摘要
采用同源序列法克隆得到了大豆根部特异性启动子、种皮特异性启动子、种子特异性启动子,核苷酸序列大小分别为2 500bp、1 832bp、1 268bp,分别具有CANNTG-motifs、GATA-box、ACGT等启动子表达元件,并分别构建了这3个组织特异性启动子的植物报告表达载体。通过农杆菌介导法将3个表达载体转入烟草NC89,通过组织化学染色和GUS基因的相对表达量分析验证3个启动子的功能。通过转化获得了根部特异性启动子转基因烟草植株11株,种皮特异性启动子转基因烟草植株4株,种子特异性启动子转基因烟草植株8株,转35S启动子启动GUS基因的阳性烟草植株7株。GUS化学组织染色结果表明,克隆得到的根、种皮、种子特异性启动子都表现为组织特异性表达,表达水平明显高于叶片、茎部、花等部位。进一步的荧光定量PCR结果显示,根部特异性启动子GUS的相对表达量在根部最高,而在茎、叶、种子、种皮、花中表达量较低;种皮部特异性启动子GUS的相对表达量在种皮最高,而在根、茎、叶、种子、花中表达量较低;种子特异性启动子GUS的相对表达量在种子最高,而在根、茎、叶、种皮、花中表达量较低,但是与35S启动子相比,这3个特异性启动子的GUS相对表达量在相应的组织均低于35S启动子。
In this study,the soybean root specific promoter,seed specific promoter and seed coat specific promoter were cloned by homologous sequence method respectively. Their sizes were 2 500 bp,1 832 bp and 1 268 bp.They had different expression elements including CANNTG-motifs,GATA-box,ACGT. Three plant expression vectors of these tissue specific promoters were constructed and transferred into Nicotiana tabacum NC89 by Agrobacterium-mediated method to prove GUS activity of different promoters. Soybean root,seed coat and seed specific promoter nucleotide sequence were isolated by PCR method. Then 11 root specific promoter transgenic plants,4 seed coat specific promoter transgenic plants,8 seed specific promoter transgenic plants,and 7 35 S-promoter-transgenic plants had been obtained. GUS activity assays indicated that GUS gene expression level in root,seed and seed coat were higher than leaf,shoot and flower. Further quantitative real time PCR results showed that the expression levels of GUS gene of root specific promoter in root were higher than those in stem,leaf,seed,seed coat and flower; the expression levels of GUS gene of seed coat specific promoter in seed coat were higher than those in root,stem,leaf,seed and flower; the expression levels of GUS gene of seed specific promoter in seed were higher than those in root,stem,leaf,seed coat and flower. But the expression levels of GUS of these three tissue specific promoters were lower than those of 35 S promoter in each tissues.
出处
《中国油料作物学报》
CAS
CSCD
北大核心
2017年第6期771-777,共7页
Chinese Journal of Oil Crop Sciences
基金
大学生创新创业训练计划项目(126)
科技创新基金项目(126)
关键词
启动子
组织特异性
基因表达
相对表达量分析
promoter
tissue specific
gene expression
relative expression analysis
