玉米ZmMYB59基因启动子的克隆及功能分析

Cloning and functional analysis for the promoter of ZmMYB59 gene from maize(Zea mays L.)

为研究ZmMYB59基因启动子的表达模式,以玉米自交系‘B73’幼苗基因组DNA为模板,克隆ZmMYB59基因2个启动子片段分别命名为MYB59-P-1、MYB59-P-2,构建GUS植物表达载体pCXGUS-MYB-1K,pCXGUS-MYB-2K,并通过农杆菌介导法转化水稻‘日本晴’获得GUS植物表达载体转基因植株。通过PlantCARE软件进行生物信息学分析,发现ZmMYB59基因启动子中具有ABRE和CCGTCC-box等重要的顺式作用元件。GUS染色结果显示:1)pCXGUS-MYB-1K的种子没有着色,表明其未驱动GUS在种子中表达,pCXGUS-MYB-2K的种子胚乳边缘着色,表明其驱动的GUS在种子胚乳边缘表达;2)种子萌发期pCXGUSMYB-1K只有芽尖着色,表明其仅驱动GUS在芽尖表达,pCXGUS-MYB-2K芽尖和根均着色,芽尖着色较深,根维管束细胞少量着色,表明其驱动的GUS主要在芽尖表达,而在根部维管束组织中表达较弱;3)苗期pCXGUSMYB-1K,pCXGUS-MYB-2K植株的根、茎、叶均能着色,但后者着色较深,表明ZmMYB59基因的启动子可能是组成型启动子,同时也说明MYB59-P-1是启动子发挥正常调控功能所必须的,但启动能力不强,推测MYB59-P-2中可能存在增强启动子表达的顺式作用元件。

To explore the expression pattern of the promoter of ZmMYB59 gene,two promoter fragments of ZmMYB59 gene,MYB59-P-1 and MYB59-P-2,were cloned by using genomic DNA of‘B73’as template in this study,respectively.pCXGUS-MYB-1 K,pCXGUS-MYB-2 K expression vectors were then constructed by using MYB59-P-1 and MYB59-P-2 to drive GUS gene expression,respectively.Bioinformatics analysis was carried out by using PlantCARE software,and the result showed that the ZmMYB59 gene promoter contains important cis-acting elements such as ABRE and CCGTCC-box.GUS staining was performed on germinated seeds,and seedling roots,stems and leaves in the T1 generation plants transformed with pCXGUS-MYB-1 Kor pCXGUS-MYB-2 K.In seeds,no GUS staining signal was observed in pCXGUS-MYB-1 Ktransformed lines,which indicated that it didn’t drive GUS expression in seeds.Some signal was observed in the endosperm edge of pCXGUS-MYB-2 Ktransformed lines indicating that the promoter drived the expression of GUS in the endosperm of seeds edge.During germination period,the GUS activity could be detected only in shoot tips in pCXGUS-MYB-1 Ktest,which indicated that pCXGUS-MYB-1 Konly drived GUS expression in shoot tips,while the GUS expression was observed in pCXGUS-MYB-2 Ktest with weak signal in roots and strong signal in shoot tips indicating that it mainly drived the GUS expression in shoot tips.At seedling stage,the GUS activity could be detected in roots,stems,leaves of pCXGUS-MYB-1 K and pCXGUS-MYB-2 k.And in the pCXGUS-MYB-2 ktest stronger expression was observed,indicating that the promoter of ZmMYB59 gene is constitutive promoter,and it also indicates that MYB59-P-1 is required for the normal regulatory function of the promoter,but the promoter ability is not strong,it is speculated that there may be cis-acting elements in MYB59-P-2 that enhance the expression of promoter.