文章摘要
史西志.海带雄性配子体差异表达基因片段的克隆及筛选[J].水产学报,2005,29(5):
海带雄性配子体差异表达基因片段的克隆及筛选
Cloning and screening of differentially expressed genes from the male gametophytes of Laminaria japonica Aresch
投稿时间:2014-03-10  修订日期:2014-03-10
DOI:
中文关键词: 海带 差异表达基因 抑制消减杂交 cDNA消减文库 雄配子体
英文关键词: Laminaria japonica differentially expressed genes suppression subtractive hybridization (SSH) subtracted cDNA library male gametophyte
基金项目:国家自然科学基金,上海市教委资助项目,上海水产大学校科研和教改项目
作者单位地址
史西志 SHI Xi-zhi SHI Xi-zhi
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中文摘要:
      利用抑制消减杂交法(suppression subtractive hybridization,SSH)获得了海带雌、雄配子体差异表达的cDNA片段,将雄配子体差异表达的cDNA片段克隆于pGEM-T载体并转化大肠杆菌(Escherichia coli)JM109,构建雄配子体差异表达的cDNA消减文库.自750个阳性克隆中随机挑选100个进行PCR扩增鉴定,电泳图谱表明插入片段大小在200~1000 bp之间.进一步经过Southern斑点杂交筛选后,选取10个阳性克隆进行测序和序列分析,有7个片段与GenBank数据库中已知基因序列无明显同源性,初步判断为新的基因序列,3个片段与已知Fcp基因和Lhcf 6基因的同源性分别达到88%和98%.
英文摘要:
      Differentially expressed cDNAs subtracted female from male gametophytes of Laminaria japonica were obtained by suppression subtractive hybridization (SSH). The differentially expressed cDNAs from the male gametophytes were ligated into the pGEM-T vector and the latter was consequentially transformed into Escherichia coli JM109 competent cells. The subtracted cDNA libraries of male gametophytes were constructed. One hundred randomly selected clones were amplified by PCR using T7 and SP6 primers for primary screening. The length of inserted fragments assayed by PCR amplification was mostly from 200 to 1000 base pairs. Ten clones obtained after dot-blot Southem hybridization were sequenced and analyzed for homology comparison using BLAST. Seven of the ten clones showed no sequence homology in the GenBank database, suggesting that they or at least some of them with longer base pairs might represent novel gene sequences. The other three clones shared 88% and 98% sequence homologies with understood Fop and Lhcfs genes, respectively.
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