微生物学报

Plasmids	Purpose and properties	Source
pEX18Km	Suicide plasmid for knockout, carrying reverse selection markers sacB, oriT, and Km^R	[19]
pEX18Km-atu4546	The atu4546 knockout box is inserted into pEX18Km for knockout of atu4546	This study
pCB301	Promoter less expression plasmid, Km^R	[19]
pCB301-Prm4546-lacZ	The upstream promoter of atu4546 was linked to the reporter gene lacZ and integrated into pCB301	This study
pCB301-Prm4547-lacZ	The upstream promoter of atu4547 was linked to the reporter gene lacZ and integrated into pCB301	This study
pCB301-Prm4547 (remove the binding site)-lacZ	pCB301-Prm4547-lacZ removes transcription factor binding sites	This study
pCB301-Prm4547 (unrelated sequence)-lacZ	The transcription factor binding site on pCB301-Prm4547-lacZ was replaced by an unrelated sequence	This study
pUCA19	For the construction of gene complement plasmid, carrying an agrobacterial replicon, Ap^R, Cr^R	[20]
pUCA19-atu4546	The coding region of atu4546 was inserted into pUCA19 to express atu4546	This study

Plasmids	Purpose and properties	Source
pEX18Km	Suicide plasmid for knockout, carrying reverse selection markers sacB, oriT, and Km^R	[19]
pEX18Km-atu4546	The atu4546 knockout box is inserted into pEX18Km for knockout of atu4546	This study
pCB301	Promoter less expression plasmid, Km^R	[19]
pCB301-Prm4546-lacZ	The upstream promoter of atu4546 was linked to the reporter gene lacZ and integrated into pCB301	This study
pCB301-Prm4547-lacZ	The upstream promoter of atu4547 was linked to the reporter gene lacZ and integrated into pCB301	This study
pCB301-Prm4547 (remove the binding site)-lacZ	pCB301-Prm4547-lacZ removes transcription factor binding sites	This study
pCB301-Prm4547 (unrelated sequence)-lacZ	The transcription factor binding site on pCB301-Prm4547-lacZ was replaced by an unrelated sequence	This study
pUCA19	For the construction of gene complement plasmid, carrying an agrobacterial replicon, Ap^R, Cr^R	[20]
pUCA19-atu4546	The coding region of atu4546 was inserted into pUCA19 to express atu4546	This study

Primers name	Primer sequences (5′→3′)	Purpose
Atu4546-U-F	GGCCAGTGCCAAGCTTCGCAGTGGTTTCGAACGGA	Amplify the upstream homologous arm of atu4546
Atu4546-U-R	TCGCACAAATGGAGCTTGAGCGGCGGTGTCAACCGCG	Amplify the upstream homologous arm of atu4546
Atu4546-D-F	CGCGGTTGACACCGCCGCTCAAGCTCCATTTGTGCGA	Amplify the downstream homologous arm of atu4546
Atu4546-D-R	CGGTACCCGGGGATCCGTGTAGAAGGCGGGAATGCCA	Amplify the downstream homologous arm of atu4546
C-atu4546-F	TGATTACGCCAAGCTTATGGCTGTCAGTGAAAGAGACATGA	Amplify atu4546
C-atu4546-R	CGGTACCCGGGGATCCTCAGACCAGCATTCTCGCCAG	Amplify atu4546
Prm4546-F	TAGAACTAGTGGATCCTCTCAAAACCTCCATCCAGC	Amplify the promoter region of atu4546
Prm4546-R	AAATGGAGCTTGAGCATGACCATGATTACG	Amplify the promoter region of atu4546
4549-502-F1	GCCAGTGCCAAGCTTGAAGCTGAAGCCGGTCAAC	Amplify the downstream homologous arm of atu4549
4549-502-R2	GTGAATCCGTAATCATGGTCATTTTCTCCTCTTTTCAGACCCGCTCCAGCG	Amplify the downstream homologous arm of atu4549
4549lacZ3	CGCTGGAGCGGGTCTGAAAAGAGGAGAAAATGACCATGATTACGGATTCAC	Amplification of lacZ containing atu4549 homologous arm
4549lacZ4	CGAATGCCGCTTTTCCCGTTATTTTTGACACCAGACCAACT	Amplification of lacZ containing atu4549 homologous arm
4550-485-F5	AGTTGGTCTGGTGTCAAAAATAACGGGAAAAGCGGCATTCG	Amplify the upstream homologous arm of atu4550
4550-485-R6	GGTACCCGGGGATCCGGCGGCTTTTGCGACATCT	Amplify the upstream homologous arm of atu4550
Prm4547-F	AGAACTAGTGGATCCGATGACCCGCAAACCCTT	Amplify the promoter region of atu4547
Prm4547-R	GTAATCATGGTCATTCTCAAAACCTCCATCCAGC	Amplify the promoter region of atu4547
LacZ-F	ATGGAGGTTTTGAGAATGACCATGATTACGGATTCAC	Amplify lacZ
LacZ-R	GGTATCGATAAGCTTTTATTTTTGACACCAGACCAACT	Amplify lacZ
Prm4547-delete-1	GATGACCCGCAAACCCTTGG	Amplify the upstream sequence of the binding site
Prm4547-delete-2	GTGCGTTATATGATTTTTAAATGGAGCTTGAGCATGG	Amplify the upstream sequence of the binding site
Prm4547-delete-3	CCATGCTCAAGCTCCATTTAAAAATCATATAACGCACAAAATCC	Amplify the downstream sequence of the binding site
Prm4547-delete-4	TCTCAAAACCTCCATCCAGC	Amplify the downstream sequence of the binding site
Prm4547-unrelated-1	GATGACCCGCAAACCCTTGG	Amplifying upstream fragment of unrelated sequence
Prm4547-unrelated-2	TTAATGGCAACTTTTAAATGGAGCTTGAGCATGG	Amplifying upstream fragment of unrelated sequence
Prm4547-unrelated-3	AAAAGTTGCCATTAAAAAAATCATATAACGCACAAAATCC	Amplify the downstream fragment of unrelated sequence
Prm4547-unrelated-4	TCTCAAAACCTCCATCCAGC	Amplify the downstream fragment of unrelated sequence

Primers name	Primer sequences (5′→3′)	Purpose
Atu4546-U-F	GGCCAGTGCCAAGCTTCGCAGTGGTTTCGAACGGA	Amplify the upstream homologous arm of atu4546
Atu4546-U-R	TCGCACAAATGGAGCTTGAGCGGCGGTGTCAACCGCG	Amplify the upstream homologous arm of atu4546
Atu4546-D-F	CGCGGTTGACACCGCCGCTCAAGCTCCATTTGTGCGA	Amplify the downstream homologous arm of atu4546
Atu4546-D-R	CGGTACCCGGGGATCCGTGTAGAAGGCGGGAATGCCA	Amplify the downstream homologous arm of atu4546
C-atu4546-F	TGATTACGCCAAGCTTATGGCTGTCAGTGAAAGAGACATGA	Amplify atu4546
C-atu4546-R	CGGTACCCGGGGATCCTCAGACCAGCATTCTCGCCAG	Amplify atu4546
Prm4546-F	TAGAACTAGTGGATCCTCTCAAAACCTCCATCCAGC	Amplify the promoter region of atu4546
Prm4546-R	AAATGGAGCTTGAGCATGACCATGATTACG	Amplify the promoter region of atu4546
4549-502-F1	GCCAGTGCCAAGCTTGAAGCTGAAGCCGGTCAAC	Amplify the downstream homologous arm of atu4549
4549-502-R2	GTGAATCCGTAATCATGGTCATTTTCTCCTCTTTTCAGACCCGCTCCAGCG	Amplify the downstream homologous arm of atu4549
4549lacZ3	CGCTGGAGCGGGTCTGAAAAGAGGAGAAAATGACCATGATTACGGATTCAC	Amplification of lacZ containing atu4549 homologous arm
4549lacZ4	CGAATGCCGCTTTTCCCGTTATTTTTGACACCAGACCAACT	Amplification of lacZ containing atu4549 homologous arm
4550-485-F5	AGTTGGTCTGGTGTCAAAAATAACGGGAAAAGCGGCATTCG	Amplify the upstream homologous arm of atu4550
4550-485-R6	GGTACCCGGGGATCCGGCGGCTTTTGCGACATCT	Amplify the upstream homologous arm of atu4550
Prm4547-F	AGAACTAGTGGATCCGATGACCCGCAAACCCTT	Amplify the promoter region of atu4547
Prm4547-R	GTAATCATGGTCATTCTCAAAACCTCCATCCAGC	Amplify the promoter region of atu4547
LacZ-F	ATGGAGGTTTTGAGAATGACCATGATTACGGATTCAC	Amplify lacZ
LacZ-R	GGTATCGATAAGCTTTTATTTTTGACACCAGACCAACT	Amplify lacZ
Prm4547-delete-1	GATGACCCGCAAACCCTTGG	Amplify the upstream sequence of the binding site
Prm4547-delete-2	GTGCGTTATATGATTTTTAAATGGAGCTTGAGCATGG	Amplify the upstream sequence of the binding site
Prm4547-delete-3	CCATGCTCAAGCTCCATTTAAAAATCATATAACGCACAAAATCC	Amplify the downstream sequence of the binding site
Prm4547-delete-4	TCTCAAAACCTCCATCCAGC	Amplify the downstream sequence of the binding site
Prm4547-unrelated-1	GATGACCCGCAAACCCTTGG	Amplifying upstream fragment of unrelated sequence
Prm4547-unrelated-2	TTAATGGCAACTTTTAAATGGAGCTTGAGCATGG	Amplifying upstream fragment of unrelated sequence
Prm4547-unrelated-3	AAAAGTTGCCATTAAAAAAATCATATAACGCACAAAATCC	Amplify the downstream fragment of unrelated sequence
Prm4547-unrelated-4	TCTCAAAACCTCCATCCAGC	Amplify the downstream fragment of unrelated sequence

根癌农杆菌转录因子PcaR的功能分析

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徐楠 , 程爽 , 王婉瑜 , 李诚昊 , 郭敏亮 ^*

微生物学报 | 研究报告 2025,65(12): 5424-5437

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微生物学报 | 研究报告 2025, 65(12): 5424-5437

根癌农杆菌转录因子PcaR的功能分析

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徐楠, 程爽, 王婉瑜, 李诚昊, 郭敏亮^*

作者信息

扬州大学生物科学与技术学院，江苏扬州

Functional analysis of transcription factor PcaR in Agrobacteriumtumefaciens

Nan XU, Shuang CHENG, Wanyu WANG, Chenghao LI, Minliang GUO^*

Affiliations

College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, Jiangsu, China

出版时间: 2025-12-04 doi: 10.13343/j.cnki.wsxb.20250383

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摘要

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根癌农杆菌(Agrobacterium tumefaciens)作为植物-微生物互作研究的经典模式生物，是重要的植物转基因工具。植物受伤后分泌的酚酸类化合物会影响根癌农杆菌侵染宿主。目的考察根癌农杆菌转录因子PcaR对简单酚酸类物质代谢的影响、对靶基因的调控作用，以及对宿主植物致瘤能力的影响。方法通过自杀质粒pEX18Km和带有强启动子的质粒pUCA19分别构建atu4546基因敲除株A. tumefaciens Δatu4546和回补菌株C-Δatu4546。评估这些菌株在以原儿茶酸和对羟基苯甲酸为唯一碳源时的生长情况，以及在胡萝卜茎块和落地生根叶片上的致瘤能力。在野生型C58和Δatu4546菌株中将报告基因原位插入到代谢靶基因atu4549下游，通过检测β-半乳糖苷酶活性研究atu4546对靶基因的调控关系；通过构建含有atu4546自身启动子与lacZ报告基因的质粒研究PcaR的自调控特性。构建含有靶基因上游启动子区域与lacZ报告基因的质粒，将生物信息学预测的结合位点去除和替换后，通过测定β-半乳糖苷酶活性确定PcaR结合位点。结果敲除atu4546基因不影响A. tumefaciens在蔗糖上的生长，但导致A. tumefaciens无法利用对羟基苯甲酸和原儿茶酸作为唯一碳源，回补atu4546后生长能力恢复。Δatu4546菌株侵染胡萝卜茎块和落地生根叶片后形成的肿瘤质量分别下降34.90%和52.58%，每0.1 g肿瘤中的菌落数分别减少72.19%和80.54%。敲除atu4546基因使其自身启动子活性增强102.04%，说明atu4546负调控自身表达。Δatu4546中靶基因下游的β-半乳糖苷酶活性比野生型降低74.86%，表明atu4546促进atu4547-atu4549基因簇表达。通过atu4547启动子区序列改造实验确定PcaR结合位点为GTGCGATATACGAAC。结论本研究表明转录因子PcaR参与酚酸分解代谢，负调控自身并促进下游基因pcaIJF转录，其对下游靶基因的结合位点为GTGCGATATACGAAC。PcaR的缺失会降低根癌农杆菌的致病性。本研究揭示了酚酸代谢-致病信号通路中的双重调控机制，拓展了对微生物-植物互作的理论认知。

关键词

根癌农杆菌 / β-酮己二酸途径 / PcaR

Abstract

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Agrobacteriumtumefaciens, a classic model organism for plant-microbe interaction research, is a valuable transgenic tool for plants. Phenolic acids secreted by plants after injury can affect the infection of the host by A. tumefaciens. Objective This study investigated the transcription factor PcaR of A. tumefaciens regarding its effects on the metabolism of simple phenolic acids, regulation of the target gene, and effect on the bacterial tumorigenicity in host plants. Methods The A. tumefaciens strain with atu4546 knockout (Δatu4546) and the complement strain C-Δatu4546 were constructed via the suicide plasmid pEX18Km and the plasmid pUCA19 with a strong promoter, respectively. Both Δatu4546 and C-Δatu4546 were tested for growth with p-hydroxybenzoic acid or protocatechuic acid as the sole carbon source and tumorigenicity on carrot stems and Kalanchoe pinnata leaves. In the wild-type strain C58 and Δatu4546, the reporter gene was in situ inserted into the downstream region of the metabolic target gene atu4549. The regulatory link between atu4546 and the target gene was examined based on the β-galactosidase activity. To investigate the self-regulation of PcaR, we constructed the atu4546 self-promoter reporter plasmid. To identify the binding sites of PcaR, we constructed the upstream promoter region reporter plasmid of the target gene to remove or replace the predicted binding sites and then determined the β-galactosidase activity. Results The knockout of atu4546 did not affect the growth of A. tumefaciens on sucrose, but led to the inability to use p-hydroxybenzoic acid or protocatechuic acid as the sole carbon source. The growth was restored after atu4546 was complemented. The tumor weights of carrot stems and K. pinnata leaves infected by Δatu4546 decreased by 34.90% and 52.58%, respectively, and the number of colonies per 0.1 g tumor decreased by 72.19% and 80.54%, respectively. The knockout of atu4546 led to a 102.04% increase in its own promoter activity, which suggested that atu4546 negatively regulated its own expression. Atu4546 boosted the expression of the atu4547-atu4549 gene cluster, as evidenced by a 74.86% decrease in β-galactosidase activity downstream of the target gene in Δatu4546 compared with that in the wild type. The promoter region sequence alteration experiment identified GTGCGATATATACGAAC as the binding site of PcaR. Conclusion This study shows that the transcription factor PcaR is involved in phenolic acid catabolism, negatively regulates itself and stimulates the transcription of the downstream gene pcaIJF. The binding site of PcaR to the target gene is GTGCGATATACGAAC. The knockout of PcaR attenuates the pathogenicity of A. tumefaciens. This study reveals the dual regulation mechanism in the phenolic acid metabolism-pathogenic signaling pathway and expands the theoretical cognition of plant-microbe interactions.

Key words

Agrobacteriumtumefaciens / β-ketoadipate pathway / PcaR

引用本文

徐楠, 程爽, 王婉瑜, 李诚昊, 郭敏亮. 根癌农杆菌转录因子PcaR的功能分析. 微生物学报, 2025 , 65 (12) : 5424 -5437 . DOI: 10.13343/j.cnki.wsxb.20250383

Nan XU, Shuang CHENG, Wanyu WANG, Chenghao LI, Minliang GUO. Functional analysis of transcription factor PcaR in Agrobacteriumtumefaciens[J]. Acta Microbiologica Sinica, 2025 , 65 (12) : 5424 -5437 . DOI: 10.13343/j.cnki.wsxb.20250383

正文

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根癌农杆菌(Agrobacterium tumefaciens)是一种广泛存在于土壤中的革兰氏阴性菌，是引发植物冠瘿瘤病的主要病原菌^[1-3]。在自然环境中，它能够通过植物根部伤口入侵植物，将自身携带的Ti质粒上的T-DNA片段整合到植物基因组中，促使植物细胞异常增殖，进而形成肿瘤^[4]。这一过程主要依赖于细菌对植物信号分子的感知与响应，酚酸类化合物在其中扮演着关键角色^[5]。植物受伤后会释放出酚酸类物质，根癌农杆菌可借助趋化系统感知这些信号，向植物伤口部位聚集^[6]。酚酸类物质能够激活根癌农杆菌的vir基因，启动T-DNA的加工、转移和整合过程，从而实现植物的遗传转化^[7]。

β-酮己二酸途径是微生物降解芳香族化合物的重要代谢途径，广泛存在于土壤细菌和真菌中。此途径包含原儿茶酸途径和儿茶酚途径 2个分支，涉及多个基因编码的多种酶的协同作用^[8]。该途径可将多种芳香族化合物，如阿魏酸、4-羟基苯甲酸等，逐步转化为β-酮己二酸，最终进入三羧酸循环(tricarboxylic acid cycle, TCA cycle)，为微生物的生长和生存提供能量与碳源^[9-10]。对A. tumefaciens C58基因组注释发现，其通过原儿茶酸途径代谢酚(酸)类化合物。原儿茶酸在3,4-双加氧酶(pcaHG)的催化作用下，通过环氧键断裂机制发生开环反应生成3-羧基顺式,顺式粘康酸酯；继而，3-羧基顺式,顺式粘康酸酯经顺式粘康酸环异构酶(pcaB)异构化作用进一步转化为4-羧基粘康内酯，4-羧基粘康内酯在4-羧基粘康内酯脱羧酶(pcaC)作用下发生脱羧反应，形成β-酮己二酸烯醇内酯；随后，β-酮己二酸烯醇内酯水解酶(pcaD)催化β-酮己二酸烯醇内酯水解，产生β-酮己二酸^[10]。最终，β-酮己二酸经由β-酮己二酸琥珀酰辅酶A转运酶(pcaIJ)和β-酮己二酸单酰辅酶A硫解酶(pcaF)的级联催化反应，生成乙酰辅酶A和琥珀酰辅酶A^[11]，进入三羧酸循环，为微生物提供能量代谢和物质合成所需的碳源及能量。目前，针对根癌农杆菌β-酮己二酸途径的研究已取得一定进展。已明确编码该途径相关酶的基因主要分布在pcaDCHGB和pcaIJF 2个操纵子中，它们的转录受到不同调控因子的影响。pcaDCHGB操纵子受LysR型调节蛋白PcaQ调控^[12-13]。此外，pobA等基因也参与了酚酸类物质向原儿茶酸的转化过程，进而与β-酮己二酸途径相联系^[14]。研究发现PobR作为一种转录激活因子不仅能够启动自身的负调控作用，还能激活下游靶基因pobA转录^[15]。除了转录因子PcaQ和PobR，PcaR (atu4546)被注释为IclR家族转录因子，可能参与到根癌农杆菌β-酮己二酸途径的调控。然而，其在根癌农杆菌中的具体功能以及与靶基因之间的调控关系尚未有研究。

本研究通过构建根癌农杆菌转录因子 PcaR(Atu4546)的基因敲除株及回补菌株，考察 atu4546 基因缺失对根癌农杆菌利用常用碳源和酚酸类碳源的影响。通过构建不同原位报告菌株和报告质粒探究 atu4546 基因对自身及下游靶基因的调控模式及具体结合位点。此外，通过在胡萝卜茎块和落地生根叶片上的致瘤实验考察atu4546 基因对根癌农杆菌致病性的影响。本研究旨在揭示转录因子PcaR在根癌农杆菌代谢调控及致病过程中的功能，为深入解析根癌农杆菌的酚酸代谢与致病机制提供理论基础。

1 材料与方法

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1.1 菌株和质粒

根癌农杆菌(A. tumefaciens)野生型菌株为基因组测序用菌株C58，其Ti质粒为胭脂碱型。根癌农杆菌的pcaR缺失菌株Δatu4546是由C58敲除该蛋白质的编码基因所得，缺失突变体采用全编码框无痕删除法获得^[16]。大肠杆菌(Escherichia coli) DH5α菌株用作构建质粒的宿主菌。大肠杆菌用LB培养基培养。根癌农杆菌用富营养培养基MG/L (mannitol-glutamate/lysogeny broth)或全合成培养基AB (Agrobacterium minimal medium)-蔗糖培养基培养，具体配方见参考文献[17-18]。本研究所用的质粒见表1。

1.2 根癌农杆菌 atu4546 缺失株及回补株的获得

使用细菌基因组DNA提取试剂盒[天根生化科技(北京)有限公司]提取C58菌株基因组DNA。以C58菌株基因组为模板，分别使用引物对Atu4546-U-F/Atu4546-U-R和Atu4546-D-F/Atu4546-D-R扩增atu4546上、下游同源臂。PCR反应体系(50 μL)：2×Hieff Canace^® Plus PCR Master Mix 25 μL，上、下游引物(10 μmol/L)各1.5 μL，C58基因组1 μL，ddH₂O 21 μL。PCR扩增条件：98 ℃ 预变性3 min；95 ℃变性10 s，60 ℃退火10 s，72 ℃延伸30 s，共30个循环；72 ℃终延伸5 min。将自杀质粒pEX18Km用BamH I、Hind III双酶切获得线性载体，分别与atu4546上、下游同源臂以同源重组的方式连接。连接体系：线性化载体2 μL，上、下游片段各1 μL，2×CE Mix 5 μL，ddH₂O 补至10 μL。连接产物经热激转化至大肠杆菌DH5α感受态中，待菌落在卡那霉素抗性平板上长出后，用引物对Atu4546-U-F/Atu4546-D-R进行菌落PCR验证。PCR反应体系(20 μL)：2×Rapid Taq Master Mix 10 μL，上、下游引物(10 μmol/L)各1 μL，以单菌落为模板，ddH₂O 8 μL。PCR反应条件：95 ℃预变性5 min；95 ℃变性15 s，60 ℃退火15 s，72 ℃延伸30 s，共30个循环；72 ℃终延伸5 min。经菌落PCR鉴定后送往北京擎科生物科技股份有限公司测序，序列正确的质粒为pEX18Km-atu4546。向根癌农杆菌C58感受态中加入测序成功的质粒10 μL，用枪头吹吸混匀，冰上放置 8 min。将混匀液加入到电转杯中，设置电转仪(Eppendorf公司)参数为1 200 V/mm，连续按start键3下，结束后快速取出电转杯，向其中加入1 mL预冷MG/L液体培养基，吹吸混匀，将混匀液吸至1.5 mL离心管中，将离心管放置在28 ℃、150 r/min缓慢复苏2 h，复苏完成后4 500 r/min离心3 min，弃部分上清，重悬沉淀，将菌液涂布在含卡那霉素抗性(100 μg/mL)的MG/L平板上，28 ℃倒置培养36 h。经过卡那霉素抗性(100 μg/mL)和蔗糖敏感性(5%蔗糖)正向和反向2次筛选，对转化子进行菌落PCR和DNA测序验证，得到atu4546缺失突变体Δatu4546。

获得atu4546回补株时使用具有强启动子的pUCA19载体。使用引物对C-atu4546-F/C-atu4546-R以C58基因组为模板扩增atu4546基因编码区。将pUCA19载体使用限制性内切酶Hind Ⅲ和BamH I进行双酶切。将atu4546编码区序列和线性化的pUCA19载体进行同源重组连接，连接产物经热激转化至大肠杆菌DH5α感受态中，经菌落PCR验证和测序结果正确的质粒命名为pUCA19-atu4546。将重组质粒pUCA19-atu4546通过电击转化方式^[18]转入Δatu4546菌株感受态中，在含有羧苄霉素(100 μg/mL)抗性的MG/L平板上28 ℃倒置培养36 h，将长出的单菌落进行纯化，随后进行菌落PCR鉴定，鉴定成功的菌落即为回补株A. tumefaciens C-Δatu4546。所用引物见表2。

1.3 含有 lacZ 报告基因菌株的获得

1.3.1 Prm4546-lacZ 菌株的获得

为了研究atu4546对自身是否具有调控作用，首先使用引物对Prm4546-F/Prm4546-R和LacZ-F/LacZ-R分别以A. tumefaciens C58和大肠杆菌BL21为模板，扩增atu4546基因上游启动子序列和lacZ编码区，然后与无启动子的pCB301载体进行同源重组连接。将连接片段化转至大肠杆菌DH5α感受态中，待菌落PCR和测序结果均正确后获得重组质粒 pCB301-Prm4546-lacZ。随后将重组质粒分别电击转化^[18]至野生型C58以及Δatu4546菌株中，在含有羧苄霉素(100 μg/mL)抗性的MG/L平板上28 ℃倒置培养36 h，将长出的单菌落进行纯化，随后进行菌落PCR鉴定。

1.3.2 atu4549 原位插入 lacZ 菌株的获得

为了研究atu4546对下游基因pcaIJF的调控作用，在C58以及atu4546突变菌株的atu4549基因下游原位插入lacZ报告基因，通过β-半乳糖苷酶活性反映pcaIJF基因的表达水平。设计3对引物，以野生型C58基因组为模板，用引物4549-502-F1、4549-502-R2扩增出atu4549基因下游502 bp的片段，用4549lacZ3、4549lacZ4扩增含有同源臂的全长lacZ基因，用4550-485-F5、4550-485-R6扩增atu4550基因上游485 bp的片段。为了精确mRNA在核糖体上的定位，在lacZ序列上游添加一段12 bp长的SD序列，该序列为5′-TTTCTCCTCTTT-3′。将上述3段序列进行扩增，随后将纯化的3个DNA片段与线性pEX18Km载体同源重组连接，接着热激转化至大肠杆菌DH5α感受态中，将菌落PCR鉴定正确的菌落送北京擎科生物股份有限公司测序，测序结果正确后提取质粒。然后将其电转入^[18]野生型C58以及Δatu4546菌株中，经过多次筛选以及PCR鉴定获得C58-4549-lacZ与Δatu4546-4549-lacZ菌株。所用引物见表2，设计原理如图1所示。

1.3.3 Prm4547-lacZ 以及去除和替换结合位点菌株的获得

为了确定atu4546与下游基因atu4547启动子区域的结合位点，获得了含有lacZ报告基因的菌株，方法如下：首先使用引物Prm4547-F/-R和LacZ-F/-R分别扩增atu4547的启动子序列和lacZ基因序列，然后将纯化后的2个DNA片段与无启动子的线性pCB301质粒同源重组连接，经热激转化至大肠杆菌DH5α感受态中。将菌落PCR鉴定正确的菌落送北京擎科生物股份有限公司测序，测序结果正确后提取质粒pCB301-Prm4547-lacZ。然后将其电转入^[18]野生型C58以及Δatu4546菌株中，获得C58-Prm4547-lacZ与Δatu4546-Prm4547-lacZ菌株。去除结合位点与结合位点替换为无关序列的菌株获得方法如下：首先利用MEME suite^[21]预测了atu4546与atu4547启动子区域的结合序列为GTGCGATATACGAAC，然后通过重叠PCR的方法将atu4547启动子区域的结合序列去除或替换成无关序列。接着将其与lacZ连接并克隆入无启动子的pCB301质粒中，获得重组质粒pCB301-Prm4547 (remove the binding site)-lacZ与pCB301-Prm4547 (unrelated sequence)-lacZ，方法与1.3.1一致。所用引物见表2。

1.4 以蔗糖为唯一碳源生长曲线的测定

将野生型C58、Δatu4546以及C-Δatu4546 这3种菌株在MG/L平板上进行三区划线，28 ℃倒置培养36 h。接着用接种环挑取单菌落于5 mL MG/L液体培养基中，28 ℃、200 r/min培养约14 h，然后4 500 r/min离心3 min收集菌体，用无菌水清洗菌体2-3次。接着将菌液OD₆₀₀调至1.0，取1 mL菌液转接到100 mL对应的AB-蔗糖培养基中，28 ℃、200 r/min培养约60 h。培养期间每4 h取1次样，测量OD₆₀₀数值，直至到达衰退期。

1.5 以对羟基苯甲酸或原儿茶酸为碳源生长曲线测定

按照1.4节的方法培养菌株，4 500 r/min离心3 min收集菌体，用AB-无碳源合成培养基清洗菌体2-3次后，将菌液OD₆₀₀调成1.0。分别取1 mL菌液接种到新鲜的100 mL AB-对羟基苯甲酸(5 mmol/L)和AB-原儿茶酸(10 mmol/L)合成培养基中。每种菌设置3个重复组。每隔4 h取样，到达衰退期结束取样。

1.6 β-半乳糖苷酶活性测定

将C58-Prm4546-lacZ、Δatu4546-Prm4546-lacZ、C58-4549-lacZ、Δatu4546-4549-lacZ、C58-Prm4547-lacZ、Δatu4546-Prm4547-lacZ以及去除结合位点和替换成无关序列的菌株在含有相应抗性的MG/L平板上活化，接着挑取单菌落于MG/L液体培养基中，28 ℃、200 r/min培养14 h，然后4 500 r/min离心3 min收集菌体，用AB-阿拉伯糖培养基清洗菌体2-3次，取200 μL转接至5 mL对应的AB培养基中培养至对数生长期(OD₆₀₀=0.6-0.8)。接着取出菌液，测定相应OD₆₀₀数值，取200 μL菌液，依次加入900 µL Z-buffer (已添加无水乙醇)、50 µL氯仿、20 µL 0.1%的SDS，旋涡振荡30 s后于37 ℃水浴15-30 min，每组设置3个重复。最后加入200 µL的ONPG，记录溶液变黄的时间。溶液变黄后立即加入终止反应剂Na₂CO₃，4 500 r/min离心3 min后将上清液吸出，测量OD₄₂₀值。 β-半乳糖苷酶活性计算如公式(1)所示。

β-半乳糖苷酶活=

1 000 × O D 420 t (m i n) × V (m L) × O D 600

(1)

式中：t、V、OD₆₀₀ 、OD₄₂₀分别为反应时间、菌液体积和吸光度、反应后样品的420 nm下吸光度。

1.7 致瘤实验

为了探究atu4546对根癌农杆菌致病性的影响，选择了胡萝卜和落地生根2种植物作为研究对象。将C58、Δatu4546及C-Δatu4546菌株28 ℃、200 r/min培养14 h。然后转接至AB-蔗糖培养基中继续培养至对数生长期(OD₆₀₀=0.6-0.8)，使用无菌水清洗2-3次，将每种菌液OD₆₀₀调至0.5。接着清洗胡萝卜，将胡萝卜、金属打孔器、手术刀在1.05%的次氯酸钠溶液中浸泡30 min杀菌。然后用金属打孔器和手术刀将胡萝卜切割为同样厚度(约为5 mm)的柱状圆盘，相隔1.5 cm间距放在1.5%的水琼脂平板上，取5 µL菌液接种于胡萝卜圆盘中央，每组菌设置15个重复，将平板于28 ℃培养。落地生根叶片用75%的乙醇棉球擦拭干净，使用针头在叶片上划相同长度的伤口，取5 µL菌液至伤口位置，室温放置在阴凉处。在培养4周以后，将胡萝卜茎块和落地生根叶片上的肿瘤取下称重。接着取0.1 g的肿瘤放入研钵中，加入无菌生理盐水进行充分研磨。然后吸取100 µL菌液稀释至10^-8后，取100 µL涂布到含蔗糖的AB平板，28 ℃培养48 h后对平板上的菌落进行计数。

1.8 数据处理与分析

采用GraphPad Prism 10.0对所有数据进行处理和绘图，采用t检验(t-test)进行统计学分析，显著差异用*表示，ns：差异不显著；*：P<0.05；**：P<0.01；***：P<0.001；****：P<0.000 1。

2 结果与分析

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2.1 根癌农杆菌 atu4546 对生长的影响

为研究atu4546的功能，首先通过自杀质粒pEX18Km同源重组获得atu4546基因敲除株A. tumefaciens Δatu4546。然后，利用质粒pUCA19在Δatu4546菌株中表达atu4546得到回补菌株C-Δatu4546。以常见碳源(蔗糖)和酚酸(对羟基苯甲酸和原儿茶酸)为唯一碳源，研究野生型菌株C58、atu4546敲除突变体Δatu4546和回补菌株C-Δatu4546的生长情况。如图2A所示，3种菌株在常用碳源蔗糖上的生长趋势相似，这表明atu4546对根癌农杆菌的中心碳代谢无影响。进一步考察根癌农杆菌在非常用碳源上的生长情况，发现野生型C58能够利用对羟基苯甲酸和原儿茶酸作为唯一碳源生长。当atu4546被敲除后，C58不能利用对羟基苯甲酸和原儿茶酸作为唯一碳源生长。相比之下，在Δatu4546中表达atu4546时其生长缺陷得以恢复(图2B、2C)。这些结果表明atu4546参与对羟基苯甲酸和原儿茶酸的分解代谢。由于atu4546被注释为转录因子，说明该基因参与根癌农杆菌β-酮己二酸途径的代谢调控。

2.2 atu4546对自身的调控作用

为探究atu4546对自身的调控作用，首先将atu4546基因上游调控区与β-半乳糖苷酶基因(lacZ)连接，并克隆入无启动子的pCB301质粒中，获得重组质粒pCB301-Prm4546-lacZ。然后将重组质粒分别转化入野生型C58和Δatu4546菌株中，通过测定β-半乳糖苷酶活性探究atu4546对自身的调控作用。如图3所示，Δatu4546中atu4546启动子区控制下的β-半乳糖苷酶活性比野生型C58高102.04%，在添加原儿茶酸和己二酸后，Δatu4546菌株中β-半乳糖苷酶活性依然显著高于未敲除atu4546的野生型菌株，结果表明atu4546对自身具有负调控作用。此外，在C58中额外添加原儿茶酸或己二酸后β-半乳糖苷酶活性较未添加时无显著性差异。

2.3 atu4546对下游基因 pcaIJF 的调控作用

为探究atu4546对下游基因的调控作用，将报告基因lacZ分别原位插入野生型C58和Δatu4546中pcaF (atu4549)下游，通过测量β-半乳糖苷酶活性探究atu4546对下游基因pcaIJF的调控作用。如图4所示，Δatu4546中的β-半乳糖苷酶活性比野生型C58低74.86%，这表明atu4546对下游基因具有促进作用，为转录激活因子。在分别添加10 mmol/L原儿茶酸和5 mmol/L对羟基苯甲酸后，野生型C58菌株中β-半乳糖苷酶活性依然显著高于Δatu4546菌株。在野生型C58中额外添加诱导物原儿茶酸和对羟基苯甲酸后，较之有碳源阿拉伯糖(Ara)组，β-半乳糖苷酶活性分别提高了32.24%和109.71%，说明2种诱导物均可诱导atu4546转录，但诱导程度不同。

2.4 atu4546与靶基因启动子区结合位点的确定

为确定PcaR对下游靶基因的调控作用及结合位点，首先将atu4547的上游调控区与β-半乳糖苷酶基因(lacZ)连接并克隆至无启动子的pCB301质粒中，获得重组质粒pCB301-Prm4547-lacZ。随后将重组质粒分别转化至野生型C58和Δatu4546菌株中，在原儿茶酸、对羟基苯甲酸和己二酸诱导下测量β-半乳糖苷酶活性。Δatu4546中atu4547启动子区控制下的β-半乳糖苷酶活性比野生型C58低21.68% (图5A)，与报告基因原位插入靶基因atu4549下游的酶活结果一致，表明atu4546对下游靶基因具有激活作用。为确定atu4546与atu4547启动子区域的转录因子结合位点(transcription factor binding sites, TFBS)，首先利用MEME suite^[21]预测了atu4546与atu4547启动子区域的结合序列为GTGCGATATACGAAC，然后将atu4547的启动子区中预测的结合位点去除，或随机突变成无关序列，通过测量β-半乳糖苷酶活性来验证预测结合位点的准确性。去除预测的结合位点后，C58和Δatu4546菌株中β-半乳糖苷酶活性结果均无显著差异(图5B)，初步验证了所预测的DNA序列确为atu4546与atu4547启动子区域的结合位点。将预测的结合序列随机突变成无关序列后，C58和Δatu4546菌株中β-半乳糖苷酶活性结果同样均无显著差异(图5C)，进一步验证了所预测的DNA序列确为atu4546与atu4547启动子区域的结合位点。

2.5 atu4546 对宿主植物致病性的影响

为探究atu4546对根癌农杆菌致病性的影响，本研究选择落地生根和胡萝卜2种植物进行侵染实验，通过野生型菌株C58、基因缺失菌株Δatu4546和基因回补菌株C-Δatu4546侵染2种植物后肿瘤的质量以及肿瘤中的菌落数评价几种菌株对宿主植物致病能力的强弱。肿瘤称重结果显示，atu4546的缺失使胡萝卜茎块和落地生根叶片上的肿瘤质量分别降低34.90%和52.58%，C-Δatu4546的肿瘤质量恢复至与野生型相似水平，表明atu4546基因缺失会影响根癌农杆菌的致瘤能力，重新表达后可恢复基因缺失株的肿瘤发生(图6C、6D)。统计每0.1 g肿瘤中的菌落数结果显示，atu4546的缺失使胡萝卜茎块和落地生根叶片上每0.1 g肿瘤中的菌落数分别降低72.19%和80.54%，C-Δatu4546的菌落数恢复至与野生型相似水平(图6E、6F)。肿瘤质量和菌落数均表明atu4546的缺失降低了根癌农杆菌的致病性。

3 讨论与结论

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本文研究了根癌农杆菌IclR家族转录因子PcaR在β-酮己二酸途径中的代谢调控作用。该基因缺失导致根癌农杆菌无法利用4-羟基苯甲酸和原儿茶酸作为唯一碳源生长。Atu4546对自身表达具有负调控作用，它能够结合下游基因atu4547启动子的调控区(结合序列为GTGCGATATACGAAC)，激活下游基因atu4547-atu4549的表达，进而促进β-酮己二酸转化为TCA循环中间物。在不同微生物中转录因子PcaR的功能和调控方式既有相似之处，又存在差异^[22-23]。在恶臭假单胞菌(Pseudomonasputida)中，PcaR作为转录激活因子能够与特定的DNA序列结合，激活原儿茶酸代谢相关基因的表达，促进原儿茶酸的降解^[24]。在苜蓿中华根瘤菌(Sinorhizobiummeliloti)中，PcaR不仅参与调控原儿茶酸代谢基因，还存在自调控机制，通过负自我调节维持自身及相关基因表达的平衡^[25]。在节杆菌(Arthrobacter)中，PcaR参与调控邻苯二甲酸分解代谢途径，对邻苯二甲酸降解相关基因的表达起到调节作用，使细菌能够有效利用邻苯二甲酸作为碳源^[26]。在不动杆菌(Acinetobacter)中，pcaR与pcaU等基因共同参与对原儿茶酸利用相关基因的调控。PcaU是原儿茶酸利用基因的转录激活因子，PcaR与PcaU在氨基酸序列上有一定的相似性，它们可能通过与相似的DNA序列结合来调控基因表达^[27]。在浑浊红球菌(Rhodococcusopacus)中，PcaR为转录抑制子调控原儿茶酸途径^[28]。在不同微生物中，PcaR对β-酮己二酸代谢的调控机制差异体现了进化的多样性，以及微生物对不同生态位的适应特征。

atu4546的缺失显著降低了根癌农杆菌在胡萝卜茎块和落地生根叶片上的结瘤能力及肿瘤中的菌落数。atu4546又是根癌农杆菌利用4-羟基苯甲酸和原儿茶酸的必需基因，可见根癌农杆菌对酚酸类物质的代谢能力很可能与致病性密切相关。酚酸类物质(如对羟基苯甲酸、原儿茶酸)既是植物信号分子，也是细菌的碳源，可作为碳源支持根癌农杆菌在植物体内的生长繁殖。根癌农杆菌PcaR通过调控酚酸代谢基因使细菌在植物体内能够利用这些碳源，从而支持其定殖。相比之下，当pcaR缺失时细菌无法有效分解酚酸，可能引起生长受限，进而影响对宿主植物的侵染和肿瘤形成(图6)。这些现象表明β-酮己二酸途径的代谢功能可能影响细菌毒力因子的表达和细胞生理状态。研究表明根癌农杆菌对酚酸类物质的有效代谢有助于其在植物体内更好地定殖和致病^[15]。此外，根癌农杆菌的致病性与毒力基因(vir)表达密切相关^[29]。本研究以实验室前期获得的C58 virB-lacZ为出发菌株，敲除atu4546，检测β-半乳糖苷酶的活性，发现基因缺失株中vir基因的启动子活性与出发菌株无明显差异，说明atu4546基因不会直接影响virB基因表达(数据未展示)。Atu4546影响农杆菌致病性的原因可能与酚酸代谢产物间接影响毒力基因的表达相关。例如，原儿茶酸的积累可能通过virA/virG信号通路激活vir基因^[30]。一方面，PcaR会激活PcaIJF表达，促进根癌农杆菌代谢酚酸；同时，PcaR负调控自身表达可能通过维持酚酸代谢平衡避免过度消耗信号分子，从而优化vir基因的激活效率。这种代谢与毒力的协同自身调控机制之前也有报道，如根癌农杆菌中PcaQ通过调控原儿茶酸代谢影响vir基因表达^[13]。因此，pcaR的缺失可能打破代谢与信号分子的平衡，削弱细菌对植物的侵染能力。

本研究首次揭示了根癌农杆菌PcaR通过负调控自身并激活下游基因转录，参与β-酮己二酸途径的代谢调控，并影响其致病性。与其他细菌相比，根癌农杆菌PcaR对自身的负调控及结合位点的特异性体现了其在适应植物宿主环境中的独特进化策略。然而，仍有许多问题有待解决。在调控网络方面，PcaR与其他调控因子(如PcaQ)之间的互作机制尚不明确，需要进一步探究它们在全局调控网络中的协同或拮抗关系，以解析根癌农杆菌复杂的调控网络。在效应分子的作用上，虽然初步验证β-酮己二酸可能作为PcaR的配体影响其DNA结合能力，但仍需通过结构生物学和生化实验进行验证，进一步明确效应分子与PcaR的结合模式和调控细节。在与毒力基因的联系上虽然发现pcaR缺失影响根癌农杆菌的致病性，但PcaR是否直接调控毒力因子仍有待深入研究。

作者贡献声明

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徐楠：实验总体设计，论文初稿撰写与修改，数据整理与管理；程爽：实验开展及论文撰写；王婉瑜：实验开展；李诚昊：协助数据处理和校对；郭敏亮：研究课题监管与指导，论文审阅与修订。

作者利益冲突公开声明

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作者声明不存在任何可能会影响本文所报告工作的已知经济利益或个人关系。

基金

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国家自然科学基金(22278350)
扬州大学高端人才支持计划

参考文献

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文献

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参考文献引证文献

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doi: 10.13343/j.cnki.wsxb.20250383

接收时间：2025-05-12
首发时间：2025-12-08
出版时间：2025-12-04

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收稿日期：2025-05-12
录用日期：2025-06-20

基金

National Natural Science Foundation of China(22278350)

国家自然科学基金(22278350)

High-end Talent Support Program of Yangzhou University

扬州大学高端人才支持计划

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扬州大学生物科学与技术学院，江苏扬州

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Plasmids	Purpose and properties	Source
pEX18Km	Suicide plasmid for knockout, carrying reverse selection markers sacB, oriT, and Km^R	[19]
pEX18Km-atu4546	The atu4546 knockout box is inserted into pEX18Km for knockout of atu4546	This study
pCB301	Promoter less expression plasmid, Km^R	[19]
pCB301-Prm4546-lacZ	The upstream promoter of atu4546 was linked to the reporter gene lacZ and integrated into pCB301	This study
pCB301-Prm4547-lacZ	The upstream promoter of atu4547 was linked to the reporter gene lacZ and integrated into pCB301	This study
pCB301-Prm4547 (remove the binding site)-lacZ	pCB301-Prm4547-lacZ removes transcription factor binding sites	This study
pCB301-Prm4547 (unrelated sequence)-lacZ	The transcription factor binding site on pCB301-Prm4547-lacZ was replaced by an unrelated sequence	This study
pUCA19	For the construction of gene complement plasmid, carrying an agrobacterial replicon, Ap^R, Cr^R	[20]
pUCA19-atu4546	The coding region of atu4546 was inserted into pUCA19 to express atu4546	This study

Plasmids

Purpose and properties

Source

pEX18Km

Suicide plasmid for knockout, carrying reverse selection markers sacB, oriT, and Km^R

[19]

pEX18Km-atu4546

The atu4546 knockout box is inserted into pEX18Km for knockout of atu4546

This study

pCB301

Promoter less expression plasmid, Km^R

[19]

pCB301-Prm4546-lacZ

The upstream promoter of atu4546 was linked to the reporter gene lacZ and integrated into pCB301

This study

pCB301-Prm4547-lacZ

The upstream promoter of atu4547 was linked to the reporter gene lacZ and integrated into pCB301

This study

pCB301-Prm4547 (remove the binding site)-lacZ

pCB301-Prm4547-lacZ removes transcription factor binding sites

This study

pCB301-Prm4547 (unrelated sequence)-lacZ

The transcription factor binding site on pCB301-Prm4547-lacZ was replaced by an unrelated sequence

This study

pUCA19

For the construction of gene complement plasmid, carrying an agrobacterial replicon, Ap^R, Cr^R

[20]

pUCA19-atu4546

The coding region of atu4546 was inserted into pUCA19 to express atu4546

This study

Primers name	Primer sequences (5′→3′)	Purpose
Atu4546-U-F	GGCCAGTGCCAAGCTTCGCAGTGGTTTCGAACGGA	Amplify the upstream homologous arm of atu4546
Atu4546-U-R	TCGCACAAATGGAGCTTGAGCGGCGGTGTCAACCGCG	Amplify the upstream homologous arm of atu4546
Atu4546-D-F	CGCGGTTGACACCGCCGCTCAAGCTCCATTTGTGCGA	Amplify the downstream homologous arm of atu4546
Atu4546-D-R	CGGTACCCGGGGATCCGTGTAGAAGGCGGGAATGCCA	Amplify the downstream homologous arm of atu4546
C-atu4546-F	TGATTACGCCAAGCTTATGGCTGTCAGTGAAAGAGACATGA	Amplify atu4546
C-atu4546-R	CGGTACCCGGGGATCCTCAGACCAGCATTCTCGCCAG	Amplify atu4546
Prm4546-F	TAGAACTAGTGGATCCTCTCAAAACCTCCATCCAGC	Amplify the promoter region of atu4546
Prm4546-R	AAATGGAGCTTGAGCATGACCATGATTACG	Amplify the promoter region of atu4546
4549-502-F1	GCCAGTGCCAAGCTTGAAGCTGAAGCCGGTCAAC	Amplify the downstream homologous arm of atu4549
4549-502-R2	GTGAATCCGTAATCATGGTCATTTTCTCCTCTTTTCAGACCCGCTCCAGCG	Amplify the downstream homologous arm of atu4549
4549lacZ3	CGCTGGAGCGGGTCTGAAAAGAGGAGAAAATGACCATGATTACGGATTCAC	Amplification of lacZ containing atu4549 homologous arm
4549lacZ4	CGAATGCCGCTTTTCCCGTTATTTTTGACACCAGACCAACT	Amplification of lacZ containing atu4549 homologous arm
4550-485-F5	AGTTGGTCTGGTGTCAAAAATAACGGGAAAAGCGGCATTCG	Amplify the upstream homologous arm of atu4550
4550-485-R6	GGTACCCGGGGATCCGGCGGCTTTTGCGACATCT	Amplify the upstream homologous arm of atu4550
Prm4547-F	AGAACTAGTGGATCCGATGACCCGCAAACCCTT	Amplify the promoter region of atu4547
Prm4547-R	GTAATCATGGTCATTCTCAAAACCTCCATCCAGC	Amplify the promoter region of atu4547
LacZ-F	ATGGAGGTTTTGAGAATGACCATGATTACGGATTCAC	Amplify lacZ
LacZ-R	GGTATCGATAAGCTTTTATTTTTGACACCAGACCAACT	Amplify lacZ
Prm4547-delete-1	GATGACCCGCAAACCCTTGG	Amplify the upstream sequence of the binding site
Prm4547-delete-2	GTGCGTTATATGATTTTTAAATGGAGCTTGAGCATGG	Amplify the upstream sequence of the binding site
Prm4547-delete-3	CCATGCTCAAGCTCCATTTAAAAATCATATAACGCACAAAATCC	Amplify the downstream sequence of the binding site
Prm4547-delete-4	TCTCAAAACCTCCATCCAGC	Amplify the downstream sequence of the binding site
Prm4547-unrelated-1	GATGACCCGCAAACCCTTGG	Amplifying upstream fragment of unrelated sequence
Prm4547-unrelated-2	TTAATGGCAACTTTTAAATGGAGCTTGAGCATGG	Amplifying upstream fragment of unrelated sequence
Prm4547-unrelated-3	AAAAGTTGCCATTAAAAAAATCATATAACGCACAAAATCC	Amplify the downstream fragment of unrelated sequence
Prm4547-unrelated-4	TCTCAAAACCTCCATCCAGC	Amplify the downstream fragment of unrelated sequence

Primers name

Primer sequences (5′→3′)

Purpose

Atu4546-U-F

GGCCAGTGCCAAGCTTCGCAGTGGTTTCGAACGGA

Amplify the upstream homologous arm of atu4546

Atu4546-U-R

TCGCACAAATGGAGCTTGAGCGGCGGTGTCAACCGCG

Amplify the upstream homologous arm of atu4546

Atu4546-D-F

CGCGGTTGACACCGCCGCTCAAGCTCCATTTGTGCGA

Amplify the downstream homologous arm of atu4546

Atu4546-D-R

CGGTACCCGGGGATCCGTGTAGAAGGCGGGAATGCCA

Amplify the downstream homologous arm of atu4546

C-atu4546-F

TGATTACGCCAAGCTTATGGCTGTCAGTGAAAGAGACATGA

Amplify atu4546

C-atu4546-R

CGGTACCCGGGGATCCTCAGACCAGCATTCTCGCCAG

Amplify atu4546

Prm4546-F

TAGAACTAGTGGATCCTCTCAAAACCTCCATCCAGC

Amplify the promoter region of atu4546

Prm4546-R

AAATGGAGCTTGAGCATGACCATGATTACG

Amplify the promoter region of atu4546

4549-502-F1

GCCAGTGCCAAGCTTGAAGCTGAAGCCGGTCAAC

Amplify the downstream homologous arm of atu4549

4549-502-R2

GTGAATCCGTAATCATGGTCATTTTCTCCTCTTTTCAGACCCGCTCCAGCG

Amplify the downstream homologous arm of atu4549

4549lacZ3

CGCTGGAGCGGGTCTGAAAAGAGGAGAAAATGACCATGATTACGGATTCAC

Amplification of lacZ containing atu4549 homologous arm

4549lacZ4

CGAATGCCGCTTTTCCCGTTATTTTTGACACCAGACCAACT

Amplification of lacZ containing atu4549 homologous arm

4550-485-F5

AGTTGGTCTGGTGTCAAAAATAACGGGAAAAGCGGCATTCG

Amplify the upstream homologous arm of atu4550

4550-485-R6

GGTACCCGGGGATCCGGCGGCTTTTGCGACATCT

Amplify the upstream homologous arm of atu4550

Prm4547-F

AGAACTAGTGGATCCGATGACCCGCAAACCCTT

Amplify the promoter region of atu4547

Prm4547-R

GTAATCATGGTCATTCTCAAAACCTCCATCCAGC

Amplify the promoter region of atu4547

LacZ-F

ATGGAGGTTTTGAGAATGACCATGATTACGGATTCAC

Amplify lacZ

LacZ-R

GGTATCGATAAGCTTTTATTTTTGACACCAGACCAACT

Amplify lacZ

Prm4547-delete-1

GATGACCCGCAAACCCTTGG

Amplify the upstream sequence of the binding site

Prm4547-delete-2

GTGCGTTATATGATTTTTAAATGGAGCTTGAGCATGG

Amplify the upstream sequence of the binding site

Prm4547-delete-3

CCATGCTCAAGCTCCATTTAAAAATCATATAACGCACAAAATCC

Amplify the downstream sequence of the binding site

Prm4547-delete-4

TCTCAAAACCTCCATCCAGC

Amplify the downstream sequence of the binding site

Prm4547-unrelated-1

GATGACCCGCAAACCCTTGG

Amplifying upstream fragment of unrelated sequence

Prm4547-unrelated-2

TTAATGGCAACTTTTAAATGGAGCTTGAGCATGG

Amplifying upstream fragment of unrelated sequence

Prm4547-unrelated-3

AAAAGTTGCCATTAAAAAAATCATATAACGCACAAAATCC

Amplify the downstream fragment of unrelated sequence

Prm4547-unrelated-4

TCTCAAAACCTCCATCCAGC

Amplify the downstream fragment of unrelated sequence