凡纳滨对虾类胰岛素肽基因的结构及表达分析

Gene structure and expression analysis of insulin-like peptide in the Pacific white shrimp Litopenaeus vannamei

中文关键词: 凡纳滨对虾  类胰岛素肽  基因结构  功能预测  表达分析

英文关键词: Litopenaeus vannamei  Insulin-like peptide  Gene structure  Functional prediction  Expression analysis

中文摘要:

      类胰岛素肽(ILP)是胰岛素超家族的成员，具有进化保守性，是影响动物生命活动的重要因子之一。本研究克隆了凡纳滨对虾(Litopenaeus vannamei) ILP1 (LvILP1)全长基因，mRNA长度为812 bp，其中开放阅读框长543 bp，编码180个氨基酸。序列分析显示，LvILP1蛋白预计分子量为20.81 kDa，理论等电点为9.45，不稳定系数为96.20，具有一个信号肽，没有跨膜结构，推导其为碱性、不稳定的分泌蛋白。结构预测显示，该蛋白具有胰岛素超家族保守的IlGF结构域，由N端信号肽、B链、C肽和A链组成，同时具有6个保守的半胱氨酸位点和2个断裂位点。系统进化分析显示，LvILP1与斑节对虾(Penaeus monodon) ILP7亲缘关系最近，与甲壳动物ILP1聚为一支，然后分别与无脊椎动物ILP7、脊椎动物松弛素(Relaxin)、胰岛素(Insulin)、胰岛素样生长因子(IGF)聚在一起。无脊椎动物ILP7类与外群海葵(Actinia tenebrosa) ILP的进化关系最近，表明这类ILP可能与胰岛素超家族的祖先较为相似。转录因子预测显示，LvILP1可能的转录因子为叉头框蛋白O3 (FoxO3)、糖皮质激素受体(GR)、CAAT区/增强子结合蛋白(C/EBP)、信号传导及转录激活蛋白(STAT)等；蛋白互作分析显示，LvILP1与细胞膜上的胰岛素受体(IR)、神经信号分子(VGLUT1、SYT 1_3)、糖蛋白激素(GPHB5)、鞣化激素(Bursicon)等相互作用；对这些转录因子和互作蛋白的生物功能进行分析，进而推测LvILP1可能具有调节生长发育、激素刺激反应、神经系统稳态、碳水化合物稳态、蜕皮后组织重建以及生殖发育等作用。分析发现，LvILP1在凡纳滨对虾早期发育阶段有较高表达，在成体各个组织中均有表达，但在眼柄中表达量最高。本研究结果为深入了解凡纳滨对虾ILP的基因结构、进化、功能及表达提供了重要信息，同时为凡纳滨对虾的分子育种和健康养殖提供线索。

英文摘要:

      Insulin-like peptide (ILP) is a member of the insulin superfamily with evolutionary conservation and is one of the most important factors affecting animal life activities. In this study, a full-length of ILP1 gene in Pacific white shrimp, Litopenaeus vannamei, was cloned, and the mRNA length consisted of 812 bp with an open reading frame (ORF) of 543 bp, encoding 180 amino acids. Sequence analysis showed that the predicted molecular weight of LvILP1 protein was 20.81 kDa, and the theoretical isoelectric point was 9.45 and the instability coefficient was 96.20. There was a signal peptide, no transmembrane structure. It was deduced that it was located outside the cell and is an alkaline unstable secreted protein. Structure prediction found that LvILP1 protein had the conserved IlGF domain of the insulin superfamily, which was composed of the N-terminal signal peptide, B-chain, C-peptide and A-chain, as well as six conserved cysteine sites and two cleavage sites. Phylogenetic analysis found that LvILP1 was most closely related to ILP7 in Penaeus monodon, and clustered with ILP1 of Crustaceans to form a branch, and then clustered with ILP7 of Invertebrate, Relaxin, Insulin and Insulin-like growth factor (IGF) of vertebrate; ILP7 of Invertebrate was evolutionarily closest to outgroup sea anemone ILP1, suggesting that it may be more similar to the ancestor gene of the insulin superfamily. Transcription factor prediction found that the possible transcription factors of LvILP1 are Forkhead box protein O3 (FoxO3), Glucocorticoid receptor (GR), CAAT region/enhancer binding protein (C/EBP) and Signal transduction and transcription activator protein (STAT); The protein interaction analysis found that LvILP1 interacted with Insulin receptor (IR) on the cell membrane, nerve signaling molecules (VGLUT1, SYT 1_3), Glycoprotein hormone beta 5 (GPHB5), Bursicon alpha, etc. By analyzing the biological function analysis of these transcription factors and interacting proteins, it is speculated that LvILP1 may play an important role in regulating shrimp growth and development, response to hormonal stimulation, nervous system homeostasis, carbohydrate homeostasis, postmolt tissue remodeling and reproductive development. The analysis showed that LvILP1 was expressed in the early developmental stage of shrimp, and expressed in all tissues of adults, but the expression level in the eye stalk was the highest. This study provides important information for in-depth understanding of the structure, evolution, function and expression of ILP in shrimp, as well as clues for molecular breeding and healthy farming of shrimp.