拟穴青蟹VIH的转录调控机制研究

Members of crustacean hyperglycemic hormone family, including crustacean hyperglycemic hormone (CHH), molt inhibiting hormone (MIH), vitellogenesis inhibiting hormone (GIH), and mandibular organ inhibiting hormone (MOIH), are mostly secreted from the X-organ sinus gland complex of the eyestalk in crustacean. Similar to the hypothalamus-pituitary-gonad axis in vertebrates, the X-organ sinus gland complex and gonad in crustaceans form a similar axis to control the maturation of gonad. Many studies have confirmed that VIH inhibits vitellin synthesis in ovarian tissue. However, up to date, the mechanism of transcriptional regulation on expression of VIH gene have not been reported in crustaceans. Supported by the National Natural Science Foundation, VIH gene from Scylla paramamosain was cloned for the first time. The full length of cDNA, genomic DNA, and partial sequence of 5'-regulatory region of VIH of mud crab were obtained. Based on the progresses we have made in previous projects, in this project, 5' upstream regulatory region will be cloned and prolonged to 5-6 kb by Tail-PCR firstly. Secondly, the activity of 5' upstream regulatory region will be analyzed and the function of cis-element will be identified. Thirdly, the important transcriptional factors of VIH will be obtained, and the interact with cis-element will be identified. The functional correction between transcriptional factor and VIH mRNA will be elicited. Fourthly, Some gonad-related genes co-regulated by transcriptional factors of VIH in X-organ sinus gland complex will also be identified using ChIP-seq method. Finally, the mechanism of transcriptional regulation of VIH mRNA in S. paramamosain will be interpreted. The completion of this project will help us to demonstrate the molecular mechanism of transcriptioanl regulation of VIH in S. paramamosain. It will also help us to develop new reproduction regulation techniques for crab aquaculture.

由X-器官窦腺复合体组成的虾蟹眼柄内分泌系统对虾蟹生殖系统的发育和分化起着至关重要的调控作用。其合成和分泌的VIH抑制性腺发育。但是VIH基因在转录前如何被调控，到目前为止尚未见报道。在国家自然科学基金的资助下，课题组获得了拟穴青蟹VIH基因的全长cDNA和gDNA，通过Tail-PCR获得了转录起始位点上游部分序列。在此基础上，本项目将进一步延长拟穴青蟹VIH 5'上游调控区序列并分析其活性，确认VIH顺式作用元件的功能。同时鉴定调控VIH表达的重要转录因子，了解VIH表达与转录因子之间的功能相关性，确认转录因子对VIH的正负调控作用。通过ChIP-seq技术分析调控VIH的重要转录因子在X-器官窦腺复合体同步调控的其它性腺发育相关基因，阐明VIH的转录调控机制。研究成果将使我们在理论上更加深入了解拟穴青蟹性腺发育的调控机制，在生产实践为建立新的生殖调控技术提供新的思路。

由X-器官窦腺复合体组成的虾蟹眼柄内分泌系统对虾蟹生殖系统的发育和分化起着至关重要的调控作用，其合成和分泌的VIH抑制性腺发育。但VIH基因在转录前如何被调控到目前为止尚未见报道。本研究通过Genome walker 和Tail-PCR克隆获得VIH基因的5’上游调控区序列，从翻译起始位点（ATG）起，共3070 bp，从预测的转录起始位点（A）起，共2398 bp。通过生物信息学预测软件和启动子活性分析表明，其核心启动子区位于-203 bp—+213 bp之间，包含在线软件预测的转录起始位点区域。在转录起始位点（A）上游-28 bp处存在TATA box。将pSpVIH-1（含核心启动子区）重组至转基因载体pEGFP-1上，在HEK293FT细胞中能驱动EGFP的表达。不同长度缺失片段启动子活性分析表明，在pSpVIH-4和pSpVIH-6之间可能同时存在正调控和负调控的转录因子。对pSpVIH-4片段中的Oct1/Oct4/Sox9结合位点进行位点突变和活性分析，发现突变该位点其活性发生显著性降低（p<0.05），表明Oct1/Oct4/Sox9可能是正调控拟穴青蟹VIH基因表达的重要转录因子。采用EMSA技术和基于SPR原理的BIAcore分析技术确定了青蟹中确实存在转录因子Oct1／Oct4／Sox9或其相关家族成员能识别并与VIH启动子相应位点结合。过表达转录因子Oct4和SoxE能促进VIH核心启动子片段的活性。同样，通过点突变方法确认了pSpVIH-5片段中转录因子T3R-alpha和Pit-1a起负调控作用，可能对VIH基因的表达起抑制作用。.本研究还分析了与VIH同属于CHH家族的MIH和CHH基因5’上游调控区的順式作用元件，并确认其活性。发现转录因子NF-κB和RAP1可能正调控MIH基因的表达。在CHH上游-286 — -46、-583 — -388和-388 — -286区域可能存在调控CHH基因表达的转录因子结合位点。.对转录因子Sox家族SoxE，Sox14，Sox14b,和Sox21的基因及转录因子OCT家族的Oct4和Oct1基因进行了克隆，序列特征分析，对它们在各组织、不同胚胎发育阶段、不同性腺发育阶段的表达进行了分析。.研究成果使我们在理论上更加深入地了解了青蟹性腺发育的调控机制，在生产实践为建立新的生殖调控技术提供了新的思路。