中华绒螯蟹卵巢发育过程中类胡萝卜素积累机制的研究

Carotenoid is an important class of nutrients deposited in the crustacean ovary, but very limited information is known for their deposition mechanisms during the ovarian development process. The Chinese mitten crab (Eriocheir sinensis) is one of the most important aquaculture species in China. Our previous studies have shown the contents of ovarian carotenoids increased significantly during the ovarian maturation of E. sinensis, and many candidate genes related carotenoids deposition were obtained and preliminarily identified by bioinformatics. The current project is designed to comprehensively investigate “the accumulation mechanism of ovarian carotenoids during the ovarian development of E. sinensis. Firstly, we will study the changes of ovarian carotenoids composition, their contents and existing forms of major carotenoids during the ovarian maturation process of E. sinensis. Further study will be conducted to investigate the localization and expression patterns of candidate genes related to carotenoids deposition. Then, using in vitro experiments (tissue culture with different carotenoids as well as different concentration of one carotenoid) and vivo experiments (oral administration of dietary carotenoids), a series of comprehensive assessments will be performed to investigate the effects of major carotenoids on ovarian development, antioxidant capability, lipid metabolism, carotenoids composition, their contents and related gene expression levels. Based on the previous findings, 1 - 2 candidate genes for carotenoids binging/transport protein will be selected/screened and further identified for their potential functions during the process of ovarian carotenoids deposition, through binding assay, site-directed mutagenesis, RNA interference, co-localization and the other methods. The expected outcomes of this project would not only improve our understanding for the nutritional physiology of carotenoids deposition and ovarian development, but also provide the valuable information for the feed development and quality improvement of female E. sinensis.

类胡萝卜素是甲壳动物卵巢中积累的一类重要营养物质，有关其在卵巢发育过程中的积累机制所知甚少。河蟹是我国最重要的养殖蟹类，本项目在发现河蟹卵巢发育过程中大量积累类胡萝卜素和获得多个类胡萝卜素积累候选基因的基础上，拟系统研究“河蟹卵巢发育过程中类胡萝卜素的积累机制”。项目首先研究河蟹卵巢发育过程中主要类胡萝卜素组成、含量和存在形式的变化；在此基础上研究河蟹卵巢发育过程中类胡萝卜素积累相关基因的定位和表达模式；进一步通过离体和活体调控研究，全面评估主要类胡萝卜素对河蟹卵巢发育、抗氧化、脂类代谢、类胡萝卜素积累及相关基因表达的影响；根据以上研究结果，筛选1-2个类胡萝卜素结合/转运蛋白的候选基因，通过结合效率分析、定点突变、RNAi和共定位等研究这些关键基因在类胡萝素积累过程中的生理功能。预期研究结果对于深入理解河蟹卵巢发育的营养生理、育肥饲料开发和营养品质调控均具有重要的理论意义和现实意义。

类胡萝卜素是甲壳动物卵巢中积累的一类重要营养物质，目前有关其在卵巢发育过程中的积累机制所知甚少。河蟹是我国最重要的养殖蟹类，河蟹卵巢组织积累的类胡萝卜素含量及比例会影响河蟹雌体营养价值和后续生殖性能。本项目首先研究河蟹卵巢发育过程中主要类胡萝卜素组成、含量和存在形式的变化，发现河蟹卵巢发育过程中优先积累虾青素，且酯化虾青素为主，双酯虾青素含量高于单酯虾青素；结合组织离体培养和活体饲料营养调控实验，全面评估了5种常见类胡萝卜素（虾青素、β-胡萝卜素、玉米黄素、叶黄素、角黄素）对河蟹卵巢发育、抗氧化能力、脂类代谢、类胡萝卜素积累及相关基因表达的影响，进一步通过饲料中单因素实验和双因素实验，探明了饲料中虾青素和β-胡萝卜素、虾青素和DHA含量均存在交互作用，建议河蟹雌体育肥饲料中适宜虾青素、β-胡萝卜素和DHA适宜添加量分别为50 mg/kg、100 mg/kg和0.15%；基于河蟹基因组和转录组等筛选了7个类胡萝卜素裂解酶、3个类胡萝卜素结合蛋白和3个虾青素结合蛋白，并对关键基因的生物信息学、表达模式和表达定位进行了研究，发现2个类胡萝卜素结合蛋白、2个类胡萝卜素裂解酶和2个虾青素结合蛋白在类胡萝卜素积累中起着重要作用；最后，通过“重组蛋白结合效率分析”、“RNAi”和“CO-IP”等研究，初步确定βCBP2蛋白可以与β-胡萝卜素优先结合在卵巢中类胡萝卜素积累起着重要作用，βCBP3可能和肝胰腺中类胡萝卜素运输有关。以上研究结果，对于深入理解河蟹卵巢发育期间的类胡萝卜素积累机制、育肥饲料开发和营养品质调控均具有重要的理论价值和现实意义。项目执行过程中，发表SCI论文3篇，获得授权发明专利3项（其中1项已经转让给企业），后续研究项目2项，已经培养博士和硕士生各1名。