实验海洋生物学重点实验室知识库

凡纳滨对虾（Litopenaeus vannamei）属于节肢动物门甲壳动物亚门十足目，是甲壳动物的典型代表之一。同时，凡纳滨对虾更是一种重要的经济价值物种，是目前世界上养殖产量最高的虾类。提高对虾的生长性能有助于增加养殖效益，传统上通过改善养殖环境和改进养殖饲料来提高对虾的产量；近年来，随着分子生物学技术的发展和对虾基因组的破译，通过分子遗传育种从基因水平上提高对虾生长性能的研究日益受到关注。在高等动物中，胰岛素（Insulin）与胰岛素样生长因子（Insulin-like growth factor，IGF）对糖代谢和生长发育有重要的作用。目前在无脊椎动物中并未发现上述两个基因，而是存在着胰岛素超家族的另一个成员类胰岛素肽（Insulin-like peptide，ILP）。基于本课题组前期对凡纳滨对虾基因组和转录组数据的研究，我们认为在对虾中也存在类似的胰岛素信号通路。本研究利用基因组和转录组数据，从中筛选出凡纳滨对虾类胰岛素肽基因（LvILP），并深入分析了LvILP的基因结构及进化特征；通过饥饿实验以及葡萄糖、重组蛋白、RNA双链注射实验研究LvILP与对虾血糖变化的关系；同时通过连续的RNA干扰（RNA interference，RNAi）结合转录组分析，研究了LvILP基因对对虾生长的影响。本文的主要研究结果如下：

1、凡纳滨对虾类胰岛素肽的基因结构、进化和表达特征分析

本研究克隆了凡纳滨对虾的LvILP基因，通过生物信息学对其进行基因结构及进化分析。结果显示LvILP基因具有胰岛素超家族保守的IIGF结构域，由一个信号肽、B链、不保守的C肽、A链组成，具有6个保守的半胱氨酸位点，并属于γ型蛋白。通过蛋白二级结构和三级结构预测发现，LvILP的A、B链均主要由α-螺旋结构构成，它们可能是LvILP发挥生物学功能的位点。系统发生树分析发现LvILP与斑节对虾ILP7亲缘关系最近，与甲壳动物ILP1聚类在一起，然后与昆虫的ILP7及脊椎动物的Relaxin聚类。ILP作为系统发生树的外支，显示其可能是胰岛素超家族的祖先基因。转录因子预测发现LvILP基因可能的转录因子为FoxO3、GR、C/EBP、STAT等；蛋白互作分析发现LvILP可能与细胞膜上的胰岛素受体（IR）、胰岛素下游基因（PI3K、Akt）、神经信号（VGLUT1、Putative SYT 1_3）、糖蛋白激素（GPHB5）、鞣化激素（Bursicon alpha）等相互作用；通过预测的转录因子及互作蛋白的生物学功能推测LvILP可能在调节对虾生长发育、对激素刺激的反应、神经系统的稳态、碳水化合物的稳态、蜕皮后组织的重建以及生殖发育中发挥重要作用。荧光定量检测发现LvILP在各个组织中均有表达，其在眼柄中表达量最高，表明LvILP基因可能主要由眼柄分泌运输到靶组织器官发挥生物学作用。

2、类胰岛素肽与对虾血糖变化的关系

目前在多种甲壳动物中均鉴定到类胰岛素肽的存在，但其在调节血糖方面是否与脊椎动物胰岛素具有相似的功能尚不清楚。本研究检测了凡纳滨对虾血糖的成分，并分析了不同处理下血糖浓度的变化以及LvILP和糖代谢基因表达量的变化。结果显示葡萄糖是对虾体内血糖的主要成分，饥饿导致血淋巴中葡萄糖浓度先升后降，外源葡萄糖注射后升高的血糖在短时间内恢复到正常水平，说明对虾体内具有维持血糖稳态的调节机制。此外，注射牛胰岛素和重组LvILP蛋白均导致血糖快速降低，表明LvILP具有降低血糖的作用。值得注意的是，LvILP基因干扰不会显著影响血糖水平，但是会抑制清除外源性葡萄糖的速率。糖代谢基因检测发现LvILP可能通过调节这些基因的表达来维持血糖的稳定。这些结果表明ILP在对虾中具有类似于脊椎动物中胰岛素的保守功能，在维持血糖稳态中起重要作用。

3、类胰岛素肽调控对虾生长的功能研究

为了研究ILP对对虾生长的影响，本研究通过持续RNA干扰LvILP基因2周后，测量对虾的生长性状，并通过转录组测序研究LvILP基因敲降对肝胰腺、肌肉、表皮组织基因表达的影响。RT-qPCR结果显示双链RNA干扰后，LvILP基因的表达量显著下降；生长性状测量显示相比于对照组，实验组对虾的体长和体重的增长速率均下降。转录组分析结果显示，LvILP基因的干扰影响了眼柄、肝胰腺、肌肉和表皮组织中胰岛素通路下游基因表达的变化。转录组差异基因分析显示LvILP基因敲降可能促进了肝胰腺中蜕皮激素调控通路基因表达，还导致保幼激素的合成减少和氨基酸代谢紊乱；促进了肌肉组织蛋白质的降解，抑制肌肉主要结构蛋白的合成和细胞的生长发育；促进了表皮中胶原纤维的形成，抑制表皮层的更新。以上的研究结果表明ILP基因可能通过胰岛素信号通路调控对虾的生长，并且对不同组织具有不同的影响。

上述实验结果表明凡纳滨对虾具有结构保守的ILP基因，该基因对维持对虾血糖稳态具有重要作用，并且在生长方面具有一定的正向调控作用。本研究为从基因水平上理解类胰岛素肽对对虾糖代谢和生长的作用机制提供了有价值的线索，同时为筛选对虾的分子遗传育种候选基因提供了重要参考。

Pacific white shrimp, Litopenaeus vannamei, belonging to Decapoda, Crustacea, Arthropoda, is one of the typical representatives of crustaceans. At the same time, it is an important species of economic value, which is the shrimp with the highest aquaculture production in the world. Improving the growth performance of shrimp contributes to increasing the efficiency of aquaculture. Traditionally, the production of shrimp has been raised by improving the breeding environment and bait optimization. With the deciphering of the shrimp genome, it has attracted more and more attention to improve the growth performance of shrimp from the genetic level through molecular genetic breeding. In vertebrates, insulin and insulin-like growth factor (IGF) play an important role in glucose metabolism and growth. So far, the above two genes have not been found in invertebrates, which exist another member of the insulin superfamily, insulin-like peptide (ILP). From previous studies in insects and our group's analysis basing on the genome and transcriptome data of L. vannamei, we believe that there is a similar insulin signaling pathway in shrimp.

In this study, we screened an ILP gene from the genome and transcriptome data of L. vannamei, deeply analyzed the structure and evolution characteristics of the LvILP gene, and studied the relationship between LvILP gene and hemolymph glucose changes by starvation experiment and injection experiment of glucose, recombinant protein and double-strand RNA. In addition, we studied the effect of LvILP gene on shrimp growth by continuous RNA interference (RNAi) experiment and transcriptome analysis. The main conclusions are as follows:

1、Gene structure, evolution and expression characteristics of insulin-like peptide of L. vannamei

In this study, the LvILP gene was cloned, and its gene structure and evolution were analyzed by bioinformatics. The results showed that LvILP gene has the conserved IIGF domain of insulin superfamily, which is composed of a signal peptide, B chain, unconserved C peptide and A chain. LvILP gene has 4 conserved cysteine​​sites, and belongs to γ-type protein. The predicted secondary and tertiary structure of the protein showed that the A and B chains were mainly composed of α-helical structures, which may have sites where LvILP exerts biological functions. Phylogenetic tree analysis found that LvILP was most closely related to ILP7 of Penaeus monodon, clustering one branch with ILP1 in crustaceans, and they co-clustered a big branch with ILP7 in insects and relaxin in vertebrates. ILP is an external branch in the phylogenetic tree, indicating that it may be the ancestral gene of the insulin superfamily. Transcription factor prediction found that FoxO3, GR, C/EBP, STAT may be the transcription factors of LvILP gene. Protein interaction analysis found that LvILP gene may interact with insulin receptor (IR) on the cell membrane, insulin downstream genes (PI3K, Akt), neural signals (VGLUT1, Putative SYT 1_3), glycoprotein hormones beta 5 (GPHB5), Bursicon alpha, etc. Through the biological functions of predicted transcription factors and interacting proteins, it is speculated that LvILP gene may play an important role in growth and development, responsing to hormonal stimulation, nervous system homeostasis, carbohydrate homeostasis, postmolt tissue remodeling, and reproductive development. RT-qPCR detection showed that LvILP was expressed in various tissues, but had the highest expression in the eyestalk, indicating that LvILP may mainly be secreted by eyestalk and transported to the target tissues to play a biological function.

2、The relationship between LvILP and hemolymph sugar changes

ILP have been identified in a variety of crustaceans, but whether they have similar functions to vertebrate insulin in regulating blood sugar is unclear. In this study, we analyzed the components of blood sugar of L. vannamei, and investigated the changes of hemolymph glucose concentration and the expressions of LvILP and glucose metabolism genes under different treatments. The results showed that glucose was the main component of hemolymph sugar in shrimp, starvation caused hemolymph glucose to rise first and then decline, and the raised hemolymph glucose after exogenous glucose injection could return to basal levels within a short time, indicating that shrimp has a regulatory mechanism to maintain hemolymph glucose homeostasis. In addition, the injection of bovine insulin and recombinant LvILP protein both resulted for a fast decline of the hemolymph glucose. Notably, RNA interference of LvILP did not significantly affect hemolymph glucose levels, but inhibited exogenous glucose clearance. Base on the detection of glucose metabolism genes, we found LvILP might maintain hemolymph glucose stability by regulating the expression of these genes. These results suggest that ILP has a conserved function in shrimp similar to insulin in vertebrates and plays an important role in maintaining hemolymph glucose homeostasis.

3、Function of LvILP gene in regulating growth

In order to study the effect of ILP on the growth of shrimp, we measured the growth traits of shrimp after continuous RNA interference of LvILP for 2 weeks, and investigated the effect of LvILP gene knockdown on gene expression in hepatopancreas, muscle and epidermis by transcriptome sequencing. The results showed that after RNA interference, the expression of LvILP was significantly decreased. Compared with the control group, the growth rate of the shrimp body length and body weight decreased in the experimental group. Transcriptome analysis found that LvILP gene interference affected the expression of insulin pathway downstream genes in eyestalk, hepatopancreas, muscle and epidermal tissues. The differential gene analysis showed that LvILP gene knockdown may promote the expression of ecdysone pathway genes in hepatopancreas, and also lead to the reduction of juvenile hormone synthesis and amino acid metabolism disorders. At the same time, it promotes the degradation of tissue proteins in muscle and inhibits the expression of major muscle structural proteins and cell growth and development. In addition, it promotes the formation of collagen fibers in the epidermis and inhibits the renewal of the epidermis. The above results suggest that the LvILP gene may regulate the growth of shrimp through the insulin signaling pathway, and has different effects in different tissues.

The above experimental results showed that the L. vannamei has a structurally conserved ILP gene, which plays an important role in maintaining hemolymph glucose homeostasis and certain positive regulatory effect on growth. This study provides valuable clues for understanding the regulatory mechanism of ILP on the glucose metabolism and growth of shrimp at the genetic level, and also provides an important reference for screening candidate genes for molecular genetic breeding of shrimp.

目 录

第1章 引 言... 1

1.1 凡纳滨对虾生长相关基因研究... 1

1.2 甲壳动物类胰岛素肽... 3

1.3 无脊椎动物中胰岛素信号通路及功能... 5

1.4 甲壳动物类胰岛素肽的糖代谢调控功能... 7

1.4.1 甲壳动物的血糖研究现状... 7

1.4.2 类胰岛素肽的糖代谢调控功能... 8

1.5 甲壳动物类胰岛素肽的生长调控功能... 9

1.5.1 甲壳动物的生长调控... 9

1.5.1.1 肝胰腺与生长代谢... 9

1.5.1.2 肌肉与对虾生长... 11

1.5.1.3 蜕皮与生长的关系... 12

1.5.2 类胰岛素肽的生长调控功能... 14

1.6 本研究的目的和意义... 15

第2章 凡纳滨对虾类胰岛素肽的基因结构、进化和表达特征分析... 17

2.1 材料与方法... 17

2.1.1 序列鉴定... 17

2.1.2 生物信息学分析... 17

2.1.3 氨基酸序列比对及进化树构建... 18

2.1.4 组织表达特征... 19

2.2 结果和分析... 22

2.2.1 LvILP基因氨基酸序列分析... 22

2.2.2 LvILP基因的基因组定位... 24

2.2.3 LvILP基因结构特征... 24

2.2.4 LvILP多序列比对及系统进化... 26

2.2.5 LvILP基因的CpG岛预测... 28

2.2.6 LvILP基因的转录因子预测... 29

2.2.7 LvILP基因的互作蛋白预测... 29

2.2.8 LvILP基因的组织表达特征... 31

2.3 讨论... 31

2.3.1 LvILP基因的结构特征及系统进化... 31

2.3.2 LvILP基因的功能预测... 33

第3章 类胰岛素肽与对虾血糖变化的关系... 35

3.1 材料与方法... 35

3.1.1 实验动物... 35

3.1.2 LvILP基因的克隆... 35

3.1.2.1 LvILP基因的扩增... 35

3.1.2.2 PCR产物纯化回收... 36

3.1.2.3 目的片段连接转化... 37

3.1.2.4 质粒提取... 38

3.1.3 双链RNA（dsRNA）合成... 39

3.1.4 LvILP重组蛋白表达... 42

3.1.4.1 LvILP表达载体的构建... 42

3.1.4.2 LvILP重组蛋白的表达... 44

3.1.5 凡纳滨对虾血糖及基因表达的检测... 46

3.1.5.1 血糖成分的检测... 46

3.1.5.2 饥饿处理后对虾血糖及糖代谢关键基因表达的检测... 47

3.1.5.3 喂食处理后对虾血糖的检测... 48

3.1.5.4 注射葡萄糖后对虾血糖及糖代谢关键基因表达的检测... 48

3.1.5.5 注射牛胰岛素后对虾血糖及糖代谢关键基因表达的检测... 48

3.1.5.6 注射重组LvILP蛋白后对虾血糖及糖代谢关键基因表达的检测... 48

3.1.5.7 注射dsLvILP后对虾血糖浓度及糖代谢关键基因表达的检测... 49

3.1.5.8 统计分析... 49

3.2 结果和分析... 49

3.2.1 对虾的血糖成分... 49

3.2.2 饥饿应激影响血糖水平以及LvILP和糖代谢基因的表达... 50

3.2.3 喂食处理后对虾血糖的变化... 51

3.2.4 对虾通过激活LvILP和糖代谢基因快速清除外源性葡萄糖... 52

3.2.5 牛胰岛素降低对虾血糖并影响糖代谢基因表达... 54

3.2.6 重组LvILP蛋白加速对虾血糖的下降... 55

3.2.7 LvILP基因干扰抑制外源性葡萄糖的清除并下调糖代谢基因的表达... 56

3.3 讨论... 57

3.3.1 对虾血糖的主要成分... 57

3.3.2 对虾体内存在维持血糖稳态的机制... 58

3.3.3 LvILP负调控对虾的血糖... 59

3.3.4 LvILP基因调节糖代谢... 60

第4章 类胰岛素肽调控对虾生长的功能研究... 63

4.1 材料与方法... 63

4.1.1 实验动物... 63

4.1.2 RNA干扰实验... 63

4.1.3 LvILP基因干扰后转录组测序... 64

4.1.3.1 样品制备... 64

4.1.3.2 文库构建与质检... 64

4.1.3.3 质量控制和组装转录组... 65

4.1.3.4 基因表达定量及差异表达分析... 65

4.1.3.5 基因集富集分析... 65

4.1.4 统计分析... 65

4.2 结果和分析... 66

4.2.1 LvILP基因干扰对生长性状的影响... 66

4.2.2 转录组数据分析... 69

4.2.2.1 数据质控... 69

4.2.2.2 Reads与参考基因组比对情况统计... 70

4.2.2.3 比对区域分布... 71

4.2.2.4 基因表达分布... 71

4.2.2.5 样本间相关性分析... 72

4.2.3 WGCNA分析... 73

4.2.3.1 样品层次聚类... 73

4.2.3.2 模块层次聚类... 74

4.2.3.3 模块间相关性热图... 75

4.2.3.4 样品和模块间相关性热图... 76

4.2.4 差异分析... 77

4.2.5 LvILP基因干扰后肝胰腺中差异表达基因分析... 78

4.2.5.1 差异表达基因统计... 78

4.2.5.2 GO富集分析... 78

4.2.5.3 KEGG富集分析... 80

4.2.5.4 LvILP基因干扰影响肝胰腺中胰岛素通路基因的表达... 81

4.2.5.5 LvILP基因干扰影响肝胰腺中激素的生物合成... 82

4.2.5.6 LvILP基因干扰影响肝胰腺中氨基酸的代谢... 83

4.2.6 LvILP基因干扰后肌肉中差异表达基因分析... 85

4.2.6.1 差异表达基因统计... 85

4.2.6.2 GO富集分析... 86

4.2.6.3 KEGG富集分析... 87

4.2.6.4 LvILP基因干扰影响肌肉中胰岛素通路基因的表达... 88

4.2.6.5 LvILP基因干扰影响肌肉中胰岛素下游信号传导通路... 89

4.2.6.6 LvILP基因干扰影响肌肉中结构蛋白相关基因的表达... 90

4.2.7 LvILP基因干扰后表皮中差异表达基因分析... 91

4.2.7.1 差异表达基因统计... 91

4.2.7.2 GO富集分析... 92

4.2.7.3 KEGG富集分析... 94

4.2.7.4 LvILP基因干扰影响表皮中胰岛素通路基因的表达... 96

4.2.7.5 LvILP基因干扰影响表皮中细胞黏附相关基因的表达... 97

4.2.8 LvILP基因干扰影响预测的转录因子基因在三个组织中的表达... 98

4.2.9 LvILP基因干扰影响预测的互作蛋白基因在三个组织中的表达... 99

4.3 讨论... 100

4.3.1 LvILP基因干扰抑制对虾的生长... 100

4.3.2 LvILP基因干扰对肝胰腺的影响... 101

4.3.2.1 LvILP基因干扰影响肝胰腺中的生物学过程... 101

4.3.2.2 LvILP基因干扰影响肝胰腺中的激素合成... 102

4.3.2.3 LvILP基因干扰影响肝胰腺中的氨基酸代谢... 102

4.3.3 LvILP基因干扰对肌肉的影响... 103

4.3.3.1 LvILP基因干扰影响肌肉中的生物学过程... 103

4.3.3.2 LvILP基因干扰抑制肌肉中的代谢... 104

4.3.3.3 LvILP基因干扰抑制肌肉组织的生长... 105

4.3.4 LvILP基因干扰对表皮的影响... 105

4.3.4.1 LvILP基因干扰影响表皮中的生物学过程... 105

4.3.4.2 LvILP基因干扰抑制对虾表皮层的更新... 107

结 论... 109

参考文献... 111

附录 Ⅰ 软件的名称及网址... 123

附录 Ⅱ 引物序列... 124

致 谢... 125

作者简历及攻读学位期间发表的学术论文与研究成果... 127