实验海洋生物学重点实验室知识库

类胰岛素超家族是动物中一个古老的基因家族，参与碳水化合物代谢和脂质代谢等多种生理过程，对个体的生殖、生长和发育起着重要的调控作用。贝类中发现了多个类胰岛素家族成员，但是其相应的功能研究还比较欠缺，目前尚不清楚这些基因参与的具体生物学过程。本研究以长牡蛎为实验对象，采用自然生长周期取样及实验室条件下的饥饿处理等方法，测定生理指标和基因表达水平，结合多元统计等分析方法研究牡蛎类胰岛素基因与生理性状的相关性，肌肉注射葡萄糖后取肝胰腺组织进行转录组测序和生物信息学分析，探究类胰岛素基因在血糖调节中的作用。具体结果如下：

为探究长牡蛎在自然生长周期内糖原含量和性腺发育等生理指标的变化，及其与类胰岛素基因（cgMIP123、cgMIP4、cgMILP7、cgILP）相对表达量的相关性，自2020年5月至2021年5月采集了山东胶南养殖海区的长牡蛎，测定了血糖、糖原含量、条件指数、目标基因相对表达量及环境因子（酸度、温度、盐度、叶绿素a含量）等相关数据，采用多元统计的方法对数据进行分析。logistic回归分析结果显示，长牡蛎配子排放前后血糖含量和内脏团糖原含量具有显著变化。构建的回归模型可以通过血糖含量和内脏团糖原含量准确判断配子是否已经排放，区分度C-index为0.903，Hosmer-Lemeshow拟合优度检验χ²值为9.06，p-value > 0.05，验证结果显示该模型可靠。多元线性回归分析结果显示：条件指数与cgMIP123、cgMIP4和cgMILP7具有相关性，R²为0.83，p-value为7.87e-04； cgMIP4和cgILP与糖原含量呈正相关；cgMIP123与糖原含量未显示出相关性；cgMILP7的相对表达量与内脏团和唇瓣组织的糖原含量及血糖含量呈负相关，推测该基因与性腺发育有关，在血糖调节中具有一定相关性但不是主要的调控因子。

长牡蛎肌肉注射葡萄糖后在短时间内血糖含量迅速升高，随后呈波动式逐渐下降，2小时左右恢复至最初水平。取注射葡萄糖后的肝胰腺组织进行转录组学分析，在差异基因集中并未鉴定到我们所关注的四条类胰岛素基因，筛选到与糖原合成和类胰岛素分泌相关的cgPPP1R3B和cgPCSK1两个差异基因，其中cgPPP1R3B在糖原合成过程中起到重要的作用，cgPCSK1在ILP多肽的加工过程中起到重要作用，表明在翻译后水平上对ILP的调控增强。由于实验周期较短、生物重复数较少，饥饿实验的结果显示血糖、糖原含量、类胰岛素基因相对表达量无显著差异。

The insulin-like superfamily is an ancient gene family in animals, which is involved in the regulation of a variety of physiological processes, including reproduction, development, growth, carbohydrate and lipid metabolism. There are many insulin-like peptide genes (ILPs) have been found in shellfish, but research on their function is scant and it is unclear what specific biological processes these genes are involved in. In this study, we selected Crassostrea gigas as experimental animal to assay the possible involvement of oyster ILPs in the regulation of glycogen content and gonad development. Physiological condition and gene expression levels were assayed on oysters collected monthly from the oyster farm, multivariate statistical analysis was then applied to study the correlations between ILP expression and physiological conditions. RNAseq was conducted on the hepatopancreas of oysters injected with glucose to assay the transcriptomic response to the blood glucose increase. The results are listed as follows:

Glycogen content variations during natural state of Crassostrea gigas and their correlations with the expression patterns of insulin-like genes (cgMIP123, cgMIP4, cgMILP7, cgILP). The blood glucose content, glycogen content, condition index and relative gene expression level were assayed on oysters cultured in a farm at Jiaonan, Shandong, while typical marine environmental factors (acidity, temperature, salinity, Chla) were collected from May 2020 to May 2021. Multivariate logistic regression analysis indicated high correlation between the spawning and variations of blood glucose content and glycogen content around pyloric process. A regression model was constructed to estimate the gonad discharging status by the two parameters. The concordance index was at 0.903, while the χ² value was at 9.06 (p-value > 0.05) for Hosmer-Lemeshow test, suggesting high goodness of fit. Application of the model to verification samples also showed high reliability. The multiple linear regression analysis showed correlations of growth or physiological values with the expression pattern of some genes: the condition index was correlated with cgMIP123, cgMIP4 and cgMILP7 (R²: 0.83, p-value: 7.87e-04); cgMIP4 and cgILP were positively correlated with glycogen content; there is no correlation between cgMIP123 and glycogen content; the relative expression of cgMILP7 was negatively correlated with glycogen content of all the assayed tissues, this gene may be related to gonadal development and has partial correlation in blood glucose regulation.

After intramuscular injection of glucose, the blood glucose level of Crassostrea gigas increased rapidly in a short period of time, and then decreased gradually in a fluctuating way, and returned to the initial level 2 hours later. Transcriptome analysis of hepatopancreas of oysters after injection of glucose stimulation showed that insulin-like genes were not concentrated in differential genes, which may indicate that there was no change in insulin-like genes at the transcriptional level. cgPPP1R3B and cgPCSK1 were selected as two differential genes from the differential gene set. cgPPP1R3b played an important role in the process of glycogen synthesis, and cgPCSK1 plays an important role in the processing of ILP polypeptides, indicating enhanced regulation of ILP at the post-translational level. The results of starvation experiment did not show significant differences in blood glucose and glycogen content, which may be related to the short experiment period and fewer biological replicates.

摘  要... I

Abstract. III

第1章 文献综述..................................................................................... 1

1.1 牡蛎的生长性状... 1

1.1.1 糖原积累... 1

1.1.2 条件指数... 2

1.1.3 性腺发育... 3

1.2 类胰岛素超家族... 3

1.2.1 类胰岛素超家族成员的结构保守性... 5

1.2.2 类胰岛素超家族成员的功能差异性... 5

1.2.3 类胰岛素超家族成员的分泌部位与进化的关系... 7

1.2.4 胰岛素的分泌及下游信号通路... 8

1.2.5 长牡蛎类胰岛素... 10

1.3 环境因素与贝类生长的关系... 11

1.4 多元回归分析... 12

1.4.1 logistic模型... 12

1.4.2 多元线性回归... 13

1.5 研究的计划及意义... 14

第2章 实验材料与方法.......................................................................... 17

2.1 实验材料... 17

2.1.1 实验动物及预处理... 17

2.1.2 水样采集及处理... 20

2.1.3 试剂耗材... 21

2.1.4 仪器设备... 22

2.2 实验方法... 23

2.2.1 血糖含量的测定... 23

2.2.2 糖原含量的测定... 24

2.2.3 基因表达的测定... 24

2.2.4 体尺性状和条件指数的测定... 26

2.2.5 性腺切片的制作... 26

2.2.6 叶绿素a含量的测定... 28

2.2.7 肌肉注射葡萄糖及血糖测定... 28

2.2.8 转录组学分析... 29

2.2.9 饥饿实验... 30

2.3 数据分析方法... 31

2.3.1 统计分析... 31

2.3.2 相关性分析... 31

第3章 实验结果..................................................................................... 33

3.1 海区环境因子的周年变化... 33

3.2 血糖含量的周年变化... 33

3.3 糖原含量的周年变化... 34

3.4 类胰岛素基因表达的周年变化... 35

3.5 体尺性状及条件指数... 38

3.6 性腺发育... 41

3.7 回归分析结果... 45

3.7.1 logistic回归分析... 45

3.7.2 多元线性回归模型... 47

3.8 肌肉注射葡萄糖后的血糖变化... 48

3.9 转录组数据分析... 49

3.9.1 血糖变化... 49

3.9.2 转录组数据分析... 50

3.9.3 饥饿实验... 56

第4章 讨论............................................................................................. 59

4.1 环境因子及长牡蛎生长性状间的相关性... 59

4.2 cgMIP123、cgMIP4与生长性状的相关性... 61

4.3 cgMILP7与性腺发育和血糖调节的相关性... 62

4.4 cgILP与血糖变化的相关性... 62

4.5 长牡蛎血糖调控的转录组学分析... 63

第5章 结论............................................................................................. 65

参考文献... 67

致  谢... 73

作者简历及攻读学位期间发表的学术论文与研究成果    75