大蒜防御素基因AsPDF1的克隆与表达分析
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摘要
植物防御素是一类低分子量、富含半胱氨酸的抗菌多肽,是植物防御生物和非生物侵害的重要物质之一。为了解大蒜中防御素的序列特征和功能,本研究利用RT-PCR方法从大蒜‘苍山四六瓣’中克隆得到防御素基因AsPDF1。序列分析结果表明,来源于大蒜的防御素基因含有1个246 bp的开放阅读框,编码81个氨基酸。推测其编码的蛋白质分子量为8.45 kDa,等电点(pI)为9.36。该蛋白含有8个保守的半胱氨酸, N端有一段23个氨基酸组成的信号肽区域,空间结构由1个α螺旋和3股反向平行的β折叠构成。在进化关系上,大蒜防御素与禾本科的小麦和高粱防御素蛋白最为接近。荧光定量PCR分析表明, AsPDF1在根、鳞茎、叶片中均有表达,在根中表达量最高,且能响应盐胁迫的诱导。本研究克隆的大蒜防御素基因为进一步鉴定防御素基因的生物学功能和表达调控机制奠定了基础。
Plant defensins, one of the important substances for plant defense against biotic and abiotic damage, are a class of low molecular weight, cysteine-rich antimicrobial peptides. To investigate the sequence characteristics and functions of defensins in garlic, a defensin gene AsPDF1 was cloned from garlic cultivar ‘Cangshan siliuban’ by RT-PCR method. Sequence analysis showed that the open reading frame of garlic defensin gene was 246 bp in length, encoding 81 amino acids. The predicted molecular weight of the garlic defensin was 8.45 kDa, and the isoelectric point pI was 9.36. AsPDF1 consists of eight conserved cysteine residues with a peptide signal of 27 amino acid length at the N terminal. Analysis on three-dimension structure found that the protein was composed of one α helix and three anti-parallel β sheets. Phylogenetic analysis indicated that garlic defensin AsPDF1 is close to defensins of wheat and sorghum in the Gramineae family. Quantitative real-time PCR analysis demonstrated that AsPDF1 was expressed in all tissues examined, with the highest level in garlic roots and lower levels in the cloves and leaves. However, the genes can respond to salt stress in all samples tested. The gene cloned in this study would provide basic information for further studies on the biology function and expression mechanism of garlic defensins.
Plant defensins, one of the important substances for plant defense against biotic and abiotic damage, are a class of low molecular weight, cysteine-rich antimicrobial peptides. To investigate the sequence characteristics and functions of defensins in garlic, a defensin gene AsPDF1 was cloned from garlic cultivar ‘Cangshan siliuban’ by RT-PCR method. Sequence analysis showed that the open reading frame of garlic defensin gene was 246 bp in length, encoding 81 amino acids. The predicted molecular weight of the garlic defensin was 8.45 kDa, and the isoelectric point pI was 9.36. AsPDF1 consists of eight conserved cysteine residues with a peptide signal of 27 amino acid length at the N terminal. Analysis on three-dimension structure found that the protein was composed of one α helix and three anti-parallel β sheets. Phylogenetic analysis indicated that garlic defensin AsPDF1 is close to defensins of wheat and sorghum in the Gramineae family. Quantitative real-time PCR analysis demonstrated that AsPDF1 was expressed in all tissues examined, with the highest level in garlic roots and lower levels in the cloves and leaves. However, the genes can respond to salt stress in all samples tested. The gene cloned in this study would provide basic information for further studies on the biology function and expression mechanism of garlic defensins.
引文
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Bendtsen JD,Nielsen H,von Heijne G,et al(2004).Improved prediction of signal peptides:SignalP 3.0.J Mol Biol,340:783-795
Bruix M,Jiménez MA,Santoro J,et al(1993).Solution structure of gamma 1-H and gamma 1-P thionins from barley and wheat endosperm determined by 1H-NMR:a structural motif common to toxic arthropod proteins.Biochemistry,32:715-724
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Carvalho AO,Gomes VM(2011).Plant defensins and defensin-like peptides-biological activities and biotechnological applications.Curr Pharm Des,17:4270-4293
Do HM,Lee SC,Jung HW,et al(2004).Differential expression and in situ localization of a pepper defensin(CADEF1)gene in response to pathogen infection,abiotic elicitors and environmental stresses in Capsicum annuum.Plant Sci,166:1297-1305
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Ganz T,Selsted ME,Szklarek D,et al(1985).Defensins.Natural peptide antibiotics of human neutrophils.J Clin Invest,76:1427-1435
Guerra-Lupián MA,Ruiz-Medrano R,Ramírez-Pool JA,et al(2018).Localized expression of antimicrobial proteins mitigates huanglongbing symptoms in Mexican lime.JBiotechnol,285:74-83
Ikeda M,Arai M,Lao DM,et al(2002).Transmembrane topology prediction methods:a re-assessment and improvement by a consensus method using a dataset of experimentally-characterized transmembrane topologies.In Silico Biol,2:19-33
Koike M,Okamoto T,Tsuda S,et al(2002).A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation.Biochem Biophys Res Commun,298:46-53
Lay FT,Anderson MA(2005).Defensins-components of the innate immune system in plants.Curr Protein Pept Sci,6:85-101
Li Z,Zhou M,Zhang Z,et al(2011).Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis.Funct Integr Genomics,11:63-70
Livak KJ,Schmittgen TD(2001).Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,25:402-408
Luo JS,Huang J,Zeng DL,et al(2018).A defensin-like protein drives cadmium efflux and allocation in rice.Nat Commun,9:645
Mendez E,Moreno A,Colilla F,et al(1990).Primary structure and inhibition of protein synthesis in eukaryotic cellfree system of a novel thionin,gamma-hordothionin,from barley endosperm.Eur J Biochem,194:533-539
Mirouze M,Sels J,Richard O,et al(2006).A putative novel role for plant defensins:a defensin from the zinc hyper-accumulating plant,Arabidopsis halleri,confers zinc tolerance.Plant J,47:329-342
Park HC,Kang YH,Chun HJ,et al(2002).Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage.Plant Mol Biol,50:59-69
Pelegrini PB,Franco OL(2005).Plant gamma-thionins:novel insights on the mechanism of action of a multi-functional class of defense proteins.Int J Biochem Cell Biol,37:2239-2253
Picart P,Pirttil?AM,Raventos D,et al(2012).Identification of defensin-encoding genes of Picea glauca:characterization of PgD5,a conserved spruce defensin with strong antifungal activity.BMC Plant Biol,12:180
Sasaki K,Kuwabara C,Umeki N,et al(2016).The cold-induced defensin TAD1 confers resistance against snow mold and Fusarium head blight in transgenic wheat.JBiotechnol,228:3-7
Shahzad Z,Ranwez V,Fizames C,et al(2013).Plant Defensin type 1(PDF1):protein promiscuity and expression variation within the Arabidopsis genus shed light on zinc tolerance acquisition in Arabidopsis halleri.New Phytol,200:820-833
Tamura K,Peterson D,Peterson N,et al(2011).MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Mol Biol Evol,28:2731-2739
Thevissen K,de Mello Tavares P,Xu D,et al(2012).The plant defensin RsAFP2 induces cell wall stress,septin mislocalization and accumulation of ceramides in Candida albicans.Mol Microbiol,84:166-180
van der Weerden NL,Anderson MA(2013).Plant defensins:common fold,multiple functions.Fungal Biol Rev,26:121-131
Vriens K,Cammue BP,Thevissen K(2014).Antifungal plant defensins:mechanisms of action and production.Molecules,19:12280-12303
Wang MX,Zhu XL,Luo MY,et al(2017).Cloning and defensive functional analysis of a wheat defensin gene TaPDF35.J Plant Genet Res,18:925-932(in Chinese with English abstract)[王敏霞,祝秀亮,罗美英等(2017).小麦防御素基因TaPDF35的克隆与功能分析.植物遗传资源学报,18:925-932]
Wang ZN,Li LH,Xu RH,et al(2018).Advances in mitochondrial proteins responding to stresses in plants.Plant Physiol J,54:221-231(in Chinese with English abstract)[王忠妮,李鲁华,徐如宏等(2018).响应植物逆境胁迫的线粒体蛋白研究进展.植物生理学报,54:221-231]
Waterhouse A,Bertoni M,Bienert S,et al(2018).SWISS-MODEL:homology modelling of protein structures and complexes.Nucleic Acids Res,46:W296-W303
Weerawanich K,Webster G,Ma JK,et al(2018).Gene expression analysis,subcellular localization,and in planta antimicrobial activity of rice(Oryza sativa L.)defensin 7and 8.Plant Physiol Biochem,124:160-166
Zeya HI,Spitznagel JK(1966).Cationic proteins of polymorphonuclear leukocyte lysosomes.I.Resolution of antibacterial and enzymatic activities.J Bacteriol,91:750-754
Zheng XM,Guo N,Gao TS,et al(2015).Cloning and expression analysis of defensin genes from Brassica napus.Acta Agron Sin,41:725-732(in Chinese with English abstract)[郑小敏,郭楠,高天姝等(2015).甘蓝型油菜防御素基因的克隆与表达分析.作物学报,41:725-732]
Bendtsen JD,Nielsen H,von Heijne G,et al(2004).Improved prediction of signal peptides:SignalP 3.0.J Mol Biol,340:783-795
Bruix M,Jiménez MA,Santoro J,et al(1993).Solution structure of gamma 1-H and gamma 1-P thionins from barley and wheat endosperm determined by 1H-NMR:a structural motif common to toxic arthropod proteins.Biochemistry,32:715-724
Carvalho AO,Gomes VM(2009).Plant defensins-Prospects for the biological functions and biotechnological properties.Peptides,30:1007-1020
Carvalho AO,Gomes VM(2011).Plant defensins and defensin-like peptides-biological activities and biotechnological applications.Curr Pharm Des,17:4270-4293
Do HM,Lee SC,Jung HW,et al(2004).Differential expression and in situ localization of a pepper defensin(CADEF1)gene in response to pathogen infection,abiotic elicitors and environmental stresses in Capsicum annuum.Plant Sci,166:1297-1305
Dong W,Wu PX,Yang N,et al(2018).Cloning and expression analysis of WRKY transcription factor involved in salinity stress in alfalfa.Plant Physiol J,54:1481-1489(in Chinese with English abstract)[董蔚,邬培祥,杨宁等(2018).紫花苜蓿盐胁迫响应WRKY转录因子的克隆及表达特征分析.植物生理学报,54:1481-1489]
Ganz T,Selsted ME,Szklarek D,et al(1985).Defensins.Natural peptide antibiotics of human neutrophils.J Clin Invest,76:1427-1435
Guerra-Lupián MA,Ruiz-Medrano R,Ramírez-Pool JA,et al(2018).Localized expression of antimicrobial proteins mitigates huanglongbing symptoms in Mexican lime.JBiotechnol,285:74-83
Ikeda M,Arai M,Lao DM,et al(2002).Transmembrane topology prediction methods:a re-assessment and improvement by a consensus method using a dataset of experimentally-characterized transmembrane topologies.In Silico Biol,2:19-33
Koike M,Okamoto T,Tsuda S,et al(2002).A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation.Biochem Biophys Res Commun,298:46-53
Lay FT,Anderson MA(2005).Defensins-components of the innate immune system in plants.Curr Protein Pept Sci,6:85-101
Li Z,Zhou M,Zhang Z,et al(2011).Expression of a radish defensin in transgenic wheat confers increased resistance to Fusarium graminearum and Rhizoctonia cerealis.Funct Integr Genomics,11:63-70
Livak KJ,Schmittgen TD(2001).Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCT method.Methods,25:402-408
Luo JS,Huang J,Zeng DL,et al(2018).A defensin-like protein drives cadmium efflux and allocation in rice.Nat Commun,9:645
Mendez E,Moreno A,Colilla F,et al(1990).Primary structure and inhibition of protein synthesis in eukaryotic cellfree system of a novel thionin,gamma-hordothionin,from barley endosperm.Eur J Biochem,194:533-539
Mirouze M,Sels J,Richard O,et al(2006).A putative novel role for plant defensins:a defensin from the zinc hyper-accumulating plant,Arabidopsis halleri,confers zinc tolerance.Plant J,47:329-342
Park HC,Kang YH,Chun HJ,et al(2002).Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage.Plant Mol Biol,50:59-69
Pelegrini PB,Franco OL(2005).Plant gamma-thionins:novel insights on the mechanism of action of a multi-functional class of defense proteins.Int J Biochem Cell Biol,37:2239-2253
Picart P,Pirttil?AM,Raventos D,et al(2012).Identification of defensin-encoding genes of Picea glauca:characterization of PgD5,a conserved spruce defensin with strong antifungal activity.BMC Plant Biol,12:180
Sasaki K,Kuwabara C,Umeki N,et al(2016).The cold-induced defensin TAD1 confers resistance against snow mold and Fusarium head blight in transgenic wheat.JBiotechnol,228:3-7
Shahzad Z,Ranwez V,Fizames C,et al(2013).Plant Defensin type 1(PDF1):protein promiscuity and expression variation within the Arabidopsis genus shed light on zinc tolerance acquisition in Arabidopsis halleri.New Phytol,200:820-833
Tamura K,Peterson D,Peterson N,et al(2011).MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Mol Biol Evol,28:2731-2739
Thevissen K,de Mello Tavares P,Xu D,et al(2012).The plant defensin RsAFP2 induces cell wall stress,septin mislocalization and accumulation of ceramides in Candida albicans.Mol Microbiol,84:166-180
van der Weerden NL,Anderson MA(2013).Plant defensins:common fold,multiple functions.Fungal Biol Rev,26:121-131
Vriens K,Cammue BP,Thevissen K(2014).Antifungal plant defensins:mechanisms of action and production.Molecules,19:12280-12303
Wang MX,Zhu XL,Luo MY,et al(2017).Cloning and defensive functional analysis of a wheat defensin gene TaPDF35.J Plant Genet Res,18:925-932(in Chinese with English abstract)[王敏霞,祝秀亮,罗美英等(2017).小麦防御素基因TaPDF35的克隆与功能分析.植物遗传资源学报,18:925-932]
Wang ZN,Li LH,Xu RH,et al(2018).Advances in mitochondrial proteins responding to stresses in plants.Plant Physiol J,54:221-231(in Chinese with English abstract)[王忠妮,李鲁华,徐如宏等(2018).响应植物逆境胁迫的线粒体蛋白研究进展.植物生理学报,54:221-231]
Waterhouse A,Bertoni M,Bienert S,et al(2018).SWISS-MODEL:homology modelling of protein structures and complexes.Nucleic Acids Res,46:W296-W303
Weerawanich K,Webster G,Ma JK,et al(2018).Gene expression analysis,subcellular localization,and in planta antimicrobial activity of rice(Oryza sativa L.)defensin 7and 8.Plant Physiol Biochem,124:160-166
Zeya HI,Spitznagel JK(1966).Cationic proteins of polymorphonuclear leukocyte lysosomes.I.Resolution of antibacterial and enzymatic activities.J Bacteriol,91:750-754
Zheng XM,Guo N,Gao TS,et al(2015).Cloning and expression analysis of defensin genes from Brassica napus.Acta Agron Sin,41:725-732(in Chinese with English abstract)[郑小敏,郭楠,高天姝等(2015).甘蓝型油菜防御素基因的克隆与表达分析.作物学报,41:725-732]