An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves.
An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves.
复制标题
烟草延伸蛋白基因终止子的无内含子形式强烈增强植物叶片中的瞬时基因表达。
DOI：
10.1007/s11103-018-0708-y
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发表时间：
2018
影响因子：
5.1
通讯作者：
Mason,HughS
中科院分区：
文献类型：
--
作者：
Rosenthal,SunHee;Diamos,AndrewG;Mason,HughS
我们发现了一个植物基因（延伸蛋白）3 '侧翼区域的有趣特征，包括当内含子被移除时非常有效的聚腺苷化，极大地提高了转基因的瞬时表达。它的应用将极大地促进植物基因表达的研究、分子生物学的研究以及重组蛋白的应用。摘要植物是生产重组蛋白的理想平台。为了在植物中高效表达高价值蛋白，使用合适的调控序列优化表达盒是至关重要的。在这里，我们通过在烟叶、烟草和生菜中的瞬时表达来表征烟草延伸蛋白（Ext）基因终止子的活性。Ext是富含羟脯氨酸糖蛋白（HRGP）超家族的成员，是细胞壁的主要蛋白质成分。目前的研究表明，与先前建立的终止子相比，去除天然内含子的Ext终止子可使瞬时基因表达增加13.5倍。转基因表达的增强与mRNA积累的增加和可读转录物水平的降低相关，这可能会损害基因表达。对转录本3 ' -末端的分析发现，大多数聚腺苷化转录本在典型AAUAAA基序和富ug区域下游的YA二核苷酸处被切割，这两个区域在相关的延伸蛋白终止子中被发现是高度保守的。删除这两个区域中的任何一个都消除了终止子的大部分活性。此外，在聚腺苷化位点上游约175 nt的45 nt多嘌呤序列也被发现是增强表达所必需的。我们得出的结论是，使用Ext终止子对植物中重组蛋白的生产有很大的潜力。
Key messageWe have found interesting features of a plant gene (extensin) 3′ flanking region, including extremely efficient polyadenylation which greatly improves transient expression of transgenes when an intron is removed. Its use will greatly benefit studies of gene expression in plants, research in molecular biology, and applications for recombinant proteins.AbstractPlants are a promising platform for the production of recombinant proteins. To express high-value proteins in plants efficiently, the optimization of expression cassettes using appropriate regulatory sequences is critical. Here, we characterize the activity of the tobaccoextensin(Ext) gene terminator by transient expression inNicotiana benthamiana, tobacco, and lettuce. Ext is a member of the hydroxyproline-rich glycoprotein (HRGP) superfamily and constitutes the major protein component of cell walls. The present study demonstrates that the Ext terminator with its native intron removed increased transient gene expression up to 13.5-fold compared to previously established terminators. The enhanced transgene expression was correlated with increased mRNA accumulation and reduced levels of read-through transcripts, which could impair gene expression. Analysis of transcript 3′-ends found that the majority of polyadenylated transcripts were cleaved at a YA dinucleotide downstream from a canonical AAUAAA motif and a UG-rich region, both of which were found to be highly conserved among related extensin terminators. Deletion of either of these regions eliminated most of the activity of the terminator. Additionally, a 45 nt polypurine sequence ~ 175 nt upstream from the polyadenylation sites was found to also be necessary for the enhanced expression. We conclude that the use of Ext terminator has great potential to benefit the production of recombinant proteins in plants.