Screening and analysis of differential expression genes in blastula and tail bud stage embryos in the orange-spotted grouper ( Epinephelus coioides )

Suppression subtractive hybridization (SSH) libraries were constructed in blastula stage and tail bud stage embryos in the orange-spotted grouper (Epinephelus coioides). The subtraction efficiency of the two subtractive cDNA libraries indicated by alpha-tubulin cDNA was approximately 2(8) and 2(7) folds, respectively. One hundred and ninety two and 960 PCR positive clones were respectively selected to perform dot blot tests; 15 and 131 dot blot positive clones were obtained from SSH plasmid libraries in the blastula and tail bud stage embryos. Sequence analysis and database searches showed that there were 11 known genes and four unknown cDNA fragments in the sequenced 15 dot blot positive clones for the blastula embryos and 123 known genes and eight unknown cDNA fragments in the sequenced 131 dot blot positive clones for the tail bud embryos. RT-PCR analysis revealed the state of partially differential expression and tissue distribution of 12 cDNA fragments. Further functional studies on the differentially expressed genes screened in this study will provide more information on the molecule regulation mechanisms of embryo development, early sex determination and gonad differentiation in groupers