脊尾白虾(Exopalaemon carinicauda)ferritin基因克隆及表达分析

CLONING AND EXPRESSION ANALYSIS OF FERRITIN GENE IN EXOPALAEMON CARINICAUDA

采用RT-PCR及RACE技术获得了总长度为755bp的脊尾白虾ferritin基因cDNA序列。该基因序列5’和3’的非编码区分别为112bp和146bp,开放阅读框497bp,推测编码169个氨基酸,预测蛋白分子量19.1kDa,等电点5.46,该基因命名为Ecfer基因。利用Neighbor-joining法构建系统进化树分析结果表明,Ecfer基因氨基酸序列与无脊椎动物中的甲壳动物聚为一支(同源性为64%—67%)。此外,Ecfer基因氨基酸序列与脊椎动物铁蛋白H链同源性为53%—57%。采用荧光定量RT-PCR研究Ecfer基因在组织中以及经WSSV刺激后血淋巴、肝胰腺中的表达变化情况。研究结果表明,Ecfer在肝胰腺、卵巢、肌肉、鳃和血淋巴中均有分布。注射WSSV后3h和6h,肝胰腺和血淋巴Ecfer基因表达较对照组均显著上调(P<0.05),并具有显著的时间差异性。说明Ecfer基因可能参与了脊尾白虾抵抗WSSV入侵的免疫反应。

The ferritin gene in Exopalamon carinicauda was first cloned using RT-PCR and RACE and a 755bp eDNA was acquired. The result of sequencing indicated that the 5＇-and 3＇-untranslated regions （UTR） of this gene were 112bp and 146bp, respectively. It had an open reading frame of 497bp that encoded a 169 amino-acid polypeptid （Mr 19. l kDa）, which had the isoelectric point （pI） of 5.46. The eDNA sequence was named Ecfer. Phylogenetic analysis using neighbor-joining on amino acid sequence from different species showed that Ecfer was in the same category as those in the crustaceans （homology was 64%--67%）. In addition, the amino acid sequence of Ecfer shares 53%--57% similarity with the ferritin H in vertebrates. We used quantitative real-time PCR to estimate mRNA expression of Ecfer in organs or tissues and its in- duced expression in haemolymph, hepatopancreas of E. carinicauda following WSSV infection. The results showed that Ecfer expression existed in all tested organs or tissues of E. carinicauda, including hepatopancreas, ovary, muscle, gills, and haemolymph. 3h and 6h after WSSV injection, the levels of Ecfer mRNA in hepatopancreas and haemolymph clearly increased, and this increase changed with time. We then concluded that Ecfer may be involved in the immune response of resisting WSSV invasion in E. carinicauda.