马氏珠母贝(Pinctada fucata martensii)Pm-CTSL基因的克隆及表达分析

Cloning and Expression Analysis of Pm-CTSL Gene in Pinctada fucata martensii

组织蛋白酶L (cathepsin L, CTSL)是无脊椎动物体内非特异性免疫的重要组成部分。为了研究马氏珠母贝CTSL (Pm-CTSL)的功能,本实验利用RACE技术克隆获得了Pm-CTSL基因,并对其结构和组织表达模式进行了分析。结果表明,Pm-CTSL基因c DNA序列全长为1 237 bp,其中开放式阅读框957 bp,5'UTR69 bp,3'UTR 211 bp,共编码氨基酸318个。结构域分析发现Pm-CTSL具有CTSL两个典型的结构域Inhibitor_I29和Pept_C1。Pm-CTSL也具有信号肽、保守序列ERFNVN和GNFD、两个潜在的N-糖基化位点和催化活性位点三联体残基(Cys, His和Asn)。多序列比对结果表明Pm-CTSL与太平洋牡蛎的同源性最高,为42%;系统进化分析发现,Pm-CTSL与虾夷扇贝等贝类聚为一支。实时荧光定量分析发现,Pm-CTSL在肝胰腺中显著高表达(p<0.05),表明Pm-CTSL可能参与马氏珠母贝的免疫应答。本研究为进一步探讨CTSL在马氏珠母贝非特异性免疫应答中的作用提供了基础资料。

Cathepsin L(CTSL) is an important component of invertebrate nonspecific immunity. To study the functions of Cathepsin L in Pinctada fucata martensii( Pm-CTSL), we cloned the Pm-CTSL gene using rapid amplification of c DNA end(RACE) methods and analyzed its structure and tissue expression pattern. The results showed that the full length of Pm-CTSL c DNA was 1 237 bp, containing 957 bp of the open reading frame, a 5’UTR of 69 bp and a 3’UTR of 211 bp, which encoded 318 amino acids. Two typical structural domains, which were Inhibitor_I29 and Pept_C1, were found in Pm-CTSL by structural domain analysis. And Pm-CTSL was also proved to have an signal peptide, two conserved sequences, ERFNVN and GNFD, two potential N-glycosylation sites, and catalytically active sites triad residues(Cys, His and Asn). The multiple alignments showed that the Pm-CTSL had the highest homology to the Crassostrea gigas with a 42% identity. Phylogenetic analysis found that Pm-CTSL was clustered with shellfish such as Patinopecten yessoensis into one branch. The real-time fluorescence quantitative analysis showed that Pm-CTSL was highly expressed in the hepatopancreas(p<0.05), which suggested that Pm-CTSL may be involved in the immune response of the P. f. martensii. This study may provide basic materials for the study of CTSL in the non-specific immunity response in P. f. martensii.