Chong D K, Langridge W H
Center for Molecular Biology and Gene Therapy, Loma Linda University, CA 92350, USA.
Transgenic Res. 2000 Feb;9(1):71-8. doi: 10.1023/a:1008977630179.
A cDNA fragment encoding human lactoferrin (hLF) linked to a plant microsomal retention signal peptide (SEK-DEL) was stably integrated into the Solanum tuberosum genome by Agrobacterium tumefaciens-mediated leaf disk transformation methods. The lactoferrin gene was expressed under control of both the auxin-inducible manopine synthase (mas) P2 promoter and the cauliflower mosaic virus (CaMV) 35S tandem promoter. The presence of the hLF cDNA in the genome of regenerated transformed potato plants was detected by polymerase chain reaction amplification methods. Full-length hLF protein was identified by immunoblot analysis in tuber tissue extracts from the transformed plants by immunoblot analysis. The hLF produced in transgenic plant tissues migrated during polyacrylamide gel electrophoresis as a single band with an approximate molecular mass equal to hLF. Auxin activation of the mas P2 promoter increased lactoferrin expression levels in transformed tuber and leaf tissues to approximately 0.1% of total soluble plant protein. Antimicrobial activity against four different human pathogenic bacterial strains was detected in extracts of lactoferrin-containing potato tuber tissues. This is the first report of synthesis of full length, biologically active hLF in edible plants.
通过根癌农杆菌介导的叶盘转化方法，将一个编码人乳铁蛋白（hLF）并与植物微粒体滞留信号肽（SEK-DEL）相连的cDNA片段稳定整合到马铃薯基因组中。乳铁蛋白基因在生长素诱导型甘露碱合成酶（mas）P2启动子和花椰菜花叶病毒（CaMV）35S串联启动子的控制下表达。通过聚合酶链反应扩增方法检测再生转化马铃薯植株基因组中hLF cDNA的存在。通过免疫印迹分析在转化植株的块茎组织提取物中鉴定出全长hLF蛋白。转基因植物组织中产生的hLF在聚丙烯酰胺凝胶电泳中迁移为一条单带，其近似分子量与hLF相等。mas P2启动子的生长素激活使转化块茎和叶片组织中的乳铁蛋白表达水平提高到总可溶性植物蛋白的约0.1%。在含乳铁蛋白的马铃薯块茎组织提取物中检测到对四种不同人类致病细菌菌株的抗菌活性。这是在可食用植物中合成全长、具有生物活性的hLF的首次报道。
