大豆GmNHX3基因克隆及遗传转化载体的构建

利用RACE(rapid amplification of cDNA end)方法, 以大豆叶片提取总的RNA为模板克隆了大豆Na⁺/H⁺逆向转运蛋白（Glycine max Na⁺/H⁺ antiporter, GmNHX）基因, 并将其连接到表达载体PBI121中, 构建重组表达载体PBI121 NHX3. 分析结果表明： 该基因的ORF为1 503 bp, 推测编码501个氨基酸. 与所选取的10种植物同类蛋白氨基酸序列进行对比, 一致性为72%～94%, 并具有真核生物单价阳离子(氢离子)反向转运蛋白典型的结构域, 将该基因命名为GmNHX3, GenBank接收号为JN872904. 通过PCR和酶切鉴定, PBI121 NHX3构建成功.

Cloning of GmNHX3 Gene from Glycine max L Merr and Construction of Its Genetic Transformation Vector

RACE(rapid-amplification of cDNAends) technique was used to clone Na+/H+ antiporter gene from Glycine max L Merr with the total RNA as the template.This gene was linked to the plasmid PBI121 and the expression vector PBI121-NHX3 was constructed.The results show its ORF(open reading frame)consists of 1 503 bp,which can be deduced to encode 501 amino acids.It was found that predicted amino acid sequence had an identification of 72%-94% compared with amino acid sequence from other ten plant species and it had a typical structural domain of the monovalent cation(proton) antiporter.The gene was named GmNHX3,(GenBank accession number: JN872904).Genetic transformation vector of PBI121-NHX3 was constructed successfully by identification method of PCR and restriction enzyme digestion.