Grant Trudi N L, De La Torre Carola M, Zhang Ning, Finer John J
Department of Horticulture and Crop Science, OARDC, The Ohio State University, 1680 Madison Ave., Wooster, OH, 44691, USA.
Mid-Florida Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida, 2725 Binion Road, Apopka, FL, 32703-8504, USA.
Planta. 2017 Apr;245(4):849-860. doi: 10.1007/s00425-016-2646-8. Epub 2017 Jan 9.
Specific sequences within the leader intron of a soybean polyubiquitin gene stimulated gene expression when placed either within a synthetic intron or upstream of a core promoter. The intron in the 5' untranslated region of the soybean polyubiquitin promoter, Gmubi, seems to contribute to the high activity of this promoter. To identify the stimulatory sequences within the intron, ten different sequential intronic sequences of 40 nt were isolated, cloned as tetrameric repeats and placed upstream of a minimal cauliflower mosaic virus 35S (35S) core promoter, which was used to control expression of the green fluorescent protein. Intron fragment tetramers were also cloned within a modified, native intron, creating a Synthetic INtron Cassette (SINC), which was then placed downstream of Gmubi and 35S core promoters. Intron fragment tetramers and SINC constructs were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Intron fragments, used as tetramers upstream of the 35S core promoter, yielded up to 80 times higher expression than the core promoter in transient expression analyses and ten times higher expression in stably transformed hairy roots. Tetrameric intronic fragments, cloned downstream of the Gmubi and 35S core promoters and within the synthetic intron, also yielded increased transient and stable GFP expression that was up to 4 times higher than Gmubi alone and up to 40 times higher than the 35S core promoter alone. These intron fragments contain sequences that seem to act as promoter regulatory elements and may contribute to the increased expression observed with this native strong promoter. Intron regulatory elements and synthetic introns may provide additional tools for increasing transgene expression in plants.
大豆多聚泛素基因前导内含子中的特定序列，当置于合成内含子内或核心启动子上游时，可刺激基因表达。大豆多聚泛素启动子Gmubi的5'非翻译区中的内含子似乎有助于该启动子的高活性。为了鉴定内含子中的刺激序列，分离了10个不同的40 nt连续内含子序列，将其克隆为四聚体重复序列，并置于最小花椰菜花叶病毒35S（35S）核心启动子上游，该启动子用于控制绿色荧光蛋白的表达。内含子片段四聚体也被克隆到一个修饰的天然内含子中，创建了一个合成内含子盒（SINC），然后将其置于Gmubi和35S核心启动子的下游。使用利马豆子叶中的瞬时表达和大豆毛状根中的稳定表达对内含子片段四聚体和SINC构建体进行了评估。在瞬时表达分析中，用作35S核心启动子上游四聚体的内含子片段产生的表达比核心启动子高80倍，在稳定转化的毛状根中高10倍。克隆到Gmubi和35S核心启动子下游以及合成内含子内的四聚体内含子片段，也产生了增加的瞬时和稳定的绿色荧光蛋白表达，分别比单独的Gmubi高4倍和比单独的35S核心启动子高40倍。这些内含子片段包含似乎充当启动子调控元件的序列，可能有助于该天然强启动子观察到的表达增加。内含子调控元件和合成内含子可能为提高植物中的转基因表达提供额外的工具。
