拟南芥K+转运蛋白AtKup1基因的DNA改组

采用同源重组法制备钾离子转运蛋白TRK1和TRK2缺失的酿酒酵母钾营养缺陷型，通过RNA 反转录PCR方法从拟南芥幼根扩增获得片段长度为2139bp 的Atkup1基因，以此片段为模板，采用DNA 改组技术,经Dnase I降解,Primerless PCR , PrimerPCR，建立Atkup1 基因突变库。将突变库和未经DNA 重排处理的Atkup1基因分别构建酵母穿梭载体导入K+转运蛋白基因TRK1和TRK2缺失的酿酒酵母中,分别在低钾(5.0mM KCl)不含色氨酸的培养基上筛选转化子, 突变基因库酵母转化子中获得2株长势明显好于Atkup1 基因转化子的突变基因转化菌株，菌株质粒上的突变Atkup1基因核苷酸测序结果发现突变基因Atkup1发生2个碱基的置换，造成2个氨基酸的改变，转化烟草烟叶化学成分分析证实突变基因的吸钾活性显著提高。

DNA shuffling of Arabidopsis thalianna K+ uptake transporter gene

The DNA fragment sized 2139bp which sequence is the same with AtKup1 gene from Arabidopsis thalianna was used as the templates to do DNA family shuffling. The shuffled AtKup1 gene library was expressed in the mutant of S. cerevisae in which potassium transporter gene TRK1 and TRK2 were knocked out by homologous recombination. Then the screening was carried out in the low potassium media containing 5.0 mM KCl and no histidine in it. it was found that both of diverse and wild AtKup1 genes can rescue the trk1 trk2 yeast mutant strain on low K+] medium. The growth of 2 clones yeast contained diverse AtKup1 were beter than that of AtKup1 wild gene transformant. The sequencing results of the shuffled Atkup1 showed that there were 2 nucleotides had changed and resulted in 2 amino acid variations in it compared with original AtKup1. The potassium uptaking capacity of shuffled Atkup1 gene increased significantly when it was transformed into tobacco.