【摘要】 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46kDa,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pmi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。更多还原

【Abstract】 Mannose-6-phosphate isomerase(PMI)is encoded by the manA/pmi gene from some bacteria as a selectable marker which has been used for the transformation of plant.The novel pmi gene was cloned from E.coli DH10B strain according to homologous sequence by PCR methods.Then,a prokaryotic vector pEPMi and a plant expression vector pCPMi both harboring pmi gene were constructed,respectively.The prokaryotic vector pEPMi with his-tag was transformed into Escherichia coli to express PMI protein.A single band on SDS-PAGE gel was presented by optimizing the concentration of imidazole in the buffers during the PMI protein isolation.Then,western blot assay confirmed that the induced target protein was PMI protein fused His tag,the size of protein is about 46 kDa.The results of analysis of enzyme reaction indicated that PMI has catalyzed the conversion of mannose-6-phosphate to fructose-6-phosphate with high efficiency.In addition,plant expression vector pCPMi was transformed into tobacco by Agrobacterium-mediated system,and 72 of 96 independent tobacco transformants were confirmed to contain the pmi gene by PCR detection.So the novel pmi gene can be directly used in plant transformation as a kind of safe selectable marker.更多还原