拟南芥AtNHX6基因启动子的克隆及表达分析

为了探明拟南芥内膜反向转运体AtNHX6基因的组织表达模式,从基因组中克隆了AtNHX6基因开放阅读框(ORF)上游侧翼调控区1 922bp序列,并成功构建AtNHX6基因启动子与GUS融合表达载体pCAM-BIA1381-proNHX6-GUS,通过农杆菌花序浸染法转化野生型拟南芥获得T3代纯合转基因拟南芥株系,经PCR检测扩增得到2 187bp目的条带。利用组织染色法鉴定转基因拟南芥的GUS表达模式发现,在子叶、下胚轴和花中GUS活性显著。在这些广泛表达的部位中,微管系统中的表达最为显著,真叶中只有局部检测到GUS表达;在根中GUS在根毛和侧根生长部位表达;在未成熟果荚中只有在果荚顶端和基部存在GUS活性,成熟果荚中只在果柄检测到GUS表达;在花中,雄蕊的花丝和花粉粒及雌蕊的柱头中检测到GUS表达。GUS染色分析结果表明,AtNHX6基因启动子与GUS的融合表达载体成功构建并正常启动GUS基因表达,且AtNHX6基因主要在拟南芥的子叶、下胚轴、根、花、果荚中的微管系统、根毛和侧根生长部位以及花丝、花粉、柱头中表达。

Cloning and Expression Analysis of AtNHX6 Gene Promoter from the Arabidopsis thaliana

In order to test the tissue expression pattern of endosomal AtNHX6 gene, we amplified the promoter sequence of AtNHX6 gene from Arabidopsis thaliana, a 1 922 bp upstream of reading frame (ORF) by PCR. The fusion expression vector pCAMBIA1381 proNHX6 GUS was successfully constructed by the AtNHX6 gene promoter and GUS gene and introduced into the A. thaliana (ecotype Columbia 0) by the floral dip procedure. The homozygous T₃ transgenic A. thaliana lines was identified by PCR amplification of a 2 187 bp fragment. The expression pattern was monitored using GUS histochemical staining. The results showed that GUS staining was preferentially observed in cotyledons, hypocotyls and flowers of transgenic seedlings carrying the AtNHX6 promoter. In these organs, the highest GUS activity was detected in the vasculature, although GUS was widely expressed. In true leaves, GUS activity was only partially detected for the AtNHX6 promoter. In the root, GUS was expressed in the root hair and lateral root growth site. In immature siliques, GUS staining was restricted to the silique tip and base for the AtNHX6 promoter, but was detected only in fruit stalks in mature siliques. In flowers, GUS staining was observed in the filament of the stamens and pollen grains within the anthers and the ovarian stigma for the AtNHX6 promoter. Results from histochemical GUS assay suggested that fusion expression vector of AtNHX6 gene promoter and GUS was successfully constructed and it drives the expression of GUS gene successfully. The results also showed that AtNHX6 gene predominantly expressed in vascular tissues of cotyledons, hypocotyls, roots, flowers and siliques and the growth site of root hair and lateral root, as well as filaments, pollen, ovarian stigma of A. thaliana.