用于马铃薯质体代谢工程的小合成基因组(Mini-Synplastome)优化版。

IF 3.7 2区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS
ACS Synthetic Biology Pub Date : 2024-12-20 Epub Date: 2024-12-10 DOI:10.1021/acssynbio.4c00724
Alessandro Occhialini, Gabriella King, Mohammad Majdi, Ivette A Fuentes Quispe, Jennifer M DeBruyn, Scott C Lenaghan
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引用次数: 0

摘要

在植物基因工程中,质体是许多生物技术应用中很有前景的目标,从用作生物反应器以过度生产有价值的分子,到安装转基因以改善植物性状,不一而足。30 多年来,质体转化的常规方法一直依赖于同源重组整合载体。然而,作为植物细胞质粒基因工程的创新工具,非整合外显子质粒最近受到了更多关注。迷你同步质粒就是这些新技术中的一种,它是一种带有叶绿体特异性复制源(ori)的表体质粒,用于在质体中表达转基因。为了通过降低虚假重组事件的频率来提高外显子序列的稳定性,我们设计了一种优化版的迷你同步质粒(Gen3)。Gen3 设计的创新之处在于大幅缩减了含有oris的质体序列的大小,只包括参与复制的结构域,并降低了整个外显子与内源质体的序列同源性。在这项工作中,我们证明了 Gen3 可用于在 Solanum tuberosum(马铃薯)叶绿体中安装多基因通路,并且外显子组在植物的所有发育阶段都能以高拷贝数的全长环状形式稳定存在,直到植物开花,其表型参数正常。预计在未来十年中,微型合成体将成为在质体中安装复杂遗传回路的重要工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
An Optimized Version of the Small Synthetic Genome (Mini-Synplastome) for Plastid Metabolic Engineering in Solanum tuberosum (Potato).

Plastids represent promising targets in plant genetic engineering for many biotech applications, ranging from their use as bioreactors for the overproduction of valuable molecules to the installation of transgenes for improving plant traits. For over 30 years, routine methods of plastid transformation have relied on homologous recombination integrating vectors. However, nonintegrating episomal plasmids have recently received more attention as an innovative tool for the plastid genetic engineering of plant cells. One of these novel technologies is the mini-synplastome, an episomal plasmid with a chloroplast-specific origin of replication (ori) used to express transgenes in plastids. In order to improve episome sequence stability overtime by reducing the frequency of spurious recombination events, an optimized version of mini-synplastome (Gen3) was designed. The innovation in the Gen3 design was to substantially reduce the size of the plastomic sequence containing oris to include only domains involved in replication and to reduce the sequence homology of the whole episome with the endogenous plastome. In this work, we have demonstrated that Gen3 can be used to install a multigene pathway in Solanum tuberosum (potato) chloroplasts, and the episome is stable in a full-length circular form at high copy number throughout all plant developmental stages to anthesis in plants with normal phenotypic parameters. It is anticipated that in the next decade the mini-synplastome will be a valuable tool for installing complex genetic circuits in plastids.

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来源期刊
CiteScore
8.00
自引率
10.60%
发文量
380
审稿时长
6-12 weeks
期刊介绍: The journal is particularly interested in studies on the design and synthesis of new genetic circuits and gene products; computational methods in the design of systems; and integrative applied approaches to understanding disease and metabolism. Topics may include, but are not limited to: Design and optimization of genetic systems Genetic circuit design and their principles for their organization into programs Computational methods to aid the design of genetic systems Experimental methods to quantify genetic parts, circuits, and metabolic fluxes Genetic parts libraries: their creation, analysis, and ontological representation Protein engineering including computational design Metabolic engineering and cellular manufacturing, including biomass conversion Natural product access, engineering, and production Creative and innovative applications of cellular programming Medical applications, tissue engineering, and the programming of therapeutic cells Minimal cell design and construction Genomics and genome replacement strategies Viral engineering Automated and robotic assembly platforms for synthetic biology DNA synthesis methodologies Metagenomics and synthetic metagenomic analysis Bioinformatics applied to gene discovery, chemoinformatics, and pathway construction Gene optimization Methods for genome-scale measurements of transcription and metabolomics Systems biology and methods to integrate multiple data sources in vitro and cell-free synthetic biology and molecular programming Nucleic acid engineering.
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