家蚕蛹期滞育关联基因的鉴定及功能研究

Diapause process is essential for life maintain and species development of insects. Diapause of F1 generation of bivoltine silkworm (Bombyx mori) strain is regulated by environmental condition of it parent during embryo stage, and oocytes of 3rd-day pupae are most sensitive to diapauses hormone (DH). According to this principle and trait, we are going to prepare the 3rd-day-pupa ovary samples for high-through sequencing by incubating activated hibernating eggs of the bivoltine strain, Qiufeng under 17℃ in darkness (treatment group) and 25℃ in natural day-night circadian rhythm (control group), respectively. After hatching, larvae will be fed with fresh mulberry leaves under 25-26℃and 70-85% relative humidity. After high-through sequencing the differential expressed genes between the treatment group and control group, and candidate key genes related to diapause will be screened by bioinformatics methods. Then spatial and temporal expression of candidate genes will be analysed by quantitative real-time PCR (qPCR) and Western blot technology. To validate the function of key genes of diapause, the high-tech method, CRISPR/Cas9 system will be applied for target gene editing in early embryo stage combined with micro injection transgenic method. Follow by identification of phenotype in G0 and F1 generations. Thereby to improve the dipause regulation network of B. mori, and provide experimental data for explanation of diapause molecular mechanism of B. mori. The results of the project will provide theoretical base for silkworm breeding, as well as reference for developing of novel agriculture and forestry pest control method.

滞育对昆虫生命的维持和种群发展有着不可或缺的作用。本项目利用家蚕二化性品种子代滞育性受亲代胚胎期环境条件调控的原理和蛹期3d卵母细胞对滞育激素最敏感的特点，以二化性品系“秋丰”活化的越年卵为材料，采用17℃黑暗催青（试验组）和25℃明催青（对照组），孵化后新鲜桑叶在25-26℃和相对湿度70-85%条件下饲养，至蛹期3d解剖卵巢组织进行高通量测序；生物信息学分析差异表达基因，初步筛选滞育关键候选基因；利用qPCR和Western blot技术分析候选基因的时空表达特性；利用CRISPR/Cas9系统，结合早期胚胎显微注射转基因技术等，靶向编辑候选基因，分析G0和F1代的表型，验证滞育关键基因的功能，从而完善家蚕滞育分子调控网络，为阐明家蚕滞育分子机制提供实验数据，研究结果将为家蚕育种提供理论依据，也为农林害虫防治新途径研究提供借鉴。

昆虫类是地球上最繁盛的生物群，经过长期的进化，形成了各种各样奇妙的生存方式，滞育对昆虫生命的维持和种群发展有着不可或缺的作用。昆虫滞育机理一直是国内外的研究热点，也取得了很大的进展，但是昆虫滞育分子机理的尚未得到阐明。. 本项目根据家蚕二化性品种子代滞育性受亲代胚胎期环境条件调控的原理和蛹期3d卵母细胞对滞育激素最敏感的特点，以二化性品系“秋丰”活化越年卵为材料，蛾区半分法分为17℃黑暗催青组（NDEP）和25℃光照催青组（DEP，对照），孵化后新鲜桑叶25-26℃饲养，制备蛹期3d卵巢样本进行转录组高通量测序，首次在全基因组分析查找获得289个在NDEP和DEP组差异表达基因（DEGs）——滞育关联基因，它们主要分布在过氧化物酶体通路、三酰甘油合成通路、蜕皮激素合成等信号通路；荧光定量PCR和Western blot分析21个DEGs的时空表达特点；分析鉴定3个家蚕滞育关键基因BmSDH、BmSDH-2a和BmSDH-2b的启动子特性，并确定了其转录起始位点；RNAi和CRISPR/Cas9技术对滞育关键基因Bmshadow、Bmark2e-like和BmSDH-2a等进行靶向编辑和功能研究，初步明确BGIBMGA003835、BGIBMGA012335、Bmark2e-like、Bmshadow、BmSDH-2a等为滞育关键基因。研究结果进一步完善了家蚕滞育调控的分子网络，有助于阐明家蚕滞育的分子机制，为家蚕育种提供理论依据，并可为农林害虫的防治新途径研究提供借鉴。