摘要
提取拟南芥总RNA,反转录合成Na＋/H＋逆向转运蛋白基因AtNHX1,构建了植物高效表达载体,用叶盘法将其导入烟草,经筛选获得了抗卡那霉素和GUS染色阳性的转化子38株。应用荧光定量PCR方法对转化材料部分含AtNHX1基因的烟草内源钾通道基因、烟草K＋转运蛋白基因和烟草焦磷酸酶基因的转录水平进行了分析。结果表明：在转化烟草中可以检测到AtNHX1基因mRNA,与未转化材料相比,烟草钾离子转运蛋白基因NtHAK1的转录降低,H＋-ATPase基因NHA1转录增加,钾通道基因NKT转录无显著变化,T1代转化烟叶中K＋含量显著增加,Na^＋、Ca^2＋无显著变化,烟叶中总糖含量降低,证明了编码拟南芥AtNHX1基因与植物的K＋吸收有关。
The vacuolar Na^＋/H^＋ antiporter gene AtNHX1 was cloned from Arabidopsis thaliana using RNA isolation and RTPCR methods. The binary vector containing AtNHX1 gene was constructed, and introduced into tobacco via Agrobacterium-mediated leaf disk transformation. Thirty-eight transgenic tobacco plants which appeared kanamycin resistance and GUS assay positive were obtained by AtNHX1 PCR screening. Transcript levels of AtNHX1 and four tobacco genes in- volved in K^＋ uptake, including NKT （K^＋ channel）, NtHAK1 （K^＋ transporter）, NHA1 （P-H^＋ -ATPase） and PPA （V- H^＋ -PPase） in the AtNHX1 modified tobacco roots were analyzed by real-time PCR. The mRNA of AtNHX1 can be detected in the transgenic plants. The transcript of P-H ^＋ -ATPase gene NHA 1 is up-regulation, and that of potassium transporter gene NtHAK1 is down-regulation. No significant alteration can be found in the transcript of potassium channel NKT1. The potassium content in the leaves of AtNHX1 transgenic tobacco T1 generation is increased by about 53.9% and the sugar content is significantly reduced, however, no significant changes was found in Na^＋and Ca^2＋contents in tobacco leaves. Our data indicate that AtNHX1 plays an important role in potassium uptake.
出处
《高技术通讯》
CAS
CSCD
北大核心
2007年第11期1174-1179,共6页
Chinese High Technology Letters
基金
国家烟草专卖局科技攻关项目（110200101008）资助.
作者简介
男，1964年生，博士；研究方向：分子生物学。
通讯作者，E-mail：yangq@hit．edu．cn
