【摘要】 以自行构建的重组南瓜韧皮部特异启动子dENP构建了植物表达载体pBII2。利用根癌农杆菌(Agrobacterium tumefaciens)LBA44O4介导转化棉花(Gossypium hirsutum L.)品种珂字312,共获得转pBII2和对照pBI121的抗卡那霉素棉花再生植株243株,筛选出172株为转基因阳性植株。Southern blot分析确证外源Gus基因插入拷贝数在1个或2个以上。对这些转基因棉花植株进行Gus染色结果表明dENP和CaMV35S启动子一样均能驱动Gus基因的表达,前者仅在棉花的韧皮部内特异表达,而CaMV35S启动子驱动的Gus基因为组成性表达。Gus酶活力测定结果进一步表明dENP和CaMV35S启动子驱动Gus基因表达水平相当,但是转pBII2棉花植株的Gus富集在韧皮部组织。证明了dENP启动子驱动的外源基因在棉花中具有韧皮部特异而高效表达的特征,从而可用于棉花抗病、抗蚜虫转基因研究。更多还原

【Abstract】 The plant expression vector pBII2 was constructed on the basis of pBI121,which contains the neomycin phosphotransferase gene(NPT II) expression cassette and the β-glucuronidase(Gus) gene driven by a recombinant pumpkin phloem-specific promoter(dENP).Cotton embyogenesis calli from aseptically cultured cv.Coker 312 plants were transformed with pBII2 and pBI121 mediated by Agrobacterium tumefaciens,respectively.243 independent transformants of kanamycin resistant cotton plants were regenerated.PCR and Southern blot analyses confirmed that the Gus gene had been integrated into the plant genome in 172 out of all independently transformed cotton plants.Integration of the transgene varied from one to over two estimated copies in the analyzed plants.Gus expression in the transgenic plants was either constitutive or tissue specific,depending on the nature of the promoter used.Results from histochemical staining confirmed that the Gus activity was specifically localized in phloem tissue of the transgenic plants if dENP promoter drives the Gus gene.The average Gus activity in pBII2 transgenic plants had no significant difference compared with that driven by CaMV35S promoter.更多还原