葱蝇滞育调控基因的表达和功能研究

Insect diapause is a unique physiological and developmental process, which involves intricate molecular regulation mechanisms. The pupal diapause can be divided into winter diapause and summer diapause, which were shown to be of inconsistent physiological processes. To date, a large number of identifications and studies of winter diapause related genes have been done; however, the comprehensive understanding of the molecular regulationary mechanisms of diapause, winter diapause and summer diapause is yet to be elucidated. In recent years, we have established a model animal system for the comparative study of winter diapause and summer diapause with the onion maggot Delia antiqua and have conducted preliminary studies on genes related to winter diapause and summer diapause. We have recently completed RNA-seq sequencing of different stages of winter diapause and summer diapause pupae and the corresponding stage of non-diapause pupae of the onion maggot. Based on the model system, the proposed project aims to: 1) identify the potential diapause regulation genes and their regulatory pathways at different developmental stages of winter diapause and summer diapause using the results of RNA-seq sequencing; 2) clarify the expression patterns and their regulations of main diapause-relative genes at different developmental stages of winter diapause and summer diapause using quantitative PCR and Northern hybridization analysis; 3) clarify the functions and regulationary pathways of main diapause regulationary genes through RNAi. The proposed study will be of scientific significance for further understanding of the molecular mechanisms of insect diapause and its regulation, organism developmental stagnation and then restarting, and physiological differences of winter diapause and summer diapause.

昆虫滞育是一个独特的生理和发育过程，涉及错综复杂的分子调控机制。蛹滞育可分为冬滞育和夏滞育，具有不同的生理过程。国内外已开展了大量冬滞育相关基因的鉴定和研究工作，但滞育、冬滞育和夏滞育分子调控机制仍缺乏全面的了解。近年来，申请者建立了葱蝇(Delia antiqua)冬滞育和夏滞育比较研究系统并对其相关基因做了初步研究。最近,我们完成了非滞育、冬滞育和夏滞育蛹各时期表达谱测序(RNA-seq)工作。本项目以葱蝇为模式种，以RNA-seq测序为基础系统鉴定冬滞育和夏滞育不同发育时期的滞育调控基因及其调控通路；通过定量PCR分析和Northern杂交，弄清主要滞育调控基因在冬滞育和夏滞育各发育时期的表达模式及其规律；通过RNAi等基因功能研究方法弄清主要滞育调控基因的功能和调控通路。以期进一步弄清昆虫滞育及其调控、有机体发育停滞和再开始、冬滞育和夏滞育生理差异的分子机理。

昆虫滞育是一个独特的生理和发育过程，涉及错综复杂的分子调控机制。蛹滞育可分为冬滞育和夏滞育，具有不同的生理过程。国内外已开展了大量冬滞育相关基因的鉴定和研究工作，但滞育、冬滞育和夏滞育分子调控机制仍缺乏全面的了解。近年来，申请者建立了葱蝇(Delia antiqua)冬滞育和夏滞育比较研究系统并对其相关基因做了初步研究。本项目以葱蝇为模式种，测序和分析了葱蝇转录组；以非滞育、冬滞育和夏滞育蛹各时期RNA-seq测序为基础系统鉴定冬滞育和夏滞育不同发育时期的滞育调控基因，表达模式及其调控通路，预测了可能的功能；通过基因克隆、定量PCR分析、生物信息学分析及酶活分析，弄清主要滞育调控基因在冬滞育和夏滞育各发育时期的表达模式、特征和可能的功能；通过RNAi等基因功能研究方法研究主要滞育调控基因的功能和调控通路；通过双向凝胶电泳比较分析，鉴定了13 个非滞育和夏滞育间差异表达的蛋白质，他们分别为胶原蛋白、 纺锤丝组装非正常蛋白6、5, 10-亚甲四氢叶酸合成酶、Bnb蛋白及其他功能未知蛋白；测序和注释了葱蝇线粒体基因组全序列，基于13个蛋白质编码基因的核苷酸序列建立13个双翅目昆虫种的系统发育关系，结果表明葱蝇所在的花蝇科和丽蝇科更近缘。这些研究对于进一步弄清昆虫滞育及其调控、有机体发育停滞和再开始、冬滞育和夏滞育生理差异的分子机理具有重要意义。