甘蓝型油菜钙离子转运蛋白CAX家族基因生物信息学及其对镉胁迫响应表达分析
|
摘要
CAXs(Cation/H~+ exchanger antiporter)是一类重要的阳离子跨膜转运蛋白,在调控植物Ca~(2+)平衡,参与转运金属离子Cd~(2+)和Mn~(2+)中发挥了重要作用,但在甘蓝型油菜中尚缺乏系统研究。本文利用生物信息学的方法对甘蓝型油菜CAX家族成员的基因拷贝数、系统进化、进化选择压力、分子特征、保守基序、染色体定位、基因结构及其启动子区域所能结合的顺式作用元件进行了预测和分析;同时采用荧光定量PCR分析了甘蓝型油菜BnaCAXs的组织表达模式及其对Cd~(2+)胁迫的响应。结果表明,甘蓝型油菜CAX家族共包含17个成员,系统进化分析结果表明BnaCAXs与拟南芥进化相似,并且明显地分为5个亚家族。BnaCAXs家族所有基因成员的K_a/K_s值均小于0.3,受到强烈的纯化选择作用。大部分BnaCAXs均属于稳定的疏水蛋白,并包含8～11个跨膜结构域。基因结构差异明显,内含子数目从6～11不等,并且Dof、MYB以及W-BOX是其启动子上丰度较大的顺式作用元件,可能参与到BnaCAX抗逆过程的调控。荧光定量PCR结果表明,甘蓝型油菜CAX基因主要在地下部表达;BnaC4.CAX1-2和BnaC3.CAX2-2是该家族的核心基因,并受到Cd~(2+)胁迫的显著诱导。
CAXs(Cation/H~+ exchanger antiporter)are a class of secondary energised ion transporter that primarily functioning in mediating Ca~(2+)transportation,which play an important role in transporting Cd~(2+)and Mn~(2+).However,they are rarely studied in Brassica napus.In this study,the bioinformatical methods were employed to predict and analyze some bioinformatical parameters of BnaCAXs,including copy number,phylogenetic analysis,evolutionary selection pressure,molecular characteristics,conserved motifs,chromosome location,gene structures and the cis-acting elements binged in the promoter regions.Moreover,the expression pattern and gene expression response to Cd~(2+)of BnaCAXs were analyzed.The results showed that 17 members were identified in BnaCAX gene family,which divided into five subfamilies as Arabidopsis thaliana.The K_a/K_s of BnaCAXs were lower than 0.3,indicating a strong purify selection was acting on all the BnaCAXs.Most of the BnaCAXs were stable and hydrophobic,which compromised 8–11 transmembrane domains.The gene structures of BnaCAXs were varied,which were disrupted by 6–11 introns.Dof,MYB and W-BOX were the most abundant cis-acting elements binding to the promoter regions of BnaCAXs,which might participate in the regulation of stress tolerance.In addition,most of the BnaCAXs were mainly expressed in roots,and BnaC4.CAX1-2 and BnaC3.CAX2-2 were identified as the core members in BnaCAX,which were significantly induced by Cd~(2+).
CAXs(Cation/H~+ exchanger antiporter)are a class of secondary energised ion transporter that primarily functioning in mediating Ca~(2+)transportation,which play an important role in transporting Cd~(2+)and Mn~(2+).However,they are rarely studied in Brassica napus.In this study,the bioinformatical methods were employed to predict and analyze some bioinformatical parameters of BnaCAXs,including copy number,phylogenetic analysis,evolutionary selection pressure,molecular characteristics,conserved motifs,chromosome location,gene structures and the cis-acting elements binged in the promoter regions.Moreover,the expression pattern and gene expression response to Cd~(2+)of BnaCAXs were analyzed.The results showed that 17 members were identified in BnaCAX gene family,which divided into five subfamilies as Arabidopsis thaliana.The K_a/K_s of BnaCAXs were lower than 0.3,indicating a strong purify selection was acting on all the BnaCAXs.Most of the BnaCAXs were stable and hydrophobic,which compromised 8–11 transmembrane domains.The gene structures of BnaCAXs were varied,which were disrupted by 6–11 introns.Dof,MYB and W-BOX were the most abundant cis-acting elements binding to the promoter regions of BnaCAXs,which might participate in the regulation of stress tolerance.In addition,most of the BnaCAXs were mainly expressed in roots,and BnaC4.CAX1-2 and BnaC3.CAX2-2 were identified as the core members in BnaCAX,which were significantly induced by Cd~(2+).
引文
Catala R,Santos E,Alonso JM,et al(2003).Mutations in the Ca2+/H+transporter CAX1 increase CBF/DREB1 expression and the cold-acclimation response in Arabidopsis.Plant Cell,15(12):2940-2951
Cheng N,Pittman JK,Shigaki T,et al(2002).Characterization of CAX4:an Arabidopsis H+/cation antiporter.Plant Physiol,128(4):1245-1254
Cheng NH,Pittman JK,Shigaki T,et al(2005).Functional association of Arabidopsis CAX1 and CAX3 is required for normal growth and ion homeostasis.Plant Physiol,138(4):2048-2060
Feng G,Wang X,Bai JY,et al(2018).Analysis of the potential remediation of oilseed rape to soils contaminated with cadmium.J Sichuan Univ,55(1):173-178(in Chinese with English abstract)[冯刚,王鑫,白九元等(2018).油菜对Cd污染土壤的修复潜力分析.四川大学学报,55(1):173-178]
Hirschi KD,Korenkov VD,Wilganowski NL,et al(2000).Expression of Arabidopsis CAX2 in tobacco altered metal accumulation and increased manganese tolerance.Plant Physiol,124(1):125-133
Hua YP,Zhou T,Song HX,et al(2018).Integrated genomic and transcriptomic insights into the two-component high-affinity nitrate transporters in allotetraploid rapeseed.Plant Soil,427:245-268
Kamiya T,Akahori T,Maeshima M(2005).Expression profile of the genes for rice cation/H+exchanger family and functional analysis in yeast.Plant Cell Physiol,46(10):1735-1740
Koren’kov V,Park S,Cheng NH,et al(2007).Enhanced Cd2+-selective root-tonoplast-transport in tobaccos expressing Arabidopsis cation exchangers.Planta,225(2):403-411
Liu S,Liu Y,Yang X,et al(2014).The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes.Nat Commun,5:3930
Mei H,Cheng NH,Zhao J,et al(2009).Root development under metal stress in Arabidopsis thaliana requires the H+/cation antiporter CAX4.New Phytolt,183(1):95-105
Nekrutenko A,Makova KD,Li WH(2002).The KA/KS ratio test for assessing the protein-coding potential of genomic regions:an empirical and simulation study.Genome Res,12(1):198-202
Ou QC,Jiang SL,Wang F,et al(2018).Genome-wide identification and expression analysis of auxin response factor(ARF)gene family in pear.Sci Agr Sin,51(2):327-340(in Chinese with English abstract)[欧春青,姜淑苓,王斐等(2018).梨全基因组生长素反应因子(ARF)基因家族鉴定及表达分析.中国农业科学,51(2):327-340]
Pittman JK,Shigaki T,Marshall JL,et al(2004).Functional and regulatory analysis of the Arabidopsis thaliana CAX2 cation transporter.Plant Mol Biol,56(6):959-971
Pittman JK,Hirschi KD(2016a).Phylogenetic analysis and protein structure modelling identifies distinct Ca2+/Cation antiporters and conservation of gene family structure within Arabidopsis and rice species.Rice(N Y),9(1):3
Pittman JK,Hirschi KD(2016b).CAX-ing a wide net:Cation/H+transporters in metal remediation and abiotic stress signalling.Plant Biol(Stuttg),18(5):741-930
Qudeimat E,Faltusz A M,Wheeler G,et al(2008).A PIIB-type Ca2+-ATPase is essential for stress adaptation in Physcomitrella patens.Proc Natl Acad Sci USA,105(49):19555-19560
Sanders D,Pelloux J,Brownlee C,et al(2002).Calcium at the crossroads of signaling.Plant Cell,14:401-417
Shigaki T,Sreevidya C,Hirschi KD(2002).Analysis of the Ca2+domain in Arabidopsis H+/Ca2+antiporters CAX1and CAX3.Plant Mol Biol,50(3):475-483
Wang DP,Zhang YB,Zhang Z,et al(2010).KaKs_Calculator2.0:a toolkit incorporating gamma-series methods and sliding window strategies.Genom Proteom Bioinf,8:77-80
Xu L,Zahid KR,He LR,et al(2013).GhCAX3 gene,a novel Ca2+/H+exchanger from cotton,confers regulation of cold response and ABA induced signal transduction.PLoSOne,8(6):e66303
Yamada N,Theerawitaya C,Cha-um S,et al(2014).Expression and functional analysis of putative vacuolar Ca2+-transporters(CAXs and ACAs)in roots of salt tolerant and sensitive rice cultivars.Protoplasma,251(5):1067-1075
Zhang M,Zhang J,Lu LL,et al(2016).Functional analysis of CAX2-like transporters isolated from two ecotypes of Sedum alfredii.Biol Plant,60:37-47
Zhang L,Zhang S,Zhu LM,et al(2015).Bioinformatics and expression analysis of CAX gene family in apple.J Nanjing Agri Uni,38(1):41-49(in Chinese with English abstract)[张璐,刘帅,龙鸣等(2015).苹果CAX基因家族生物信息学和表达分析.南京农业大学学报,38(1):41-49]
Zhao J,Barkla BJ,Marshall J,et al(2008).The Arabidopsis cax3 mutants display altered salt tolerance,pH sensitivity and reduced plasma membrane H+-ATPase activity.Planta,227(3):659-669
Zhou XD,Zhao CL,Sun XG,et al(2015).Research on phytoremedeation of cadmium polluted soil by Capsella bursa-pastoris and Oilseed rape.Northern Hortic,(6):167-172(in Chinese with English abstract)[周旭丹,赵春莉,孙晓刚等(2015).荠菜和油菜对镉污染土壤的修复效应研究.北方园艺,(6):167-172]
Cheng N,Pittman JK,Shigaki T,et al(2002).Characterization of CAX4:an Arabidopsis H+/cation antiporter.Plant Physiol,128(4):1245-1254
Cheng NH,Pittman JK,Shigaki T,et al(2005).Functional association of Arabidopsis CAX1 and CAX3 is required for normal growth and ion homeostasis.Plant Physiol,138(4):2048-2060
Feng G,Wang X,Bai JY,et al(2018).Analysis of the potential remediation of oilseed rape to soils contaminated with cadmium.J Sichuan Univ,55(1):173-178(in Chinese with English abstract)[冯刚,王鑫,白九元等(2018).油菜对Cd污染土壤的修复潜力分析.四川大学学报,55(1):173-178]
Hirschi KD,Korenkov VD,Wilganowski NL,et al(2000).Expression of Arabidopsis CAX2 in tobacco altered metal accumulation and increased manganese tolerance.Plant Physiol,124(1):125-133
Hua YP,Zhou T,Song HX,et al(2018).Integrated genomic and transcriptomic insights into the two-component high-affinity nitrate transporters in allotetraploid rapeseed.Plant Soil,427:245-268
Kamiya T,Akahori T,Maeshima M(2005).Expression profile of the genes for rice cation/H+exchanger family and functional analysis in yeast.Plant Cell Physiol,46(10):1735-1740
Koren’kov V,Park S,Cheng NH,et al(2007).Enhanced Cd2+-selective root-tonoplast-transport in tobaccos expressing Arabidopsis cation exchangers.Planta,225(2):403-411
Liu S,Liu Y,Yang X,et al(2014).The Brassica oleracea genome reveals the asymmetrical evolution of polyploid genomes.Nat Commun,5:3930
Mei H,Cheng NH,Zhao J,et al(2009).Root development under metal stress in Arabidopsis thaliana requires the H+/cation antiporter CAX4.New Phytolt,183(1):95-105
Nekrutenko A,Makova KD,Li WH(2002).The KA/KS ratio test for assessing the protein-coding potential of genomic regions:an empirical and simulation study.Genome Res,12(1):198-202
Ou QC,Jiang SL,Wang F,et al(2018).Genome-wide identification and expression analysis of auxin response factor(ARF)gene family in pear.Sci Agr Sin,51(2):327-340(in Chinese with English abstract)[欧春青,姜淑苓,王斐等(2018).梨全基因组生长素反应因子(ARF)基因家族鉴定及表达分析.中国农业科学,51(2):327-340]
Pittman JK,Shigaki T,Marshall JL,et al(2004).Functional and regulatory analysis of the Arabidopsis thaliana CAX2 cation transporter.Plant Mol Biol,56(6):959-971
Pittman JK,Hirschi KD(2016a).Phylogenetic analysis and protein structure modelling identifies distinct Ca2+/Cation antiporters and conservation of gene family structure within Arabidopsis and rice species.Rice(N Y),9(1):3
Pittman JK,Hirschi KD(2016b).CAX-ing a wide net:Cation/H+transporters in metal remediation and abiotic stress signalling.Plant Biol(Stuttg),18(5):741-930
Qudeimat E,Faltusz A M,Wheeler G,et al(2008).A PIIB-type Ca2+-ATPase is essential for stress adaptation in Physcomitrella patens.Proc Natl Acad Sci USA,105(49):19555-19560
Sanders D,Pelloux J,Brownlee C,et al(2002).Calcium at the crossroads of signaling.Plant Cell,14:401-417
Shigaki T,Sreevidya C,Hirschi KD(2002).Analysis of the Ca2+domain in Arabidopsis H+/Ca2+antiporters CAX1and CAX3.Plant Mol Biol,50(3):475-483
Wang DP,Zhang YB,Zhang Z,et al(2010).KaKs_Calculator2.0:a toolkit incorporating gamma-series methods and sliding window strategies.Genom Proteom Bioinf,8:77-80
Xu L,Zahid KR,He LR,et al(2013).GhCAX3 gene,a novel Ca2+/H+exchanger from cotton,confers regulation of cold response and ABA induced signal transduction.PLoSOne,8(6):e66303
Yamada N,Theerawitaya C,Cha-um S,et al(2014).Expression and functional analysis of putative vacuolar Ca2+-transporters(CAXs and ACAs)in roots of salt tolerant and sensitive rice cultivars.Protoplasma,251(5):1067-1075
Zhang M,Zhang J,Lu LL,et al(2016).Functional analysis of CAX2-like transporters isolated from two ecotypes of Sedum alfredii.Biol Plant,60:37-47
Zhang L,Zhang S,Zhu LM,et al(2015).Bioinformatics and expression analysis of CAX gene family in apple.J Nanjing Agri Uni,38(1):41-49(in Chinese with English abstract)[张璐,刘帅,龙鸣等(2015).苹果CAX基因家族生物信息学和表达分析.南京农业大学学报,38(1):41-49]
Zhao J,Barkla BJ,Marshall J,et al(2008).The Arabidopsis cax3 mutants display altered salt tolerance,pH sensitivity and reduced plasma membrane H+-ATPase activity.Planta,227(3):659-669
Zhou XD,Zhao CL,Sun XG,et al(2015).Research on phytoremedeation of cadmium polluted soil by Capsella bursa-pastoris and Oilseed rape.Northern Hortic,(6):167-172(in Chinese with English abstract)[周旭丹,赵春莉,孙晓刚等(2015).荠菜和油菜对镉污染土壤的修复效应研究.北方园艺,(6):167-172]