脊尾白虾14-3-3基因cDNA全长的克隆和表达分析

Cloning and expression analysis of 14-3-3 gene from Exopalaemon carinicauda

利用本实验室构建的脊尾白虾(Exopalaemon carinicauda)血细胞cDNA文库中脊尾白虾14-3-3巨球蛋白基因EST序列,采用cDNA末端快速扩增(RACE)技术,克隆获得脊尾白虾14-3-3基因c DNA全长,命名为Ec14-3-3。该基因全长2905 bp,包含744 bp的开放阅读框,编码247个氨基酸组成的蛋白质,分子量为27.95 kD,理论等电点为4.65。同源性分析表明,脊尾白虾Ec14-3-3氨基酸序列与斑节对虾(Penaeus monodon)14-3-3的同源性最高,达到98%。荧光定量PCR分析结果表明,Ec14-3-3基因在血细胞、卵巢、肝胰腺等组织中均有表达,其中血细胞中Ec14-3-3相对表达量最高。感染鳗弧菌和WSSV后6 h,脊尾白虾血细胞和肝胰腺中Ec14-3-3基因的表达量较对照组均显著增加(P<0.05),且具有明显的时间差异性。本研究结果表明,Ec14-3-3基因在脊尾白虾免疫反应中发挥重要作用。

Exopalaemon carinicauda is the most commercially important shrimp in China based on reproduction, a long growing season, and ecological flexibility. However, the frequency of disease outbreaks has increased as production has increased, resulting in huge economic losses. E. carinicauda relies on the innate immune response to defend against invading pathogens. The 14-3-3 family of proteins is highly conserved and present in all eu- karyotic cells. The 14-3-3 proteins are involved in many cellular pathways, including those for metabolism, the cell cycle, differentiation, signaling, apoptosis, and neoplastic transformation. Based on the 14-3-3 expressed se- quence tag from a hemocyte cDNA library ofE. carinicauda, the full-length cDNA of 14-3-3 from E. carinicauda （named Ec14-3-3） was cloned using the rapid amplification of cDNA ends technique. The Ec14-3-3 cDNA was 2 905 bp in length and contained an open reading frame of 744 bp, encoding a mature protein of 247 amino acids, with a molecular mass of 27.95 kD and an estimated pI of 4.65. Homology analysis revealed that the Ec14-3-3 amino acid sequence was highly conserved with homologs from other crustaceans, and amino acid sequence simi- larity was 98% with the 14-3-3 from Penaeus monodon. Ec14-3-3 expression levels were measured in different tissues by quantitative real-time polymerase chain reaction analysis. Ec14-3-3 expression was detected in hemo- cytes, gill, hepatopancreas, muscle, ovary, intestine, stomach, and the eyestalk ofE. carinicauda, with the highest expression level in hemocytes. Ec14-3-3 expression was upregulated in hemocytes and the hepatopancreas after challenges with Vibrio anguillarum and white spot syndrome virus. These results suggest that Ec14-3-3 may play an important role in the prawn immune response.