大豆种子形态建成期与成熟期正反抑制消减文库构建及差异表达基因分析

大豆种子形态建成期与成熟期正反抑制消减文库构建及差异表达基因分析 Analysis of Differentially Expressed Genes at Pattern Formation and Maturation Stages of Soybean Seed 颜克亮 YAN Keliang1 1 first-author 陈波 CHEN Bo2 2 姚家玲 YAO Jialing1 1 周新安 ZHOU Xinan2 2 张会芳 ZHANG Huifang2 2 魏文辉 WEI Wenhui2 2 华中农业大学生命科学技术学院 华中农业大学生命科学技术学院 College of Life Sciences and Technology,Huazhong Agricultural University College of Life Sciences and Technology,Huazhong Agricultural University 中国农业科学院油料作物研究所 中国农业科学院油料作物研究所 Insititute of Oil Crops,Chinese Academy of Agricultural Sciences Insititute of Oil Crops,Chinese Academy of Agricultural Sciences 为了分析大豆种子形态建成期与成熟期基因差异表达情况,获得在发育过程中可能影响大豆种子形态建成及蛋白质、油脂合成代谢等调控的重要基因,利用抑制性消减杂交技术（suppression subtractive hybridization,SSH）构建了高含油量大豆品种中豆32开花后15天胚和36天胚正反消减文库.从正反消减文库中挑取克隆,经PCR及点杂交筛选后,从15天胚消减库（正向）和36天胚消减库（反向）中分别挑选了476个和471个有效克隆测序,经BLAST比对归并后,根据测序结果设计基因特异引物,通过半定量PCR和荧光定量PCR检测了13个差异表达基因,其中正向库5个,反向库8个.对这些差异表达基因的组织特异性检测表明,仅发现4-662（蔗糖结合蛋白基因）在胚中特异表达.对这些基因在大豆种子发育过程中的作用进行了讨论. The gene regulation of seed oil and protein synthesis and ovular development in soybean is largely unknown.In this study,we compared the gene expression between different development stages of a high oil soybean line,Zhongdou 32.Based on the measurement of fatty acid and protein contents at different development stages of soybean seed,the immature seeds were sampled 15 and 36 days after flowering（DAF） and differentially expressed gene between these two different developmental stages were obtained by subtractive suppression hybridization（SSH）.All SSH clones were arrayed and screened by dot blot hybridization,followed by PCR analysis,476 clones in forward library and 471 clones in reverse library were sequenced,respectively.The sequences were analysed on line by BLAST in the GeneBank dbase,and then design the gene special primers with Primer permier 5.Finally 13 differential expressed genes were detected by real-time quantitative PCR.Tissue-specific detection of these 13 genes showed that only sucrose-binding protein gene was found to be specially expressed in the seed.All the results provide important information on the gene regulation of seed oil/protein synthesis and ovular development.The possible role of these genes is discussed during the development of soybean seed. 抑制消减杂交 大豆 种子发育 差异表达基因 实时定量PCR subtractive suppression hybridization（SSH） Soybean seed development differentially expressed genes real-time quantitative PCR Q943.2;S565.1 中国农业科学院油料作物研究所所长基金（200602）资助项目 2008-02-01 2021-04-01 2