琯溪蜜柚及其早熟红肉突变体成熟果实SSH 文库的构建及初步分析.pdfVIP

园 艺 学 报 2013 ，40 （7 ）：1359 –1368 http: // www. ahs. ac. cn Acta Horticulturae Sinica E-mail: yuanyixuebao@126.com 琯溪蜜柚及其早熟红肉突变体成熟果实 SSH 文 库的构建及初步分析 * 刘顺枝，黄 忠，胡位荣，江月玲，王小兰 （广州大学生命科学学院，广州 510006 ） 摘 要：分别以琯溪蜜柚及其突变体红肉蜜柚（成熟期早 20 ~ 25 d ）的成熟果肉cDNA 作驱赶子和 检测子，利用结合分子镜像选择技术的 SSH 技术，构建了红肉蜜柚和琯溪蜜柚的正向差减 cDNA 文库。 通过 PCR 检测 cDNA 克隆外源插入片段，其大小介于 100 ~ 1 000 bp。通过点杂交差异筛选文库，得到 102 个表达增强 2 倍或以上的克隆并测序，结果表明这 102 个克隆代表 44 个 Unigenes ，通过序列同源性 比对分析，发现获得的Unigenes 在功能上主要涉及信号传导、蛋白质合成、应激反应、转运等代谢反应。 关键词：琯溪蜜柚；突变体；抑制差减杂交；差异表达基因 中图分类号：S 666 文献标志码：A 文章编号：0513-353X （2013 ）07-1359-10 Construction and Analysis of Suppression Subtractive Hybridization Library of Red-fleshed Sweet Pomelo * LIU Shun-zhi ，HUANG Zhong ，HU Wei-rong ，JIANG Yue-ling ，and WANG Xiao-lan （School of Life Sciences ，Guangzhou University，Guangzhou 510006 ，China） Abstract ：A forward subtractive cDNA library was constructed through suppression subtractive hybridization（SSH） method with mirror orientation selection ，which was performed using the cDNA from wild type ‘Guanxi ’pomelo （Citrus grandis）as driver，those from its red-flesh mutant as tester. PCR analysis showed that most of the clones with insertions ranged from 100 bp to 1 000 bp. After differential screening through dot hybridization，a total of 102 transcripts represented 44 Unigenes with signal ratios stronger than two times were selected for 2 additional screenings. Homology analysis showed that they were found to be involved in diverse biological processes ，such as signal transduction，protein synthesis ， stress response as well as transport metabolism.