脊尾白虾组织蛋白酶L基因的克隆及其表达分析

Cloning and expression analysis of Cathepsin L cDNA of Exopalaemon carinicauda

根据本实验室构建的脊尾白虾(Exopalaemon carinicauda)血细胞全长cDNA文库获得的EST序列,利用RACE技术克隆获得脊尾白虾组织蛋白酶L基因的cDNA全长,命名为EcCatL基因。该序列全长1136bp,包括5'非编码区24bp,开放阅读框960bp和3'非编码区152bp,开放阅读框共编码319个氨基酸,预测相对分子量为35.30×103,理论等电点为5.27。同源性分析表明,脊尾白虾组织蛋白酶LEcCatL氨基酸序列与其它甲壳动物高度保守,与变色小长臂虾(Palaemonetes varians)及北极甜虾(Pandalus borealis)CatL的同源性分别为92%和76%。系统进化分析表明,EcCatL基因氨基酸序列与变色小长臂虾的CatL聚为一支。荧光定量PCR分析结果表明,EcCatL基因在血细胞、鳃、肝胰腺、肌肉、卵巢、肠、胃及眼柄中均有表达,其中肝胰腺中的相对表达量最高。感染鳗弧菌及WSSV后6h和12h,脊尾白虾血细胞和肝胰腺中EcCatL的表达量较对照组均极显著增加(P<0.01),且具有明显的时间差异性,表明EcCatL基因在脊尾白虾免疫反应中具有重要作用。

Based on the EST sequence from a hemocyte eDNA library, the cathepsin L eDNA of Exopalaemon carinicauda （EcCatL） was cloned by rapid amplification of eDNA ends （RACE）. The EcCatL eDNA was 1136 bp in length, which contains an open reading frame （ORF） of 960 bp, encoding a 319 amino-acid polypeptide. Homology analysis revealed that the amino acid sequence of EcCatL was highly conserved with its homologs in other crustaceans. The similarities of EeCatL with the CarL of Palaemonetes varians and Pandalus borealis were 92% and 76%, respectively. Phylogenetic analysis showed that EcCatL was in the same branch as that of Palaemonetes varians. The expression levels of EcCatL in different tissues were analyzed by quantitative real-time PCR. Expression of EcCatL was detected in all tested tissues of E. carinicauda, including hemocytes, gill, hepatopancreas, muscle, ovary, intestine, stomach and eyestalk, with the highest expression level in hepatopancreas. After challenged with Vibrio anguillarum or white spot syndrome virus, the expression of EcCatL were up-regulated in the hemocytes and hepatopancreas orE. carinicauda. Our results implied that EcCatL might play an important role in the prawn immune response.