牡蛎营养品质等重要经济性状的遗传定位与基因解析

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	牡蛎营养品质等重要经济性状的遗传定位与基因解析
	李春燕
学位类型	博士
导师	张国范
	2017-05-20
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	水产养殖
关键词	长牡蛎品质性状全基因组关联分析 Qtl定位 Cgcaspase8-2
摘要	牡蛎素有“海洋牛奶”之美称，其富含糖原、谷氨酸、牛磺酸、锌、硒、二十碳五烯酸、二十二碳六烯酸等极具营养价值的物质，市售的各种牡蛎营养制品更是充分体现了牡蛎的营养价值。然而，种质退化、养殖管理不善等造成的牡蛎品质低下问题已经严重影响了牡蛎产业的发展，导致牡蛎养殖产业整体效益长期在低水平徘徊。因此，培育生长快、肥满度高、高营养价值的牡蛎新品种是亟需解决的问题。除了生长与营养品质性状，牡蛎夏季大规模死亡等现象也不容忽视，由此凋亡过程因其在内环境稳态、机体免疫防御等过程中的重要作用也备受关注。作为起始凋亡酶，caspase-8在脊椎动物外源性凋亡通路中发挥承上启下作用，然而无脊椎动物诱导凋亡的方式却大相径庭。相关生物学问题，如牡蛎中是否存在外源性凋亡通路、牡蛎caspase-8是否具有与脊椎动物caspase-8类似的功能以及其是否具有免疫功能等都有待研究。长牡蛎（Crassostrea gigas），又名太平洋牡蛎，是世界范围内重要的经济养殖贝类，也是我国黄渤海区的主养贝类。本次研究把牡蛎生长和营养品质性状以及凋亡酶caspase-8基因作为研究对象，以长牡蛎为代表物种展开研究。一方面，通过群体与家系构建，通过全基因组关联分析与连锁分析的方法，将生长、糖原、氨基酸、脂肪酸等表型性状与SNP、单倍型等分子标记建立联系，为相应品质性状的遗传改良提供大量候选标记与关键基因，以期对品质性状遗传机制进行解析，为分子育种奠定基础；另一方面，针对长牡蛎caspase-8基因，检测其是否与外源性凋亡通路接头蛋白FADD相互作用，并分析其在不同时空（不同组织、发育时期）和不同病理条件下（细菌、病毒刺激）的表达模式，以期对长牡蛎caspase-8基因的凋亡及免疫功能作出解释，并为无脊椎动物凋亡信号规律提供理论基础和科学价值。论文的主要内容及结论如下： 1. 牡蛎生长与营养品质性状的遗传解析（1）生长与营养品质性状全基因组关联分析将采自世界5个国家23个地点的长牡蛎，与青岛当地的一个父本杂交，同步构建了427个半同胞家系，子一代在青岛同海域同环境养殖。一方面，通过全基因组重测序对母本进行基因分型，另一方面用子一代的表型均值代表母本，在牡蛎1.5岁时采集生长、糖原、氨基酸、脂肪酸等表型信息，通过全基因组关联分析，其中19性状一共找到33个阳性信号，进一步通过LD (Linkage disequilibrium)区间内的基因解读，鉴定出一批与性状相关的关键基因，尤其是脂肪酸性状关联到的直接参与脂肪酸代谢及调控的相应基因，如NFYA、CYP7A1等，具极大的分子育种价值。（2）遗传图谱构建与生长、营养品质性状QTL（Quantitative trait locus）定位分别以昌黎和青岛长牡蛎个体为母本和父本，构建全同胞家系，并从中随机选取169个体，结合双酶切GBS（Genotyping by sequencing）等方法构建遗传图谱。5024个单倍型标记均匀分布于整合图谱的10条连锁群，图谱总长1982.07 cM，独特位点平均间距0.68 cM，是目前牡蛎中上图标记最多、密度最大图谱。同时，图谱与基因组共线性很高，利用图谱首次将约70%的基因组scaffold定位于连锁群上。此外，QTL定位结果显示，其中17个性状共检测到41个QTL，区间内既鉴定出已报道与生长性状显著相关的基因，也找到许多直接参与氨基酸、脂肪酸代谢调控和转运的基因，其中，生长、氨基酸和脂肪酸性状共6个候选基因（AMY、BMP1、P5CS、GR、ADAMTS和DYRK）经验证可以通过mRNA表达水平的变化来影响相应性状，这6个基因可作为生长和营养品质性状分子育种的重要目标。总结来说，生长与营养品质性状的全基因组关联分析与连锁分析均为性状的遗传改良提供大量候选标记与关键基因，两者相辅相成，相互补充。结合基因功能研究手段，候选基因影响性状的遗传机制将进一步坐实。牡蛎虽具极高的营养价值，但氨基酸、脂肪酸等营养物质含量的遗传解析实属首次，所作工作具极大的产业价值，尤其GWAS中找到的脂肪酸性状相关4个基因以及连锁分析鉴定的6个在mRNA表达水平上显著影响性状的基因，都将作为营养品质等重要经济性状分子育种的重要目标，加速分子育种进程。 2. 长牡蛎CgCaspase8-2基因功能研究本研究克隆了首个长牡蛎caspase-8基因CgCaspase8-2，其具有典型的caspase-8结构域及保守的“QACQG”凋亡酶活性位点。序列比对及进化分析表明，CgCaspase8-2与其他物种caspase-8之间的一致性较低，在进化树上属于无脊椎动物分支。亚细胞定位结果显示，CgCaspase8-2蛋白在胞质中表达发挥活性，且HEK293T细胞转染实验表明，过表达CgCaspase8-2会诱导细胞死亡。但因为CgCaspase8-2不与上游接头分子CgFADD相互作用，所以推测其并不参与外源性凋亡通路。需要特别指出的是，CgCaspase8-2基因在免疫器官鳃和血淋巴中显著高表达，揭示其潜在的免疫功能，进而通过该基因在长牡蛎经受溶菌弧菌和poly (I:C)病菌刺激后的表达分析，证明其具有特异性的抗病毒而非抗菌免疫功能。
其他摘要	Oysters are known as “The Milk of the sea”, they are rich in nutrition, such as glycogen, glutamate, taurine, zinc, selenium, EPA, and DHA. Various nutritional products of the oyster further verified its nutritional value. However, problems like germplasm degradation and unreasonable farming mode, had been a barrier, and resulted in the low profits of the oyster industry. Because of which, breeding process of oysters towards fast-growing, high condition index, and high nutrtion, should be paid more attention. Apart from growth and nutritional traits, summer mortality also limited the development of the oyster industry. Because of which, apoptosis caused wide attention as its role in immune defense, homeostasis, etc.. As an initiator caspase, caspase-8 plays a key role in the extrinsic apoptotic pathway of the vertebrate. For the invertebrate, the apoptotic pathway varied with the species. The problem that whether extrinsic apoptotic pathway is conserved in Crassostrea gigas (C. gigas) and what’s the role of caspase-8 in C. gigas needs further study. Crassostrea gigas, also named the Pacific oyster, is a vital economic shellfish that cultured all over the world, and meantime the major cultivated shellfish of Huanghai and Bohai Sea of China. In this study, we take growth and nutritional traits, as well as caspase-8 gene of C. gigas as research object. On one hand, we constructed specific population and family, and detected the association between molecular markers and traits by genome-wide association study (GWAS) and linkage analysis. Detected traits include growth and nutritional traits, the latter includes glycogen, amino acids, and fatty acids. Our purpose is providing candidate loci and causative genes for molecular breeding, and laying the foundation for the genetic parsing of growth and nutritional traits in C. gigas. On the other hand, we studied the function of caspase-8 gene in C. gigas. We detected its interaction with upper protein in the pathway, analyzed its expression pattern among different tissues and developmental stages, and towards bacteria and virus challenge. This work will help explain the apoptosis and immune function of caspase-8 in C. gigas, and provide basis for the studing of apoptotic process of the invertebrate. The main contents and conclusions are as follows: 1. Genetic parsing of growth and nutritional traits of the oyster. (1) Genome-wide association study of growth and nutritional traits. We constructed 427 sub-families by hybridzing the maternal oysters collected from five countries and 23 locations with the same paternal oyster of Qingdao, Shandong Province. All the families were cultivated in the same environment in Qingdao. We genotyped all the maternal oysters by whole-genome-resequencing, and detected the average phenotype values of the F1 family to represent the maternal oyster. Finally, we identified 33 significant positive signals of 20 traits, and found some causative genes that were associated with the traits by gene annotation of LD (Linkage disequilibrium) interval. Candidate genes associated with fatty acid traits, like NFYA and CYP7A1, can directly participate in the fatty acid metabolism and regulation, and show great value of molecular breeding. (2) Genetic map construction and quantitative triat loci (QTL) mapping of growth and nutritional traits. We randomly selected 169 individuals from the full-family constructed by hybridzing maternal oyster from Changli and paternal oyster from Qingdao. Using double-digest genotyping by sequencing (ddGBS) method, we constructed a high-density genetic map. The consensus map contained 5024 evenly distributed markers on ten linkage groups (LG). The map length was 1982.07 cM, with an average distinct marker interval of 0.68 cM, thus representing the densest genetic map produced for C. gigas. According to the high collinearity between the consensus map and the oyster genome, 1574 scaffold (about 70%) of the genome of C. gigas was first anchored to the LGs of the consensus map. Using this map, we also conducted QTL analysis for growth and nutritional traits. Overall, 41 QTLs were detected for 17 traits. Candidate genes identified in the QTL interval include both genes related to growth traits and genes participating in amino acid and fatty acid metabolism and regulation. Of which, six candidate genes could influence phenotype by regulating mRNA expression levels, and thus are key targets for molecular breeding of growth and nutritional traits. These six genes include AMY、BMP1、P5CS、GR、ADAMTS, and DYRK. In summary, both GWAS and QTL mapping for growth and nutritional traits provided lots of valuable markers and candidate genes for molecular assisted selection (MAS). Combined with gene function studying method, genetic mechanism underlying related traits will be clarified. Besides, it’s the first time to perform genetic parsing of amino acid and fatty acid traits in C. gigas, despite its high nutritional value. Our work can directly provide benefits for the oyster industry. Candidate genes both identified in fatty acid peak SNP linkage interval by GWAS and identified by linkage analysis that can influence phenotype by changing mRNA expression levels are key objects of molecular breeding of important economic traits of the oyster, and will promote the process of molecular breeding. 2. Functional characterization of CgCaspase8-2. In this study, we cloned the first caspase-8-like gene (CgCaspase8-2) of C. gigas. The deduced CgCaspase8-2 protein contained typical domains and the conserved pentapeptide motif ‘QACQG’ of caspase-8. Phylogenetic analysis indicated that CgCaspase8-2 was clustered with initiator caspases in the invertebrate subgroup, but the similarity between CgCaspase8-2 and other invertebrate caspase-8s was low. CgCaspase8-2 protein was localized in the cytoplasm, and over-expression of CgCaspase8-2 in HEK293T cells induced cell death, suggesting a role in apoptosis. However, CgCaspase8-2 didn’t combine with the upper adapter protein (CgFADD) of the pathway, so we suggested it didn’t take part in the extrinsic apoptotic pathway. Quantitative real-time PCR results demonstrated that CgCaspase8-2 was highly expressed in the hemolymph and gill, suggesting its underlying immune function. Furthermore, CgCaspase8-2 gene expression analysis in response to bacterial and poly (I:C) challenge showed that CgCaspase8-2 was specifically involved in immune response against virus rather than bacteria.
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/136525
专题	实验海洋生物学重点实验室
作者单位	1.中国科学院海洋研究所 2.中国科学院大学
推荐引用方式 GB/T 7714	李春燕. 牡蛎营养品质等重要经济性状的遗传定位与基因解析[D]. 北京. 中国科学院大学,2017.

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