关键酶FAR分别可以生成28烷醇、28烷醇和24烷醇，WS的体外表达可以生成24酸24酯等。FAR的最适底pH值是3.0，最适温度是35℃。对关键酶基因分别进行RNA干涉，结果表明基因在第9天后表达量下降，3个基因进行RNA干涉，白蜡虫泌蜡量均显著降低。该研究分析了白蜡虫泌蜡关键酶基因的体外活性，阐述了白蜡的生物合成通路。在白蜡生产中主要利用湖南四川等地的高温和高湿环境提高白蜡虫的泌蜡量，本项目研究表明，酶的最适温度是35℃，为解释白蜡生产中高温环境促进白蜡虫泌蜡提供了分子证据。本研究鉴定的关键酶基因，对于白蜡虫优良品种选育也具有重要的参考价值。