山葡萄VaCBF3基因克隆及生物信息学分析
|
摘要
为预测山葡萄VaCBF3基因的功能,利用RT-PCR方法从低温处理的山葡萄中克隆出抗寒转录因子VaCBF3基因。利用生物信息学方法对其结构域及功能进行预测,包括开放阅读框分析、基本理化性质分析、保守结构域分析、信号肽预测、蛋白修饰位点分析、疏水性分析、系统进化分析、蛋白质二级结构及三级结构预测等。结果表明:经测序VaCBF3基因序列全长为854 bp,编码239个氨基酸,序列提交至GenBank中(登录号为:EU672969;蛋白登录号为:ACD45468.1)。该基因属于AP2 superfamily家族,具有AP2保守结构域;蛋白相对分子量为25.9 kDa,分子式为C1109H1754N332O364S11,原子总数为3 570,理论等电点为7.1;为不稳定亲水蛋白,不含信号肽;共计43个磷酸化位点,1个N-糖基化位点,39个O-糖基化位点;二级结构中α-螺旋占25.94%,β-转角占4.60%,不规则卷曲链56.07%,延伸链占13.39%;三级结构含有1个α-螺旋和3个β-折叠。系统发育树分析表明,VaCBF3蛋白与Vitis amurensis聚为一支,亲缘关系最近,与V. vinifera、V. riparia亲缘关系较远。
In order to predict the function of the VaCBF3 gene in Vitis amurensis, by using RT-PCR method, a cold resistance gene VaCBF3 was cloned from mountain grape which had been treated at low temperature. Bioinformatics method was used to predict its domain and function, including open reading frame analysis, basic physical and chemical properties analysis, conservative domain analysis, signal peptide prediction, protein modification site analysis, hydrophobicity analysis, phylogenetic analysis, protein secondary structure and tertiary structure prediction, etc. The results were as follows: the total length of VaCBF3 gene was 854 bp(GenBank accession number: EU672969), encoded 239 amino acids(GenBank accession number: ACD45468.1). VaCBF3 belonged to the AP2 superfamily family and had an AP2 conservative domain. The relative molecular weight of protein was 25.9 kDa, molecular formula was C1109 H1754 N332 O364 S11, total number of atoms was 3 570 and theoretical isoelectric point was 7.1; Protein is unstable and hydrophilic, had no signal peptides, contained 43 phosphorylated sites, 1 N-glycosylated site and 39 O-glycosylated sites. In its secondary structure,the percentage of α-helix was 25.94 %, β-angle was 4.60 %, irregular curled chain was 56.07 % and extended chain was13.39 %. Its tertiary structure contained one alpha-helix and three beta-folds. Phylogenetic analysis showed that the VaCBF3 protein was closely related to that of Vitis amurensis, but distantly related to that of V. vinifera and V. riparia.
In order to predict the function of the VaCBF3 gene in Vitis amurensis, by using RT-PCR method, a cold resistance gene VaCBF3 was cloned from mountain grape which had been treated at low temperature. Bioinformatics method was used to predict its domain and function, including open reading frame analysis, basic physical and chemical properties analysis, conservative domain analysis, signal peptide prediction, protein modification site analysis, hydrophobicity analysis, phylogenetic analysis, protein secondary structure and tertiary structure prediction, etc. The results were as follows: the total length of VaCBF3 gene was 854 bp(GenBank accession number: EU672969), encoded 239 amino acids(GenBank accession number: ACD45468.1). VaCBF3 belonged to the AP2 superfamily family and had an AP2 conservative domain. The relative molecular weight of protein was 25.9 kDa, molecular formula was C1109 H1754 N332 O364 S11, total number of atoms was 3 570 and theoretical isoelectric point was 7.1; Protein is unstable and hydrophilic, had no signal peptides, contained 43 phosphorylated sites, 1 N-glycosylated site and 39 O-glycosylated sites. In its secondary structure,the percentage of α-helix was 25.94 %, β-angle was 4.60 %, irregular curled chain was 56.07 % and extended chain was13.39 %. Its tertiary structure contained one alpha-helix and three beta-folds. Phylogenetic analysis showed that the VaCBF3 protein was closely related to that of Vitis amurensis, but distantly related to that of V. vinifera and V. riparia.
引文
[1]贾会霞,李建波,孙佩,等.胡杨CBF基因家族的鉴定及表达特性分析[J].分子植物育种,2017,15(2):492-500.
[2]StockingE J,Gilmour S J,Thomashow M F. Arabidopsis thalianaCBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cisacting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J].Proceedings of the National Academy of Sciences,1997,94:1035-1040.
[3]Novillo F,Medina J,Rodríguezfranco M,et al. Genetic analysis reveals a complex regulatory network modulating CBF gene expression and Arabidopsis response to abiotic stress[J].Journal of Experimental Botany, 2012,63(1):293-304.
[4]Zhou M, Hu C, Wei D, et al. Arabidopsis CBF3 and DELLAs positively regulate each other in response to low temperature[J].Scientific Reports, 2017(7):39819.
[5]Thomashow M F. Molecular basis of plant cold acclimation:insights gained from studying the CBF cold response pathway[J].Plant Physiol, 2010, 154(2):571-577.
[6]Wang Z, Wang F, Tang J, et al. C-repeat binding factor gene family identified in non-heading Chinese cabbage is functional in abiotic and biotic stress response but different from that in Arabidopsis[J]. Acta Physiologiae Plantarum, 2014, 36(12):3217-3229.
[7]Allen M D, Yamasaki K, Ohme-Takagi M, et al.A novel mode of DNA recognition by a[beta]-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[J].Embo J,1998,17(18):5484-5496.
[8]Tian Q, Chen J, Wang D, et al. Overexpression of a Populus euphratica CBF4 gene in poplar confers tolerance to multiple stresses[J].Plant Cell Tissue&Organ Culture,2017,128(2):391-407.
[9]Nakano T, Suzuki K, Fujimura T, et al. Genome-wide analysis of the ERF gene family in Arabidopsis and rice[J].Plant Physiology,2006,140(2):411-432.
[10]丁咚,陈亚娟,崔进荣,等.毛果杨CBF/DREB1基因家族生物信息学分析[J].西南农业学报,2018,31(3):457-461.
[11]李月,代培红,刘超,等.海岛棉5个CBF/DREB基因的克隆与表达分析[J].棉花学报,2016,28(1):42-51.
[12]Xiong Y, Fei SZ.Functional and phylogenetic analysis of a DREB/CBF-like gene in perennial ryegrass(Lolium perenne L.)[J].Planta, 2006, 224(4):878-888.
[13] Kidokoro S, Watanabe K, Ohori T, et al. Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression[J].The Plant J., 2015, 81(3):505-518.
[14]Ryu JY, Hong SY, Jo SH, et al.Molecular and functional characterization of cold-responsive C-repeat binding factors from Brachypodium distachyon[J]. BMC Plant Biol, 2014, 14(15):1-15.
[15]Lei X, Xiao Y, Xia W, et al. RNA-seq analysis of oil palm under cold stress reveals a different C-repeat binding factor(CBF)mediated gene expression pattern in Elaeis guineensis compared to other species[J]. PLoS One, 2014, 9(12):e114482.
[16]Lee SC, Lim MH, Yu JG, et al. Genome-wide characterization of the CBF/DREB1 gene family in Brassica rapa,Plant Physiol[J].Biochem,2012,61:142-152.
[17]马刘峰,陈芸,任羽,等.棉花CBF2基因克隆和超表达CBF2棉花增强抗冷性[J].植物生理学报,2018,54(2):255-264.
[18]高启明,王斌,赛买提·吐尔逊,等.农杆菌介导的CBF基因转化哈密大枣[J].北方园艺,2016(18):94-98.
[19]王沛文,朱文哲,刘阳,等.多毛番茄冷诱导转录因子CBF1转化番茄的研究[J].江苏农业科学,2015,43(4):30-35.
[20]谭克,赵福顺,吴慧杰,等.冷诱导基因转录因子CBF1转入黄瓜的研究[J].北方园艺,2015(9):79-82.
[21]孟平红,万发香,王永清,等.冷诱导转录因子CBF3转化茄子的初步研究[J].中国蔬菜,2013(10):36-43.
[22]徐春波,王勇,赵海霞,等.冷诱导转录因子AtCBF1转化紫花苜蓿的研究[J].草业科学,2012,21(4):168-174.
[23]王玉成,薄海侠,杨传平.胡杨、柽柳总RNA提取方法的建立[J].东北林业大学学报,2003,31(5):99-100.
[24]刘雪梅.白桦木质素生物合成酶基因分离及遗传转化的研究[D].哈尔滨:东北林业大学,2005.
[25]Sakuma Y, Liu Q, Dubouzet J G, et al.DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved indehydration and cold-inducible gene expression[J].Biochem Biophys Res Commun,2002,290(3):998-1009.
[26]柏星轩,闫雪,要晨宇,等. DREB/CBF转录因子在植物非生物胁迫中的作用及研究进展[J].生物学杂志,2017,34(4):88-93.
[27]高世庆,徐惠君,程宪国,等.转大豆GmDREB基因增强小麦的耐旱及耐盐性[J].科学通报,2005,50(23):2617-2625.
[28]Yamaguchi S K, Shinozaki K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses[J].Annu Rev Plant Biol,2006,57(1):781-803.
[29]Chen M, Wang Q, Cheng X,et al. GmDREB2, a soybean DRE binding transcription factor, conferred drought and high-salt tolerance in transgenic plants[J].Biochem Biophys Res Commun,2007,353(2):299-305.
[30]Chen M, Xu Z, Xia L, et al.Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene,GmDREB3,in soybean(Glycine max L.)[J]. J Exp Bot,2009,60(1):121-135.
[2]StockingE J,Gilmour S J,Thomashow M F. Arabidopsis thalianaCBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cisacting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[J].Proceedings of the National Academy of Sciences,1997,94:1035-1040.
[3]Novillo F,Medina J,Rodríguezfranco M,et al. Genetic analysis reveals a complex regulatory network modulating CBF gene expression and Arabidopsis response to abiotic stress[J].Journal of Experimental Botany, 2012,63(1):293-304.
[4]Zhou M, Hu C, Wei D, et al. Arabidopsis CBF3 and DELLAs positively regulate each other in response to low temperature[J].Scientific Reports, 2017(7):39819.
[5]Thomashow M F. Molecular basis of plant cold acclimation:insights gained from studying the CBF cold response pathway[J].Plant Physiol, 2010, 154(2):571-577.
[6]Wang Z, Wang F, Tang J, et al. C-repeat binding factor gene family identified in non-heading Chinese cabbage is functional in abiotic and biotic stress response but different from that in Arabidopsis[J]. Acta Physiologiae Plantarum, 2014, 36(12):3217-3229.
[7]Allen M D, Yamasaki K, Ohme-Takagi M, et al.A novel mode of DNA recognition by a[beta]-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[J].Embo J,1998,17(18):5484-5496.
[8]Tian Q, Chen J, Wang D, et al. Overexpression of a Populus euphratica CBF4 gene in poplar confers tolerance to multiple stresses[J].Plant Cell Tissue&Organ Culture,2017,128(2):391-407.
[9]Nakano T, Suzuki K, Fujimura T, et al. Genome-wide analysis of the ERF gene family in Arabidopsis and rice[J].Plant Physiology,2006,140(2):411-432.
[10]丁咚,陈亚娟,崔进荣,等.毛果杨CBF/DREB1基因家族生物信息学分析[J].西南农业学报,2018,31(3):457-461.
[11]李月,代培红,刘超,等.海岛棉5个CBF/DREB基因的克隆与表达分析[J].棉花学报,2016,28(1):42-51.
[12]Xiong Y, Fei SZ.Functional and phylogenetic analysis of a DREB/CBF-like gene in perennial ryegrass(Lolium perenne L.)[J].Planta, 2006, 224(4):878-888.
[13] Kidokoro S, Watanabe K, Ohori T, et al. Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression[J].The Plant J., 2015, 81(3):505-518.
[14]Ryu JY, Hong SY, Jo SH, et al.Molecular and functional characterization of cold-responsive C-repeat binding factors from Brachypodium distachyon[J]. BMC Plant Biol, 2014, 14(15):1-15.
[15]Lei X, Xiao Y, Xia W, et al. RNA-seq analysis of oil palm under cold stress reveals a different C-repeat binding factor(CBF)mediated gene expression pattern in Elaeis guineensis compared to other species[J]. PLoS One, 2014, 9(12):e114482.
[16]Lee SC, Lim MH, Yu JG, et al. Genome-wide characterization of the CBF/DREB1 gene family in Brassica rapa,Plant Physiol[J].Biochem,2012,61:142-152.
[17]马刘峰,陈芸,任羽,等.棉花CBF2基因克隆和超表达CBF2棉花增强抗冷性[J].植物生理学报,2018,54(2):255-264.
[18]高启明,王斌,赛买提·吐尔逊,等.农杆菌介导的CBF基因转化哈密大枣[J].北方园艺,2016(18):94-98.
[19]王沛文,朱文哲,刘阳,等.多毛番茄冷诱导转录因子CBF1转化番茄的研究[J].江苏农业科学,2015,43(4):30-35.
[20]谭克,赵福顺,吴慧杰,等.冷诱导基因转录因子CBF1转入黄瓜的研究[J].北方园艺,2015(9):79-82.
[21]孟平红,万发香,王永清,等.冷诱导转录因子CBF3转化茄子的初步研究[J].中国蔬菜,2013(10):36-43.
[22]徐春波,王勇,赵海霞,等.冷诱导转录因子AtCBF1转化紫花苜蓿的研究[J].草业科学,2012,21(4):168-174.
[23]王玉成,薄海侠,杨传平.胡杨、柽柳总RNA提取方法的建立[J].东北林业大学学报,2003,31(5):99-100.
[24]刘雪梅.白桦木质素生物合成酶基因分离及遗传转化的研究[D].哈尔滨:东北林业大学,2005.
[25]Sakuma Y, Liu Q, Dubouzet J G, et al.DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs,transcription factors involved indehydration and cold-inducible gene expression[J].Biochem Biophys Res Commun,2002,290(3):998-1009.
[26]柏星轩,闫雪,要晨宇,等. DREB/CBF转录因子在植物非生物胁迫中的作用及研究进展[J].生物学杂志,2017,34(4):88-93.
[27]高世庆,徐惠君,程宪国,等.转大豆GmDREB基因增强小麦的耐旱及耐盐性[J].科学通报,2005,50(23):2617-2625.
[28]Yamaguchi S K, Shinozaki K. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses[J].Annu Rev Plant Biol,2006,57(1):781-803.
[29]Chen M, Wang Q, Cheng X,et al. GmDREB2, a soybean DRE binding transcription factor, conferred drought and high-salt tolerance in transgenic plants[J].Biochem Biophys Res Commun,2007,353(2):299-305.
[30]Chen M, Xu Z, Xia L, et al.Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene,GmDREB3,in soybean(Glycine max L.)[J]. J Exp Bot,2009,60(1):121-135.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |