林烟草Shaker钾通道基因NsylNKT6启动子的克隆与功能分析

Cloning and Functional Analysis of the Promoter of Shaker Potassium Channel Gene NsylNKT6 from Nicotiana sylvestris

Shaker家族钾离子通道在植物钾素的吸收转运中发挥重要作用。前期从林烟草(Nicotiana sylvestris)中克隆到Shaker家族GroupⅡ的一个成员NsylNKT6,但其具体的组织表达模式尚不清楚。本研究以林烟草基因组DNA为模板,克隆获得NsylNKT6上游长1981 bp的启动子序列。生物信息学分析表明,NsylNKT6启动子含有多个光、激素和胁迫等响应元件及分生组织表达元件。利用叶盘转化法,获得转NsylNKT6启动子驱动GUS报告基因的林烟草材料。GUS染色结果显示,正常培养条件下,在林烟草出苗期的叶片保卫细胞检测到GUS活性;在林烟草团棵期,叶片的叶脉维管组织、腺毛及保卫细胞均检测到GUS活性。上述结果表明NsylNKT6在保卫细胞、叶脉维管组织和腺毛细胞中表达。推测该基因可能通过调节钾吸收参与气孔运动,或介导维管组织和腺毛中的钾离子吸收转运过程,且其表达可能受光、激素和胁迫等的调控。本研究为解析NsylNKT6钾通道的生理功能提供理论基础。

Shaker family potassium channels play an important role in potassium uptake and transport in plants. A Shaker potassium channel gene, NsylNKT6, was cloned from Nicotiana sylvestris in the early stage and phylogenetic analysis indicated that it belongs to the Group Ⅱ subfamily, but its expression pattern is unclear. In this study, a 1 981 bp promoter sequence upstream of NsylNKT6 was cloned from N. sylvestris. Bioinformatics analysis showed that NsylNKT6 promoter contains light-, hormone-, stress-responsive elements and meristem expression elements. NsylNKT6 promoter driving GUS reporter gene was then transformed into N. sylvestris by Agrobacterium-mediated leaf disc method to obtain the transgenic tobacco plants, and GUS staining assay was performed.The results showed that, under the normal growth condition, GUS activity was detected in the stomatal guard cells of the leaves at the emergence stage. During the rosette stage, GUS activity was detected in vein vascular tissue,glandular hair cells and guard cells of the leaves. The results indicated that NsylNKT6 is expressed in stomatal guard cells, vein vascular tissue and glandular hair cells. It is speculated that NsylNKT6 might participate in stomatal movement by regulating K+absorption or participate in the absorption and transport of K+in vascular tissues and glandular hairs, and its expression might be regulated by light, hormones, and some environmental stresses.This study provided the theory foundation for analyzing the physiological functions of the potassium channel,NsylNKT6.