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园艺学报 ›› 2008, Vol. 35 ›› Issue (2): 189-194.

• 果树 • 上一篇    下一篇

小金海棠MxIRT1基因定点突变的研究

张学宁;孔 瑾;王 忆;韩振海;许雪峰*
  

  1. (中国农业大学果树逆境生理与分子生物学实验室,北京100094)
  • 收稿日期:2007-07-03 修回日期:2007-08-17 出版日期:2008-02-25 发布日期:2008-02-25
  • 通讯作者: 许雪峰

Studies of Site Direct Mutagenesis of MxIRT1 Gene of Malus xiaojinensis

ZHANG Xue-ning,KONG Jin,WANG Yi,HAN Zhen-hai,and XU Xue-feng*
  

  1. (Stress Physiology and Molecular Biology Laboratory of Fruit Tree, China Agricultural Universit, Beijing 100094, China)
  • Received:2007-07-03 Revised:2007-08-17 Online:2008-02-25 Published:2008-02-25
  • Contact: XU Xue-feng

摘要:

采用PCR技术对小金海棠MxIRT1基因的定点突变体系进行了探讨。根据MxIRT1开放阅读框(ORF)两端和突变位点序列各设计一对引物。通过PCR扩增,获得带有突变位点且在突变位点相互重叠的两个PCR片段。对以上两个片段的浓度进行调整后,将其用作PCR反应的模板。对退火温度和延伸时间进行优化后,利用ORF两端引物,将带有突变位点的两个片段拼接起来,从而获得了含有所要突变位点的MxIRT1,并将其插入pEASY-T2载体中。DNA序列分析表明在预期位点上已经发生了突变:MxIRT1编码的第186位密码子已由组氨基酸残基变为丙氨酸残基,证明用PCR技术已成功地突变了MxIRT1基因。整个突变过程不需要任何回收与纯化步骤,是一个高效、快捷的定点突变体系。

关键词: 小金海棠, 基因, 定点突变, MxIRT1, PCR

Abstract:

PCR were applied for point mutagenesis of MxIRT1 gene. Two pairs of primers were designed according to the Opening Read Frame (ORF) and sequence for mutation of MxIRT1. Two fragments overlapping at the target mutated base were amplified by PCR. The fragments were amplified after dilution with the primers designed with the two ends of ORF, and the two mutated fragments were stringed together. The annealing temperature and extension time were optimized for successful amplification. The mutated MxIRT1 were ligated into pEASY-T2. Sequencing result showed that we mutated MxIRT1 successfully. DNA recollection and purification were not necessary for this experiment. We establish an efficient, simple gene mutagenesis system.

Key words: Malus xiaojinensis, MxIRT1, PCR, Site Direct Mutagenesis

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