文蛤类胡萝卜素代谢关键基因鉴定及其在壳色变异中的表达调控机制研究

Shell color, one of the most significant phenotypic traits of cultured shellfish, is of great value in breeding because it is always related to economic traits such as growth, survival and stress resistance. Discovery and identification of key genes involved in carotenoid metabolism and in-depth study on the molecular mechanism are the important prerequisites for the accurate use of shell color. Funded by the former National Natural Science Foundation, we have identified the red pigment on shell of Meretrix meretrix as carotenoids, and cloned and analyzed the correlation between 4 genes (BCMO1, BCDO2, SR-BI and SCD) for carotenoid metabolism and pigment accumulation. However, the key genes involved in carotenoid metabolism and their mechanism of regulation are still unclear to date. In this study, we intend to screen and identify key genes associated with carotenoids metabolism at the DNA level using association of SNP markers in candidate genes and genome-wide association study (GWAS). By qRT-PCR, western bolt and Immunofluorescence methods, we analyze the gene expression patterns, protein change and cellular localization of proteins. The gene functions were verified by RNAi technology. From the epigenetics, we research the network regulation mechanism of miRNA on the gene expression of carotenoid metabolism. The main aim of this project is to analyze the signal pathway and molecular regulation mechanism of carotenoid metabolism in M. meretrix for providing theoretical basis and technical support for molecular marker-assisted selection.

壳色是养殖贝类最显著的表型性状，因常与生长、存活、抗逆等经济性状相关而在育种中具有重要利用价值。发掘壳色控制的关键基因并解析其表达调控机制，是精准利用壳色性状的重要前提。申请人前一个国家自然科学基金已将文蛤壳表红色色素鉴定为类胡萝卜素，克隆并分析了4个类胡萝卜素代谢基因与色素积累的相关性，然而类胡萝卜素代谢的关键基因及其表达调控机制至今仍不清楚。本项目拟用候选基因SNP关联分析法和全基因组关联分析（GWAS）技术，筛查和验证类胡萝卜素代谢的关键基因和关联SNPs；然后用qRT-PCR、Western bolt和免疫荧光技术，检测关键基因mRNA和表达蛋白的变化，用RNA干扰技术验证基因功能；从表观遗传学角度，研究miRNA对类胡萝卜素代谢基因表达的网络调控机制。本项目旨在解析文蛤类胡萝卜素代谢的信号通路和分子调控机制，为深入开展分子辅助育种提供必要的理论依据和技术支撑。

壳色是养殖贝类最显著的表型性状，且常与生长、抗逆等经济性状相关而在育种中具有重要利用价值。发掘壳色控制的关键基因并解析其表达调控机制，是精准利用壳色性状的重要前提。前期已将文蛤壳表红色色素鉴定为类胡萝卜素，然而类胡萝卜素代谢的关键基因及其表达调控机制仍不清楚。本研究以红壳文蛤为试材，用候选基因SNP关联分析法和GWAS技术，筛查和验证类胡萝卜素代谢的关键基因和关联SNPs；用qRT-PCR、Western bolt和免疫荧光技术，检测关键基因mRNA和表达蛋白的变化，用RNA干扰技术验证基因功能；从表观遗传学角度，研究miRNA对类胡萝卜素代谢基因表达的网络调控机制。项目实施三年来，取得了一系列重要成果，① 系统开展了文蛤类胡萝卜素代谢3个关键基因BCMO1、BCDO2、SR-BI的克隆、表达、细胞定位及红壳性状关联性分析，筛选到21个关联SNPs标记，为分子辅助育种提供了候选标记；② 首次成功破译文蛤基因组，获得组装到染色体水平的高质量基因组参考图谱，为壳色性状全基因组关联分析奠定了重要基础；③ 首次开展文蛤壳色的多组学（转录组+蛋白组+代谢组）联合分析，筛选到一批差异显著的基因、蛋白和代谢物，并对关键基因mRNA和表达蛋白的变化进行检测分析，用RNA干扰技术验证了基因的生物功能，为壳色形成提供了有力的分子证据；④ 从文蛤红壳和白壳差异miRNAs中预测到4个与类胡萝卜素代谢相关的基因（SR-C、SCD、LDLR、LRP 2），为红壳色形成的表观遗传学调控机制提供了重要参考。项目研究成果已在国内外重要学术期刊上发表研究论文6篇，其中SCI论文4篇、一级学报2篇，授权发明专利2项，获国审文蛤壳色新品种1个。研究结果可为文蛤红壳色形成分子机制研究以及壳色新品种培育提供理论基础和技术支持，也对其他贝类壳色分子遗传基础研究和育种实践深入开展具有理论指导作用。