人工合成的植物化猪α乳清蛋白基因在转基因拟兰芥的表达(英文)

Expression of Synthetic Porcine Alpha-Lactalbumin in Transgenic Arabidopsis

将一个398 bp的植物化的猪α乳清蛋白基因(Lactalbumin, LA)编码区克隆到带有花椰菜花叶病毒的35S启动子的质粒中。对它们进行PCR检测和序列分析,证实这些阳性克隆是实验预期的重组质粒。随即将35S启动子- α-乳清蛋白基因-终止子这一表达单元克隆到双元载体pCG-CB中,用该重组质粒双元载体pCG-CB-Lact转化农杆菌V3101后,以农杆菌法进行拟兰芥植物转化,用除草剂Finale对转化植物进行抗除草剂基因筛选,得到一些抗除草剂的转化植株。对这些抗除草剂植株进行猪α乳清蛋白基因PCR分析,成功筛选到带有猪α乳清蛋白基因并且可以在后代稳定遗传的转基因植物。Western blot蛋白质表达分析,表明猪α乳清蛋白在拟兰芥中成功表达,并且猪α乳清蛋白被正确加工成天然蛋白。

A 398-bp plantified encoding fragment of porcine α -lactalbumin, was cloned into plasmid under the control of the cauliflower mosaic virus 35S promoter. The expression cassette was introduced into Arabidopsis with Agrobacterium mediated transformation. Some herbicide-resistant lines of transformed Arabidopsis wereselected. The gene integration of chromosomal DNA level were confirmed in regenerated plants. All of them are positive for porcine α-lactalbumin gene via genomic DNA PCR. The expression at the protein level could beconfirmed only in the transgenic Arabidopsis. The arabidopsis-expressed lactalbumin (LA) migrated in SDS-PAGE with identical mobility to human LA prepared from human milk, indicating that the LA was correctly processed to yield a mature protein in plants