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对病毒启动子进行转基因表达分析,以及 5'-UTR 对. 中翻译起始位点选择的影响分析。

Analysis of Viral Promoters for Transgene Expression and of the Effect of 5'-UTRs on Alternative Translational Start Sites in .

机构信息

Molecular Biotechnology & Systems Biology, RPTU Kaiserslautern-Landau, Paul-Ehrlich-Straße 23, 67663 Kaiserslautern, Germany.

Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA.

出版信息

Genes (Basel). 2023 Apr 21;14(4):948. doi: 10.3390/genes14040948.

Abstract

Microalgae biotechnology has the potential to produce high quality bioproducts in a sustainable manner. Here, has shown great potential as a host for biotechnological exploitation. However, low expression of nuclear transgenes is still a problem and needs to be optimized. In many model organisms, viral promoters are used to drive transgene expression at high levels. However, no viruses are known to infect , and known viral promoters are not functional. Recently, two different lineages of giant viruses were identified in the genomes of field isolates. In this work, we tested six potentially strong promoters from these viral genomes for their ability to drive transgene expression in . We used , , and as reporter genes, and three native benchmark promoters as controls. None of the viral promoters drove expression of any reporter gene beyond background. During our study, we found that mCherry variants are produced by alternative in-frame translational start sites in . We show that this problem can be overcome by mutating the responsible methionine codons to codons for leucine and by using the 5'-UTR of instead of the 5'-UTRs of or . Apparently, the 5'-UTR promotes the use of the first start codon. This could be mediated by the formation of a stem-loop between sequences of the 5'-UTR and sequences downstream of the first AUG in the reporter, potentially increasing the dwell time of the scanning 40S subunit on the first AUG and thus decreasing the probability of leaky scanning.

摘要

微藻生物技术有可能以可持续的方式生产高质量的生物制品。在这里,已被证明是生物技术开发的宿主具有巨大潜力。然而,核转基因的低表达仍然是一个问题,需要进行优化。在许多模式生物中,病毒启动子被用于驱动转基因的高水平表达。然而,目前还没有已知的病毒感染 ,并且已知的病毒启动子不起作用。最近,在野外分离株的基因组中鉴定出了两种不同的巨型病毒谱系。在这项工作中,我们测试了这些病毒基因组中的六个潜在强启动子,以确定它们在 中驱动转基因表达的能力。我们使用 、 和 作为报告基因,并以三个天然的基准启动子作为对照。没有任何病毒启动子能够驱动任何报告基因的表达超过背景水平。在我们的研究过程中,我们发现 mCherry 变体是由 中的替代框架内翻译起始位点产生的。我们表明,通过将负责的甲硫氨酸密码子突变为亮氨酸密码子,并使用 5'-UTR 代替 或 的 5'-UTR,可以克服这个问题。显然,5'-UTR 促进了第一个起始密码子的使用。这可能是通过 5'-UTR 与第一个 AUG 下游序列之间形成茎环来介导的,这可能增加了扫描 40S 亚基在第一个 AUG 上的停留时间,从而降低了渗漏扫描的概率。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1079/10138193/5dbd264bd66d/genes-14-00948-g001.jpg

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