华贵栉孔扇贝橙色个体富集类胡萝卜素的关键基因研究

Carotenoid is a large group of fat soluble chemicals with biological activity, which plays important roles in human and animals health, and affects the quality of aquaculture animals. Being lack of ideal animal models, the mechanism of accumulating carotenoids in bivalves is still unclear. Funded by the previous NSFC program, we found that the noble scallops Chlamys nobilis with orange shells and orange muscles contain higher carotenoids content than those with brown shells and white muscles, and confirmed that these traits are steadily inheritable. Meanwhile, some orange strains (total carotenoids content higher) and brown strains (total carotenoids content lower) have been established by our laboratory. These strains provide a superior model for studying the accumulating carotenoids in shellfish, and these different strains will be used in the present study. First, some candidate genes in related with absorption, transportation and cleavage of carotenoids will be screened by the Digital Gene Expression Profiling (DGE) and Roche 454 sequencing data. Meanwhile, these candidate genes will be also screened by RACE、qRT-PCR and mass spectral analysis (MS). Next, these candidate genes will be further functionally identified using RNAi and expression system in Saccharomyces cerevisiae. Finally, the association between gene polymorphism, expression profile of key genes and total carotenoids content will be also analyzed. Our works will not only help clarify the exact mechanism of accumulating carotenoids in the orange individuals of noble scallop C. nobilis, but also provide a guide for improving quality and tolerance to environment in shellfish genetic breeding.

类胡萝卜素（CAR）是一类具有生物活性的脂溶性物质，不但对人类与动物健康具有重要作用，还影响水产动物的品质。由于缺乏合适的动物研究模型，人们一直不清楚软体动物是如何富集CAR的。在前期基金项目资助下，我们不但证实了华贵栉孔扇贝橙色个体富集的CAR能够稳定遗传，并培育出富含CAR的橙色品系，为本申请项目奠定了良好的基础。本项目拟以选育的华贵栉孔扇贝橙色品系（CAR含量高）和褐色品系（CAR含量低）为材料，首先结合RACE、Qrt-PCR、质谱等技术，利用数字基因表达谱从454转录组数据中筛选与CAR吸收、转运、代谢等相关基因并获得候选的关键基因，再通过酿酒酵母表达系统和RNAi技术验证关键基因的生物学功能，最后研究这些基因位点多态性、表达量与CAR含量间的相关性。研究成果有助于阐明华贵栉孔扇贝橙色个体富集CAR的分子机制，具有重要的理论意义；为品质高、抗性强的贝类新品种培育提供理论指导。

类胡萝卜素是一类具有生物活性的脂溶性物质，不但对人类与动物健康具有重要作用，还影响水产动物的品质。由于缺乏合适的动物研究模型，人们一直都不清楚软体动物是如何累积类胡萝卜素的。本课题以华贵栉孔扇贝富含类胡萝卜素的橙色品系和类胡萝卜素含量低的褐色品系为材料，利用454平台对它们的全组织(闭壳肌、外套膜、鳃、性腺) 转录组测序，获得111,670条unigene；利用HigSeq 2000对它们的闭壳肌、血液、肠道分别进行了转录组测序，差异分析后，获得94,120条Unigenes。利用数字基因表达谱筛选出SRB-like-1、SRB-like-2、SRB-like-3、SRB-like-4、StAR-like-3、StAR-like-5、Vg、BCMO1、BCMO2等9个与类胡萝卜素积累相关的基因，克隆了它们的全长cDNA。并对候选的关键基因，通过酿酒酵母表达系统和RNAi技术进行验证，获得了一个橙色个体富集类胡萝卜素的关键基因SRB-like-3。构建华贵栉孔扇贝BAC文库，筛选了含有类胡萝卜素关键基因SRB-like-3的阳性克隆，并进行了高通量测序。采用原核表达，获得候选基因StAR-3的重组蛋白，并对其进行功能验证以及蛋白在橙、褐扇贝不同组织中的表达差异，阐明该基因在扇贝中的功能。研究了不同光照下类胡萝卜素含量的变化规律及相关基因表达量间的关系以及抗生素干扰实验后，血液中的类胡萝卜素含量与RAR（视黄酸受体）基因表达量间的关系。同时研究了类胡萝卜素与抗性、免疫基因间的关系，克隆了cold shock domain protein、toll like receptor-1、big defensin、cathepsin D、Cnlec-1、peroxiredoxin、Cu-Zn SOD、MnSOD、CAT、thioredoxin-like protein等10个基因，发现类胡萝卜素能够上调等基因的表达。目前结果表明，华贵栉孔扇贝橙色个体富集类胡萝卜素需要一些关键基因的参与。研究成果不但具有重要的理论意义，也为华贵栉孔扇贝“南澳金贝”新品种的育成提供了理论指导。