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

doi:10.13560/j.cnki.biotech.bull.1985.2024-0446

Abstract

Abstract:

【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.

Key words: Bacillus pumilus, cis-acting elements, transcriptome, UgRNA/Cas9 multi-gene editing, metabolome

LI Ming-kun, BI Mei-ying, ZHANG Tian-hang, WU Xiang-yu, YANG Pei-ru, YING Ming. Restoration of Agricultural Function of Rhizobacteria by UgRNA/Cas9 Multi-gene Editing[J]. Biotechnology Bulletin, 2024, 40(10): 275-287.

Figures/Tables 11

References 33

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名称Name	UgRNA序列 UgRNA sequence（5'-3'）	相似度 Similarity/%
Cac:	AAACTGTAAGCGTTATCAATACGC	70
Cac:	AAAAGCGTATTGATAACGCTTACA	70
Cpt :	AAACCGCAACCAAAGCTTCAGATT	70
Cpt :	AAAAAATCTGAAGCTTTGGTTGCG	70
Cax :	AAACGAAAGCGTTACCAGCAATAG	55
Cax :	AAAACTATTGCTGGTAACGCTTTC	55

名称Name	UgRNA序列 UgRNA sequence（5'-3'）	相似度 Similarity/%
Cac:	AAACTGTAAGCGTTATCAATACGC	70
Cac:	AAAAGCGTATTGATAACGCTTACA	70
Cpt :	AAACCGCAACCAAAGCTTCAGATT	70
Cpt :	AAAAAATCTGAAGCTTTGGTTGCG	70
Cax :	AAACGAAAGCGTTACCAGCAATAG	55
Cax :	AAAACTATTGCTGGTAACGCTTTC	55

质粒Plasmid	特性Characterization	来源Source
pCas9	Bacterial expression of Cas9 nuclease, tracrRNA and crRNA guide, E.coli	Addgene（http://www.addgene.org/42876/）
pCas9-Cax	pCas9 derivate, containing crRNA of Cax	本实验This study^[13]
pCas9-Cac	pCas9 derivate, containing crRNA of Cac	本实验This study^[13]
pCas9-Cpt	pCas9 derivate, containing crRNA of Cpt	本实验This study^[13]

质粒Plasmid	特性Characterization	来源Source
pCas9	Bacterial expression of Cas9 nuclease, tracrRNA and crRNA guide, E.coli	Addgene（http://www.addgene.org/42876/）
pCas9-Cax	pCas9 derivate, containing crRNA of Cax	本实验This study^[13]
pCas9-Cac	pCas9 derivate, containing crRNA of Cac	本实验This study^[13]
pCas9-Cpt	pCas9 derivate, containing crRNA of Cpt	本实验This study^[13]

引物Primer	序列 Sequence（5'-3'）	引物Primer	序列 Sequence（5'-3'）
glpFKD-F	GTGCTATGGAAAAAAGCATGGAGT	glpFKD-R	TAAATGCGCTCCGCTGATTCC
ptsGHI-F	CTGCAGATTCTGGAATTCACGCAC	ptsGHI-R	AGATAGTGATTGTAGCACCTTTCGC
ykgB-F	TCCACTGTTCCTTTTGGTCTTCC	ykgB-R	TTCTCAATCTGCTGCTTGTCCG
crr-F	GCTCAAGCCCTGAGAGTGTT	crr-R	GCTCGAATACGAGGAAGTTGT
fsa-F	CTGCACTGACAGAACCTGATACCA	fsa-R	GGAAGAAATCGGTAAAATGACAGGTC
fbaA-F	ATGTAGCGTCCTGCTCCTTCA	fbaA-R	CCGTGTCATCATTATGACAGCC
eno-F	CAGGATCGTTCTTTTCTTCACCA	eno-R	GAAGGCAGCATGGTGAAAGTC
gap-F	GCATAACTGTGAGAAGAATCTGGC	gap-R	CTTGTTTTCCGTACACCGCTTT
tkt-F	GAGACCGGTTTGTCAGACGC	tkt-R	GATGCTCAGGTTGTATCCGCTTA
pdhABCD-F	CCAAGTTGAAGACAGTGAACCGT	pdhABCD-R	TACGACTTCGCCTTTTTCGTTT
pgi-F	GGGCAAATTCTTCGCGATCA	pgi-R	CATACACTCGATCACAATGTGG
guaB-F	TTCTCGATGTTACACCATGCGC	guaB-R	GATACCAAGACCACCTTGACGT
tenA-F	CATGCTCAGGGAGTTTCCGTTTA	tenA-R	CAATATCTCTTCGAATCGTCCGC
ribA-F	CCTGGTCGATGGATCAGGGA	ribA-R	GGTGCGAAGGCATACCACTTA
ribBD-F	CGCTTAGGGCCTGTCAGAAG	ribBD-R	TTCCGCCACCTTTCAGTTCT
crtNIM-F	ACGGAAGGAGCTTCCTTTCA	crtNIM-R	ATGAAAGCTGCGGAAGTGCT
crtP-F	CAGGCTTCTATCCGTTTCTCCT	crtP-R	GGTATGGCTTTTTCCACAGGGG
glk-F	CCATTCCAATCCCAATCAGCG	glk-R	GGAGAGACAGCGAAAGCAGAG
glmS-F	GATCACCCGCTACATTGTGATTGAC	glmS-R	TTTGATGCGGATGCGCATTTAGA
glmM-F	GAAGCAGCCGAATCTCGTCG	glmM-R	CGAGATACGCACTTCCCATC
glmU-F	GGAGAGTTTCGCGACATTGCA	glmU-R	CCCAGCTGTTCTTCTTGAAGTG
murA-F	GGCGAGCGGAGAAGATTCTT	murA-R	GAGGGAACCAGTTCTCCGTA
murC-F	GGACAGTATACCCAGTATCATG	murC-R	CGAACAGGAGGAATTAATGCT
murD-F	GGCTCAAATCGCTGTGAAATATG	murD-R	CCGTAACCAAGTGTGACAAATAAC
murE-F	GGGATAGCCTGCCTGAGATG	murE-R	GCCTGTGATCCCTATGAGATTC
murGB-F	GGAGAGGCATACGAATAGCACT	murGB-R	TAGAAGCCGCATAAACCACAGG
accAD-F	GGTTTTCTGAGATCAGGTGC	accAD-R	AGTGTGCACGTTCTTTCTGA
srfA-F	GCGGTATTTAGTCGATTATTTTCGG	srfA-R	AAGGGAGTCAGTTTGACGAATGTAC
bacABCDF-F	GCAGGACTACTGATCATGGCT	bacABCDF-R	GCCGAGTGTAGTGGTGGAATG
bacG-F	CTATTGCCGCAAAGGCCTCA	bacG-R	GCGTGAGGGTTCTCCTACAAAG
aroA-F	CGCTGCTATGACACCTGACT	aroA-R	CCAACGTCTTTACGTATTACGGT
aroBC-F	GCGCTGATTGCAGCACTTTATC	aroBC-R	GCCAAGGTGTTCATCAAATGAGC
aroG-F	CTTCTTGAAGTTCTAGACCTGCTT	aroG-R	CAAGAGTTTGAAGCATTTGCGTCT
liaGFRS-F	GCTTCCACCCGTAATCTTCACAT	liaGFRS-R	GCAGCAGTTAGAAACGAAGC
liaHI-F	GCCACTTCACCATTGATCATCG	liaHI-R	GGCGCAGCTCTCTTACATGT