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

Wnt1 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 . WISP 1a, WISP 1b, WISP 2 and WISP 3 were identified respectively.ORF of the four isforms of WISP were cloned and sequenced in common carp. WISP 1a was 1 089 bp encoding polypeptides of 362 amino acids with a calculated Mw of 41.2 ku and P I of 8.66. WISP 1b was 1 077 bp encoding a polypeptide of 358 amino acids with a calculated Mw of 41.0 ku and PI of 8.42. WISP 2 was 1 038 bp encoding polypeptides of 345 amino acids with a calculated Mw of 38.4 ku and PI of 8.22. WISP 3 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 WISP 1, WISP 2 and WISP 3 were highly homologous and were clustered to their corresponding subgroup respectively. WISP 1a, WISP 1b and WISP 2 all contained 4 conservative domains and WISP 3 lost the second domain in common carp.RT-PCR analysis revealed WISP 1a to be expressed in most common carp tissues,with the principal expression in skin and weakest expression in blood cells. WISP 1b was predominantly expressed in testicle,brain,skin and ovary. WISP 2 was highly observed in blood cells and gill. WISP 3 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 WISP 3 and all lowest at 36 hpf and then increased before 6th d.