茄科作物高效双碱基编辑新工具的开发与应用

doi:10.7668/hbnxb.20195725

摘要/Abstract

摘要：

旨在开发一款高效适配茄科作物的双碱基编辑工具,以突破现有碱基编辑系统在茄科作物中适配性差、编辑效率低等问题。研究将核酸酶功能部分丧失的 Cas9 缺口酶(nCas9)与高效腺嘌呤脱氨酶 ABE8e、优化型胞嘧啶脱氨酶 sdd7-mini 及尿嘧啶糖基化抑制剂 UGI 等元件进行融合,依据茄科作物的启动子偏好性和密码子使用频率进行系统性优化,最终构建双碱基编辑器 TPDBE-S。在番茄和茄子原生质体瞬时转化试验中,该编辑器表现出极高的编辑效率。在本氏烟草稳定转化体系中,通过靶向 PDS 基因验证,5 个阳性转基因家系中有 4 个出现明显白化表型,证实其稳定编辑能力。该编辑器的编辑窗口与 ABE8e和 sdd7-mini的特性高度一致,且对茄科作物密码子偏好性和启动子活性的优化显著提升了编辑效率与适配性。TPDBE-S 的构建采用模块化设计,基于 tRNA 自切割机制的多靶点表达盒可通过一步基因合成实现快速组装,成本低且操作简便。该工具填补了茄科作物专用双碱基编辑空白。

关键词: 茄子, 基因编辑, 双碱基编辑, 番茄, 编辑效率

Abstract:

This study seeks to develop a highly efficient dual-base editing tool for solanaceous crops to address the challenges of poor compatibility and low editing efficiency associated with existing base editing systems in these plants. The research involved fusing the nickase Cas9 enzyme (nCas9),which has partially lost its nuclease function,with components such as the highly efficient adenine deaminase ABE8e,the optimized cytosine deaminase sdd7-mini,and the uracil glycosylation inhibitor UGI. Systematic optimization was conducted based on the promoter preferences and codon usage frequencies specific to solanaceous crops. Ultimately,the dual-base editor TPDBE-S was constructed. In transient transformation experiments using tomato and eggplant protoplasts,this editor exhibited exceptionally high editing efficiency. In the stable transformation system of tobacco,targeting the PDS gene verification revealed that four out of five positive transgenic families exhibited pronounced albinism phenotypes,thereby confirming the stable editing capability. The editing window of this editor aligns closely with the characteristics of ABE8e and sdd7-mini. Furthermore,optimizing codon preference and promoter activity in Solanaceae crops significantly enhances editing efficiency and adaptability. The construction of TPDBE-S employs a modular design. The multi-target expression cassette,based on the tRNA self-cleaving mechanism,can be rapidly assembled through one-step gene synthesis,which is both cost-effective and straightforward. This tool addresses the gap in double-base editing specifically for Solanaceous crops.

Key words: Eggplant, Gene editing, Dual-base editing, Tomato, Editing efficiency

中图分类号:

田鹏, 李亚栋, 张敬敬, 田哲娟, 康忱, 高秀瑞, 李冰, 刘伟, 武彦荣, 吴志明. 茄科作物高效双碱基编辑新工具的开发与应用[J]. 华北农学报, 2026, 41(1): 39-48. doi: 10.7668/hbnxb.20195725.

TIAN Peng, LI Yadong, ZHANG Jingjing, TIAN Zhejuan, KANG Chen, GAO Xiurui, LI Bing, LIU Wei, WU Yanrong, WU Zhiming. Development and Application of a High-Efficiency Dual-Base Editing Tool for Solanaceous Crops[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(1): 39-48. doi: 10.7668/hbnxb.20195725.

http://www.hbnxb.net/CN/Y2026/V41/I1/39

图/表 7

表1 PCR引物列表

Tab.1 List of PCR primers

引物名称 Name	引物序列(5'—3') Primer sequence(5'—3')	用途 Purpose
T-A-180F	CCTCCAAACCCTCCACCCCA	检测编辑位点T-A
T-A+400R	CTCAGGCATAGCTGTTTCCTGCC	检测编辑位点T-A
T-B-60F	CGGAGGGGAGAGCTGAAATGGCT	检测编辑位点T-B
T-B+500R	AACCAGTCAGTCCACTGCTCCA	检测编辑位点T-B
T-C-150F	TGGGACGGGCGGAGATGGTT	检测编辑位点T-C
T-C+200R	CACCGCTCGAACCTGTCACAT	检测编辑位点T-C
E-2350F	TGCGTGTCGGATTCGCCAAAA	检测编辑位点E-A/B/C/D
E-4350R	CCTGCGAAAGGGCTTCCTCTGC	检测编辑位点E-A/B/C/D
Cas9-440F	TCCTCTAGCAAGTGGACCAACGTA	检测烟草转基因阳性植株
Cas9-440R	ACAAAGGCTCCTCTTTCAGCTTCTA	检测烟草转基因阳性植株

图1 双碱基编辑器TPDBE-S结构示意图 35S.花椰菜花叶病毒35S RNA启动子;NLS.核定位信号;Sdd7-mini.胞嘧啶脱氨酶;TadA8e,腺嘌呤脱氨酶;32aa/39aa.长度为32aa/39aa的连接肽;DBD.DNA结合基团;nCas9.核酸酶功能部分丧失的Cas9缺口酶;UGI.尿嘧啶糖基化抑制剂;P2A.一种来源于猪捷申病毒的自我剪切序列;GFP.绿色荧光蛋白;Tnos.转录终止子;SiU6.番茄SiU6 RNA酶Ⅲ启动子;tRNA.转运RNA序列(77 bp);gRNA.引导RNA序列(96 bp);Poly T.转录终止信号;HygR.潮霉素磷酸转移酶;LB/RB.双元载体插入植物基因组的左、右边界。红色虚线框.编辑目标基因时需合成的片段。

Fig.1 Schematic diagram of the double-base editor TPDBE-S structure 35S.Cauliflower mosaic virus 35S RNA promoter;NLS.Nuclear localization signal;Sdd7-mini.Cytosine deaminase;TadA8e.Adenine deaminase;32aa/39aa.Linker peptides of lengths 32aa/39aa;DBD.DNA-binding domain;nCas9.Cas9 nickase with partially lost nuclease function;UGI.Uracil glycosylase inhibitor;P2A.Self-cleaving sequence derived from porcine teschovirus;GFP.Green fluorescent protein;Tnos.Transcription terminator;SiU6.Tomato SiU6 RNase Ⅲ promoter;tRNA.Transfer RNA sequence(77 bp);gRNA.Guide RNA sequence(96 bp);Poly T.Transcription termination signal;HygR.Hygromycin phosphotransferase;LB/RB.Left and right borders for binary vector insertion into the plant genome.The red dashed box.Fragment that needs to be synthesized when editing the target genes.

图2 双碱基编辑器TPDBE-S 结构图及酶切验证 A.双碱基编辑器TPDBE-S 结构示意图。该示意图通过软件SnapGene V6.0.2绘制,关键酶切位点已标出。B.双碱基编辑器TPDBE-S酶切检测图:M.Marker,1.TPDBE-S质粒,2.TPDBE-S质粒Spe Ⅰ酶切,3.TPDBE-S质粒SmaⅠ酶切,4.TPDBE-S质粒BamH Ⅰ酶切。

Fig.2 Schematic diagram of the double-base editor TPDBE-S structure and enzyme digestion verification A.Schematic diagram of the double-base editor TPDBE-S structure.This diagram was drawn using SnapGene V6.0.2 software,with key restriction sites highlighted.B.Enzyme digestion verification of the double-base editor TPDBE-S: M.Marker,1.TPDBE-S plasmid,2.TPDBE-S Plasmid Spe Ⅰ digestion,3.TPDBE-S plasmid Sma Ⅰ digestion,4.TPDBE-S plasmid BamH Ⅰ digestion.

图3 双碱基编辑器TPDBE-S gRNA表达盒序列及结构示意图首末端下划线部分.TPDBE-S载体上Spe Ⅰ酶切位点两侧同源臂序列;红色字体.Spe Ⅰ酶切位点;蓝色阴影部分.tRNA序列;黄色阴影部分.sgRNA中tracrRNA的编码序列;T1~T3.敲除靶标序列;灰色阴影部分.Poly T终止信号。

Fig.3 Schematic diagram of the double-base editor TPDBE-S gRNA expression cassette sequence and structure The underlined portions at the start and end of the sequences.The homologous arm sequences flanking the Spe Ⅰ restriction site on the TPDBE-S vector,Red font.The Spe Ⅰ restriction site;Blue shaded portions.The tRNA sequences;Yellow shaded portion.The tracrRNA sequences;T1—T3.CRISPR-Cas9-targeted sequences;The gray shaded portion.The Poly T termination signal.

表2 不同位点编辑效率统计

Tab.2 Statistics on editing efficiency at different sites

位点 Site	物种 Species	基因号 Gene number	靶标序列 Target sequence	总编辑效率 Overall editing efficiency	A-G的编辑效率 Editing efficiency of A-G	C-T的编辑效率 Editing efficiency of C-T	A-G&C-T的编辑效率 Editing efficiency of A-G&C-T
T-A	番茄	Solyc01g105270	TCAAAGATGGGGCGAACTCG TGG	86.7%(26/30)	66.7%(20/30)	60.0%(18/30)	40.0%(12/30)
T-B	番茄	Solyc02g080370	ACGTCAACGAATCCGTTCAG TGG	80.0%(24/30)	53.3%(16/30)	60.0%(18/30)	33.3%(10/30)
T-C	番茄	Solyc01g007900	CAACGCCGTAGAGAACAGCA AGG	53.3%(16/30)	0(0/30)	53.3%(16/30)	0(0/30)
E-A	茄子	Smechr0602754	ACTTCAAAGGGCTAGCTCTC AGG	66.7%(20/30)	36.7%(11/30)	46.7%(14/30)	16.7%(5/30)
E-B	茄子	Smechr0602754	TGTTTGACATGGAATATTCA AGG	55.3%(16/30)	53.3%(16/30)	3.3%(1/30)	3.3%(1/30)
E-C	茄子	Smechr0602754	TTGTAATGCAGTTCGAAGCT CGG	90.0%(27/30)	70.0%(21/30)	56.7%(17/30)	36.7%(11/30)
E-D	茄子	Smechr0602754	GAACGCTGCTTCCTTTGGAT GGG	63.3%(19/30)	0(0/30)	63.3%(19/30)	0(0/30)

图4 双碱基编辑器TPDBE-S在烟草稳转体系中的编辑效果 A.双碱基编辑器TPDBE-S 经烟草农杆菌介导转化体系所得的阳性植株表型(编号#1~#5),其中白化表型所出现的位置已用红色箭头标出。标尺.1 cm。B.烟草转基因家系PCR检测电泳图:M.Marker,CK+.阳性对照(TPDBE-S质粒做模板),CK-.阴性对照(H₂O做模板),#1~#5.5个转基因家系检测结果。

Fig.4 Editing efficiency of the dual-base editor TPDBE-S in the stable transformation system of Nicotiana tabacum A.Phenotypes of positive transgenic plants(lines #1—#5)obtained via Agrobacterium-mediated transformation with TPDBE-S.The albinotic sectors,indicative of targeted mutagenesis,are indicated by red arrows.Scale bar.1 cm.B.PCR-based genotyping of transgenic N.tabacum T-DNA insertion events: M.DNA molecular-weight Marker,CK+.Positive control(TPDBE-S plasmid),CK-.Negative control(H₂O),#1—#5.PCR products corresponding to the five independent transgenic lines.

图5 TPDBE-S在不同位点编辑窗口及编辑占比总览图蓝色字符.PAM位点;红色字符.被编辑后的位点。

Fig.5 Overview of editing window and frequency of TPDBE-S at various target sites Blue characters.PAM sites;Red characters.The edited sites.

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