Screening And Cloning Of Metamorphosis Related Genes In Flatfishes

Vertebral asymmetry in flatfish is the most significant among vertebrates,with both eyes being located on only one side(the ocular side) of the body.Changes in body proportion occur during flatfish metamorphosis,which exhibit extensive morphological and functional changes in various organs.There is little molecular reason to help understand the mechanism of flounders' eye migration,but still need to be proved.The hybrids between Japanese flounder and stone flounder showed mixed eye-location:with both dextral type and sinistral type,and thus become good samples for studying the eye-migration problems,mRNAs from 22dph sinistral and dextral hybrids larvae were screened with the classical differential display RT-PCR (DD-RT-PCR) and representational difference analysis of cDNA(cDNA-RDA),and 23 and 47 putative fragments were isolated respectively.Most of the fragments isolated were not involved into eye migration specifically.But cDNA fragments of creatine kinase and trypsinogen 2 precursor genes isolated by cDNA-RDA exhibited stage specific expression patterns,indicating that they were related with energy supply and physiological changes during metamorphosis of flatfish.The present results showed that cDNA-RDA is one of the capable methods in isolating differentially expressed genes.Creatine kinase(CK) catalyzes the reversible reaction of phosphorylation of creatine by ATP,which produces phosphorycreatine,a high-energy phosphagen in all vertebrates.CK-M1 had been cloned and sequenced from the cDNA-RDA pool of Japanese flounder during metamorphosis.It shows conserved sequences with other vertebrate and high similarity and identity with other teleost CK-M.CK-M1 expresses in various tissues of adult flounder,including skeletal muscle,purtenance and gill. Whole mount hybridization and Real-time PCR showed that the CK-M1 levels changed in a stage-specific manner during larval development and metamorphosis, with increasing at first and reach the climax at the beginning of metamorphosis,then decreasing as the morphological changes going,finally come back to the pre-metamorphosis level,suggesting the CK-M1 is one of the important part of development of flounder larvae. Trypsinogen 2 precursor(TR2P) is the precursor of a major proteolytic enzyme precursor produced by pancreatic acinar cells.TR2P was selected by RDA between sinistral and dextral hybrid between Japanese flounder and stone flounder.It had been cloned and sequenced in Japanese flounder and stone flounder.Both of them showed high similarity and identity with other teleost TRP.TR2P expresses expecially in intestine.Real-time PCR showed that the TR2P levels increasd sharply at the beginning of Japanese flounder's metamorphosis,and kept higher expression than before till after metamorphosis,suggesting the TR2P is related with development of flounder larvae.Real-time PCR verified that the TR2P isolated in dextral pool showed more relativity to the dextral stone flounders than sinistral Japanese flounder, suggesting that TR2P's expression is also related to the genes that direct flatfishes' eye-position.