葡萄VviHKT1;1基因启动子的克隆与表达分析

Cloning and expression analysis of grape VviHKT1;1 gene promoter

HKT1;1转运蛋白是一类具有Na^(+)转运功能的蛋白,在植物抗盐中起关键作用。为了探究葡萄中VviHKT1;1基因在盐胁迫下的表达模式,用荧光定量PCR技术检测了VviHKT1;1在不同盐浓度,不同组织及不同处理时间的表达量,发现VviHKT1;1在葡萄茎中的表达量远高于根和叶;不同盐浓度处理后,VviHKT1;1在根和茎中的表达量随着盐浓度的升高而显著降低,叶中除100 mmol·L^(-1)NaCl外,其余盐浓度下表达量稍有升高;盐处理2 d后,VviHKT1;1表达量在葡萄根和茎中明显降低,但在葡萄叶片中随着盐胁迫时间延长显著升高。随后,克隆了VviHKT1;1上游1620 bp启动子序列,通过PlantCARE预测发现该区域有多个激素和逆境响应元件。用该启动子与GUS报告基因融合构建植物表达载体,并转化拟南芥,通过GUS染色与荧光定量分析了启动子对盐胁迫的响应。结果一致显示,GUS主要分布在地上部分,并且在盐胁迫后GUS在叶中的表达量比根中有明显增加。因此,我们推测盐处理后,地上部分可以加强VviHKT1;1表达将地上部分较多的Na+再回流到根,以降低地上部分的Na+积累缓解盐害。

HKT1;1 transporter has Na^(+)transport function,which plays a key role in plant salt resistance.In this study,the expression profile of Vvi HKT1;1 in grapes was investigated in different tissues and different treatment times of NaCl treatment by qPCR.The results showed that the expression level of VviHKT1;1 in grape stems was much higher than roots and leaves.Moreover,with salt concentration increasing,the expression level of VviHKT1;1 in roots and stems decreased significantly,while the expression level increased slightly in leaves,except for 100 mmol·L^(-1)NaCl.After 2 days of treatment,the expression level of VviHKT1;1 decreased significantly in grape roots and stems,but significantly increased in grape leaves.Furtherly,the 1620 bp upstream sequence of Vvi HKT1;1 was cloned,and it was found that there were multiple hormone and stress response elements in this region by PlantCARE analysis.The promoter was fused with GUS reporter gene to construct a plant expression vector and transformed into Arabidopsis thaliana.The response of the promoter to salt stress was analyzed by GUS staining and expression level in transgenic plants.The results showed that GUS mainly distributed in the shoots,and GUS expression had a significant increase in leaves than in roots in salt stress.Therefore,we suggested that the increase of VviHKT1;1 expression level in shoot was benefit to Na+recycle back from shoot to root for dropping Na+accumulation in shoot,relieving salt harm.