实验海洋生物学重点实验室知识库

随着生活水平的提高，消费者对贝类海产品的品质和口感要求也日益提高， 这对文蛤产业提出了更高的挑战和机遇，文蛤呈味物质的遗传改良是提升文蛤品 质的一个重要方面。本研究通过对文蛤群体、不同壳色文蛤和文蛤家系呈味物质 种类及含量的特征分析，最终聚焦文蛤中两种游离氨基酸作为主要的研究性状， 一种是味道强度值（Taste Activity Value, TAV）最高，对呈味贡献最大的游离谷 氨酸（Free Glutamate, Glu）；另一种为含量较高，且在不同壳色文蛤间具有显著 差异的游离甘氨酸（Free Glycine，Gly）。本研究克隆了文蛤谷氨酸脱氢酶基因 （MpGDH）的全长 cDNA，发现了与高 MpGDH 基因表达水平和较高游离谷氨 酸含量显著相关的单倍型 H2；通过全基因组关联分析，挖掘到 103 个与游离甘 氨酸含量相关的 SNP 遗传位点和 97 个候选基因。主要实验结果如下：

1、以文蛤群体为研究对象，探究文蛤肌肉组织中游离氨基酸（Free amino acid， FAA）的种类及含量分布规律，结果显示在文蛤足肌肉中包含 15 种 FAAs，其中 游离谷氨酸具有最高的味道强度值（Taste Activity Value, TAV）为 6.46，5 种呈鲜 味氨基酸（Ala、Glu、Lys、Asp、Gly）在 15 种 FAAs 中占比最高，为 66%，变 异系数范围在 21.88%-44.82%，且呈正态分布。在此研究基础上，进一步检测了 同一群体中三种壳色文蛤的游离氨基酸、呈味核苷酸和甜菜碱含量差异，发现黑 斑壳色文蛤的总 FAA 含量和甜菜碱含量最高，且味精当量（Equivalent Umami Concentration, EUC）和 TAV 最高，暗示文蛤壳色和呈味物质的含量存在一定的 联系。

2、以文蛤的 11 个全同胞家系为研究对象，在对文蛤家系生长性状特点和文 蛤 FAA 的种类和含量进行测定的基础上，通过主成分分析、TOPSIS 分析开展了 文蛤生长和游离氨基酸含量等性状的综合评价。结果显示，家系生长性状和游离 氨基酸含量在不同家系间存在显著差异。通过主成分分析在 4 种生长性状和 15 种 FAAs 含量性状中提取到 3 个主成分，其累积方差贡献率为 92.01%，可代表 文蛤中生长性状和 FAA 含量的综合信息。T 值较高的家系依次是 22A8、22A5、 22A6、22A9、22A1，与 TOPSIS 分析结果一致。结果表明上述 5 个文蛤家系可作 为生长较快、游离氨基酸含量高的优良种质资源用于文蛤的选育工作。

3、GDH 是 Glu 代谢过程中的关键酶，并决定了 Glu 代谢途径中反应的速度 和方向。本研究克隆了文蛤 MpGDH 基因的全长 cDNA，并进一步研究了 MpGDH 基因多态性、基因表达水平、酶活性和 Glu 含量之间的关系。实验发现，MpGDH 基因表达与 Glu 含量呈显著正相关（r=0.62，P<0.05）；5 个 SNPs 与 MpGDH 基 因表达显著相关（P<0.05），基于连锁不平衡现象，我们从中构建了两个主要的单倍型。结果表明，单倍型 H2 可能是与 MpGDH 基因高表达和 Glu 含量较高相 关的有效单倍型。通过不同的家系材料验证了不同单倍型的遗传效应，结果表明 H1H2 杂合型的 MpGDH 基因表达水平显著高于纯合型 H1H1（P<0.05），研究结 果揭示了 MpGDH 基因多态性对 Glu 含量的潜在影响，为文蛤高游离谷氨酸含量 的选育提供了分子生物学信息。

4、以文蛤群体为研究对象，对 300 个文蛤进行重测序后通过全基因组关联 分析，筛选出与游离 Gly 含量显著相关的 103 个遗传位点和 97 个候选基因。本 研究重点关注了落在基因（GLDC、RB40B、OPSO、RC3H1、DPYS、MIB2、CONT、 DBF4A、CE022）编码区附近的 9 个 SNP。在 9 个基因的基因分型与个体游离 Gly 含量的相关性分析中发现，特定 SNP 个体对应的 Gly 含量显著高于其他个 体，可作为重要的候选基因进行深入研究。

With the improvement of living standards, consumers' demands for the quality and taste of shellfish and seafood products are also increasing. This presents both higher challenges and opportunities for the clam Meretrix petechialis industry. Genetic improvement of flavor substances in clams is an important aspect of enhancing clam quality. This study focused on the characterization of flavor substances and their levels in clam populations, different shell-colored clams, and biparental families of clams. Ultimately, the study narrowed down to two free amino acids as the main research trait. One is free glutamate (Glu), which has the highest TAV value and contributes the most to taste, the other is free glycine (Gly), which has a high content and significant differences between clams of different shell colors. The full-length cDNA of the clam glutamate dehydrogenase gene (MpGDH) was cloned, and a haplotype H2 significantly associated with high MpGDH gene expression levels and higher free glutamate content was discovered. Through genome-wide association analysis, 103 SNP genetic loci associated with glycine content were identified along with 97 candidate genes. The main experimental results are as follows:

1. The research subjects are clam populations; this study explores the distribution patterns of the types and levels of free amino acids (FAA) in clam muscle tissues. The results indicate that there are 15 types of FAAs present in the foot muscles of clams. Among these, glutamate exhibits the highest Taste Active Value (TAV) at 6.46. Five umami amino acids (Ala, Glu, Lys, Asp, Gly) account for 66% of the total FAA, with a coefficient of variation ranging from 21.88% to 44.82%, and they exhibit a normal distribution. Building upon this research, further investigations were conducted to detect differences in the levels of free amino acids, taste nucleotides, and betaine among three shell-colored clams within the same population. It was found that black-spotted clams have the highest total FAA and betaine content, as well as the highest Equivalent Umami Concentration (EUC) and TAV. This suggests a certain connection between clam shell color and the content of taste substances.

2. The research focuses on eleven different biparental families of clams; aimed to evaluate the growth traits and the types and levels of free amino acids (FAA) in clams. Through principal component analysis (PCA) and TOPSIS analysis, a comprehensive evaluation of clam growth and free amino acid content traits was conducted. The results revealed significant differences in growth traits and free amino acid content among different families. PCA extracted three principal components from four growth traits and fifteen FAA content traits, contributing to a cumulative variance of 92.01%, representing comprehensive information on growth traits and FAA content in clams. The families with higher T-values were ranked as follows: 22A8, 22A5, 22A6, 22A9, and 22A1, consistent with the results of the TOPSIS analysis. These findings indicate  that these five clam families can serve as excellent germplasm resources with fast growth and high free amino acid content for clam breeding.

3. GDH is involved in the production of glutamate from a-ketoglutarate and determines the speed and direction of reaction in the glutamate metabolic pathway. In this study, we cloned a full-length cDNA of MpGDH homologues in the clam Meretrix petechialis and further investigated the relationship among MpGDH gene polymorphisms, gene expression levels, enzyme activity and glutamate content traits. A significant positive correlation of MpGDH gene expression with free glutamate content was observed (r=0.62, P<0.05). The relationship of variation in MpGDH gene expression with its SNP genotype and SNP haplotype was assessed. Five SNPs were found to be significantly associated with MpGDH gene expression (P<0.05), from which we constructed two main haplotypes due to linkage disequilibrium. The results showed that haplotype H2 may be an effective haplotype associated with high MpGDH gene expression and high free glutamate content. The genetic effect of different haplotypes was validated by different family materials, which showed that MpGDH gene expression level of the H1H2 heterozygous genotype were significantly higher than those of the homozygous genotype H1H1 (P<0.05). In summary, our findings revealed the potential influence of the MpGDH polymorphism on the free glutamate content and provided molecular biological information for the selective breeding of high free glutamate content traits of M. petechialis.

4. This study using a population of clams as the research subjects, resequencing was conducted on 300 clams. Through genome-wide association analysis, 103 SNPs and 97 candidate genes significantly associated with the content of free Glycine (Gly) were identified. This study focused on 9 SNPs located near the coding regions of genes (GLDC, RB40B, OPSO, RC3H1, DPYS, MIB2, CONT, DBF4A, CE022). In the correlation analysis between genotyping of 9 genes and individual free glycine content, it was found that individuals corresponding to specific SNPs had significantly higher Glycine content compared to others, making them important candidate genes for intensive study.

第 1 章 绪论 ............................................................................................................... 1 
1.1 文蛤生物学特征及产业现状 ............................................................................. 1 
1.2 双壳贝类呈味物质和品质评价方法的研究进展 ............................................. 2 
1.2.1 双壳贝类呈味物质的种类 ................................................................................2 
1.2.2 双壳贝类品质评价方法 ....................................................................................3 
1.3 双壳贝类遗传育种方法的发展 ......................................................................... 4 
1.4 单核苷酸多态性（SNP）标记 ......................................................................... 5 
1.4.1 SNP 分型与检测 .......................................................................................... 5 
1.4.2 双壳贝类 SNP 的开发与应用 .................................................................... 6 
1.5 GWAS 在双壳贝类遗传育种中的应用 ............................................................. 9 
1.6 研究内容、目的和意义 ..................................................................................... 9 
第 2 章 文蛤群体呈味性状分析 ............................................................................11 
2.1 研究背景 ........................................................................................................... 11 
2.2 材料与方法 ....................................................................................................... 12 
2.2.1 实验材料 .................................................................................................... 12 
2.2.2 游离氨基酸含量的测定 ............................................................................ 13 
2.2.3 呈味核苷酸和甜菜碱含量的测定 ............................................................ 13 
2.2.4 味道强度值与味精当量的计算 ................................................................ 14 
2.2.5 数据统计分析 ............................................................................................ 14 
2.3 结果 ................................................................................................................... 15 
2.3.1 文蛤游离氨基酸组成与含量的特征分析 ................................................ 15 
2.3.2 不同壳色文蛤游离氨基酸含量的差异分析 ............................................ 17 
2.3.3 不同壳色文蛤其他呈味物质与味精当量的差异分析 ............................ 20 
2.4 讨论 ................................................................................................................... 20 
第 3 章 文蛤家系游离氨基酸含量和生长性状的分析与综合评价 ..................23 
3.1 研究背景 ........................................................................................................... 23 
3.2 材料与方法 ....................................................................................................... 24 
3.2.1 实验材料 .................................................................................................... 24 
3.2.2 生长性状的测量 ........................................................................................ 24 
3.2.3 游离氨基酸含量的测定 ............................................................................ 24 
3.2.4 味道强度值的计算 .................................................................................... 24 
3.2.5 数据统计分析 ............................................................................................ 24 
3.3 结果 ................................................................................................................... 25 

3.3.1 文蛤家系生长性状动态变化的特征和比较 ............................................ 25 
3.3.2 文蛤家系游离氨基酸组成与含量和生长性状的特征分析 .................... 26 
3.3.3 文蛤家系游离氨基酸含量和生长性状的差异分析 ................................ 28 
3.3.4 文蛤家系游离氨基酸含量和生长性状之间的相关性分析 .................... 29 
3.3.5 文蛤家系游离氨基酸含量和生长性状的主成分分析与综合评价 ........ 31 
3.3.6 文蛤家系游离氨基酸含量和生长性状的 TOPSIS 分析 ........................ 33 
3.4 讨论 ................................................................................................................... 34 
第 4 章 文蛤谷氨酸脱氢酶（MpGDH）基因 SNP 多态性、表达水平
与游离谷氨酸含量差异的分析 ..............................................................................36 
4.1 研究背景 ........................................................................................................... 36 
4.2 材料与方法 ....................................................................................................... 37 
4.2.1 MpGDH 基因的鉴定与序列分析 .............................................................. 37 
4.2.2 实验材料 .................................................................................................... 37 
4.2.3 游离谷氨酸含量的测定 ............................................................................ 38 
4.2.4 总 RNA 提取和 cDNA 合成 ..................................................................... 39 
4.2.5 基因组 DNA 的提取 ................................................................................. 40 
4.2.6 实时荧光定量 PCR 检测 MpGDH 基因表达 .......................................... 41 
4.2.7 SNP 检测和基因分型 ................................................................................ 42 
4.2.8 MpGDH 酶活性的检测 ............................................................................. 43 
4.2.9 数据统计分析 .................................................................................................43 
4.3 结果 ................................................................................................................... 44 
4.3.1 MpGDH 基因的分析 .....................................................................................44 
4.3.2 游离谷氨酸含量与 MpGDH mRNA 表达水平的关系 ........................... 48 
4.3.3 SNP 鉴定与连锁不平衡分析 ...................................................................... 49 
4.3.4 MpGDH mRNA 表达与基因多态性的关系 .............................................51 
4.3.5 MpGDH 酶活性与基因多态性的相关性 ................................................. 53 
4.3.6 文蛤家系中 MpGDH 不同基因型遗传效应的验证................................ 54 
4.4 讨论 ................................................................................................................... 55 
第 5 章 文蛤游离甘氨酸含量的全基因组关联分析 ..........................................57 
5.1 研究背景 ...........................................................................................................57 
5.2 材料与方法 .......................................................................................................58 
5.2.1 基因组 DNA 的提取、测序及质控 ......................................................... 58 
5.2.2 序列比对、SNP 鉴定和群体结构分析 ................................................... 58 
5.2.3 GWAS 与候选基因注释 ............................................................................ 58 
5.3 实验结果 ...........................................................................................................59 
5.3.1 游离甘氨酸含量的特征 ..............................................................................59 
5.3.2 测序数据质量控制及分析 .........................................................................59 
5.3.3 主成分分析和连锁不平衡分析 .................................................................59 
5.3.4 全基因组关联分析结果 .............................................................................60 

5.3.5 候选基因注释 ..............................................................................................60 

5.4 讨论 ..................................................................................................................64 

第 6 章 结论与展望................................................................................................66 

参考文献 ..................................................................................................................67 

附 录 .........................................................................................................................83 

致 谢 .........................................................................................................................85 

作者简历及攻读学位期间发表的学术论文与其他相关学术成果 ..................86