马铃薯StmiR166b靶基因的鉴定及其表达分析

Identification and Expression Analysis of StmiR166b and Its Target Genes in Potato

为阐明马铃薯(Solanum tuberosum L.)StmiR166b的生物学功能,以栽培品种‘甘农薯2号’试管苗为试验材料,采用定量PCR技术检测StmiR166b及其靶基因StHB14、StHB15在不同组织中的差异表达;利用pRS300质粒为模板,通过over-lapping PCR技术克隆了包含StmiR166b-StmiR166b^*成熟体序列的前体结构,构建了其人工表达载体,通过根癌农杆菌(Agrobacterium tumefaciens)介导法转化马铃薯获得了转基因植株。结果表明,StmiR166b及其靶基因StHB14和StHB15在马铃薯根、茎和叶中均有表达,在转基因株系L1、L3中靶基因不同程度地下调表达;与非转基因对照相比,转基因株系的株高和根长变短,生长受到抑制。

In order to elucidate the biological function of StmiR166 b of potato(Solanum tuberosum L.),plantlets in vitro of potato cultivar‘Gannongshu 2’were used as dornors,the differential expression of StmiR166 b and its target genes StHB14 and StHB15 were determined in different tissues by real-time quantitative PCR. Furthmore,the pre-structure of StmiR166 b-StmiR166 b^* muture sequence was cloned using pRS300 plasmid as a template and over-lapping PCR technology. The artificial expression vector was constructed and further the transgenic potato plants were obtained via Agrobacterium tumefaciensmediated method. The results showed that StmiR166 b and its target genes StHB14 and StHB15 were expressed in roots,stems and leaves,which were down-regulated expression in the transgenic lines L1 and L3. The growth of the transgenic lines was inhibited,and their plant height and root length were shorter cmopared with the untransgenic plants under normal growth conditions.