转拟南芥AtKup1基因高含钾量烟草获得

提取拟南芥幼根总RNA,进行RTPCR逆转录,产物测序,将其构建到含Km(带内含子)筛选标记的植物双元表达载体上,根癌农杆菌介导法将AtKup1基因导入烟草,对转化子进行PCR扩增、GUS染色、Southern杂交和AtKup1基因mRNA荧光定量PCR分析,并进行烟叶内在成分化验分析。PCR获得2100bp左右扩增产物,测序结果证实扩增产物序列与拟南芥AtKup1基因(GenbankNo:AF029876)一致;得到GUS染色阳性的AtKup1基因转化烟草材料29株。经PCR扩增、分子杂交检测证实AtKup1基因已整合到转基因烟草的基因组,荧光定量PCR分析可以在幼根中检测到AtKup1基因的mRNA转录。烟叶含钾量化验结果表明导入的AtKup1基因在转化材料中成功表达,使烟叶含钾量提高约45%。

Transgenic Tobacco with Arabidopsis thaliana AtKup1 Gene Has High Potassium Content in Leaves

Total RNA was isolated from the roots of Arabidopsis thaliana, and AtKup1 gene was amplified using RT-PCR methods. The PCR product was cloned into pMD18-T, which named YF5501. After sequencing, the DNA fragment which containing AtKup1 gene was cloned into the plant binary expression vector containing intron kanamycin gene, and introduced into tobacco varieties by Agrobacterium mediated transformation. YF5501 contained the same sequence of AtKup1 gene (Genebank No:AF029876). 29 transgenic plants of tobacco were obtained and proved by PCR, GUS, Southern blot and realtime PCR analysis. The results of chemical content assays confirmed that AtKup1 gene had been expressed effectively in the modified plants and the potassium content in the tobacco leaves had been increased up to 45%.