Molecular cloning,expression and phylogenetic tree's construction of WISP gene family in common carp

Abstract

Wnt-1 inducible signaling pathway proteins(WISP)constitute the CCN family with CYR61,CTGF and NOV.We used BLAST program at the NCBI to identify near matches in the common carp(Cyprinus carpio)genome database to each zebrafish(Danio rerio)WISP.WISP1a,WISP1b,WISP2 and WISP3 were identified respectively.ORF of the four isforms of WISP were cloned and sequenced in common carp.WISP1a was 1 089 bp encoding polypeptides of 362 amino acids with a calculated Mw of 41.2 ku and PI of 8.66.WISP1b was 1 077 bp encoding a polypeptide of 358 amino acids with a calculated Mw of 41.0 ku and PI of 8.42.WISP2 was 1 038 bp encoding polypeptides of 345 amino acids with a calculated Mw of 38.4 ku and PI of 8.22.WISP3 was 1 026 bp encoding polypeptides of 341 amino acids with a calculated Mw of 37.9 ku and PI of 9.10.They all have 5 exons and 4 introns.Phylogenetic relationship analysis indicated that WISP1,WISP2 and WISP3 were highly homologous and were clustered to their corresponding subgroup respectively.WISP1a,WISP1b and WISP2 all contained 4 conservative domains and WISP3 lost the second domain in common carp.RT-PCR analysis revealed WISP1a to be expressed in most common carp tissues,with the principal expression in skin and weakest expression in blood cells.WISP1b was predominantly expressed in testicle,brain,skin and ovary.WISP2 was highly observed in blood cells and gill.WISP3 was most strongly expressed in blood cells and also expressed in brain,liver,spleen,head kidney and not detected in other tissues.Quantitative real-time PCR were used to analyse the expression of WISP in common carp development,and the result indicated that WISP transcriptions are expressed highly at the initial stage except WISP3 and all lowest at 36 hpf and then increased before 6th d.