Construction and analysis of subtractive cDNA library of recovery body wall in sea cucumber Apostichopus japonicus

A subtracted cDNA library of sea cucumber Apostichopus japonicus(body weight 65-90 g) was constructed by suppression subtractive hybridization technology(SSH) to screen EST associated with recovery body wall.The cDNA library of the test group has been constructed by the mRNA of the body walls 24,48,72,96 and 120 h after the operation,and those with no operation as the control group.Differential EST from the subtracted cDNA library have been identified by both PCR technology and dot blot hybridization.Two hundred and ninety-two positive clones were observed from total 768 clones,and 224 positive ones were sequenced.Two hundred and eight EST were found and analyzed by BlastX tool in GenBank database,in which 171 EST were homologous with sequences in the database,and 18 were high homologous with known function genes including: beta-tubulin,tubulin alpha-1 chain,actin,tRNA pseudouridine synthase A,GTPase,cell division cycle 2-like,Mitogen-activated protein kinase,homeobox protein,elongation factor 1A,ribosomal protein L30 and L17,60S acidic ribosomal protein P0,26S protease regulatory subunit,ubiquitin specific peptidase 24,serologically defined colon cancer antigen 3 and scavenger receptor cysteine-rich protein type 12 which upregulated in wounded and recovery body walls of the sea cucumber.In this study,the subtracted cDNA library was successfully constructed by SSH and a number of EST related to recovery body wall were identified,which laid a foundation for the molecular basis and the process of regeneration of body wall in the sea cucumber.