UgRNA/Cas9多基因编辑法恢复根际细菌农用功能的研究  

Restoration of Agricultural Function of Rhizobacteria by UgRNA/Cas9 Multi-gene Editing

作　　者：李明坤 毕美营 张天航 吴翔宇 杨培儒 应明[1] LI Ming-kun;BI Mei-ying;ZHANG Tian-hang;WU Xiang-yu;YANG Pei-ru;YING Ming(School of Chemistry and Chemical Engineering,Tianjin University of Technology,Tianjin 300384)

机构地区：[1]天津理工大学化学化工学院,天津300384

出　　处：《生物技术通报》2024年第10期275-287,共13页Biotechnology Bulletin

基　　金：天津市高等学校大学生创新创业训练计划(202210060077)。

摘　　要：【目的】长期的化学耕作导致根系微生物的农用功能退化,以植物根部分离得到的一株无功能短小芽胞杆菌(Bacillus pumilus)为研究对象,探究恢复其农用功能的方法。【方法】采用一种新型的CRISPR/Cas多基因编辑系统UgRNA/Cas9,突变B.pumilus全基因组碳分解代谢物阻遏顺式作用元件CRE,改变该菌的碳源选择性。【结果】通过基因测序发现,LG3145的碳代谢和次级代谢途径的部分基因CRE位点发生了缺失、增添、转换、颠换等类型的突变。通过比较代谢组学和转录组学分析,LG3145所吸收的大部分碳源通过磷酸戊糖和氨基酸代谢途径进入了次级代谢通路,使细胞产生了细胞色素、表面活性素和杆菌素等非核糖体肽类抗菌素。经过UgRNA/Cas9编辑后,LG3145可以定殖于植物根系,促进小麦等农作物生长,提高植株的抗病菌能力。【结论】LG3145全基因组CRE序列的突变改变了菌体碳代谢流方向,使该细菌与植物之间形成了有益的互作关系。【Objective】Due to the negative impact of long-term synthetic fertilizer application on the agricultural functions of rhizosphere microorganisms,we aim to develop an effective approach to enhance these functions in a wild-type Bacillus pumilus isolated from plant roots.【Method】A novel CRISPR/Cas multi-gene editing system,UgRNA/Cas9,was used to disrupt the genome-scale cis-acting catabolite-responsive element(CRE),aiming to alter the carbon source selectivity of B.pumilus.【Result】DNA sequencing results revealed that CRE-like sites in the partial genes of the carbon and secondary metabolic pathways underwent mutations including deletions,insertions,transitions,and transversions.Comparative metabolomic and transcriptomic analyses suggest a potential for the biosynthesis of pigments,surfactin,and bacilysin through the pentose phosphate and amino acid pathways.The introduction of UgRNA/Cas9-mediated edits into the CREs enhanced the ability of the strain to better adhere to the root,thereby promoting plant growth and strengthening resistance to pathogens.【Conclusion】Mutations in the genome-wide CRE sequence of LG3145 altered the direction of carbon metabolism flow in the bacterium,resulting in the establishment of a mutually beneficial relationship between the bacterium and plants.

关 键 词：短小芽孢杆菌 顺式作用元件 转录组 UgRNA/Cas9多基因编辑 代谢组 

分 类 号：Q78[生物学—分子生物学] S154.3[农业科学—土壤学]