实验海洋生物学重点实验室知识库

牡蛎是全球范围内广泛分布的一种重要海产资源，是目前全球贸易量最大的双壳类软体动物。因其营养价值丰富，高蛋白低脂肪，富含牛磺酸、氨基酸、维生素等多种营养物质；味道鲜美，质地柔软细嫩而成为世界流行性消费贝类。中国沿海有30多种牡蛎分布，长牡蛎（Crassostrea gigas）是黄渤海区域的主要养殖贝类。其中糖原作为长牡蛎“蛎味”的主要呈味物质，其含量不仅是牡蛎相对营养价值评估的重要指标之一，还与长牡蛎的繁殖、生长、抗逆等各种生理活动密切相关。长牡蛎糖原含量在个体间差异非常大，然而，迄今为止，长牡蛎糖原含量差异的遗传机制研究却鲜有报道。本研究首次利用多组学联合分析的手段绘制了长牡蛎糖原含量差异的调控网络，并结合糖原含量关键调控基因的功能研究进一步解析了长牡蛎糖原含量差异的分子基础，从而为高品质长牡蛎分子育种提供新思路。

借助实验室已经构建完成的同质化养殖群体，从185只长牡蛎中筛选出糖原极端表型个体，高糖原和低糖原牡蛎各15只，对两组牡蛎进行转录组和代谢组学比较分析。同质化养殖群体可以尽可能的减少环境因素的影响。经过分析在高糖原组和低糖原组中鉴定出1888个差异基因和75个差异代谢物。通过转录组和代谢组的联合分析，富集到了27个代谢通路，绘制了长牡蛎糖原含量差异的调控网络，并得到以下四个结论。（1）长牡蛎糖原含量与其脂肪酸降解能力有关。通过相关性分析发现长牡蛎糖原和脂肪酸含量呈正相关性。在高糖原牡蛎中参与脂肪酸生物合成的水解酶OLAH和PPT高表达，以及棕榈酸和肉豆蔻酸等脂肪酸的高含量说明高糖原牡蛎具有更高的脂肪酸累积水平；另一方面，脂肪酸降解限速酶CPT在高糖原牡蛎中的低表达，诱导了长链脂酰-CoA含量的降低，进而通过提升糖原合酶的活性促进糖原的积累。（2）长牡蛎氨基酸含量和其转化为葡萄糖的能力与糖原含量显著相关。磷酸烯醇丙酮酸羧激酶(PEPCK)和丙酮酸高表达表明了高糖原牡蛎具有更高糖异生能力，同时生糖氨基酸的高含量，增加了生糖氨基酸通过转化成相应的酮酸进而进入糖异生途径的水平；进一步增加了葡萄糖的从头合成，从而促进高糖原牡蛎中糖原的积累。（3）高糖原个体具有更高的能量代谢水平。糖酵解过程中限速酶己糖激酶(HK)和丙酮酸激酶(PK)的高表达，以及TCA循环过程中苹果酸脱氢酶(MDH)和丙酮酸酯羧化酶(PYC)的高表达与各种中间代谢产物的高含量，提示着高糖原含量牡蛎有着更高的能量代谢水平和抗逆能力。（4）高糖原个体拥有更高的抗氧化能力。已经有文献报道，高糖原牡蛎具有更高的抗氧化和抗逆能力，但是其中的机制尚不明确。转录组学分析结果显示现高糖原牡蛎中拥有高的GPX5的表达水平，这可能提示着，GPX5抗氧化酶是参与长牡蛎抗逆反应的一个重要因子。

通过转录组分析结果筛选出一个候选基因——生糖蛋白（glycogenin），在高低糖原两组间表达量差异显著。尽管长牡蛎中参与糖原代谢的糖原合酶以及糖原磷酸化酶已经有了基本的研究，但是生糖蛋白作为糖原合成起始酶在长牡蛎中的研究是空白的。因此本研究对长牡蛎生糖蛋白（CgGN）与糖原含量的关系进行了探究，并初步阐释了CgGN在长牡蛎糖原合成中发挥的作用。我们通过分子克隆技术获得了生糖蛋白的全长，发现长牡蛎生糖蛋白在mRNA加工过程中发生外显子跳跃，形成了3种可变剪切形式。哺乳动物中存在两种生糖蛋白（Glycogenin-1和Glycogenin-2），通过系统进化树分析鉴定其为Glycogenin-1并且3种可变剪切形式均聚类到无脊椎动物中。之后，利用q-RT-PCR技术，发现CgGN在闭壳肌中表达量最高，在闭壳肌和性腺中的相对表达量随季节变化而变化，并且和糖原含量呈现出明显的相关性。随后，为了鉴定长牡蛎生糖蛋白是否能够发生自糖基化进而起始糖原的合成，本研究借助基因的定点突变技术研究CgGN的自糖基化位点。序列比对表明CgGN的Tyr-200和哺乳动物报道的Tyr-194糖基化位点相似，通过Western blot技术发现Tyr-200和邻近的Tyr-202突变成Phe（苯丙氨酸）都会造成蛋白条带分子量的减小，这意味着点突变后的CgGN发生了去糖基化，表明Tyr-200和Tyr-202为CgGN的糖基化位点，对CgGN的自糖基化具有非常重要的作用。长牡蛎生糖蛋白发生自糖基化后，是否会和糖原合酶发生相互作用继续完成糖原的合成。本研究借助哺乳动物细胞系，利用免疫共沉淀技术，证明了CgGN三种可变剪切形式的重组蛋白能够在体外和糖原合酶CgGS重组蛋白发生蛋白之间的直接相互作用。此外，通过亚细胞定位技术发现CgGN和CgGS都在细胞质中表达，这提示着长牡蛎糖原合成是由CgGN自糖基化后合成一个短的糖链引物，再和CgGS互作由CgGS继续完成后续的糖原合成。

之前的数据显示长牡蛎糖原含量和脂肪酸含量密切相关，但是其背后的分子机制还不清楚。在哺乳动物中游离脂肪酸受体可以调控糖原含量，在长牡蛎中存在唯一的游离脂肪酸受体（CgFFAR4），本研究对其基本功能及其与糖原含量的关系进行了探究。CgFFAR4全长为1098bp并且包含7次跨膜的G蛋白藕联受体结构域。本研究利用q-RT-PCR技术对CgFFAR4在长牡蛎不同发育时期和不同组织的表达模式进行了探究，发现CgFFAR4在长牡蛎面盘发育关键时期D-型幼虫时期表达水平骤升，面盘在长牡蛎幼虫中发挥协助摄食的作用；CgFFAR4在各个组织中都有表达，但是在肝胰腺、肠、胃等消化器官中表达水平最高；在面对饥饿刺激时CgFFAR4的mRNA水平显著下降。这些数据都表明CgFFAR4在长牡蛎摄食和消化中发挥重要作用。为了对CgFFAR4的功能进行更深层次的研究，通过siRNA对CgFFAR4进行体内干扰实验，结果显示CgFFAR4 mRNA表达水平的降低，会造成糖原含量的下降。而CgFFAR4KD牡蛎中胰岛素受体（CIR）和胰岛素受体底物（IRS）表达水平，以及糖原合成中的糖原合酶表达水平的降低，说明CgFFAR4可能在胰岛素通路的参与下调控糖原合酶表达从而调节糖原的合成。游离脂肪酸不仅仅参与营养调控，部分脂肪酸还可以抑制机体的炎症反应。长牡蛎受到LPS刺激后，CgFFAR4的表达水平显著下降，这说明CgFFAR4参与到长牡蛎的抗逆境反应中。FFAR4的免疫功能在无脊椎动物和哺乳动物中都是保守的，尽管在进化过程中，FFAR4在不同的组织中获得了更多样的功能，但是依然保留了原始的免疫功能。

综上所述，本研究首次利用转录组和代谢组学联合的手段绘制了长牡蛎糖原含量差异的调控网络；筛选出候选基因CgGN并对其起始糖原合成的功能进行了全面研究，进一步加深了对长牡蛎糖原含量调控机制的研究。同时，本研究首次对无脊椎动物的游离脂肪酸受体在营养调控和免疫应答中的功能进行研究。本研究的的结果不仅为揭示贝类糖原合成和积累机制提供了研究基础，为揭示糖原、氨基酸、脂肪酸相关性水平提供了分子基础，同时也为软体动物营养性状的研究提供了新的思路，成为贝类分子育种基础研究的有力手段。

Oyster is an important marine fishery resource cultivated globally and the largest sector of bivalve in global trades. Oysters become the most popular shellfish because of its soft flavor and its rich nutritional value with high quality protein, taurine, amino acids, vitamins and other nutrients. There are more than 30 oysters along the coast of China, while the Pacific oyster, Crassostrea gigas is the major cultivated oysters distributed in the coast of Bohai Sea and Yellow Sea. Glycogen, which contributes to the flavor of oysters, is not only one of the important evaluation index of oyster nutritional value, but also closely associated with physiological activities such as growth, reproduction and stress tolerance. Oysters have great glycogen variation between individuals, but little is known about the molecular and chemical mechanisms underlying glycogen content differences in Pacific oysters. In this study, the molecular basis of glycogen content difference in Pacific oysters were revealed based on regulatory network via multiple-omics analysis and functional study of pivotal genes. The mechanism research of the oyster glycogen regulation can provide new insights for high-quality oyster molecular breeding.

We selected the top 10–15 high and low glycogen content individuals from 185 half--sibling oyster families raised under a consistent environmental condition which were used to conduct transcriptome and metabolome analysis. Homogeneous culture helps minimize the impact of environment factors. We identified 1888 differentially-expressed genes, seventy-five differentially-abundant metabolites, which are part of twenty-seven signaling pathways that were enriched using an integrated analysis of the interaction between the differentially-expressed genes and the differentially-abundant metabolites. Accordingly, the following findings were based on the oyster glycogen regulation network constructed by transcriptome and metabolome analysis. (1). Oyster glycogen content is close related to the fatty acid degradation capacity. A correlation analysis showed oyster glycogen content had a positive correlation with the free fatty acids. Simultaneously, in high-glycogen content oysters, high expression of OLAH and PPT, which participate in the release of fatty acids from the fatty acid synthase complex, together with abundant levels of myristic acid and palmitic acid implicate the higher fatty acid accumulation ability in high glycogen oysters. While in low-glycogen oysters, a high level of CPT2 expression increases flux through the fatty acid degradation pathway, causes a lowering of the free fatty acid levels and a reciprocal increase in long chain fatty acyl- CoA levels that then suppress glycogen accumulation. (2). Amino acid content and conversion efficiency to glucose play crucial role in glycogen content. We observed an increase in the abundance of glucogenic amino acids, as well as increase in the level of pyruvic acid and PEPCK expression which catalyzes the first committed step in gluconeogenesis. Taken together, we speculate that this increase in abundance of glucogenic amino acids (i.e. gluconeogenic substrates) coupled with an increase in gluconeogenic capacity leads to increased de novo production of glucose and ultimately to increased glycogen synthesis. (3). Oysters with higher glycogen content exhibited increased energy metabolism. The high level of expression of the  HK(Hexokinase) and PK (Pyruvate kinase) genes involved in glycolysis and Malate dehydrogenase (MDH) and pyruvate carboxylase (PYC) that participate in the TCA cycle in high-glycogen oysters reveals that these oysters have a higher energy metabolism compared to low-glycogen content oysters and implies that they have a greater resistance to stress.(4). Higher glycogen content oyster manifests as a higher anti-adversity ability. Although it has been widely recognized that glycogen participates in stress resistance in the oyster, the molecular mechanism behind this has not been identified. Making use of integrated analysis of transcriptome and metabolome, we suggest that GPX5 may be an important enzyme in the mechanism by which glycogen contributes to stress resistance in oysters.

We selected glycogenin as candidate gene through transcriptome analysis. Although the glycogen synthase and glycogen phosphorylase involved in glycogen metabolism in Pacific oysters have been basically studied, the study of glycogenin as the initiator of glycogen synthesis is absent. Therefore, we identified and cloned C. gigas glycogenin (CgGN) to investigate its role in the initiation of glycogen synthesis and the correlation with glycogen content. Alignment of CgGN cDNA with the genomic sequence revealed that CgGN have three isoforms containing alternative exon regions. The phylogenetic tree showed that vertebrate and invertebrate glycogenin clustered separately in two distinct groups and CgGN belongs to glycogenin-1. With the aid of q-RT-PCR, we demonstrate that CgGN expression varied seasonally in the adductor muscle and gonadal area and was the highest in the adductor muscle. CgGN expression was significantly related with glycogen content in oysters. Furthermore, a site-directed mutagenesis experiment was performed to investigate the glycosylation site of CgGN. Sequence alignment indicates that Tyr-200 in CgGN corresponding to Tyr-195 of mammalian glycogenin-1, known to be the site of carbohydrate attachment. Differences in molecular weight among the Western blotting bands revealed that the Tyr200Phe and Tyr202Phe mutations could affect CgGN autoglycosylation. After autoglycosylation, CgGN can interact with glycogen synthase (CgGS) to complete glycogen synthesis. Additionally, subcellular localization analysis showed that CgGN isoforms and CgGS were located in the cytoplasm. In conclusion, CgGN has a glycosylation site corresponding to mammalian glycogenin-1 and can interact with CgGS to complete glycogen synthesis.

We used q-RT-PCR to detect the expression patterns of CgFFAR4 in different developmental stages and tissues of Pacific oysters. CgFFAR4 expression increased sharply at the D-larval stage which is the critical moment of velum development. CgFFAR4 express in various tissues while was highly expressed in hepatopancreas, stomach, and intestine, which are associated with the oyster digestion tract. Additionally, relative expression of FFAR4 to EF (elongation factor) in C. gigas hepatopancreas and nerves significantly declined after fasting. All these data demonstrated that CgFFAR4 have a critical role in oyster ingestion and digestion. To further elucidate CgFFAR4 function, we performed CgFFAR4 knock down in oysters. Reduced glycogen content after CgFFAR4 knockdown revealed that it is involved in regulation of fatty acid and glycogen content. Decreased gene expression of insulin receptor, insulin receptor substrate and glycogen synthase in oysters indicates that FFAR4 is involved in the regulation of glycogen and FFA content via insulin pathway. Additionally, FFAs were not only nutrients but also contributes to the anti-inflammatory response. In Pacific oysters, we found that LPS stimulation decreased the expression of CgFFAR4, which may contribute to the animal’s immune response. The immune function of FFAR4 is conserved in both invertebrates and mammals.

In conclusion, this is the first time to map the regulation networks of oyster glycogen integrating transcriptome and metabolome analysis. Furthermore, we screened the candidate gene CgGN and comprehensively explain its function of glycogen synthesis initiation. Meanwhile, FFAR4 function was firstly studied on nutrition regulation and immune response in invertebrate. Our study has not only revealed the molecular basis of the relationship between amino acids, fatty acids, and glycogen, but also provides a molecular explanation for stress resistance in oysters. These findings provide new insights into the study of quality traits and could promote research into the molecular breeding of oysters.

第1章 引言... 1

1.1 牡蛎养殖现状... 1

1.2 牡蛎营养特征... 2

1.3 糖原研究进展... 3

1.3.1 糖原合成关键基因... 4

1.3.2 生糖蛋白研究进展... 5

1.3.3 糖原分解及相关基因... 7

1.3.4 牡蛎糖原研究进展... 8

1.3.5 糖原与其他营养性状的关系探究... 9

1.4 多组学联合分析... 9

1.4.1 基因组学研究... 10

1.4.2 转录组学研究... 10

1.4.3 蛋白质组学研究... 10

1.4.4 代谢组学研究... 11

1.4.5 转录组学和代谢组学联合分析... 11

1.5 游离脂肪酸受体4（FFAR4）... 12

1.5.1 游离脂肪酸概述及功能... 12

1.5.2 双壳贝类脂肪酸研究进展... 12

1.5.3 游离脂肪酸受体（FFARs）分类... 13

1.5.4 游离脂肪酸受体4（FFAR4）的功能... 14

1.6 基因的可变剪切... 15

1.6.1 可变剪切概述... 15

1.6.2 可变剪切的分类... 16

1.6.3 基因可变剪切的功能... 17

1.7 研究的目的和意义... 18

1.7.1 本研究主要目的... 18

1.7.2 研究的内容... 18

1.7.3 研究意义... 18

第2章 利用转录组和代谢组联合分析揭示长牡蛎糖原含量调控机制... 20

2.1 实验材料与方法... 20

2.1.1 实验材料... 20

2.2.2 糖原含量测定... 20

2.2.3 转录组RNA提取... 21

2.2.4 转录组文库构建与测序... 22

2.2.5 序列比对和基因表达量计算... 22

2.2.6 差异表达基因的功能分析... 23

2.2.7 代谢物的提取... 24

2.2.8 代谢物的上机检测... 24

2.2.9 代谢物的多元统计分析... 25

2.2.10 高低糖原牡蛎之间差异代谢物的鉴定和通路分析... 25

2.2.11 转录组和代谢组的联合分析... 25

2.2 实验结果... 25

2.2.1 长牡蛎糖原含量描述性统计分析... 26

2.2.2 长牡蛎糖原含量差异的转录组学分析... 27

2.2.3 长牡蛎糖原含量差异的代谢组学分析... 29

2.2.4 长牡蛎糖原含量差异的代谢组学和转录组学联合分析... 35

2.3讨论... 40

2.3.1 长牡蛎糖原含量与其脂肪酸降解能力密切相关... 40

2.3.2 氨基酸向葡萄糖的转化效率在糖原含量调控中发挥重要作用... 41

2.3.3 高糖原牡蛎具有更强的能量代谢水平... 41

2.3.4 高糖原牡蛎有更强的抗逆能力... 42

2.4 本章小结与展望... 42

第3章 长牡蛎生糖蛋白（CgGN）的克隆鉴定及其对糖原含量调控机制研究... 44

3.1 实验材料... 44

3.1.1 实验动物... 44

3.1.2 菌株、细胞及载体... 44

3.1.3 抗体与试剂... 44

3.1.4 实验仪器... 45

3.1.5 引物信息... 46

3.2实验方法... 48

3.2.1 长牡蛎不同组织、不同季节、不同发育时期样本获取... 48

3.2.2 长牡蛎组织RNA提取及cDNA获取... 48

3.2.3 利用RACE技术获得目的基因cDNA全长序列... 49

3.2.4 目的基因序列分析... 49

3.2.5 长牡蛎基因表达水平分析... 50

3.2.6 糖原含量测定... 50

3.2.7 真核表达载体构建与质粒纯化... 51

3.2.8 目的基因载体定点突变... 51

3.2.9 哺乳动物细胞系的培养、传代与转染... 52

3.2.10 重组蛋白的亚细胞定位... 52

3.2.11 样品蛋白质的提取与免疫印迹检测（Western blot）... 53

3.2.12 免疫共沉淀（Co-IP）... 53

3.3 实验结果... 54

3.3.1 长牡蛎生糖蛋白基因（CgGN）克隆与多种可变剪切形式的鉴定分析... 54

3.3.2 CgGN基因序列分析... 58

3.3.3 CgGN组织特异性和季节特异性表达分析... 59

3.3.4 CgGN mRNA表达模式与长牡蛎糖原含量和糖原合酶的表达相关... 61

3.3.5 CgGN的自糖基化及关键糖基化位点... 63

3.3.6 CgGN定位于细胞质中并可以于CgGS相互作用... 65

3.4 讨论... 68

3.5 本章小结与展望... 71

第4章 游离脂肪酸受体4（CgFFAR4）在长牡蛎免疫应答和营养调控中的作用... 72

4.1 实验材料... 72

4.1.1 实验动物... 72

4.1.2 菌株与载体... 72

4.1.3 实验试剂与仪器... 72

4.1.4 引物及siRNA序列信息... 72

4.2 实验方法... 73

4.2.1 长牡蛎LPS刺激与饥饿刺激... 73

4.2.2 长牡蛎不同组织、不同发育时期取样... 74

4.2.3 RNA提取与cDNA获得... 74

4.2.4 目的基因全长获得... 74

4.2.5 利用siRNA对长牡蛎进行基因干扰... 74

4.2.6 长牡蛎基因表达水平分析... 74

4.2.7 糖原含量测定... 74

4.2.8 统计分析... 74

4.3 实验结果... 75

4.3.1 CgFFAR4的全长扩增与序列分析... 75

4.3.2 CgFFAR4系统发育分析... 76

4.3.3 CgFFAR4在不同组织和不同发育时期的表达模式分析... 77

4.3.4 饥饿处理对CgFFAR4表达模式影响... 78

4.3.5 LPS处理对CgFFAR4表达模式影响... 79

4.3.6 CgFFAR4调控糖原含量水平... 80

4.3.7 CgFFAR4通过胰岛素通路调控长牡蛎糖原合成... 82

4.4 讨论... 83

4.5 本章小结与展望... 86

4.5.1 本章小结... 86

4.5.2 展望... 87

参考文献... 89

附录 缩略词表... 99

作者简历及攻读学位期间发表的学术论文与研究成果... 101