昆虫表皮几丁质有序排列关键基因研究

Insect cuticle serves as an important component of the integument and plays protective roles against the mechanical damages and environmental pathogens. Although chitin is a major component of insect cuticle, current research on insect chitin focused primarily on chitin metabolism. It is virtually unknown as to how chitin is organized in insect cuticle. Our group has extensively studied genes involved in chitin metabolism and recently identified 7 genes putatively involved in chitin organization in Locusta migratoria. Based on our preliminary studies, we propose to advance our research to study the molecular mechanism of insect chitin organization in the model insects, Drosophila melanogaster and L. migratoria. The major objectives of our project are to: 1) clone the full-length cDNA sequences of the genes potentially involved in chitin organization by RACE-PCR, analyze expression patterns of the genes in different developmental stages and tissues of the insect, and localize the mRNAs and proteins of these genes by in situ hybridization and immunohistochemistry; 2) detect the effect of suppression/over-expression of target genes on the chitin assembly using Gal4-UAS system of Drosophila, and examine the effect of RNAi on the chitin assembly using electron microscope and chitin staining methods; and 3) understand the networking of proteins involved in the process of chitin assembly by using co-immunoprecipitation discover new genes by performing GST pull-down combined with mass spectrometry analytic techniques. Results from this study are expected to help us better understand the mechanism of insect chitin organization and discover novel molecular targets for insect pest control.

表皮是昆虫的组织结构和保护性屏障，几丁质是表皮的重要组成成分，昆虫表皮几丁质研究主要集中在几丁质的合成代谢，然而几丁质如何形成有序排列的表皮层状结构？即表皮几丁质组装的分子机制尚缺乏系统研究。本课题组围绕飞蝗表皮开展了系列研究工作，已鉴定出7个与表皮几丁质排列相关的基因。本项目拟在此基础上，以飞蝗和模式生物果蝇为材料，开展如下研究内容：1）分析表皮几丁质排列关键基因序列及其表达模式；采用原位杂交和免疫组化技术研究基因的细胞组织定位；2）利用果蝇的Gal4-UAS系统，区域化干扰或超表达关键基因，研究果蝇表皮超微结构的变化；应用RNAi技术沉默飞蝗关键基因，在细胞层面分析表皮结构的变化及对昆虫蜕皮发育的影响；3）采用co-IP探索表皮几丁质排列关键蛋白的互作；GST pull-down结合质谱技术挖掘新蛋白。研究结果将揭示昆虫表皮几丁质组装的分子机制，获得针对表皮几丁质排布的害虫防治新靶标。

表皮是昆虫的组织结构和保护性屏障，几丁质是表皮的重要组成成分，昆虫表皮几丁质研究主要集中在几丁质的合成代谢，然而几丁质如何形成有序排列的表皮层状结构？即表皮几丁质组装的分子机制尚缺乏系统研究。本项目以飞蝗和模式生物果蝇为材料，开展了如下研究工作：1）分析了表皮几丁质排列关键基因几丁质脱乙酰基酶（Chitin deacetylase，CDA）和Knickkopf（Knk）家族基因的序列及其表达模式；采用免疫组化技术研究基因的细胞组织定位，发现飞蝗的LmCDA1定位于表皮细胞质和原表皮，LmCDA2主要表达在原表皮顶部连接上表皮的位置；采用RNAi、壳聚糖染色、几丁质含量测定和电镜技术研究了基因的生物学功能，发现LmCDA1主要负责几丁质的脱乙酰基过程，LmCDA2对表皮螺旋结构形成具有支撑和骨架作用。对飞蝗Knk家族基因的研究揭示了LmKnk 和LmKnk3-5’对飞蝗的蜕皮发育具有重要作用；干扰LmKnk表皮层状结构消失，而LmKnk3-5’的沉默并不影响表皮层状结构。2）利用果蝇的Gal4-UAS系统，在果蝇翅上区域化干扰或超表达几丁质排列关键基因CDA1（serp）和CDA2（verm）发现仅仅干扰serp后，翅表皮中几丁质转化为脱乙酰基壳聚糖的能力显著降低，表明CDA1在昆虫中主要负责表皮几丁质的脱乙酰基过程；果蝇翅的电镜结果进一步证实了昆虫CDA2主要参与表皮几丁质层状结构的维持。采用knk过表达和干扰的方法对果蝇翅发育表型进行观察，结果表明干扰knk影响羽化和正常展翅；使用原子力显微镜（AFM）扫描果蝇翅膜表面的纳米级结构，发现干扰knk后，突包显著变小。而过表达knk后，翅的表型及超微结构均无显著变化。研究结果揭示了昆虫表皮几丁质组装的分子机制，获得针对表皮几丁质排布的害虫防治新靶标。