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叶片是植物光合作用器官,在能量固定和利用中具有重要作用,研究光诱导型茎叶特异表达启动子的作用元件及其功能对于其调控基因的表达研究具有重要的理论意义和应用价值。本研究用PCR技术从马铃薯(Solanum tuberosum L.)基因组中分离了光诱导型茎叶特异表达启动子ST-LS1的1556bp序列,序列分析表明,该片段与已报道的ST-LS1启动子(Gen Bank accession No.X04753.1)有99.68%的同源性,包括参与芽的特定表达和光反应顺式作用元件as-2-box、光效应顺式作用元件G-box等。将该片段与GUS基因融合,构建了植物表达载体pBⅠ121-ST-LS1,应用根癌农杆菌(Agrobacterium tumefaciens)介导法转化烟草(Nicotiana tabacum)获得了转基因植株。用qRT-PCR和GUS活性组织染色检测转基因烟草植株,结果表明,在ST-LS1启动子驱动的GUS转基因烟草植株的叶和茎中能够检测到GUS基因的表达,根中则检测不到;对黑暗、恒温光照培养、自然光条件处理20d后的转基因植株分析表明,在黑暗处理的转基因植株中GUS基因无表达,而在自然光条件处理转基因植株的叶和茎中GUS基因的表达高于恒温光照培养的转基因植株。结果可为应用基因工程改良农作物品种提供理论和应用依据。
Leaf is a plant photosynthesis organ, which plays an important role in energy fixation and utilization. Studying the action elements and their functions of light-induced stem-leaf-specific promoters has important theoretical significance and application value. In this study, a 1556 bp sequence of the light-induced stem-leaf-specific promoter ST-LS1 was isolated from the genome of Solanum tuberosum L. The sequence analysis showed that this fragment was homologous to the previously reported ST-LS1 promoter Bank accession No.X04753.1) has 99.68% homology, including the specific expression and light-responsive cis-acting element as-2-box, light-effect cis-acting element G-box and so on. The fragment was fused with GUS gene to construct the plant expression vector pBⅠ121-ST-LS1. The transgenic plants were obtained by transformation of tobacco Nicotiana tabacum with Agrobacterium tumefaciens. The transgenic plants were detected by qRT-PCR and GUS staining. The results showed that the expression of GUS gene was detected in leaves and stems of ST-LS1 promoter-driven GUS transgenic tobacco plants, but not in roots. Dark and constant light cultivation, analysis of transgenic plants after 20 days of natural light treatment showed that GUS gene was not expressed in the dark-treated transgenic plants, but GUS gene expression in leaves and stems of the transgenic plants treated with natural light was higher than that of the constant light Of transgenic plants. The results can provide theory and application basis for applying genetic engineering to improve crop varieties.