酿酒酵母NHX1基因克隆与烟草表达分析

Cloning of NHX1 Gene from Saccharomyces cerevisia and its Heterologous Expression in Tobacco

提取酿酒酵母DNA,PCR法合成片段长度为1902bp的NHX1基因,构建植物高效表达载体转化烟草,经PCR扩增、GUS酶染色分子鉴定后,应用荧光定量PCR分析转化材料幼根NHX1基因的转录水平,并将各转化材料T1代烟苗移栽至中间试验隔离圃中,化验分析烟叶内在成分。获得抗卡那霉素、NHX1基因PCR扩增和GUS染色阳性的转化子42株,在转化材料幼根中可检测到外源基因NHX1转录的mR-NA分子;烟叶内在成分化验结果表明T1代转化材料烟叶含钾量增加30%,总糖含量显著降低,证明NHX1基因除了与Na+运输有关还与K+的吸收和糖分运输有关。

The DNA fxagment sized 1 902bp which sequence was the same as NHX1 gene was amplified from Saccharomyces cerevisia using RT-PCR methods. NHX1 gene was cloned into the plant binary expression vector containing intron kanamycin gene and SAR sequence from tobacco, and introduced into tobacco varieties by A - grobacterium mediated transformation. 42 transgenic plants of tobacco were obtained and approved by PCR, GUS, and realtime PCR analysis： The results of chemical content assays confirmed that NHX1 gene had been expressed effectively in the modified tobacco. The potassium content in the tabacco leaf oftransformant T1 generation had increased about 30% and the sugar content in the leaf was reduced significantly. It indicated that NHX1 gene had the function of K^＋ uptake.