墨吉明对虾CHH家族神经肽对卵黄蛋白原基因的调控

The eyestalk X-organ sinus gland complex of the shrimp can produce the crustacean hyperglycemic hormone (CHH), molt inhibiting hormone (MIH)and gonad inhibiting hormone (GIH) that regulate many important physiological processes. They belong to the CHH/MIH/GIH neuropeptide family (or CHH family) members. GIH can suppress ovary maturation of the females through inhibition of vitellogenin expression. In shrimp aquaculture, the most common method to induce female shrimp maturation is by unilateral eyestalk ablation. This technique can reduce the amount of GIH in the hemolymh. However, this approach is harmful to the females and shrimp progenies produced by this method are of inferior quality compared to those produced by wild caught females. In this study, we propose to use Fenneropenaeus merguiensis as the research animal. We aim to study the basic mechanism of CHH members in vitellogenin regulation and provide possible explanations for the low egg quality produced by unilateral eyestalk ablation. As CHH family consists of many members that share similar structures and amino acid sequences, most of them show similar and overlapping functions. In this proposal, we begin by cloning all the CHH family members, study their expression patterns during ovary maturation cycle, identify 2-3 members that show strong association with vitellogenin gene expression and examine their functions using recombinant protein and RNA interference technology. We will also apply RNA transcriptome technique to study the expression patterns of CHH family members and other reproduction-related genes in the eyestalk of naturally matured females and artificially induced females. The results of this study will be able to illustrate that the CHH family members are important endocrine regulators of vitellogenesis and it may provide clues for the development of shrimp gonad maturation technique without the use of eyestalk ablation.

对虾眼柄X-器官窦腺复合体(XOSG)能产生CHH家族神经肽，其中包括性腺抑制激素（GIH），抑制卵巢成熟。目前主要采用单眼柄切除降低GIH浓度诱导雌虾成熟，此方法催熟的雌虾所产子代质量较差，且不能保障对虾的福利。为解决此问题，本项目将以墨吉明对虾为研究对象，针对其CHH家族基因数目众多、序列相似、功能重叠等特点，进行如下研究：1.克隆XOSG中所有CHH家族成员的基因；2.研究CHH家族成员基因在自然成熟和人工催熟的雌虾在性腺发育过程中的表达规律；3.筛选与卵黄蛋白原基因表达相关的CHH家族成员，通过重组蛋白和RNAi技术，研究CHH家族成员对卵黄蛋白原基因的表达调控机制；4.利用眼柄转录组学信息，研究不同成熟方式雌虾CHH家族成员及其它与繁殖有关的重要基因表达差异。为阐明CHH家族对雌虾繁殖内分泌调控机理奠定理论基础，并进一步开发非眼柄切除方式的对虾人工催熟技术提供理论指导和技术保障。

（1）主要研究内容。.甲壳类动物的眼柄神经存在一个名为X-器官窦腺复合体(XOSG) 的重要内分泌器官，能产生多种不同类别的神经肽。其中，甲壳动物高血糖激素(CHH),蜕皮抑制激素(MIH)及卵巢成熟抑制激素(GIH)组成一个CHH/MIH/GIH神经肽家族，其成员能控制包括功能、能量、蜕皮、生长及繁殖不同重要生理过程。项目负责人，向来以研究此家族成员对雌虾内分泌调控繁殖为主，并集中研究此家族中卵巢成熟抑制激素(GIH)调控雌虾卵巢成熟的分子机制，并希望得掌握雌虾在繁殖期，GIH对卵巢成熟时卵黄蛋白的合成的影响。申请人根据初期克隆CHH/MIH/GIH家族成员的前期研究，结果表明此家族成员，可粗分为二大亚类。但由于此家族成员众多，他们无论在分子、蛋白结构极为相似外，并有可能存在功能重叠现象。加上目前仍未能在同一品种中，分离所有的成员。导致研究此家族困难重重。本项目将在前期工作基础上，通过构建多个对虾眼柄神经转录组，建立具有深度的转录组数据库，并筛选可能参与控制卵巢成熟或调控卵黄蛋白基因表达的几个CHH/MIH/GIH神经肽家族成员，作深入分析。此研究将采用注射重组蛋白及双键RNA技术，从而进行促进及抑制的拮抗性的实验，相互引证个别或多个成员的调控繁殖功能。在墨吉明对虾的不同眼柄神绖组织及其他非神绖组织转录组测序中，筛选出约廿个墨吉明有CHH/MIH/GIH家族基因特征的转录子，其中属于CHH I 亚类的有14 ，属于CHH II 亚类的基因有6个。采用基因组DNA PCR步移法、基因组DNA PCR法证明CHH/MIH/GIH家族基因结构CHH可分三类。同时根据翻译出的神经肽蛋白结构、基因转录子序列分析，首次证明此家族基因成员可分为三个进化演变组合。显示这些演化成员与功能有关，并筛选几个与繁殖有关基因。在注射实验中，重组蛋白和双键RNA实验表明，其中一个基因可能是性腺抑制激素（GIH），另一个些可能是卵巢刺激激素（GSH）。 这一重要发现将为些虾人工催熟策略提供重要指导，并减低对雌虾切除单眼柄后的損害。