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

摘要

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.%以水稻根系cDNA为模板,PCR扩增含His标签的Lsi1基因开放阅读框,并将该基因克隆到巴斯德毕赤酵母表达载体pPIC9k中,PCR及测序验证重组质粒pPIC9k-Lsi1目的基因序列.将验证正确的重组质粒pPIC9k-Lsi1通过SacⅠ酶切线性化,电转到毕赤酵母GS115感受态细胞中,通过MD/MM板筛选出甲醇利用快型的酵母转化子,再通过不同浓度梯度的G418平板筛选多拷贝转化菌株.提取酵母基因组,PCR验证正确的菌株即为工程菌(GS115/pPIC9k-Lsi1).用1%甲醇诱导该工程菌72 h后,收集上清,经SDS-PAGE检测到位于32.8 ku处的目的蛋白即为Si转运蛋白(OsLsi1).