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

Abstract

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.