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大肠杆菌甘露糖-6-磷酸异构酶基因(manA?pmi)的克隆及功能鉴定

DOI: 10.7668/hbnxb.2011.02.023, PP. 107-110

Keywords: 甘露糖-6-磷酸异构酶,大肠杆菌,果糖,选择标记

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Abstract:

甘露糖-6-磷酸异构酶基因(pmi?manA)是当前植物转基因工程的重要选择标记基因,在生物安全上被认为是无害的.根据同源序列从大肠杆菌DH10B基因组中克隆出pmi基因.通过诱导原核表达获得PMI目的蛋白,对粗提和经过镍柱纯化的PMI蛋白进行Western分析和酶活反应鉴定.以上结果表明,PMI蛋白成功诱导,其分子量为6kDa,纯化过的PMI具有高的酶活性,能催化甘露糖-6-磷酸发生异构反应生成果糖-6-磷酸.同时利用pmi基因作为选择基因构建植物表达载体pCPMi,通过农杆菌介导法转化烟草获得转基因植株,PCR检测结果表明96个再生植株有72株为阳性.以上结果表明,克隆的pmni基因具有高的酶活性,并能作为转基因植物的一种选择标记,同时也为转基因植物提供安全的选择标记基因,便于转基因植物的推广.

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