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 Brachypodium distachyon, Setaria italica, and Zea mays. 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 Arabidopsis. 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 Arabidopsis. 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 Arabidopsis 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 via genome editing, paving the way for expression modulation of endogenous genes.

在嵌合构建物中选择与高表达组成型启动子配对的转录终止子（TTs）对于在植物中传递最佳转基因表达至关重要。在这项研究中，使用四个多聚泛素基因启动子和相应的TTs的天然组合导致玉米中转基因表达的增加高达3倍以上。在使用的八种多聚泛素启动子和TT调控元件中，有七种是新颖的，并从短枝曲霉， 狗尾草和 玉米。此外，通过将木薯花叶病毒启动子驱动的基因表达与衍生自木薯花叶病毒高表达基因的独特TT配对进行研究。拟南芥s。在研究的三个TT中，polyubiquitin10基因TT在玉米中产生最高的转基因表达。使用3'-RACE和下一代测序分析了来自八个不同TT的聚腺苷酸化模式和mRNA丰度。结果显示在TT中有1-3个独特的聚腺苷酸化位点。当相同的TT部署在嵌合构建物中时，无论使用的报告基因和启动子如何，StPinII TT的poly（A）位点模式都是一致的。根据先前报道的在水稻和水稻中的诱变和生物信息学研究，在poly（A）位点的远端，在TTs的近上游区域鉴定出推定的聚腺苷酸化信号。拟南芥。推定的聚腺苷酸化信号的长度为9至11个核苷酸。八个TT中的六个包含推定的聚腺苷酸化信号，该信号与规范的AAUAAA或类似AAUAAA的聚腺苷酸化信号和AUGAAU（水稻和大豆的最高六聚体）重叠拟南芥基因近上游区域。三个多聚泛素基因TTs包含相同的9个核苷酸重叠，即AUGAAUAAG，强调了这种重叠在mRNA 3'末端加工中的功能意义。除了确定用于玉米的高组成性状基因表达的调控元件的新组合外，这项研究还证明了TT对优化植物基因表达的重要性。从这项研究中学到的知识可以应用于其他双子叶和单子叶植物物种进行转基因表达。对TTs的研究不仅限于转基因表达，还可以扩展到将适当的突变引入TTs中通过 基因组编辑，为内源基因的表达调控铺平了道路。