银鲫原肠胚差异表达基因的筛选

SCREENING OF DIFFERENTIALLY EXPRESSED GENES AT GASTRULA STAGE DURING EMBRYOGENESIS OF CARASSIUS AURATUS GIBELIO

胚胎发育是基因组中各个基因在时间和空间上选择性表达的结果。为了鉴定参与鱼类早期胚胎发育和胚层分化的调控因了,分别构建了银鲫(Carassiusauratusgibelio)原肠胚和成熟卵子的SMARTcDNA文库,并采用差异筛选的方法,从银鲫原肠胚SMARTcDNA文库中筛选不同于成熟卵子的差异表达基因。通过菌斑和PCR产物的两轮斑点杂交,从大约1500个克隆中筛选出131个阳性克隆,从中选择58个克隆测序并将测得的序列进行了数据库比对分析,结果显示这些差异表达基因大部分为参与转录和翻译的调控因子和核糖体蛋白以及一些在胚胎早期大量表达的参与脂类代谢的脂蛋白和一些未知的新基因。接着采用RTPCR技术,对其中6个基因在胚胎发育不同阶段的表达特征进行了分析,进一步证实这些基因在胚胎发育过程中存在表达差异;研究还从分析的基因中揭示出4类不同的表达模式。通过本研究,已筛选出一批在银鲫胚胎发育早期开始表达的调控基因,为开展鱼类胚胎发育早期表达基因的功能研究奠定了前期工作基础。

Embryogenesis is a progressive and complicated process that is regulated by differential gene activity. In order to identify some differentially expressed genes in early embryogenesis,two kinds of SMART cDNAs were respectively synthesized from mature eggs and gastrula embryos of Carassius auratus gibelio,and the gastrula embryo SMART cDNA library was constructed. Following this program,131 positive clones were screened from about 1500 clones of the gastrula embryo SMART cDNA library by two rounds of dot-blot differential hybridizations using the synthesized SMART cDNAs as probes. Furthermore,58 positive clones were selected for sequencing. Searching GenBank by the nucleotide sequences indicated that most of the sequenced cDNAs encoded ribosome proteins and regulators involved in translation and transcription,and some encoded lipoproteins involved in the metabolism of fat. And,some unknown new genes were also identified from the screening. Moreover,we investigated the expression patterns of 6 genes during embryogenesis by RT-PCR analysis,and confirmed their differential expression during embryogenesis. Four different expression patterns,such as transcribing again at gastrula based on the maternal products,beginning to transcribe at blastula stage, beginning to transcribe at gastrula stage,and transcribing only during embryogenesis, were revealed in the analyzed genes. Through this study,we cloned and identified some genes expressed differentially at early stage during embryogenesis,which will be a solid base for further researches on their functions and regulative mechanisms during embryogenesis.