水母雪莲愈伤组织cDNA文库的构建.pdfVIP

植物学通报 2004, 21 (1): 61~65 Chinese Bulletin of Botany 水母雪莲愈伤组织cDNA 文库的构建① 1 1 ② 2 1 金治平 赵德修 乔传令 付春祥 1 （中国科学院植物研究所 北京 100093) 2 ( 中国科学院动物研究所 北京 100090 ） 摘要 用TRIZOL Reagent 提取水母雪莲红色系1~15 d 愈伤组织总RNA ，用SMART cDNA Library 6 11 Construction Kit 构建cDNA 文库。经测定原始文库滴度达到1.5~4 ×10 ，扩增总文库滴度达到10 ， 重组率达到98% ，插入片段在0.5 kb 到3 kb 之间，多在1 kb 左右。通过PCR 检测，从总文库中检 测到了雪莲CHS 、DFR 及SmP 基因的特异片段。SmP 基因是转录调控因子，其表达丰度很低。各项 指标都表明，已获得高质量的cDNA 文库，为雪莲基因资源保存，雪莲类黄酮次生代谢分子调控奠 定了坚实的基础。 关键词 水母雪莲, 愈伤组织, cDNA 文库 Construction of cDNA Library from the Callus of Saussrea medusa Maxim 1JIN Zhi-Ping 1ZHAO De-Xiu② 2QIAO Chuan-Ling 1FU Chun-Xiang 1 (Institute of Botany, the Chinese Academy of Sciences , Beijing 100093) 2 (Institute of Zoology, the Chinese Academy of Sciences, Beijing 100090) Abstract Total RNA of 1~15 d red line callus of S.medusa was extracted by TRIZOL Reagent. cDNA library was constructed with SMART cDNA Library Construction Kit. The primary library has a high 6 6 titer from 1.5 ×10 to 4 ×10 , in which 98% clones are recombinant and the insert cDNAs are from 0.5 kb to 3 kb. The amplified library has a titer of 1011. The positive signals of CHS, DFR and SmP gene were detected by PCR in the amplified library, though SmP gene was expressed at low abundance . This high quality cDNA library provides a useful tool for the study of the molecular mechanisms of the secondary metabolism of the flavonoids of S.medusa. Key words Saussrea medusa Maxim, Callus, cDNA library 水母雪莲(Saussrea medusa Maxim)是我国珍稀传统中药材，其主要有效成分为类黄酮类次 生代谢产物，其中高车前素（hispidulin ）和金合欢素（jaceosidin ）两种3-脱氧类黄酮具