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

Expression of Synthetic Porcine Alpha-Lactalbumin in Transgenic Arabidopsis

摘要:

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。

关键词:猪α乳清蛋白; 拟兰芥; 转化; 蛋白质表达

Abstract:

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 A rabidopsis with Agrobacterium mediated transformation． Some herbicide-resistant lines of transform ed A rabidopsis were selected． 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 be confirmed only in the transgenic A rabidopsis．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.

Key words:Porcine α -lactalbumin; Arabidopsis; Transformation; Expression