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

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

• Research Article • Previous Articles

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)

Abstract

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

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

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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