拟南芥K^+转运蛋白AtKup1基因的DNA改组

DNA Shuffling of Arabidopsis thalianna K+ Uptake Transporter Gene

采用同源重组法制备钾离子转运蛋白基因TRKI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2 139bp的AtKup1基因,以此片段为膜板,采用DNA重排技术,经DNase I降解,Primerless PCR和Primer PCR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,并导入K+转运蛋白基因TRK1和TRK2缺失的酿酒酵母中,分别在低钾(5．0mmoL／L KC1)不合色氨酸的培养基上筛选转化子,从突变基因库酵母转化子中获得2株长势明显优于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表明突变基因AtKup1发生了2个碱基的置换,造成了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。

The DNA fragment sized 2 139bp, the same Sequence with AtKupl gene from Arabidopsis thalianna was used as the templates for DNA family shuffling. The shuffeld AtKupl 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 mmol/L KCl and no histidine in it. it was found that both of diverse and wild AtKupl gene can rescues the trk1Δ trk2Δ yeast mutant strain in low [ K^＋ ] medium. The growth of 2 clones yeast containing diverse AtKupl were beter than that of AtKupl wild gene transformant. The sequencig results of the shuffeld AtKupl showed that there were 2 nucleotide changed, which resulted in 2 amino acid variations in it compared with the original AtKupl. The potassium uptaking capacity of shuffled AtKupl gene increased significantly when it was transformed into tobacco.