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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。
According to the EST sequence obtained from the full-length cDNA library of Exopalaemon carinicauda in our laboratory, the full-length cDNA of Cathepsin L gene was cloned by RACE technology and named as EcCatL gene. The full length of this sequence is 1136bp, including 24bp in 5 ’untranslated region, 960bp open reading frame and 152bp in 3’ noncoding region. The open reading frame encodes a polypeptide of 319 amino acids with a predicted relative molecular mass of 35.30 × 103 and a theoretical isoelectric point of 5.27. Homology analysis showed that the amino acid sequence of LEcCatL was highly conserved among other crustaceans and 92% and 76% homologous to CatL of Palaemonetes varians and Pandalus borealis, respectively %. Phylogenetic analysis showed that the amino acid sequence of EcCatL was clustered with CatL of C. przewalskii. Fluorescent quantitative PCR analysis showed that EcCatL gene was expressed in blood cells, gills, hepatopancreas, muscle, ovary, intestine, stomach and eyelid, and the relative expression level in hepatopancreas was the highest. At 6h and 12h after infection with V. anguillarum and WSSV, the expression of EcCatL in blood cells and hepatopancreas of P. przewalskii significantly increased compared with that of the control group (P <0.01), and had obvious time difference, indicating that EcCatL gene was expressed in the tail Shrimp immune response has an important role.