日本囊对虾(Marsupenaeus japonicus)组织蛋白酶D基因克隆及表达分析

Molecular cloning and expression analysis of cathepsin D from kuruma shrimp(Marsupenaeus japonicus)

组织蛋白酶D是溶酶体天冬氨酸蛋白酶家族的主要成员,广泛参与动物机体细胞内蛋白质的降解过程,对维持细胞稳态和正常代谢具有重要的作用。为研究组织蛋白酶D在甲壳动物非特异性免疫和幼体发育过程中的作用,本研究采用RACE技术首次克隆得到日本囊对虾组织蛋白酶D基因cDNA序列,命名为MjCatD,其中开放阅读框长为1 161bp,编码386个氨基酸残基。序列分析和同源建模显示该基因编码的蛋白含有保守的N-糖基化位点、天冬氨酸蛋白酶签名序列、酶活化位点和非消化性组织蛋白酶D的特征序列,并且呈保守的双叶形结构。同源性比较和系统进化分析发现,MjCatD与斑节对虾、美洲螯龙虾和脊尾白虾相似性较高,并且与它们紧密聚为一支。实时荧光定量PCR结果显示,MjCatD基因在日本囊对虾多个组织中均有表达,其中肝胰腺中表达量最高。在白斑综合征病毒（white spot syndrome virus,WSSV）感染后3～24h日本囊对虾肝胰腺中MjCatD的表达量逐渐下降,而在48h急剧上调至最高表达量并且与对照组差异极显著（P〈0.01）。此外,MjCatD基因在幼体发育不同阶段中也表现出明显的变化趋势。以上研究表明,MjCatD基因可能参与日本囊对虾先天免疫反应和幼体发育过程。

Cathepsin D is the principal member of lysosomal aspartic proteinase family which participates in various degradations of intracellular protein and plays important roles in normal metabolism for the maintenance of cellular homeostasis.In this current study,the cDNA sequence of kuruma shrimp（Marsupenaeus japonicus）cathepsin D（MjCatD）was cloned for the first time through RACE technology,including a 1 161 bp open reading frame encoding 386 amino acids.Both sequence analysis and homology modeling revealed that the deduced protein of MjCatD contained well conserved N-glycosylation site,aspartic proteinase signature sequence,active site and the characteristic sequence of non-digestive cathepsin D,as well as presented a conserved bilobal structure.MjCatD shared high similarity with those fromPenaeus monodon,Homarus americanus,Palaemon carinicauda,and clustered together with above three cathepsin Ds.Real-time quantitative PCR demonstrated that MjCatD was ubiquitously expressed in all the examined tissues,predominantly in hepatopancreas.After challenging with white spot syndrome virus（WSSV）,the expression level of MjCatD in the hepatopancreas was gradually down regulated during 3hto 24 h,then very significantly increased（P〈0.01）and peaked at 48 hcompared to the control.MjCatD also showed obvious changes during different larval stages.These findings indicated that MjCatD may play key roles in the innate immune response and larval development of kuruma shrimp.