Knockout of the Agrobacterium ILVC gene generates a valine-isoleucine auxotrophic strain for plant transformation.

IF 2.7 3区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Transgenic Research Pub Date : 2025-05-02 DOI:10.1007/s11248-025-00442-z

Yanhui Yang, Jun Lu, Tianrui Xue, Zhenli Cao, Mingjie Li, Zhongyi Zhang, Rong Wang, Tongyu Wu

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引用次数: 0

Abstract

The Agrobacterium tumefaciens mediated transformation is the prevailing methodology for plant genetic manipulation; however, A. tumefaciens overgrowth is a common constraint in the process. Exploring auxotrophic A. tumefaciens could reduce overgrowth and enhance plant transformation efficiency. The ILVC gene, which encodes the ketol-acid isomeroreductase, is critical for Valine (Val) and isoleucine (Ile) biosynthesis in some microorganisms. However, its function in A. tumefaciens is unclear. To ascertain the function of ILVC and generate an auxotrophic A. tumefaciens, this study employed an allelic exchange to disrupt the ILVC in A. tumefaciens strain GV3101. This resulted in the loss of ketol-acid isomeroreductase activity and the prevention of Val and Ile biosynthesis, creating a dual-auxotrophic GV3101_∆ILVC. Transient expression assays in Nicotiana benthamiana transformation demonstrated that the GV3101_∆ILVC was capable of T-DNA transfer. Moreover, stable genetic transformation analysis in N. benthamiana indicated that the introduction of GV3101_∆ILVC led to a reduction in overgrowth within infected plant tissues. Additionally, an enhancement in transformation efficiency was observed with the prolongation of the co-cultivation time of the explant-infected strain. This study revealed the function of ILVC and explored a dual-auxotrophic A. tumefaciens for Val and Ile, potentially broadening the utilization of auxotrophic strains in plant genetic transformation.

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敲除农杆菌ILVC基因可产生用于植物转化的缬氨酸-异亮氨酸营养不良菌株。

农杆菌介导的转化是植物遗传操作的主流方法；然而，在这一过程中，大肠杆菌的过度生长是一个常见的制约因素。探索营养不良的瘤胃芽胞杆菌可以减少过度生长，提高植物转化效率。ILVC基因编码酮酸异构还原酶，在一些微生物中对缬氨酸（Val）和异亮氨酸（Ile）的生物合成至关重要。然而，其在大肠杆菌中的作用尚不清楚。为了确定ILVC的功能并产生营养不良的瘤胃拟酵母菌，本研究采用等位基因交换来破坏瘤胃拟酵母菌GV3101的ILVC。这导致酮酸异构还原酶活性丧失，Val和Ile的生物合成受到阻碍，形成了双营养不良的GV3101∆ILVC。瞬时表达实验表明，GV3101∆ILVC能够转移T-DNA。此外，对N. benthamiana的稳定遗传转化分析表明，引入GV3101∆ILVC可减少受感染植物组织内的过度生长。此外，随着外植体感染菌株共培养时间的延长，转化效率也有所提高。本研究揭示了ILVC的功能，并探索了Val和Ile两种营养不良菌株，有望拓宽营养不良菌株在植物遗传转化中的应用。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Transgenic Research 生物-生化研究方法

CiteScore

5.40

自引率

0.00%

发文量

审稿时长

4-8 weeks

期刊介绍： Transgenic Research focusses on transgenic and genome edited higher organisms. Manuscripts emphasizing biotechnological applications are strongly encouraged. Intellectual property, ethical issues, societal impact and regulatory aspects also fall within the scope of the journal. Transgenic Research aims to bridge the gap between fundamental and applied science in molecular biology and biotechnology for the plant and animal academic and associated industry communities. Transgenic Research publishes -Original Papers -Reviews: Should critically summarize the current state-of-the-art of the subject in a dispassionate way. Authors are requested to contact a Board Member before submission. Reviews should not be descriptive; rather they should present the most up-to-date information on the subject in a dispassionate and critical way. Perspective Reviews which can address new or controversial aspects are encouraged. -Brief Communications: Should report significant developments in methodology and experimental transgenic higher organisms