长牡蛎糖原等品质性状的遗传基础与分子机制研究

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IOCAS-IR > 实验海洋生物学重点实验室

	长牡蛎糖原等品质性状的遗传基础与分子机制研究
	刘圣
学位类型	博士
导师	张国范
	2019-05-10
学位授予单位	中国科学院大学
学位授予地点	中国科学院海洋研究所
学位名称	农学博士
关键词	牡蛎营养品质糖原遗传基础分子机制
摘要	长牡蛎（Crassostrea gigas），亦称太平洋牡蛎，是世界上重要的经济贝类。虽然我国的牡蛎养殖规模和产量多年都稳居世界首位，占世界产量的4/5以上，但出口量却不足世界贸易量的5%。其主要原因是我国牡蛎产品品质偏差。牡蛎产品品质的评价指标除体尺规格和壳型等外，最重要的指标就是肥满度。肥满度实际上就是牡蛎软体部营养物质的相对含量，包括蛋白质、糖原、脂肪及一些微量元素，如锌（Zn）和硒（Se）等，而其中的糖原，既直接与牡蛎的肥满度有关，也与牡蛎软体部色泽和“蛎”味密切相关。过去人们大多仅了解牡蛎的糖原等物质含量会受到营养环境和生殖过程等的影响，但对其遗传基础却知之甚少。本研究1）首先探讨了短期饥饿对全软体部营养物质含量的影响及全软体部和单独的组织营养成分季节变化，还在不同的组织中检测了糖原代谢相关基因的表达，以阐明这些基因如何参与到糖原代谢途径，可以为牡蛎糖原含量的改良提供更多信息。2）其次，评估了营养品质性状的遗传力，探究了不同营养品质性状之间的遗传和表型相关性，以及生长性状与营养品质性状之间的相关性，以确定品质性状合适的的选择育种策略，并分析了通过生长性状对品质性状进行间接选育的可行性。3）再次，结合正向遗传学和反向遗传学的手段以研究糖原含量全基因组关联分析得到的候选基因（蛋白磷酸酶1调节亚基3B（PPP1R3B））的功能以及其与糖原含量的关系。4）最后，基于长牡蛎高密度芯片对肥满度、壳型、生长等重要经济性状进行了全基因组关联分析，以期找到影响这些性状的重要基因及SNP位点。主要结果及结论如下： 1. 长牡蛎营养品质性状的特征研究本研究分析了牡蛎短期饥饿和不同季节样品的营养物质含量的变化，发现短期饥饿（50小时）没有显著改变糖原，蛋白质或脂肪含量（p> 0.05）。不同季节样品分析表明，秋季和冬季是糖原和脂肪积累的主要阶段，海水温度以及蛋白质含量与糖原含量呈负相关。2月到4月，整个软体部的糖原含量较高且稳定，并且与条件指数正相关。与鳃或闭壳肌相比，性腺，唇瓣和外套膜中的糖原含量较高。参与糖原代谢的蛋白质编码基因（糖原合成酶，糖原磷酸化酶，糖原脱支酶和糖原分支酶）的相对表达量与相应组织中的糖原含量密切相关。性腺中的糖原含量受糖原代谢和糖酵解途径基因（果糖6-磷酸激酶，磷酸甘油酸激酶，丙酮酸激酶，己糖激酶和葡萄糖转运蛋白）的调节，并且储存的糖原很可能是配子发生的主要能量来源。这些发现有益于牡蛎水产养殖管理和品质性状的改良，并扩大我们对牡蛎中糖原代谢的认知。 2．长牡蛎营养品质的遗传参数估计使用巢式平衡设计构建了64个全同胞家系。最后，通过18个全同胞家系，包含了9个半同胞家系，其中每个包含2个全同胞家系的个体取样，进行了遗传力估计。糖原，蛋白质，脂肪，Zn和Se含量的狭义遗传力估计值分别为0.29±0.02,0.38±0.02,0.58±0.08,0.02±0.02和0。糖原和蛋白质含量（-0.95±0.004）之间以及脂肪和蛋白质含量（-0.59±0.05）之间存在强的负遗传相关，且脂肪和糖原含量之间存在正相关（0.16±0.06）。壳高和营养品质性状之间观察到弱的遗传和表型相关性（r < 0.2）。这些数据表明糖原，蛋白质和脂肪含量可以通过选育加以改良，但糖原及脂肪不能与蛋白质一起选择选育。此外，通过选择生长性状来进行营养品质性状的间接选择育种是困难的,该研究为牡蛎品质性状育种策略的开发提供了重要信息。 3. PPP1R3B在长牡蛎调控糖原含量中的功能研究首先，克隆了CgPPP1R3B全长并对其功能进行了研究。CgPPP1R3B在不同组织和不同季节样品中的基因表达量与糖原含量密切相关。该基因体内RNA干扰（RNAi）实验表明，实验组降低了的CgPPP1R3B表达水平可能导致糖原含量低于对照组。免疫共沉淀（Co-IP）和酵母双杂交（Y2H）实验结果表明CgPPP1R3B可以与其催化亚基CgPPP1C以及糖原合酶（CgGS）和糖原磷酸化酶（CgGP）相互作用。体外蛋白-糖原共沉淀分析表明，CgPPP1R3B蛋白可直接与糖原分子结合，这些结果表明CgPPP1R3B蛋白密切参与糖原代谢。此外，在该基因中精确定位了13个SNP位点的相对位置。在一个独立的野生群体中，13个SNP中的10个被证实与糖原含量显著关联（n = 288，p <0.05）。具有高糖原含量的牡蛎个体的CgPPP1R3B表达水平显著高于具有低糖原含量的牡蛎（n=20），并且四个关联的SNP的不同基因型个体与CgPPP1R3B基因表达水平与显著相关（p <0.05），该数据表明关联的SNP可能通过调节CgPPP1R3B表达来控制糖原含量。这些结果表明CgPPP1R3B是糖原代谢调节的重要基因，并且该基因的相关SNP和优势基因型组合可用于牡蛎糖原含量的分子标记辅助育种。 4.长牡蛎壳型等经济性状的全基因组关联分析利用青岛胶南海域采集的288个野生个体，测量壳型性状（壳高/壳长），出肉率（软体重/全湿重）,条件指数（CI, 软体干重/干壳重）以及壳高，壳长，壳宽，全湿重，软体重，软体干重等性状。使用190K高密度芯片对SNP位点进行分型，使用R中GAPIT软件包进行全基因组关联分析。 GWAS结果表明壳型、壳宽、条件指数关联到一批SNP位点，部分位点位于能量代谢相关基因上。壳高，湿重和软体重均关联到一个显著SNP（scaffold731_92759），该位点位于一个丝氨酸蛋白酶抑制剂基因的蛋白质编码区，为非同义突变（Glu→Val）, 丝氨酸蛋白酶抑制剂被认为参与代谢过程。此项结果为壳型等品质性状的选育提供了潜在的分子标记
其他摘要	Pacific oyster （Crassostrea gigas）is an important economic shellfish in the world. Although China's oyster farming scale and output have been ranked first in the world for many years, accounting for more than 4/5 of world production, but the export volume is less than 5% of world trade. The main reason is that the overall quality of oyster products in China is low. In addition to body size specifications and shell shape, the most important indicator of oyster product quality is the fatness. The fatness is actually the relative content of nutrients in the oyster soft body, including protein, glycogen, lipid and some trace elements such as zinc (Zn) and selenium (Se). And the glycogen is not only directly related to degree of oysters’ fatness, but also to the flesh color and taste of oysters. In the past, most researchers thought that the content of glycogen and other substances in oysters were affected by the nutrient environment and reproductive process, but their genetic basis was poorly understood. In this study， 1）we firstly explored the effects of short-term starvation on nutrient content and seasonal changes in nutrients in the whole soft body and separate tissues. In addition, the expression of glycogen metabolism-related genes was detected in different tissues to elucidate how these genes are involved in the glycogen metabolic pathway, providing more information on the improvement of oyster glycogen content. 2）Secondly, we estimated the heritability of nutritional quality traits, explored the genetic and phenotypic correlations between nutritional quality traits, and the correlation between growth traits and nutritional quality traits to determine appropriate breeding strategies for quality traits. And the feasibility of indirect selection of quality traits by growth traits was analyzed. 3）Then, this study combines forward genetics and reverse genetics methods to study the function of the candidate gene (protein phosphatase 1 regulatory subunit 3B (PPP1R3B)) obtained by genome-wide association analysis for glycogen content. 4）Finally, based on the high-density DNA chip of the Pacific oyster, a genome-wide association analysis was carried out on important economic traits such as shell shape and condition index, in order to find important genes and SNP locus that affect these important traits. The main results and conclusions are as follows 1. Characteristics of nutritional quality traits in the Pacific oyster We conducted corresponding studies and found that short time starvation (50 hours) did not significantly (p>0.05) affected the content of glycogen, protein and lipid. The seasonal variation assay shown that autumn and winter were vital stage for glycogen and lipid accumulation in the study area, seawater temperature and protein content were nearly reversely related to glycogen content. Glycogen content of the whole flesh were higher from Feb to Apr and it is positively related to condition index. Glycogen content were relatively higher in gonad, labial palp and mantle instead of gill or adductor muscle, relatively expression of glycogen metabolic gene (glycogen synthase, glycogen phosphorylase, glycogen debranching enzyme and glycogen branching enzyme) were closely related to glycogen content of corresponding tissues. Glycogen content in gonad were regulated by known glycogen metabolic and glycolysis pathway genes (6-phosphofructo kinase, phosphoglycerate kinase, pyruvate kinase, hexokinase, glucose transporters), stored glycogen were main energy utilized for gametogenesis. These finding are beneficial for oyster aquaculture management and glycogen improvement as well as expanding our understanding of glycogen metabolism in oysters. 2. Heritability estimates for nutritional quality-related traits of the Pacific oyster In this study, 64 full-sib families were generated using a nested mating design. Finally 18 full-sib families, of which nine half-sib families, with each containing two full-sib families, were sampled for heritability estimate. The narrow-sense heritabilities of glycogen, protein, lipid, Zn, and Se contents were 0.29±0.02, 0.38±0.02, 0.58±0.08, 0.02±0.02, and 0, respectively. Negative genetic correlations existed between both glycogen and protein contents (−0.95±0.004) and between lipid and protein contents (-0.59±0.05), whereas a positive correlation was observed between lipid and glycogen contents (0.16±0.06). Weak genetic and phenotypic correlations (r <0.2) were observed between shell height and nutritional quality traits. These data revealed that glycogen, protein, and lipid contents can be chosen in selective breeding program, but glycogen and lipids cannot be selected together with protein. Furthermore, performing indirect selective breeding for quality traits by selecting growth related traits is impossible. This study provides information for the development of breeding strategies for oyster quality traits. 3. Association and functional analyses of PPP1R3B in the regulation of glycogen content Firstly, full-length CgPPP1R3B was cloned and its function was characterized. The gene expression profiles of CgPPP1R3B in different tissues and seasons showed a close relationship to glycogen content. RNA interference (RNAi) experiments of target gene in vivo showed that decreased CgPPP1R3B levels resulted in lower glycogen contents in the experimental group than in the control group. Co-immunoprecipitation (Co-IP) and yeast two-hybrid (Y2H) assays indicated that CgPPP1R3B can interact with CgPPP1C, glycogen synthase (CgGS) and glycogen phosphorylase (CgGP), thus participating in glycogen metabolism. Co-sedimentation analysis in vitro demonstrated that the CgPPP1R3B protein can bind to glycogen molecules directly, and these results indicated the conserved function of the CgPPP1R3B protein compared to that of mammals. In addition, thirteen SNPs were precisely mapped in this gene. Ten of the thirteen SNPs were confirmed to be significantly (p<0.05) related to glycogen content in an independent wild population (n=288). The CgPPP1R3B levels in oysters with high glycogen content were significantly higher than those of oysters with low glycogen content, and gene expression levels were significantly associated with various genotypes of four associated SNPs (p<0.05). The data indicated that the associated SNPs may control glycogen content by regulating CgPPP1R3B expression. These results suggest that CgPPP1R3B is an important gene for glycogen metabolic regulation and the associated SNPs of this gene are potential markers for oyster molecular breeding for glycogen content. 4. Genome-wide association analysis of important economic traits based on high-density DNA chips of the Pacific oyster A wild population including 288 individuals were collected along the coast of Qingdao. Shell shape, meat yield, condition index (CI), shell height, shell length, shell width, body weight, soft body weight, dry soft body weight and were measured. SNP were genoyped using a 190K high-density DNA chip and genome-wide association analysis was performed using the GAPIT toolkit in R. The GWAS results indicated that several SNP were associated with the shell shape, shell width and condition index, and some loci were located on energy metabolism related genes.Shell height, body weight and soft body weight were all associated with a significant SNP (scaffold731_92759), which located in the protein coding region of a Kazal type serine protease inhibitor. It is believed that Kazal type serine protease inhibitor participated in metabolic processes. This result provides potential molecular marker for the selection of economic traits such as shell shape and condition index.
学科领域	水产养殖学
学科门类	农学::水产
URL	查看原文
语种	中文
引用统计
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/156822
专题	实验海洋生物学重点实验室
推荐引用方式 GB/T 7714	刘圣. 长牡蛎糖原等品质性状的遗传基础与分子机制研究[D]. 中国科学院海洋研究所. 中国科学院大学,2019.

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刘圣-0516-长牡蛎糖原等品质性状的遗（7729KB）	学位论文		暂不开放	CC BY-NC-SA