Cloning and expression analysis of cathepsin B gene from Pinctada maxima

【Objective】Analyzed the expression patterns of cathepsin B gene(CatB) in different tissues of Pinctada maxima, and clarified the functional role of CatB in P. maxima, in order to provide basic information for cultivating fastgrowing and stress-resistant P. maxima.【Method】The CatB gene in P. maxima(designated as PmCatB) was obtained by RACE with ExPASy ProtParam, ExPASy ProtScale, NPS@SOPMA, SWISS-MODEL and SignalP 4.1 online softwares for bioinformatics analysis and quantitative real-time PCR was used to detect the expression level of PmCatB in different tissues including the mantle (marginal zone of mantle, pallial zone of mantle and central zone of mantle), hepatopancreas, gill, foot and adductor muscle.【Result】The full length of PmCatB cDNA sequence was 1365 bp, with an open reading frame(ORF) of 1026 bp, 5' untranslated region(UTR) was 81 bp, 3' UTR was 258 bp, and encoded 341 amino acids residues. The molecular weight of PmCatB protein was 37.73 kD and the theoretical isoelectric point(pI) was 6.6. The fat solubility coefficient was 67.48, the instability index was 31.18, and the average hydrophilicity coefficient(GRAVY) was -0.451, which was a stable hydrophilic protein;there was a Pept-C1 domain at amino acids 89-337. The secondary structure prediction of PmCatB protein showed that random coils were dominant, accounting for 51.03%, α-helix accounted for 26.98%, β-turn accounted for 9.09%, and extended strand accounted for 12.90%; the tertiary structure was similar to the CatB protein structure of P. fucata martensii. The similarity between the CatB amino acid sequence of P. maxima and the P. f. martensii (ADX32985.1) was as high as 90.91%; and the similarities of the amino acid sequence of the Crassostrea gigas(XP_011428258.1), Argopecten irradians(ANG56311.1) and Cristaria plicata(AEF32260.1) compared with P. maximawere 79.47%, 65.38% and 62.18% respectively. PmCatB was expressed in tissues including the marginal zone of mantle, pallial zone of mantle, central zone of mantle, hepatopancreas, gill, foot and adductor muscle, the relative expression in hepatopancreas was the highest, and the relative expression level wassignificantly higher than the relative expression in other tissues(P<0.05), and followed with the marginal zone of the mantle and the central zone of the mantle.【Conclusion】PmCatB shows high expression in the hepatopancreas, and followed by the pallial zone of mantle and central zone of mantle, so it is speculated that PmCatB may participate in the process of growth and metabolism process bytaking part in the digestion and absorption of P.maxima.