池蝶蚌组织蛋白酶L基因的组织表达及免疫应激分析

EXPRESSION ANALYSIS AND IMMUNE RESPONSE OF THE CATHEPSIN L FROM FRESHWATER PEARL MUSSEL,HYRIOPSIS SCHLEGELII

应用cDNA文库筛查及同源片段克隆拼接技术,克隆了池蝶蚌组织蛋白酶L(Hs-CtsL)cDNA基因全长序列(GenBank注册号为JN604558)。其cDNA全长1152 bp,5′-非翻译区(Untranslated Region,UTR)长1 bp,3′-UTR长149 bp包括1个多聚腺苷信号AATAAA和Poly(A)尾巴,开放阅读框(Open reading frame ORF)为1002 bp,编码333个氨基酸组成的多肽链。其分子量约37.7 kD,理论等电点为7.16,包含信号肽、前体域和成熟域。系统进化分析显示,Hs-CtsL同无脊椎动物组织蛋白酶L聚为一支,且同三角帆蚌亲缘关系最近,其次为褶纹冠蚌。组织表达分析结果显示,池蝶蚌组织蛋白酶L在肠、鳃、性腺、外套膜、斧足、闭壳肌、血细胞、肝胰腺、肾和心脏均有表达,其中血细胞中表达量最高。应激实验表明,经嗜水气单胞菌刺激后,Hs-CtsL在血细胞、鳃、肝胰腺和外套膜中的表达量显著上调。其中在肝胰腺中刺激后6h表达量到达峰值,在血细胞、鳃和外套膜中的表达模式近似,表现为一个波动变化,在4h、12h和48h被上调。结果暗示着Hs-CtsL除参与了池蝶蚌血细胞的先天性免疫防御以外,还参与了其消化腺免疫器官的免疫应答反应。

Hyriopsis schlegelii is a fresh water mussel species farmed intensively and is chosen mainly to produce pearls in China. Cathepsin L is a member of the cysteine protease family and plays important roles in many physiological processes. In this paper, we report the full-length cDNA of cathepsin L （GenBank accession No. JN604558） from H. schlegelii based on cDNA library screening, homologous cloning strategies, and its immune response, cDNA of H. schlegelii （denoted as Hs-CtsL） was 1152 bp long and consisted of a Y-untranslated region （UTR） of 1 bp, a 3＇-UTR of 149 bp with one cytokine RNA instability motif （AATAAA） and one Poly （A） tail, and an open reading frame of 1002 bp, encoding a polypeptide of 333 amino acids, with an estimated molecular mass of 37.7 kD and a theoretical isoelectric point of 7.16. The protein sequence contained a typical signal peptide sequence with 20 amino acids, a propeptide in- hibitor domain and a mature domain. The phylogenetic analysis showed that a clear clade division was observed be- tween vertebrates and invertebrates cathepsin L proteins, and the Hs-CtsL ofH. schlegelii clustered with the invertebrate cathepsin L cysteine proteases and was closely related to corresponding sequences in Hyriopsis cumingii and Cristaria plicata. Tissue expression detection with real-time fluorescence quantitative PCR demonstrated that the Hs-CtsL tran scripts in healthy three-year adult H. schlegelii was expressed in intestine, gill, gonad, mantle, foot, adductor muscle, haemocytes, hepatopancrease, kidney and heart, with the highest expression level in haemocytes. After pathogenic Aeromonas hydrophila challenge, the Hs-CtsL mRNA expression was significantly up-regulated in haemocytes, gill, mantle and hepatopancrease, and the highest expression in hepatopanerease was observed at 6h after stimulation whereas its increased expression was detected at 4h, 12h and 48h in haemocytes, gill and mantle, showing a fluctuant change during the early stage of bacterial challenge. These results suggested that the Hs-CtsL was not only involved in the immune defense of hemocytes but also involved in the immune response of hepatopancrease in H. schlegelii.