利用胞嘧啶碱基编辑技术创制耐草甘膦水稻

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

摘要/Abstract

摘要：

草甘膦是目前使用最广泛的广谱灭生性除草剂，培育耐草甘膦基因编辑水稻将有助于改善稻田杂草化学防控效果、减少用药量和简化防控措施。本研究利用胞嘧啶碱基编辑技术对水稻内源草甘膦靶标蛋白基因OsEPSPS的第177位脯氨酸密码子进行碱基定向替换，通过农杆菌介导的遗传转化获得了靶碱基C定向替换为T的OsEPSPS（P177L）编辑材料，在其自交后代中分离鉴定获得了无外源转基因成分的纯合编辑水稻材料OsEPSPS（P177L）。载药平板试验和喷雾塔喷施试验结果表明OsEPSPS （P177L）纯合编辑水稻材料对草甘膦具有明显的抗性，可耐受四倍的草甘膦田间推荐剂量浓度。进一步调查发现P177L突变并不会对株高和发芽率等经济性状造成干扰。本研究获得的耐草甘膦水稻碱基编辑新种质OsEPSPS（P177L）为水稻的草甘膦抗性改良和稻田的杂草防治开辟了一条新的途径。

关键词: 水稻, OsEPSPS, 胞嘧啶碱基编辑, 草甘膦抗性, 农艺性状

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

卢振万, 李雪琪, 黄金光, 周焕斌. 利用胞嘧啶碱基编辑技术创制耐草甘膦水稻[J]. 生物技术通报, 2023, 39(2): 63-69.

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.

图/表 7

表1 载体与菌株

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

表2 引物信息

Table 2 Primer information

图1 水稻OsEPSPS和牛筋草EiEPSPS的氨基酸序列比对 EiEPSPS-R（CAD01095.1）和EiEPSPS-S（CAD01096.1）分别来自抗性和敏感性牛筋草[22]。EiEPSPS-P106位点用红框标记

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

图2 T0代碱基编辑材料的鉴定 A：水稻OsEPSPS基因靶位点设计；B：T0代植株OsEPSPS基因靶碱基编辑的检测结果。PAM序列标记绿色显示，靶标序列用加粗标注，红色区域为靶碱基以及靶氨基酸，蓝色区域代表已突变预期的碱基及相应的氨基酸，测序峰图中的突变碱基用下划线标记

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

图3 无外源转基因成分且纯合突变的碱基编辑材料OsEPSPS（P177L）鉴定 A：碱基编辑材料#105的自交后代的外源T-DNA元件（sgRNA、Hyg 和Cas9）分离的检测结果。Kit为野生型Kitaake，泳道1-15为T1代植株；B：部分无转基因成分的T1代植株靶位点测序结果。PAM序列标记绿色显示，靶标序列用加粗标注，红色区域为靶碱基以及靶氨基酸，蓝色区域代表已突变预期的碱基及相应的氨基酸，测序峰图中的突变碱基用下划线标记

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

图4 碱基编辑材料OsEPSPS（P177L）对草甘膦的抗性检测 A：OsEPSPS（P177L）种子在含草甘膦1/2MS培养基上培养14 d后的萌发生长情况，Bar=5 cm；B：利用喷雾塔对OsEPSPS（P177L）水稻2-3叶期幼苗进行喷施草甘膦处理14 d后的情况，以草甘膦田间推荐剂量为1 350 g a.i./ha作为1×

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×

图5 P177L纯合突变体经济性状测定

Fig. 5 Assay of P177L homozygous mutants’ economic traits

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