福建农林大学学报（自然科学版）2016，Vol.45Issue(5)：561-566,6.DOI:10.13323/j.cnki.j.fafu(nat.sci.).2016.05.014
His标签的水稻硅转运蛋白表达载体的构建及在巴斯德毕赤酵母中的表达
Construction of eukaryotic expression vector of OsLsi1 with His-tag and its expression in Pichia pastoris
摘要
Abstract
To investigate the mechanism of silicon ( Si) on improving stress resistance of rice, open reading frame ( ORF) of Lsi1 with His-tag was amplified from cDNA of rice root, and followed by being cloned into vector pPIC9k expressed by Pichia pastoris. Then the recombinant plasmid of pPIC9k-Lsi1 was verified by PCR and DNA sequencing, among which the correct recombinant plasmid was digested with restriction enzyme SacⅠ, and transformed into competent cells from P.pastoris GS115 by electroporation. Mut+ transformants were obtained by MD/MM plates, and followed by being screened with different concentrations of G418 plates to obtain transformants with multiple copies. Genome of transformants was further extracted and amplified by PCR. The results con-firmed that the positive transformant was the target strain GS115/pPIC9k-Lsi1. Lastly, 1% methanol was used to induce Si transport-er ( OsLsi1) expression, and after 72 h the supernatant was collected and detected by SDS-PAGE. The result showed the Si trans-porter ( OsLsi1) was detected at the molecular weight of 32.8 ku.关键词
巴斯德毕赤酵母/Si转运蛋白/pPIC9k/异源表达Key words
Pichia pastoris/OsLsi1/pPIC9k/heterogenous expression分类
农业科技引用本文复制引用
林红梅,方长旬,林瑞余,何建宇,林伟伟,李颖哲,林文雄..His标签的水稻硅转运蛋白表达载体的构建及在巴斯德毕赤酵母中的表达[J].福建农林大学学报（自然科学版）,2016,45(5):561-566,6.基金项目
国家自然科学基金项目(31271670,31300336) （31271670,31300336）
教育部博士点基金项目(20133515130001). （20133515130001）
