Acta Hydrobiologica Sinica (水生生物学报) 2012/36:6 PP.1128-1134
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 5′-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 inhibitor domain and a mature domain. The phylogenetic analysis showed that a clear clade division was observed between vertebrates and invertebrates cathepsin L proteins, and the Hs-CtsL of H. 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 transcripts 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 hepatopancrease 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.