十字花科黑腐病菌中转录因子HpaR1与Clp调控一个糖苷水解酶基因表达的分析

doi:10.16288/j.yczz.21-171

遗传 ›› 2021, Vol. 43 ›› Issue (9): 910-920.doi: 10.16288/j.yczz.21-171

• 研究报告 • 上一篇

十字花科黑腐病菌中转录因子HpaR1与Clp调控一个糖苷水解酶基因表达的分析

刘国芳¹(), 任沛东², 叶文新², 陆光涛²()

1. 广西民族大学海洋与生物技术学院,广西多糖材料与改性重点实验室,南宁 530008
2. 广西大学生命科学与技术学院,南宁 530004

收稿日期:2021-05-11 修回日期:2021-07-05 出版日期:2021-09-20 发布日期:2021-07-14
通讯作者: 陆光涛 E-mail:lgf8411@126.com;lugt@gxu.edu.cn
作者简介:刘国芳,博士,副教授,研究方向：植物保护。E-mail: lgf8411@126.com
基金资助:
广西民族大学科研基金资助项目编号(2019KJQN004);国家自然科学基金项目编号(31860021);广西自然科学基金项目编号(2020GXNSFBA297140);广西科技重大专项资助编号(AA18242026)

Analysis of transcriptional regulators HpaR1 and Clp regulating the expression of glycoside hydrolase-encoding gene in the Xanthomonas campestris pv. campestris

Guofang Liu¹(), Peidong Ren², Wenxin Ye², Guangtao Lu²()

1. Guangxi Key Laboratory for Polysaccharide Materials and Modifications,School of Marine Sciences and Biotechnology, Guangxi University for Nationalities, Nanning 530008, China
2. College of Life Science and Technology, Guangxi University, Nanning 530004, China

Received:2021-05-11 Revised:2021-07-05 Online:2021-09-20 Published:2021-07-14
Contact: Lu Guangtao E-mail:lgf8411@126.com;lugt@gxu.edu.cn
Supported by:
Supported by Guangxi University for Nationalities Scientific Research Fund No(2019KJQN004);the National Natural Science Foundation of China No(31860021);Guangxi Natural Science Foundation of China No(2020GXNSFBA297140);Science and Technology Major Project of Guangxi No(AA18242026)

摘要/Abstract

摘要：

十字花科黑腐病菌(Xanthomonas campestris pv. campestris, Xcc)是一种维管束致病菌,能够引起寄主的黑腐病,是研究植物病原细菌与植物互作的一种重要模式菌株。在Xcc中,GntR家族的全局性转录调控因子HpaR1参与调控Xcc的运动、胞外多糖和胞外酶的合成等许多细胞过程,并与Xcc的过敏反应(hypersensitive response, HR)和致病相关;全局性转录调控因子Clp则参与调控胞外酶和胞外多糖的分泌与合成,并与黄单胞菌的致病相关。前期研究发现,Xcc中的转录调控因子HpaR1和Clp均能与糖苷水解酶(glycoside hydrolase)编码基因(命名为ghy基因)的启动子区结合。为探究转录调控因子HpaR1和Clp共同调控ghy基因表达的分子机理,本研究首先通过凝胶阻滞分析(electrophoresis mobility shift assay, EMSA)发现HpaR1和Clp在体外能够结合在ghy基因的启动子区;利用染色质免疫共沉淀(chromatin immunoprecipitation, ChIP)方法,进一步证实HpaR1和Clp在细胞内能够结合在ghy基因启动子区。通过5ʹ-cDNA末端快速扩增(rapid amplication of 5ʹ-cDNA ends, 5ʹ- RACE)和DNase I保护实验(DNase I footprinting)确定HpaR1和Clp均结合在ghy基因启动子的-35区上游,并且HpaR1的结合位点位于Clp结合位点的上游。通过实时荧光定量PCR(real time fluorescence quantitative PCR, RT-qPCR)和体外转录的方法,发现HpaR1抑制ghy基因的转录,而Clp激活ghy基因的转录。当二者共同存在时,HpaR1能够抑制Clp对ghy基因的转录激活作用。HpaR1和Clp单独存在时,分别负调控和正调控ghy基因的转录,推测HpaR1尽管位于ghy基因启动子-35区上游,但可能通过抑制RNA聚合酶的活性来调控ghy基因的表达。

关键词: HpaR1, Clp, ghy基因, 转录调控, 十字花科黑腐病菌

Abstract:

Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen that causes black rot in host. It is an important model strain for studying the interaction between the phytopathogen and plants. In Xcc, global transcription regulator HpaR1 that belongs to the GntR family regulates many cellular processes such as the movement and synthesis of extracellular polysaccharides and extracellular enzymes, and is associated with hypersensitive response (HR) and pathogenicity. On the other hand, the global transcriptional regulator Clp regulates the secretion and synthesis of extracellular enzymes and extracellular polysaccharides, and is associated with the pathogenicity of Xanthomonas. Previous studies have shown that both HpaR1 and Clp bind to the promoter region of the glycoside hydrolase encoding gene (named ghy gene). This study investigates the molecular mechanism of the co-regulation of HpaR1 and Clp on the expression of ghy gene. Through electrophoresis mobility shift assay (EMSA), we found that both HpaR1 and Clp bind to the promoter regions of gene ghy in vitro. Both HpaR1 and Clp also bind to the promoter regions of gene ghy in vivo by chromatin immunoprecipitation (ChIP) assays. DNase I footprinting and 5ʹ-RACE assays showed that both HpaR1 and Clp bind to the -35 region upstream of the ghy promoter. The HpaR1 binding site was located upstream of the Clp binding site. RT-qPCR and in vitro transcription assays showed that HpaR1 negatively while Clp positively regulates the transcription of gene ghy. Furthermore, HpaR1 inhibits the activation of Clp on the transcription of gene ghy in vitro. Our findings indicate that HpaR1 and Clp exhibit opposite effect on the transcription of gene ghy. It is speculated that HpaR1 may regulate the expression of gene ghy by inhibiting the activity of RNA polymerase.

Key words: HpaR1, Clp, gene ghy, transcriptional regulation, Xanthomonas campestris pv. campestris

刘国芳, 任沛东, 叶文新, 陆光涛. 十字花科黑腐病菌中转录因子HpaR1与Clp调控一个糖苷水解酶基因表达的分析[J]. 遗传, 2021, 43(9): 910-920.

Guofang Liu, Peidong Ren, Wenxin Ye, Guangtao Lu. Analysis of transcriptional regulators HpaR1 and Clp regulating the expression of glycoside hydrolase-encoding gene in the Xanthomonas campestris pv. campestris[J]. Hereditas(Beijing), 2021, 43(9): 910-920.

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菌株和质粒	相关特征
大肠杆菌
DH5α	F^-,φ80d/lacZ△M15,Δ(lacZYA-argF)U169,deoR,recA1,endA1,hsdR17(r_k^-, m_k⁺),phoA,supE44, λ^-,thi-1,gyrA96,relA1
M15	Nal^s,tr^s,Rif^s,Thi^-,Lac^-,Ar⁺a,Gal⁺,Mtl^-,F^-,RecA⁺,Uvr⁺,Lon⁺harboring pREP4 plasmid,Km^r
ED8767	RecA,met,含有帮助质粒 pRK2073,Spc^r
pQE-HpaR1/M15	M15中含有质粒pQE-HpaR1,Km^r,Amp^r
pQE-Clp/M15	M15中含有质粒 pQE-Clp,Km^r,Amp^r
pL3-HpaR1::3×Flg/ DH5α	DH5α中含有质粒pL3-HpaR1:: 3×Flg,Tc^r
pL3-Clp:: 3×Flg/ DH5α	DH5α中含有质粒pL3-Clp:: 3×Flg,Tc^r
十字花科黑腐病菌
8004	野生型,Rif^r
ΔhpaR1	hpaR1基因的缺失突变体,Rif^r
Δclp	Clp基因的缺失突变体,Rif^r
8004/pLAFR3	8004中含有质粒pLAFR3,Rif^r,Tc^r
ΔhpaR1/pL3-HpaR1::3×Flg	ΔhpaR1中含有质粒 pL3-HpaR1::3×Flg,Rif^r,Tc^r
Δclp/pL3-Clp:: 3×Flg	Δclp中含有质粒 pL3-Clp:: 3×Flg,Rif^r,Tc^r
质粒
pK18mob	黄单胞菌属中的自杀质粒,Km^r
pRK2073	帮助质粒,Tra⁺,Mob⁺,ColE1,Spc^r
pQE30	表达载体,Amp^r
pQE30-HpaR1	pQE30上含有hpaR1基因的360 bp ORF,Amp^r
pQE30-Clp	pQE30上含有 clp基因的690 bp ORF,Amp^r
pLAFR3	PK2 replicon;Mob⁺,Tra^-;cos⁺,Tc^r
pL3-HpaR1::3×Flg	pLAFR3上含有 hpaR1::3×Flg,Tc^r
pL3-Clp::3×Flg	pLAFR3上含有 clp::3×Flg,Tc^r

用途	引物名称	引物序列(5′→3′)
蛋白表达	Clp-OF	ACAGTTGGATCC ATGAGCCTAGGGAACAGCAC
蛋白表达	Clp-OR	ACAGTTAAGCTT TTAGCGCGTGCCGTACAACA
凝胶阻滞分析	0026 F	ACGGCAATCGATCAGTTCGC
凝胶阻滞分析	0026 R	CATGGAAACCACTCCTTCGACG
DNase I保护实验	0026(HpaR1)F	GACAAGCCGCAGATGAAAACCC
	0026(HpaR1)R	GTTACGCTGCATCTGCCCGT
	0026(Clp)F	GCAGCCGGCGCACGCAGTAGACCTGTACT
	0026(Clp)R	CGACGGAAAAGATGCATCGCAGCCC
5ʹ-RACE	AAP	GGCCACGCGTCGACTAGTACGGGGGGGGGG
	AUAP	GGCCACGCGTCGACTAGTAC
	0026GSP1	GTTCCCACAGGATCGGCAGG
	0026GSP2	TCCAGTCCCAGCGAAATAGC
	0026GSP3	TGTTGAGGACGCCAGGTTTT
	0026GSPF	ATGTCGTGTTCTCTGTGGTTGC
体外转录	0026ivtF	GCGACAAGCCGCAGATGAAAACCCT
	0026ivtR	GATGACGCAAATTCCGCACCCGCC
	3077ivtF	TCTCACTCTGTCTTGCAAACTGCGA
	3077ivtR	AATGGGCATCGAAAACCAGAAGC
染色质免疫共沉淀	Clp(ChIP)F	CGGGATCCGACTACAAAGACCATGACGGTGATTATAAAGATCATGATATCGACTACAA-AGATGACGACGATAAAATGAGCCTAGGGAACACGACG
	Clp(ChIP)R	CCAAGCTTTTAGCGCGTGCCGTACAACA
	HpaR1(ChIP)F	CGGGATCCGACTACAAAGACCATGACGGTGATTATAAAGATCATGATATCGACTACAA-AGATGACGACGATAAAATGACTGACATCCAGTGGAGC
	HpaR1(ChIP)R	CCAAGCTTTCATGGTGTTTTCCCCTGTG
	0026(ChIP)F	CGTCACGCTTTCGTCGCCTA
	0026(ChIP)R	AACTGCGTTGTGGCCTGCAC
实时荧光定量PCR	0026qRT-F	ATGTCGTGTTCTCTGTGGTTGC
实时荧光定量PCR	0026qRT-R	TGTTGAGGACGCCAGGTTTT

十字花科黑腐病菌中转录因子HpaR1与Clp调控一个糖苷水解酶基因表达的分析

Analysis of transcriptional regulators HpaR1 and Clp regulating the expression of glycoside hydrolase-encoding gene in the Xanthomonas campestris pv. campestris

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