Expression Pattern of Ａ Recombinant Phloem-specific Promoter from Pumpkin in Transgenic Potato Plants

以本实验室构建的重组南瓜(Cucurbita moschata)韧皮部特异启动子dENP构建了植物表达载体pBdENP。利用根癌农杆菌(Agrobacterium tumefaciens)LBA44O4介导转化马铃薯(Solanum tuberosum)品种Favorita，经过抗生素筛选，共获得转pBdENP和对照pBI121的抗卡那霉素马铃薯再生植株106株。通过PCR初步筛查，筛选出65株为转基因阳性植株。通过Southern blot对部分植株进一步分析,确证外源gus基因已经插入到转基因马铃薯植株的基因组中，插入拷贝数在1个或2个以上。对这些转基因马铃薯植株进行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，前者仅在马铃薯的韧皮部内特异表达，而CaMV35S启动子驱动的gus基因为组成型性表达。GUS酶活力测定结果进一步表明dENP和CaMV35S启动子驱动gus基因表达水平没有明显区别。以上结果证明dENP启动子驱动的外源基因在马铃薯中也具有韧皮部特异而高效表达的特征，从而可用于马铃薯抗病、抗蚜虫转基因研究。

Expression Pattern of A Recombinant Phloem-specific Promoter from Pumpkin in Transgenic Potato Plants

The plant expression vector pBdENP was constructed on the basis of pBI121, which contains the neomycin phosphotransferase gene (nptⅡ) espression cassette and the β-glucuronidase (gus) gene driven by a recombinant pumpkin ( Cucurbita moschata ) phloem-specific promoter (dENP ). Potato (Solanum tuberosum ) leaf discs from aseptically cultured cv. Favorita plants were transformed with pBdENP and pBI121 mediated by Agrobacterium tumefaciens, respectively．And one hundred and six independent transformants of kanamycin resistant potato plants were regenerated. PCR and Southern blotting analyses confirmed that the gus gene had been integrated into the plant genome in 65 out of all independently transformed potato 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 in a tissue specific manner, depending on the nature of the promoter used. Results from histochemical staining confirmed that the GUS activity was localized specificly in phloem tissue of the transgenic plants if the gus gene is driven by dENP promoter. The average GUS activity in pBdENP transgenic plants had no significant difference compared with that driven by CaMV35S promoter. These results indicate that the recombinant dENP promoter can achieve a highly efficient and phloem-specific expression of a foreign gene, and can be practically useful in developing transgenic potato plants for improvement of aphid-resistence or disease-resistence.