拟南芥NSP家族基因序列分析及响应干旱胁迫表达分析
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摘要
芥子油苷是十字花科植物中的一种重要的次生代谢产物,它被酶水解的降解过程与植物响应逆境胁迫有关。NSP蛋白是一类能与黑芥子酶结合从而改变芥子油苷降解途径最终生成腈类物质的特异蛋白,其与ESP家族蛋白在功能上存在冗余。本研究以拟南芥NSP家族基因为对象,对家族成员基因序列、蛋白质序列、启动子序列进行生物信息学分析,并使用实时荧光定量PCR技术检测了该家族基因在干旱胁迫下的表达量。结果表明:该家族成员基因分布于3条染色体上,外显子数目2～4个;AtNSP5蛋白亚细胞定位于细胞核,其他成员定位于细胞质;结构域预测显示,该家族蛋白均含有4个Kelch结构域,除AtNSP5外,其他四个成员还含有1～2个Jacalin结构域;启动子序列中均含有CAAT-box、TATA-box核心元件及数量不一的逆境响应元件;干旱胁迫表达量分析显示,该家族中AtNSP5基因受干旱胁迫诱导表达,其他成员响应干旱胁迫不显著。本研究为进一步研究NSP蛋白在植物响应干旱胁迫中的分子机制提供依据,并为植物抗旱基因工程提供潜在候选基因。
The glucosinola te is an important secondary metabolite in Cruciferae plants, and its degradation process is related to the response of plants to stress. NSP protein is a kind of specific protein that can bind to myrosinase and change the degradation pathway of glucosinolate to produce nitriles. It has a functional redundancy with ESP family protein. In this study, NSP family genes of Arabidopsis thaliana were used as the objects. The bioinformatics analysis of gene sequences, protein sequences and promoter sequences of family members was performed. The expression characteristics of the family genes under drought stress were analyzed by using real-time fluorescence quantitative PCR technology. The results showed that the genes of the family members were distributed on 3 chromosomes and the number of exons was 2～4. Subcellular localization of AtNSP5 protein was in the nucleus, other members was in the cytoplasm. Domain prediction showed that all the family proteins contained 4 Kelch domains. In addition to At NSP5, the other four members also contained 1～2 Jacalin domains.The promoter sequence contained CAAT-box, TATA-box core elements and a variety of stress response elements.Drought stress expression analysis showed that the AtNSP5 gene was induced by drought stress in the family and the other members were not significantly responsive to drought stress. This study might provide basis for further studying the molecular mechanism of NSP protein response to drought stress in plants and provide potential candidate genes for plant drought-resistance genetic engineering.
The glucosinola te is an important secondary metabolite in Cruciferae plants, and its degradation process is related to the response of plants to stress. NSP protein is a kind of specific protein that can bind to myrosinase and change the degradation pathway of glucosinolate to produce nitriles. It has a functional redundancy with ESP family protein. In this study, NSP family genes of Arabidopsis thaliana were used as the objects. The bioinformatics analysis of gene sequences, protein sequences and promoter sequences of family members was performed. The expression characteristics of the family genes under drought stress were analyzed by using real-time fluorescence quantitative PCR technology. The results showed that the genes of the family members were distributed on 3 chromosomes and the number of exons was 2～4. Subcellular localization of AtNSP5 protein was in the nucleus, other members was in the cytoplasm. Domain prediction showed that all the family proteins contained 4 Kelch domains. In addition to At NSP5, the other four members also contained 1～2 Jacalin domains.The promoter sequence contained CAAT-box, TATA-box core elements and a variety of stress response elements.Drought stress expression analysis showed that the AtNSP5 gene was induced by drought stress in the family and the other members were not significantly responsive to drought stress. This study might provide basis for further studying the molecular mechanism of NSP protein response to drought stress in plants and provide potential candidate genes for plant drought-resistance genetic engineering.
引文
Kissen R.,and Bones A.M.,2009,Nitrile-specifier proteins involved in glucosinolate hydrolysis in Arabidopsis thaliana,J.Biol.Chem.,284(18):12057-12070
Kong X.Y.,Kissen R.,and Bones A.M.,2012,Characterization of recombinant nitrile-specifier proteins(NSPs)of Arabidopsis thaliana:dependency on Fe(Ⅱ)ions and the effect of glucosinolate substrate and reaction conditions,Phytochemistry,84:7-17
Miljkovic D.,Stare T.,Mozetic I.,Podpecan V.,Petek M.,Witek K.,Dermastia M.,Lavrac N.,and Gruden K.,2012,Signalling network construction for modelling plant defence response,PLoS One,7(12):8646-8655
Mithen R.,Bennett R.,and Marquez J.,2010,Glucosinolate biochemical diversity and innovation in the brassicales,Phytochemistry,71(17-18):2074-2086
Mostafa I.,Zhu N.,Yoo M.J.,Balmant K.M.,Misra B.B.,Dufresne C.,Abou-Hashem M.,Chen S.,and El-Domiaty M.,2016,New nodes and edges in the glucosinolate molecular network revealed by proteomics and metabolomics of Arabidopsis myb28/29 and cyp79B2/B3 glucosinolate mutants,J.Proteomics,138:1-19
Verslues P.E.,Agarwal M.,Katiyar-Agarwal S.,Zhu J.H.,and Zhu J.K.,2006,Methods and concepts in quantifying resistance to drought,salt and freezing,abiotic stresses that affect plant water status,Plant J.,45(4):523-539
Weidenbach D.,Esch L.,Mller C.,Hensel G.,Kumlehn J.,H-fle C.,Hückelhoven R.,and Schaffrath U.,2015,Polarized defense against fungal pathogens is mediated by the jacalin-related lectin domain of modular poaceae-specific proteins,Mol.Plant,15(4):3240-3251
Zhang J.Y.,Wang Q.J.,and Guo Z.R.,2012,Progresses on plant AP2/ERF transcription factors,Hereditas,34(7):835-847
Kong X.Y.,Kissen R.,and Bones A.M.,2012,Characterization of recombinant nitrile-specifier proteins(NSPs)of Arabidopsis thaliana:dependency on Fe(Ⅱ)ions and the effect of glucosinolate substrate and reaction conditions,Phytochemistry,84:7-17
Miljkovic D.,Stare T.,Mozetic I.,Podpecan V.,Petek M.,Witek K.,Dermastia M.,Lavrac N.,and Gruden K.,2012,Signalling network construction for modelling plant defence response,PLoS One,7(12):8646-8655
Mithen R.,Bennett R.,and Marquez J.,2010,Glucosinolate biochemical diversity and innovation in the brassicales,Phytochemistry,71(17-18):2074-2086
Mostafa I.,Zhu N.,Yoo M.J.,Balmant K.M.,Misra B.B.,Dufresne C.,Abou-Hashem M.,Chen S.,and El-Domiaty M.,2016,New nodes and edges in the glucosinolate molecular network revealed by proteomics and metabolomics of Arabidopsis myb28/29 and cyp79B2/B3 glucosinolate mutants,J.Proteomics,138:1-19
Verslues P.E.,Agarwal M.,Katiyar-Agarwal S.,Zhu J.H.,and Zhu J.K.,2006,Methods and concepts in quantifying resistance to drought,salt and freezing,abiotic stresses that affect plant water status,Plant J.,45(4):523-539
Weidenbach D.,Esch L.,Mller C.,Hensel G.,Kumlehn J.,H-fle C.,Hückelhoven R.,and Schaffrath U.,2015,Polarized defense against fungal pathogens is mediated by the jacalin-related lectin domain of modular poaceae-specific proteins,Mol.Plant,15(4):3240-3251
Zhang J.Y.,Wang Q.J.,and Guo Z.R.,2012,Progresses on plant AP2/ERF transcription factors,Hereditas,34(7):835-847