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凡纳滨对虾胰岛素信号通路相关基因鉴定及其对生长影响的初步研究
逄颖
Subtype硕士
Thesis Advisor张晓军研究员
2021-05-20
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name工程硕士
Degree Discipline生物工程
Keyword凡纳滨对虾 胰岛素信号通路 基因 生长 蜕皮
Abstract

胰岛素信号(insulin/IGF signaling,IIS)通路在有机体的生长、发育、代谢、繁殖和寿命等生命活动中发挥着重要作用。本研究通过对凡纳滨对虾(Litopenaeus vannamei)基因组和转录组数据进行分析,筛选得到凡纳滨对虾IGFBP、IR、ALS、GSK3、FoxO等一系列IIS通路相关基因序列,证明在凡纳滨对虾体内可能存在一条与脊椎动物类似的IIS信号通路,在对虾生长、发育、代谢、存活等方面发挥重要的调控作用。由于甲壳动物与脊椎动物在生理功能上差异较大,且缺少模式生物,导致关于甲壳动物IIS通路构成和作用机制方面的研究非常缺乏。凡纳滨对虾作为最重要的海水养殖物种之一,其生长性状令人关注。本研究针对对虾IIS通路相关基因,运用生物信息学方法分析相关基因的结构特点、系统进化和表达特征,同时采用活体实验方法验证其在生长方面的功能。本研究结果为阐明IIS通路相关基因对对虾生长的作用,及培育优良品种提供了理论基础,同时为深入研究甲壳动物IIS通路提供了新的线索。论文主要包括以下两个方面:

对虾IIS通路相关基因的结构和表达分析

运用生物信息学方法从凡纳滨对虾基因组和转录组数据中筛选出注释为IGFBP、IR、ALS、GSK3和FoxO等基因的相关序列;然后对目的序列的基因结构、系统发育进行分析,依据序列的保守结构域和同其他物种相关序列在发育树上的位置来鉴定各基因;通过多序列比对确定保守氨基酸位点和motif,从而进一步分析各目的基因的功能;根据各基因在不同发育阶段、不同蜕皮时期、不同组织中的表达模式,筛选出潜在关键功能基因进行后续的dsRNA活体干扰实验。

经过筛选,在凡纳滨对虾基因组中得到3条IGFBP、9条IR、13条ALS、1条GSK3和1条FoxO完整序列。通过序列结构分析发现,LvIGFBP包含3个结构域:IB(insulin growth factor binding)结构域、Kazal型丝氨酸蛋白酶抑制剂(Kazal)结构域和免疫球蛋白C2(IGc2)结构域;LvIR属于酪氨酸激酶受体家族,具有L1-Cys-L2酪氨酸激酶结构域、3个纤连蛋白Ⅲ区域(FN3),以及一个胞内酪氨酸激酶催化(TyrKc)结构域;LvALS含有10-20个LRR结构域;LvGSK3属于丝氨酸/苏氨酸激酶家族成员,含有高度保守的丝氨酸/苏氨酸蛋白激酶催化(S_TKc)结构域;LvFoxO在N端具有高度保守的Forkhead DNA-binding (FH)结构域,在C端含有高度保守的Transactivation结构域(TAD)。

基因表达模式分析发现,LvIGFBP1在大多数组织和不同早期发育阶段中表现出较高的表达水平,而LvIGFBP2和LvIGFBP3仅在血细胞中略有表达;LvIR2在仔虾P1期的表达量达到最高,并且在除性腺(卵巢和精巢)之外的所有组织中表达量都较高;LvALS8从胚胎期就开始高表达一直持续到P1期后,并且在成体阶段的肝胰腺、肌肉、血细胞、卵巢中表达量较高;LvGSK3在不同早期发育阶段、大多数成体组织和蜕皮前(D3)阶段均高表达;LvFoxO在不同组织中均有表达,且在肌肉中表达量最高。

IIS通路相关基因的双链RNA活体干扰实验

为了研究IIS通路相关基因在对虾生长中的作用,我们以成体养殖阶段的对虾为实验材料,选取在肌肉和P1期表达量高的基因(LvIGFBP1、LvIR2、LvALS8、LvGSK3和LvFoxO)进行后续的活体双链RNA干扰实验。对干扰后的实验样品进行体重和体长进行测量,以及用实时荧光定量PCR检测干扰效率。数据分析显示,dsLvIGFBP1干扰实验中实验组的体重增加量为对照组的60%左右,明显低于对照组;dsLvIR2干扰实验中实验组的体重增加量为对照组的76%,略低于对照组;dsLvALS8干扰实验中实验组的体重增加量是对照组的119%,略高于对照组;对LvGSK3进行RNA干扰不仅导致体重增加量显著低于对照组(P < 0.05),而且影响蜕皮相关基因的表达;而dsLvFoxO干扰则导致实验对虾死亡率升高,两次干扰的死亡率分别达到75%和50%,均显著高于对照组。

上述实验结果表明,LvIGFBP、LvGSK3 以及LvIR 对凡纳滨对虾的生长有一定的正向调控作用;LvALS 则可能对其生长具有一定负向调控作用;而LvFoxO对凡纳滨对虾的存活具有重要影响。

本论文阐明了凡纳滨对虾IIS通路中的5个相关基因(IGFBP、IR、ALS、GSK3、FoxO)的结构以及它们对对虾生长的影响,为今后甲壳动物相关基因的研究提供了数据基础,也为分子育种提供了一定的理论依据。

Other Abstract

Insulin signaling (IIS) pathway plays an important role in the growth, development, metabolism, reproduction and longevity of the different organism. By analyzing the genomics and transcriptomics data from Litopenaeus vannamei, we screened a series of IIS-related genes, such as IGFBP, IR, ALS, GSK3 and FoxO sequences in vivo. These results indicated that a signaling pathway which is similar to the IIS of vertebrates exist in shrimp. This signaling pathway could regulate the growth, development, metabolism and survival in shrimp, as well as that of in vertebrates. Due to the large differences in physiological function between crustaceans and vertebrates and the lack of model organisms, it is difficult to research the composition and functional mechanism of IIS in crustaceans. L. vannamei is one of the most important mariculture species, its growth traits become the most compelling characteristics. In this study, we analyzed the structural evolution and expression characteristics of IIS pathway related genes in L. vannamei by using bioinformatics methods, and verified their growth functions in vivo experiment. The results of this study provide a theoretical basis for elucidating the effects of IIS pathway related genes on the growth of L. vannamei and breeding superior varieties, and also provide new clues for further research on the crustacean IIS pathway. The thesis mainly research on the following two aspects:

1. The structure and expression analysis of IIS pathway related genes.

Some sequences annotated as IGFBP, IR, ALS, GSK3 and FoxO are screened by using bioinformatics technology from the genome and transcriptome of L. vannamei. Then, we analyse these sequences’ structure and phylogeny, and identify the accuracy of annotated sequence according to the conserved domain and the position of sequences related to other species on the phylogenetic tree. Analysing conserved amino acid sites and motif by using multi-sequence alignment to analyse the function of these sequences. According to the expression profiles of different early development stages, different molting stages and different tissues, we screen the most appropriate sequence to carry out dsRNA interference expression in vivo.

Under the preliminary screening, we verify 3 IGFBPs, 9 IRs, 13 ALSs, 1 GSK, and 1 FoxO complete gene sequences, respectively. Sequence structure analyse showed that LvIGFBPs consisted of three domains : from N-terminal to C-terminal is IB (insulin growth factor binding) domain, Kazal-type serine protease inhibitor (Kazal) domain and immunoglobulin C2 (IGc2) domain in turn; LvIRs belong to tyrosine kinase receptors superfamily, they contain L1-Cys-L2 domain, three fibronectin Ⅲ domain (FN3), and tyrosine kinase catalysis (TyrKc) domain; LvALSs include 10-20 LRR domains; LvGSK3 belongs to Ser/Thr kinase, and it contains a highly conserved Ser/Thr protein kinase catalysis (S_TKc) domain; LvFoxO has a conserved N-terminal Forkhead DNA-binding (FH) domain and a C-terminal Transactivation domain (TAD).

Gene expression profiles analysis showed that LvIGFBP1 exhibit high expression in most tissues and different developmental stages, while LvIGFBP2 and LvIGFBP3 are only slightly expressed in hemocytes; the expression level of LvIR2 peak at P1 stage, and it is highly expressed in most tissues, except for the gonads; LvALS8 is began expressing from zygote to P1 stage, and it is highly expression in hepatopancreas, muscles, hemocytes and ovary; LvGSK3 exhibit high expression in different early developmental stages, most adult tissues and pre-molting (D3) stage; LvFoxO is highly expressed in most tissues, especially in muscles.

2. The dsRNA interference experiment of IIS-related genes.

This thesis aims to research the effect of IIS pathway related genes on shrimp growth. We use adult shrimp as the experimental material, so we choose the gene which highly expressed in P1 stage and muscles (LvIGFBP1、LvIR2、LvALS8、LvGSK3 and LvFoxO) to conduct dsRNA interference experiment. Each individual was injected with optimal dosage. The experiment lasted for two weeks. We measured the weight and length traits of the samples, and detected the interference efficiency by using real-time quantitative PCR. Data analysis indicated that after two weeks dsRNA interference experiment, the increment of body weight in the dsLvIGFBP1 group was about 60% of the control groups, which was significantly lower than two control groups; the increment of body weight in the dsLvIR2 group was 76% of control groups, which was slightly lower than that of control groups; in contrast, the increment of body weight in the dsLvALS8 group was 119% of control groups, which was slightly higher than that of control groups; the RNA interference of LvGSK3 resulted in a significantly smaller increment of body weight than that of control groups, and affect the expression of molting related genes; the LvFoxO RNAi resulted in a high fatality, the fatality rates of the two interferences were 75% and 50% respectively, which were significantly higher than those of the control groups.

The final results suggested that LvIGFBP, LvGSK3 and LvIR have a positive effect on growth to some extend; LvALS could inhibit the growth of shrimp; and LvFoxO is essential for shrimp’s survival.

In this thesis, we elucidated the structure of five IIS pathway related genes (IGFBP, IR, ALS, GSK3, FoxO) in L. vannamei and their effects on the growth of L. vannamei, which provided the data basis for the research of related genes in crustaceans in the future, and also provided some theoretical basis for molecular breeding.

Subject Area分子生物学
MOST Discipline Catalogue工学::生物工程
Pages127
Language中文
Table of Contents

目 录

摘  要

Abstract

目 录

第一章 绪 论

1.1  胰岛素超家族及相关基因

1.1.1  胰岛素超家族

1.1.2  无脊椎动物的胰岛素超家族基因

1.1.3  胰岛素受体家族

1.1.4  胰岛素生长因子结合蛋超家族

1.1.5 IGFBP酸不稳定亚基

1.2  胰岛素信号通路

1.2.1  胰岛素受体底物蛋白

1.2.2 Akt

1.2.3  糖原合成激酶3(GSK3)

1.2.4 FoxO

1.3  对虾胰岛信号通路相关基因研究的目的和意义

第二章 凡纳滨对虾类胰岛素生长因子结合蛋白基因的结构和功能研究

2.1  材料与方法

2.1.1  实验动物

2.1.2  序列鉴定及分析

2.1.3  多序列比对和系统发生树构建

2.1.4  不同发育时期和不同组织中的表达模式

2.1.5  总RNA提取和cDNA合成

2.1.6  基因克隆

2.1.7  实时荧光定量PCR

2.1.8  IGFBP1重组蛋白表达

2.2  结果和分析

2.2.1  IGFBP基因的序列特征

2.2.2  IGFBP系统进化树

2.2.3  多序列比对

2.2.4  LvIGFBPs三维结构预测

2.2.5  LvIGFBP基因表达谱

2.2.6  LvIGFBP1基因RNA干扰

2.2.7  LvIGFBP1重组蛋白表达

2.3  讨论

2.3.1  LvIGFBP基因的结构特征

2.3.2  LvIGFBPs 系统发生

2.3.3  LvIGFBPs的功能

第三章  凡纳滨对虾类胰岛素受体和类胰岛素生长因子结合蛋白酸性不稳定亚基基因的结构和功能研究

3.1  材料与方法

3.1.1  实验动物

3.1.2  序列鉴定及分析

3.1.3  多序列比对和系统发生树构建

3.1.4  不同发育时期和不同组织中的表达模式

3.1.5  总RNA提取和cDNA合成

3.1.6  基因克隆

3.1.7  实时荧光定量PCR

3.1.8  统计分析

3.2  结果和分析

3.2.1  IR和ALS基因序列特征

3.2.2  IR和ALS系统发育进化树

3.2.3  IR和ALS多序列比对

3.2.4  IR和ALS基因表达谱

3.2.5  IR和ALS基因的RNA干扰

3.3  讨论

第四章  凡纳滨对虾糖原合成酶激酶3基因的结构和功能研究

4.1  材料与方法

4.1.1  实验动物

4.1.2  序列鉴定及分析

4.1.3  多序列比对和系统发生树构建

4.1.4  不同发育时期和不同组织中的表达模式

4.1.5  转录因子预测

4.1.6  总RNA提取和cDNA合成

4.1.7  基因克隆

4.1.8  实时荧光定量PCR

4.1.9  统计分析

4.2  结果和分析

4.2.1  GSK3基因序列特征

4.2.2  GSK3s序列系统进化发育树

4.2.3  多序列比对

4.2.4  LvGSK3三维结构预测

4.2.5  LvGSK3基因表达谱

4.2.6  LvGSK3基因的转录因子预测

4.2.7  LvGSK3基因RNA干扰

4.3  讨论

4.3.1  LvGSK3基因的结构特征

4.3.2  LvGSK3的系统发生

4.3.3  LvGSK3的功能

第五章  凡纳滨对虾FoxO基因的结构和功能

5.1  材料与方法

5.1.1  实验动物

5.1.2  序列鉴定及分析

5.1.3  多序列比对与系统发育树构建

5.1.4  不同发育时期和不同组织中的表达模式

5.1.5  总RNA提取和cDNA合成

5.1.6  基因克隆

5.1.7  实时荧光定量PCR

5.1.8  统计分析

5.2  结果和分析

5.2.1  FoxO基因结构特征

5.2.2  FoxOs系统进化发育树

5.2.3  FoxOs多序列比对分析

5.2.4  LvFoxO基因表达谱分析

5.2.5  LvFoxO基因RNA干扰

5.3  讨论

结 论

参考文献

附录 I 引物序列

致 谢

作者简历及攻读学位期间发表的学术论文与研究成果

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/170665
Collection实验海洋生物学重点实验室
Recommended Citation
GB/T 7714
逄颖. 凡纳滨对虾胰岛素信号通路相关基因鉴定及其对生长影响的初步研究[D]. 中国科学院海洋研究所. 中国科学院大学,2021.
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