凡纳滨对虾IRS1基因的克隆、表达特征及其功能分析

Cloning, Expression Characteristics, and Functional Analysis of IRS1 in Litopenaeus vannamei

摘要:

为探究胰岛素受体底物1(insulin receptor substrate 1,IRS1)基因在凡纳滨对虾(Litopenaeus vannamei)生长调控中的作用,本研究通过RACE技术克隆获得了IRS1全长cDNA序列,并对其序列特征、系统进化关系、组织表达模式及RNA干扰后的分子响应进行了分析。结果显示,IRS1基因 cDNA序列全长为6240 bp,其中开放阅读框长4893 bp,5’ UTR非编码区(UTR)1194 bp,3’ UTR 153 bp,编码1630个氨基酸。结构域分析显示,其N端含有PH和PTB结构域。系统进化树中凡纳滨对虾与斑节对虾聚为一支。IRS1在肝胰腺和胃中表达较高,其次是肠、肌肉和腹索神经。长期干扰结果显示,IRS1沉默显著抑制对虾生长,进一步分析发现,IRS1沉默后肌肉生长抑制素(myostatin,MSTN)和蜕皮抑制激素(molt-inhibiting hormone, MIH)表达显著上调。蛋白激酶B (protein kinase B,AKT)表达显著下调,而叉头框O类转录因子(forkhead box transcription factor,FOXO)显著上调。在能量代谢方面,糖酵解关键酶己糖激酶(hexokinase,HK)表达显著降低,而糖异生关键酶果糖-1,6-二磷酸酶(fructose-1,6-bisphosphatase,FBP)表达持续升高。本研究为开展凡纳滨对虾精准分子育种工作提供了新的靶向基因。

Abstract:

Insulin receptor substrate 1 (IRS1) is a key adaptor protein in the insulin/insulin-like signaling (IIS) pathway and plays an important role in regulating growth and energy metabolism in animals. However, its function in crustaceans remains poorly understood. In this study, the full-length cDNA of the IRS1 gene from Pacific white shrimp, Litopenaeus vannamei was cloned using rapid amplification of cDNA ends, and its sequence characteristics, phylogenetic relationships, tissue expression patterns, and functional roles in growth regulation were systematically analyzed. The full-length cDNA of IRS1 was 6240 bp, containing a 4893 bp open reading frame (ORF) encoding 1630 amino acids, a 1194 bp 5’untranslated region (UTR), and a 153 bp 3’UTR. The predicted protein possessed conserved Pleckstrin homology (PH) and phosphotyrosine-binding (PTB) domains at the N-terminus, which are typical structural features of IRS family proteins. No signal peptide was identified, suggesting that IRS1 functions as a cytoplasmic protein. Phylogenetic analysis revealed that L. vannamei IRS1 clustered closely with homologs from other crustaceans, especially Penaeus monodon, forming a distinct invertebrate branch. Quantitative real-time PCR (qRT-PCR) analysis showed that IRS1 was widely expressed in different tissues, with the highest expression levels observed in the hepatopancreas and stomach, followed by the intestine, muscle, and ventral nerve cord, while the lowest expression was detected in hemolymph. These results indicate that IRS1 may be involved in nutrient metabolism and energy regulation. To investigate its biological function, RNA interference (RNAi) experiments were performed. Long-term RNAi results showed that silencing IRS1 significantly inhibited shrimp growth. From day 7 to day 21, the body weight of the RNAi group was significantly lower than that of the control group (P < 0.05), and the final body weight of the control group was 1.27 times that of the RNAi group. Short-term RNAi experiments further demonstrated that IRS1 knockdown significantly upregulated the expression of myostatin (MSTN) and molt-inhibiting hormone (MIH), both of which are negative regulators of muscle growth and molting. In addition, IRS1 silencing led to significant changes in the IIS signaling pathway and energy metabolism-related genes. The expression of protein kinase B (AKT) was significantly downregulated, whereas forkhead box O (FOXO) was significantly upregulated, indicating inhibition of the IIS pathway. Meanwhile, the expression of hexokinase (HK), a key enzyme in glycolysis, was significantly decreased, whereas fructose-1,6-bisphosphatase (FBP), a key enzyme in gluconeogenesis, was significantly increased. These results suggest that IRS1 knockdown induces a metabolic shift from glycolysis to gluconeogenesis, thereby affecting energy allocation. In conclusion, this study successfully cloned and characterized the IRS1 gene in L. vannamei and demonstrated that IRS1 plays a positive role in regulating shrimp growth. IRS1 may regulate growth by modulating the IIS signaling pathway, influencing energy metabolism, and suppressing the expression of growth- and molting-related inhibitory factors. These findings provide new insights into the molecular mechanisms of growth regulation in crustaceans and offer a potential candidate gene for molecular breeding in shrimp.