【摘要】 以授粉后27d的种子mRNA为目标群体,授粉后7d的种子mRNA为对照,构建甘蓝型油菜种子发育期基因差异表达文库.文库包括560个克隆,经菌落PCR检测,456个菌落含有插入片段,占全部克隆的91.2%,片段大小200～600bp.对PCR阳性克隆进行斑点杂交,得到201个上调的阳性斑点杂交阳性克隆,对其进行测序,得到有效ESTs序列198个.BLASTN结果显示,134个ESTs序列没有同源序列,可能是新基因,另外64个ESTs序列在油菜或拟南芥核酸数据库中存在同源序列.这64个ESTs序列和已知功能(主要是能量代谢、物质代谢、基因调控、衰老及抗性等)的蛋白有高度相似性.更多还原

【Abstract】 A forward subtractive cDNA library was constructed using suppression subtractive hybridization (SSH) method, which was performed using the cDNA from seeds at 27 days after pollination as the tester and the cDNA from seeds at 7 DAP as driver. 560 clones were obtained. These clones were verified by colony PCR. The recombined efficiencies were 91.2 %. These inserted fragment size mostly ranged from 200 bp to 600 bp. PCR positive clones were screened by dot hybridization,a total of 201 clones were found to be up-regulated in seeds at 27 DAP. These clones were sequenced,and followed by BLASTN and BLASTX for sequences dates. 198 uniESTs were obtained,134 ESTs showed no significant matches to any other sequences in NCBI nucleotide sequence database therefore,it were regarded as novel genes. 64 ESTs carried with remarkable identity to proteins with known function in the datebase,which fell into several functional categories including energy and matter metabolism,gene regulation,senescence and resistance.更多还原