小金海棠二价铁转运蛋白基因MxIRT1定点突变的研究  

Studies of Site Direct Mutagenesis of MxIRT1 Gene of Malus xiaojinensis

作　　者：张学宁[1] 孔瑾[1] 王忆[1] 韩振海[1] 许雪峰 

机构地区：[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

出　　处：《园艺学报》2008年第2期189-194,共6页Acta Horticulturae Sinica

基　　金：北京市教育委员会共建项目(JD100190532)

摘　　要：采用PCR技术对小金海棠(Malus xiaojinensis)二价铁转运蛋白基因MxIRT1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位密码子已由组氨酸残基变为丙氨酸残基,证明用PCR技术已成功地使MxIRT1基因发生突变。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。PCR was applied for point mutagenesis of MxIlRT1 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 MxlRTI was ligated into pEASY-T2. Sequencing result showed that we mutated MxlRTI successfully. DNA recollection and purification were not necessary for this experiment. We establish an efficient, simple gene mutagenesis system.

关 键 词：小金海棠 基因突变 定点突变技术 二价铁转运蛋白基因 缺铁病 

分 类 号：S665.2[农业科学—果树学]