不同含油量大豆成熟期胚抑制消减cDNA文库构建及其差异表达基因分析.pdfVIP

2007年 12月 中国油料作物学报 2007，29(4)：365— 371 Chinese journal of oil crop sciences 不同含油量大豆成熟期胚抑制消减 CDNA文库构建及其差异表达基因分析 程金朋 §，陈 波 ，徐有明 ，周新安 ，卢长明 ，魏文辉孙 (1．华中农业大学园艺林学学院，湖北 武汉 430070； 2．中国农业科学院油料作物研究所基因组与分子生物学研究室，湖北 武汉 430062) 摘要：利用抑制性消减杂交(suppression subtractive hybridization，SSH)技术，以高含油量大豆品种中豆32和 低含油量大豆品种油春02—6受精后36d胚为实验材料构建消减cDNA文库。文库的插入片段集中在500bp左右，挑 取766个克隆进行PCR筛选获得700个阳性克隆，经过点杂交筛选后选择了575个阳性克隆进行测序。经过同源性 比对归并后得到237个差异表达基因，除1O个未知基因外，其余涉及到蛋白贮藏、蛋白质降解、激素调节等多个方 面。本文还对部分可能与油脂合成相关的差异表达基因进行了RT—PCR半定量和荧光定量PCR检测，并简要讨论 了它们的功能，为进一步研究这些基因在大豆油脂合成过程中的作用提供依据。 关键词：大豆；抑制性消减杂交；cDNA文库；RT—PCR；实时荧光定量PCR 中图分类号：$565．103 文献标识码：A 文章编号：1007—9084(2007)04—0365一O7 Construction of suppression subtractive cDNA library and analysis of differentially expressed genes during maturing period seed of high and low oil content soybean variety CHENG Jin—peng ，CHEN Bo ，XU You—ming ，ZHOU Xin—an ，LU Chang—ming ，WEI Wen—hui (1．College of Horticulture and Forestry Science，Huazhong Agricultural University，Wuhan 430070，China；2．Department of Genomics and Molecular Biology，0il Crops Research Institute， Chinese Academy of Agricultural Sciences，Wuhan 430062，China) Abstract：The 36 day after fertilization(DAF)embryoes of high oil content variety Zhongdou 32 and low oil content variety Youchun 02—6 were used to construct suppression subtractive cDNA library．The inserted fragment sizes focus on 500 bp，and 766 clones were verified by PCR，among which 700 clones were used to perform dot blot hybridization．575 clones were obtained and sequenced．237 differentially expressed genes were obtained by homologous BI AST search．Besides 1 0 unknown function genes，the others related to protein storing，protein degradation，auxin regulation and SO on．Parts of differentially expre