Creation of Glyphosate-tolerant Rice by Cytosine Base Editing

doi:10.13560/j.cnki.biotech.bull.1985.2022-0848

Abstract

Abstract:

Glyphosate is the most widely used broad-spectrum herbicide. Breeding glyphosate-resistant rice(Oryza sativa L.)will help to improve the chemical control effect of weeds in rice fields, reduce the dosage of herbicide and simplify the control measures. In this study, cytosine base editing technology was used to conduct base directional substitution on the proline codon at position 177 of rice endogenous glyphosate target protein gene OsEPSPS. OsEPSPS（P177L）with base C replaced by T was obtained by Agrobacterium-mediated genetic transformation as editing material. A homozygous edited rice material OsEPSPS（P177L）was isolated and identified from its self-crossing progenies. The results showed that OsEPSPS（P177L）presented significant resistance to glyphosate and tolerated four times of the recommended concentration of glyphosate in the field. Further investigation discovered that P177L mutation did not interfere with plant height, germination rate and other economic traits. The new glyphosate-resistant rice base editing germplasm OsEPSPS（P177L）obtained in this study provides a new approach for improving glyphosate-resistant rice and weed control in rice fields.

Key words: Oryza sativa L., OsEPSPS, cytosine base editing, glyphosate-tolerance, agronomic traits

LU Zhen-wan, LI Xue-qi, HUANG Jin-guang, ZHOU Huan-bin. Creation of Glyphosate-tolerant Rice by Cytosine Base Editing[J]. Biotechnology Bulletin, 2023, 39(2): 63-69.

Figures/Tables 7

Table 1 Bacterial strains and plasmids

	名称 Name	抗性/菌株 Resistance/Species	来源 Source
Plasmid	pENTR4:sgRNA5	Kan^R	Lab stock
Plasmid	pUbi:rBE22	Kan^R	Lab stock
Strain	JM109	Escherichia coli	Lab stock
Strain	EHA105	Agrobacterium tumefaciens	Lab stock

Table 2 Primer information

Fig. 1 Amino acid sequence alignment of rice OsEPSPS and Eleusine indica EiEPSPS EiEPSPS-R（CAD01095.1）and EiEPSPS-S（CAD01096.1）were isolated from the resistant（R）and sensitive（S）Eleusine indica biotypes, respectively[22]. EiEPSPS-P106 site is highlighted in red box

Fig. 2 Identification of base editing materials in T0 A: Design of the rice OsEPSPS gene target site. B: Detected results of the OsEPSPS target base editing in T0 transgenic plants. PAM sequences, target sequences, the target bases and target amino acids, and the mutated predicted bases and corresponding amino acids are highlighted in green, bold, red, and blue, respectively. The mutated bases in the sequencing chromatograms are underlined

Fig. 3 Identification of base editing materials OsEPSPS（P177L）without exogenous components and homozygous mutations A: Separation detection of exogenous T-DNA elements（sgRNA, Hyg and Cas9）from self-progeny of base editing material #105. Kit is wild-type Kitaake, and lane 1-15 is the T1 plants of #105. B: Sequencing results of the target sites in transgene-free T1 progeny plants. PAM sequences, target sequence, the target bases and target amino acids, and the mutated predicted bases/corresponding amino acids are highlighted in green, bold, red, and blue, respectively. The mutated bases in the sequencing chromatograms are underlined

Fig. 4 Glyphosate-tolerance assay of base editing materials OsEPSPS（P177L）plants A: Germination assay of OsEPSPS（P177L）seeds in cylinders containing 1/2MS with glyphosate, and photos were taken after 14 d treatment. Bar=5 cm. B: Glyphosate-tolerance assay of OsEPSPS（P177L）seedling by spraying glyphosate with spray tower, photographs were taken at 14 d respectively，the recommended dose of glyphosate in the field was 1 350 g a.i./ha as 1×

Fig. 5 Assay of P177L homozygous mutants’ economic traits

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