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作物杂志,2026, 第3期: 147–154 doi: 10.16035/j.issn.1001-7283.2026.03.020

• 遗传育种·种质资源·生物技术 • 上一篇    下一篇

农杆菌碱基编辑技术的建立与recA基因精准编辑型菌株应用

孙梦琳1,2(), 符晓2, 祁显涛2, 刘昌林2, 谢传晓2, 郭晋杰1(), 朱金洁2()   

  1. 1 河北农业大学农学院， 071000， 河北保定
    2 中国农业科学院作物科学研究所， 100081， 北京
  • 收稿日期:2025-03-20 修回日期:2025-04-30 出版日期:2026-06-15 发布日期:2026-06-17
  • 通讯作者: 朱金洁，研究方向为玉米基因编辑技术研发与育种应用，E-mail：zhujinjie@caas.cn；郭晋杰为共同通信作者，研究方向为玉米性状遗传改良，E-mail：guojinjie512@163.com
  • 作者简介:孙梦琳，研究方向为玉米基因编辑，E-mail：2437824406@qq.com
  • 基金资助:
    河北省现代种业科技创新团队(21326319D);河南省重点研发计划(241111112300-02)

Establishment of Base Editing Technology in Agrobacterium and Application of recA Gene Precisely Edited Strains

Sun Menglin1,2(), Fu Xiao2, Qi Xiantao2, Liu Changlin2, Xie Chuanxiao2, Guo Jinjie1(), Zhu Jinjie2()   

  1. 1 College of Agronomy, Hebei Agricultural University, Baoding 071000, Hebei, China
    2 Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2025-03-20 Revised:2025-04-30 Online:2026-06-15 Published:2026-06-17

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摘要：

农杆菌介导的遗传转化是玉米等作物生物育种体系建立的核心技术瓶颈，当前针对农杆菌菌株的工程化改良研究仍存在显著不足。本研究基于碱基编辑技术构建农杆菌高效遗传操作平台，通过“基因组精准编辑结合三元载体”策略提高农杆菌的侵染能力，显著提高了玉米幼胚的瞬时转化效率。首先，研究构建农杆菌的腺嘌呤碱基编辑器（ABE）和胞嘧啶碱基编辑器（CBE）。基于ABE靶向recA基因，TadA8e-dCas9系统的编辑效率达95%，显著高于TadA8.20-dCas9（50%）和TadA8r-dCas9（20%）。基于CBE靶向编辑recA基因，在recA基因Q26位点（CAG→TAG）引入终止密码子，成功创制recA功能缺失型EHA105（ΔrecA）菌株，编辑效率达100%。进一步构建三元载体系统，整合携带pVS1/pRiA4复制子的辅助质粒（增强Vir基因表达）以及双元表达载体（含GUS/CRC报告基因）。结果表明，携带pRiA4复制子辅助质粒的三元系统在玉米幼胚瞬时转化中表现最优，转化效率较EHA105野生型菌株显著提升28%~35%。该研究将碱基编辑技术应用于农杆菌基因组改良，通过“recA精准敲除+三元载体协同优化”策略，为突破玉米遗传转化瓶颈提供了高效可拓展的技术平台。

关键词: 农杆菌EHA105, 碱基编辑, recA基因, 三元载体, 玉米瞬时转化

Abstract:

Agrobacterium-mediated genetic transformation is the core technical bottleneck for establishing biological breeding systems for crops such as maize, and current research on the engineering improvement of Agrobacterium strains remains significantly insufficient. This study constructed a high-efficiency genetic manipulation platform for Agrobacterium based on base editing technology, and improved the infectivity of Agrobacterium through a “precise genome editing combined with ternary vector” strategy, significantly enhancing the transient transformation efficiency of maize immature embryos. First, adenine base editors (ABE) and cytosine base editors (CBE) for Agrobacterium were constructed. Based on ABE targeting the recA gene, the editing efficiency of the TadA8e-dCas9 system reached 95%, which was significantly higher than that of TadA8.20-dCas9 (50%) and TadA8r-dCas9 (20%). Based on CBE targeting the recA gene, a stop codon was introduced at the Q26 site (CAG→TAG) of the recA gene, successfully creating a recA function-deficient EHA105 (ΔrecA) strain with an editing efficiency of 100%. A ternary vector system was further constructed by integrating helper plasmids carrying pVS1/pRiA4 replicons (to enhance vir gene expression) and a binary expression vector (containing GUS/CRC reporter genes). The results showed that the ternary vector system carrying the pRiA4 replicon helper plasmid performed best in the transient transformation of maize immature embryos, with the transformation efficiency significantly increased by 28%-35% compared with that of the wild- type EHA105 strain. This study applied base editing technology to the genome improvement of Agrobacterium, providing an efficient and scalable technical platform for breaking through the bottleneck of maize genetic transformation through the “precise recA knockout + ternary vector synergistic optimization” strategy.

Key words: Agrobacterium tumefaciens EHA105, Base editing, RecA gene, Ternary vector system, Maize transient transformation

表1

相关引物序列

引物名称Primer name 引物序列（5’-3’）Primer sequence (5’-3’)
Q26-HF1 CCTAGGTATAATACTAGTTCCCAGATCGAACGGTCGTTGTTTCAGAGCTATGCTGGAAA
Q26-HF2 TCCTCTAGAGTCGACCTGCAGGTTGACAGCTAGCTCAGTCCTAGGTATAATACTAGTTC
Q26-HF3 TTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGG
ScaffT2T-HR TTAAAAAAAGGCCATCCGTCAGGATGGCCTTCTGCACCGACTCGGTGCCACTTTTTCA
Lac-flag-HF1 CTTTATGCTTCCGGCTCGTATGTTAGGAGGTCTTTATCATGGACTATAAGGACCACGAC
Lac-flag-HF2 CCATCCTGACGGATGGCCTTTTTTTAAGCTTTTTACACTTTATGCTTCCGGCTCGTATG
Flag-CDA1-HR GAATCCGCACGTACTCGGCGTCGGTCTTATCGTCATCGTCTTTGTAA
Flag-CDA1-HF GATGACGATAAGACCGACGCCGAGTACGTGCGGATTCAC
CDA1-cas9-HR GAGGCCGATGGAGTACTTCTTGTCCACGGCGGGAGACTTTGTGGTGTGC
Cas9-HF GACAAGAAGTACTCCATCGGCCTCGCCAT
UGI-T3T-HR1 GAAAGAAGAAAGGGGCAATAACGCCCCACATTTTATACGAGCATCTTAATCTTGTTCTC
T3T-MaubI-HR2 ATTCCCGATCTAGTAACATAGATGACACCGCGCGCGAGAAAGAAGAAAGGGGCAATAAC
T2-virB-HF CCATCCTGACGGATGGCCTTTTTTTAAGCTTCGAGCATTTTCGTTCTGAGCCTGACCTG
VirB-flag-HR GTCGTGGTCCTTATAGTCCATACCTTATCTCCTTAGCTCGCAACTA
VirB-flag-HF AGTTGCGAGCTAAGGAGATAAGGTATGGACTATAAGGACCACGA
CDA1-cas9-HR GAGGCCGATGGAGTACTTCTTGTCCACGGCGGGAGACTTTGTGGTGTGC
Lac-flag-HF1 CTTTATGCTTCCGGCTCGTATGTTAGGAGGTCTTTATCATGGACTATAAGGACCACGAC
Lac-flag-HF2 CCATCCTGACGGATGGCCTTTTTTTAAGCTTTTTACACTTTATGCTTCCGGCTCGTATG
Flag-T8e-HR ACTCGTGGGAAAACTCCACCTCAGACTTATCGTCATCGTCTTTGTAA
TadA8e-HF ACTCGTGGGAAAACTCCACCTCAGACTTATCGTCATCGTCTTTGTAA
Cas9-T3T-HR AAAGAAGAAAGGGGCAATAACGCCCCACATTTTATACGTCGCCGCCGAGCTGGGAGAGG
TadA8e-HF TCTGAGGTGGAGTTTTCCCACGAGT
TadA-cas9-HR GAGGCCGATGGAGTACTTCTTGTCGTCTGTGGAGCTCTGGGCCTTCTTCTGA
JCF-3301 CACTCATTAGGCACCCCAGG
JCR-Cas9 AAAATCTCCTGGAGGTAGCAG
JCF-H840A AAGAAGGGCATCCTCCAGAC
JCR-3301 CTTTGATCCCGAGGGGAACC
JCF-Q26 CAAATCCACACCGAGCTTGC
JCR-Q26 GGCACGGAAAAACGAAAGCA

表2

不同腺嘌呤脱氨酶变体

变体
Variant		 位点Site
23 26 27 28 36 47 48 51 61 73 76 82 84 96 106 108 109 110 111
TadAWT W R E V H R P R M Y I V L H A D A K T
TadA8e R L A L F W N
TadA8.20 R L A L Y S F W N
TadA8r R L K A L Y S W G R H
变体
Variant		 位点Site
114 119 122 123 126 127 146 147 149 152 154 155 156 157 158 161 166 167
TadAWT A D H H M N S D F R Q E I K A K T D
TadA8e Y C Y P V F N
TadA8.20 C R P R V F N
TadA8r V N Y I K C R P R V F

图1

不同ABE碱基编辑器介导的农杆菌EHA105精准编辑

图2

CBE碱基编辑器介导的农杆菌EHA105 recA基因精准敲除

图3

EHA105（ΔrecA）突变型菌株的DNA损伤测试 (a) 卡那霉素敏感性测试；(b) 载体特异性引物扩增PCR；(c) recA突变位点Sanger测序验证；(d) DNA损伤（0.005%MMS）测试。

图4

基于EHA105（ΔrecA）菌株的三元载体系统对玉米幼胚的瞬时侵染 (a) pVS1或pRiA4复制子辅助质粒载体结构示意图；(b) 表达报告基因的双元载体p3301-GUS和p3301-CRC的结构示意图；(c) 基于EHA105（ΔrecA）菌株的三元载体系统对玉米幼胚的瞬时侵染；(d) 基于EHA105(ΔrecA)菌株的三元系统侵染效率（n=18）；“****”表示差异极显著（P < 0.0001）。

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