药材甲几丁质脱乙酰酶家族基因的鉴定及其生理功能分析

Chitin is the main component of the integument and peritrophic matrix of the midgut in insects. The growth and development of insect reliances on rigorously controlling the synthesis and degradation of chitin. Chitin deacetylases (CDAs) are essential enzymes involved in the processes of chitin degradation. It has been documented that CDAs are responsible for numerous important physiological processes in many insects, such as regulating the behavior, molting, metamorphosis and reproduction. Due to chitin is not present in vertebrates, the insect chitin deacetylase has been paid more attention because of its potential as specific target for developing novel insecticides in pest control. Therefore, this study will be foucsing on Stegobium paniceum, a major insect pest in stored Chinese medicine material, cloning several full-length cDNA sequences of CDA family genes based on the obtained transcriptome database; detecting the expression patterns of CDA family genes by qPCR in different developmental stages and tissues, as well as in response to different stress; determining subcellular localization of those genes by in situ hybridization; elucidating the specific function of each gene during insect growth and development by RNAi. Meanwhile, the biochemical characteristics of their expression products in vitro will also be taken into consideration. The results will expand our knowledge on the functions of insect CDAs, facilitate our understanding of the development, reproduction and their regulation mechanisms in beetles, and provide basic information for the development of novel eco-friendly insecticides.

几丁质是昆虫表皮和中肠围食膜的主要成分，昆虫的生长发育依赖于体内几丁质合成和降解的精确控制。几丁质脱乙酰酶（Chitin deacetylases，CDAs）是参与几丁质降解的关键酶，调节昆虫的行为、变态发育及生殖等。由于几丁质不存在于高等动物中，故昆虫CDAs作为靶标研发新型杀虫剂备受关注，具有重要的研究价值。因此，本研究以中药材储藏期主要害虫药材甲为研究对象，在转录组测序基础上克隆CDA家族基因，利用qPCR技术对CDAs基因的时空表达谱以及应对胁迫的差异表达调控效应进行分析，并利用原位杂交确定其组织的基因定位，采用RNAi技术明确其在昆虫生长发育中的作用，同时结合关键基因体外表达产物的生化特性来揭示CDAs在药材甲体内的重要生理功能。研究结果将丰富我们对昆虫CDAs功能的认识，促进理解药材甲及其它甲虫种类变态、发育、繁殖的调控机制，为新型环境友好杀虫剂的创制提供理论依据。

几丁质降解是昆虫的一个重要生理生化过程，几丁质降解酶在昆虫蜕皮、发育等生命活动中起着关键作用。由于高等动物体内不含有几丁质，因此几丁质降解中的关键酶被认为是安全有效杀虫剂的潜在靶标。本项目以中药材储藏期严重危害的药材甲和烟草甲为研究对象，运用分子生物学技术对几丁质降解酶在昆虫体内的生物学功能进行了研究。主要研究结果如下：（1）克隆获得药材甲2个几丁质脱乙酰酶基因SpCDA1和SpCDA2的全长序列，并运用相关软件对其特性进行了分析；发现2个基因在高龄幼虫和蛹期高表达，20E和饥饿胁迫均能诱导其表达；分别向4龄幼虫注射2个基因的dsRNA后，SpCDA1基因沉默后虫体出现表皮黑化、皱缩现象，总死亡率达到72%，而SpCDA2基因沉默后60%试虫出现蜕皮困难而死亡的表型；通过原核表达技术获得了重组蛋白SpCDA2，该重组蛋白分子量为63 kD，与预测结果一致，且具有CDA蛋白的催化活性，20E对SpCDA2酶促反应有激活作用，呈现先升高后降低的趋势。（2）在烟草甲转录组测序基础上，利用RT-PCR技术克隆获得几丁质脱乙酰酶LsCDA1基因的全长序列，发现LsCDA1在高龄幼虫表达最高，且20E能够明显诱导其表达；LsCDA1基因的沉默导致80%虫体出现蜕皮异常而死亡。（3）获得烟草甲beta-N-乙酰氨基葡萄糖苷酶LsNAG1和LsNAG2基因，分析其在不同发育阶段以及20E处理后基因的表达变化，利用RNA干扰分析2个基因在幼虫—蛹和蛹—成虫转化过程中的重要作用，结果发现2个基因在烟草甲变态发育过程中参与蜕皮和翅膀发育。研究结果丰富了昆虫几丁质降解酶的理论体系，为害虫防治提供高效安全的候选基因，为实现仓储害虫的有效控制提供科学依据。本项目在国内外学术期刊发表研究论文4篇，其中SCI论文3篇，国内核心期刊1篇，授权发明专利1项，培养硕士生2名。