家蚕胚胎滞育发动及滞育解除的关联蛋白研究

With the analytic strategies of proteomics and bioinformatics, the relevant proteins of diapause origination and termination are attempted to be screened to identified from embryo in Bombyx mori. These genes will be cloned and their function will be analysed. The total proteins will be extracted from the embryo during the stage of diapause origination or diapause standing of diapause termination. By the method of LC-MS/MS, the protein composition each stage will be identified. Through difference comparison, identified analysis, querying in protein database and bioinformatics analysis, the relevant proteins of diapause origination and diapause termination will be obtained. Therefore, their biological properties, temporal variation, differential expression will be determined. According to the nucleotide sequence deduced from the amino acid sequence of candidate proteins, the primers will be designed and the genes related with diapause origination and diapause termination. With the methods of real-time quantitative PCR and Western blot, differential expression of these genes will be analysed during diapause origination and diapause termination of embryo in Bombyx mori. Their function and connected effect between diapause degree and mRNA expression level will be investigated. These result will elucidated furtherly the mechanism about embryo diapause and diapause termination in molecullar level in Bombyx mori.

利用蛋白质组学和生物信息学的研究策略，筛选和鉴别与家蚕胚胎滞育和滞育解除有关的功能蛋白，克隆相关的基因，查明其作用功能。试验将从滞育发动初期、滞育维持阶段和滞育解除后等时期获得家蚕胚胎总蛋白质，采用液相色谱串联质谱联用(LC-MS/MS)的技术方法鉴定各时期蛋白质种类，通过差异比对，鉴别分析，蛋白质数据库查询和生物信息学分析，得到与滞育和滞育解除相关的关键蛋白，研究其生物学性质，探究它在滞育发动、滞育解除过程中的时序变化、差异表达，查明蛋白质的相关功能。根据候选蛋白质的氨基酸序列预测其核苷酸序列，设计引物，通过基因工程的技术方法，克隆获得调控滞育和滞育解除相关的候选基因，利用实时荧光定量PCR与Western blot的方法，分析其在滞育发生和滞育解除中的差异表达，mRNA表达水平与滞育强度的联动效应，探索其功能作用，从分子水平的角度进一步阐明家蚕滞育及其解除的机理。

（1）利用shotgun质谱分析技术，分析鉴定了932品种的赤豆色卵浸酸前后的蛋白差异情况。通过搜索对比家蚕库、昆虫库，赤豆色卵在浸酸前后相同的蛋白有288种、218种，浸酸前的特异蛋白分别有267种、370种，浸酸后的特异蛋白分别有338种、299种。.（2）通过荧光定量PCR分析了6个候选基因BmeIF、BmADH、BmADK、BmFDS、BmPEBP和BmCSP7在932蚕品种产后1~9d的蚕卵、产后于第3d浸酸解除了滞育后1~7d的发育卵、冷藏10~90d的滞育卵以及冷藏40d经浸酸解除滞育后1~9d的发育卵中的转录表达情况。结果表明，BmeIF、BmADH可能与滞育有关联；BmADK、BmFDS、 BmPEBP和BmCSP7可能与滞育解除启动有关联。.（3）对浸酸处理前后的赤豆色卵进行了miRNA建库。并利用生物信息学工具RNAhybird等对蚕卵经浸酸后发生显著上调或下调的miRNA进行分析，从中筛选出2个与家蚕滞育解除密切关联的bmo-miR-3384-3p与bmo-miR-2761-3p，及其靶标基因BmNLK和BmSDH。.（4）用qRT-PCR技术对滞育性卵与赤豆色浸酸卵在卵龄3-7d 时bmo-miR-2761-3p、bmo-miR-3384-3p、BmSDH和BmNLK的表达水平进行定量分析，蚕卵处在滞育状态时，bmo-miR-2761-3p、bmo-miR-3384-3p表达水平较高，当蚕卵浸酸后则下降。相反BmSDH、BmNLK在蚕卵处在滞育状态时，会低水平表达，当蚕卵浸酸后则显著上调。显示该miRNA在靶基因的表达中可能起着负调控作用。.（5）构建了含有BmSDH 3’UTR、BmNLK 3’UTR的载体，并经DLR法检测验证，证实了bmo-miR-2761-3p会抑制BmSDH的表达；bmo-miR-3384-3p 会抑制BmNLK 的表达。.（6）借助ELISA方法测定了浸酸前后、冷藏前后胚胎发育过程中BmSDH和BmNLK酶以及4种辅酶(乙酰辅酶A，黄素腺嘌呤二核苷酸，辅酶Ⅰ和辅酶Ⅱ)的活性。