Wang Po-Hao, Kumar Sandeep, Zeng Jia, McEwan Robert, Wright Terry R, Gupta Manju
Applied Science & Technology, Corteva Agriscience, Johnston, IA, United States.
Data Science & Informatics, Corteva Agriscience, Indianapolis, IN, United States.
Front Plant Sci. 2020 Oct 14;11:570778. doi: 10.3389/fpls.2020.570778. eCollection 2020.
The selection of transcription terminators (TTs) for pairing with high expressing constitutive promoters in chimeric constructs is crucial to deliver optimal transgene expression in plants. In this study, the use of the native combinations of four polyubiquitin gene promoters and corresponding TTs resulted in up to >3-fold increase in transgene expression in maize. Of the eight polyubiquitin promoter and TT regulatory elements utilized, seven were novel and identified from the polyubiquitin genes of , , and . Furthermore, gene expression driven by the Cassava mosaic virus promoter was studied by pairing the promoter with distinct TTs derived from the high expressing genes of s. Of the three TTs studied, the polyubiquitin10 gene TT produced the highest transgene expression in maize. Polyadenylation patterns and mRNA abundance from eight distinct TTs were analyzed using 3'-RACE and next-generation sequencing. The results exhibited one to three unique polyadenylation sites in the TTs. The poly(A) site patterns for the StPinII TT were consistent when the same TT was deployed in chimeric constructs irrespective of the reporter gene and promoter used. Distal to the poly(A) sites, putative polyadenylation signals were identified in the near-upstream regions of the TTs based on previously reported mutagenesis and bioinformatics studies in rice and . The putative polyadenylation signals were 9 to 11 nucleotides in length. Six of the eight TTs contained the putative polyadenylation signals that were overlaps of either canonical AAUAAA or AAUAAA-like polyadenylation signals and AUGAAU, a top-ranking-hexamer of rice and gene near-upstream regions. Three of the polyubiquitin gene TTs contained the identical 9-nucleotide overlap, AUGAAUAAG, underscoring the functional significance of such overlaps in mRNA 3' end processing. In addition to identifying new combinations of regulatory elements for high constitutive trait gene expression in maize, this study demonstrated the importance of TTs for optimizing gene expression in plants. Learning from this study could be applied to other dicotyledonous and monocotyledonous plant species for transgene expression. Research on TTs is not limited to transgene expression but could be extended to the introduction of appropriate mutations into TTs genome editing, paving the way for expression modulation of endogenous genes.
在嵌合构建体中选择与高表达组成型启动子配对的转录终止子（TTs）对于在植物中实现最佳转基因表达至关重要。在本研究中，使用四个多聚泛素基因启动子和相应TTs的天然组合，使玉米中的转基因表达增加了高达3倍以上。在所利用的八个多聚泛素启动子和TT调控元件中，有七个是新的，是从、和的多聚泛素基因中鉴定出来的。此外，通过将木薯花叶病毒启动子与来自高表达基因的不同TTs配对，研究了该启动子驱动的基因表达。在所研究的三个TTs中，多聚泛素10基因TT在玉米中产生了最高的转基因表达。使用3'-RACE和下一代测序分析了八个不同TTs的多聚腺苷酸化模式和mRNA丰度。结果显示TTs中有一到三个独特的多聚腺苷酸化位点。当相同的TTs用于嵌合构建体时，无论使用何种报告基因和启动子，StPinII TT的多聚（A）位点模式都是一致的。在多聚（A）位点的远端，根据先前在水稻和中报道的诱变和生物信息学研究，在TTs的近上游区域鉴定出了推定的多聚腺苷酸化信号。推定的多聚腺苷酸化信号长度为9至11个核苷酸。八个TTs中有六个包含推定的多聚腺苷酸化信号，这些信号是典型的AAUAAA或AAUAAA样多聚腺苷酸化信号与AUGAAU（水稻和基因近上游区域的顶级六聚体）的重叠。三个多聚泛素基因TTs包含相同的9核苷酸重叠序列AUGAAUAAG，强调了这种重叠在mRNA 3'末端加工中的功能重要性。除了鉴定用于玉米中高组成型性状基因表达的调控元件新组合外，本研究还证明了TTs对优化植物基因表达的重要性。从本研究中学到的知识可应用于其他双子叶和单子叶植物物种的转基因表达。对TTs的研究不仅限于转基因表达，还可扩展到在基因组编辑中对TTs引入适当的突变，为内源基因的表达调控铺平道路。
