斜带石斑鱼囊胚期胚胎和尾芽期胚胎差异表达基因的筛选及克隆

Screening and Analysis of Differential Expression Genes in Blastula and Tail Bud Stage Embryos in the Orange-spotted Grouper (Epinephelus coioides)

以斜带石斑鱼囊胚期胚胎和尾芽期胚胎分别作为检验组和驱动组,构建了石斑鱼囊胚期胚胎和尾芽期胚胎的抑制性差减杂交cDNA文库。以α-tubulin作为检测指标,显示差减效率分别高达28和27。分别取囊胚期胚胎和尾芽期胚胎各192和960个PCR阳性克隆进行斑点杂交,得到15个囊胚期和131个尾芽期的斑点杂交阳性克隆。测序和数据库比对分析表明,囊胚期15个阳性克隆中有11个已知基因的cDNA片段和没有同源性的4个cDNA片段;而在尾芽期的131个阳性克隆中,有123个已知基因的cDNA片段和8个没有同源性的cD-NA片段。用半定量RT-PCR技术分析了部分基因片段在胚胎发育过程中的表达规律和和组织分布情况。这些差异表达片段的呈现为进一步揭示石斑鱼胚胎发育、早期性别决定和性腺分化的分子机制奠定了基础。

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 α-tubulin cDNA was approximately 2^7 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 differentia- tion in groupers.