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中国农学通报

中国农学通报 ›› 2005, Vol. 21 ›› Issue (3): 77-77.

所属专题： 生物技术

• 目次 • 上一篇    下一篇

小金海棠抑制性消减cDNA文库的构建及文库质量分析

张玉刚,许雪峰,李天忠,韩振海   

  • 出版日期:2005-03-05 发布日期:2005-03-05

Construction of Subtracted cDNA Library of Malus xiaojinensis with Suppression Subtractive Hybridization and Their Quality Analysis

Zhang Yugang, Xu Xuefeng, Li Tianzhong Han Zhenhai,   

  • Online:2005-03-05 Published:2005-03-05

摘要： 构建小金海棠抑制性消减文库，为进一步大量筛选、克隆苹果铁高效基因、果树转基因工程奠定基础。试验是在提取水培条件下小金海棠缺铁(EDTA-Fe2＋，4μmol/L)和正常供铁(EDTA-Fe2＋，40 μmol/L)的根的总RNA，经过LD-PCR扩增双链cDNA后，利用抑制性消减杂交(SSH)方法，通过两轮杂交和两次抑制性PCR，构建了小金海棠缺铁条件下包含1200个独立克隆的消减cDNA文库。用菌落PCR检测，结果显示插入片段大小范围在300~800bp之间，提取质粒进行PCR和质粒RsaⅠ酶切同样也得到一致性的结果。

Abstract: Construction of subtracted cDNA library of Malus xiaojinensis is the base of screening and cloning genes under Fe-deficiency stress. Using LD-PCR technique, cDNA was synthesized from total RNA of Malus xiaojinensis roots treated under Fe-deficiency stress(EDTA-Fe2＋，4μmol/L)which was used as tester and Fe-sufficiency(EDTA-Fe2＋，40μmol/L)as driver. A subtracted cDNA Library was constructed using suppression subtractive hybridization(SSH). After two hybridizations and two suppression PCRs, the amplified library contained 1200 positive clones. Plasmid inserts were PCR amplified and showed 300~800bp inserts. There is the same result with RsaⅠdigestion of plasmid