利用SSH技术鉴定黄瓜胚胎发育相关基因.pdfVIP

中国农业科学 2010,43(11):2292-2299 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2010.11.012 利用SSH技术鉴定黄瓜胚胎发育相关基因 盛 慧，秦智伟，周秀艳，武 涛 （东北农业大学园艺学院黄瓜分子育种实验室，哈尔滨 150030 ） 摘要：【目的】寻找黄瓜胚胎发育早期和胚胎发育晚期优势表达基因，研究其胚胎发育的分子机制。【方法】 利用抑制性消减杂交（suppression subtractive hybridization, SSH ）技术构建胚胎发育早期和晚期差异表达 cDNA文库。【结果】经反向Northern blot杂交检测，共得到97个阳性克隆。利用分子生物学软件对测序后的序 列进行质量检测和聚类、拼接，共得到 93 个 UniESTs，其中包括 86 个 singletons 和 7 个 contigs。将上述 EST 序列在 GenBank 上进行 BLAST 比对，有 83 个 EST 片段找到了与之匹配的同源序列，有 10 个 EST 片段未找到同源 序列，推测可能是新基因。【结论】其中很多EST与拟南芥、水稻或玉米蛋白质数据库中的热激蛋白具有很高的同 源性。将以上序列应用GeneOntology进行功能分类，发现在胚胎发育早期细胞防御和代谢过程占主要位置；在胚 胎发育晚期细胞防御和抗渗透胁迫功能是非常重要的。 关键词：黄瓜；SSH；胚胎发育；cDNA文库 Identification of Embryo Development-Related Genes in Cucumber Using SSH SHENG Hui, QIN Zhi-wei, ZHOU Xiu-yan, WU Tao (Cucumber Molecular Breeding Laboratory, College of Horticulture, Northeast Agricultural University, Harbin 150030) Abstract: 【Objective 】 The aim of this experiment is to investigate the embryo development-related genes in cucumber, and study its molecular mechanism. 【Method 】 A subtracted cDNA library was constructed by using Suppression Subtractive Hybridzation (SSH). 【Result 】 A total of 97 positive clones were screened using reverse Northern blot hybridization. After the removal of duplication and sequencing of low-quality sequences, 93 UniESTs were identified, including 86 singletons and 7 contigs. The 93 unique ESTs were used to BLAST in the GenBank non-redundant database. There were 83 ESTs were found matching homologous sequences, 10 ESTs homologous sequence were not found, presumably may be new genes. 【Conclusion 】 Many of these ESTs were highly homologous with the