摘要
【目的】研究早期胚胎发育基因,筛选猪早期体内发育胚胎和体外孤雌激活胚胎的差异表达基因,探讨影响胚胎发育的分子基础。【方法】分别采用孤雌激活和手术(猪体内发育)的方法收集2细胞、4细胞、8-16细胞时期的胚胎,利用SPEDDRT-PCR挑选不同时期的差异表达产物,将所获得的产物按照片段大小分离纯化后通过反向Northern杂交去除假阳性条带。将阳性条带克隆入T载体中,经过PCR鉴定后挑选其中的阳性克隆进行测序。【结果】筛选了11个代表不同时期表达差异的cDNA片段,编号为DDD1-DDD11。经过与GenBank数据进行同源性分析,发现其中DDD3、DDD11没有同源序列信息,提交数据库获得GenBank登录号(EU597224、EU597228),其余序列发现相似性较高的数据。【结论】对体内正常发育胚胎和孤雌激活胚胎进行对比、分析,发现体内4细胞期和体内8-16细胞期各有1个片段为新基因片段,为进一步分析发育相关基因及其功能奠定基础。
【Objective】 This study was conducted to explore the molecular mechanism of early porcine development and find new candidate genes in the development of in vivo and parthenogenetically activated early porcine embryos.【Method】 The single preimplantation embryo differential display reverse transcription polymerase chain reaction （SPEDDRT-PCR） was used to identify differentially expressed genes in 2-cell,4-cell,8-16-cell in in vivo and parthenogenetically activated early porcine embryos.Differentially expressed genes were characterized by reverse Northern dot blot.【Result】 A total of 11 ESTs （expressed sequences tags） were found using SPEDDRT-PCR and reverse Northern dot blot approach,three of which in 2-cell in vivo,five in 4-cell,one in 8-cell embryos,and the other two in 2-cell parthenogenetically activated embryos.All 11 ESTs were compared with nucleotide sequences deposited in the nr and dbEST database of Genbank using BLAST.DDD3 and DDD11 ESTs had no significant similarity with exisiting genes or ESTs,and were regarded as new ESTs.The two new ESTs were submitted to GenBank （accession numbers：EU597224,EU597228）.The other ESTs had their highly similar nucleotide sequences with ESTs existing in nucleotide databases （but with unknown functions）.【Conclusion】 These results can provide information on gene expression patterns and lay a foundation for further study on the biological mechanisms controlling gene expression during preimplantation porcine embryo development.
出处
《中国农业科学》
CAS
CSCD
北大核心
2010年第11期2388-2396,共9页
Scientia Agricultura Sinica
基金
国家自然科学基金(30571339)
中国农业科学院优秀科技创新团队
关键词
胚胎
孤雌激活
发育相关基因
猪
embryos
parthenogenetic activation
development-related genes
pig
