草莓KEA家族基因的克隆、鉴定及表达分析
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摘要
K~+/H~+逆向转运体(KEA)介导细胞中K~+和H~+的动态平衡,在维持植物体内的离子平衡、生长发育和信号转导中起重要作用,然而,相关研究主要体现在模式作物拟南芥中,果树中KEA家族基因的功能依然未知。本研究以Yellow Wonder 5AF8草莓为材料,筛选并克隆KEA家族基因,并对其进行生物信息学鉴定和表达特征分析,为研究果树K~+/H~+平衡及钾素动态平衡机制提供基因资源和理论依据。结果表明,在草莓基因组中检索并克隆到5个KEA家族基因,命名为FveKEA1～FveKEA5,属于典型的植物K~+/H~+逆向转运体基因;编码的蛋白质与7种已报道的不同科属植物的KEA家族蛋白在氨基酸水平上具有25.00%的一致性,并可分为2个亚族(Group I和Group II),其中,草莓FveKEA1和FveKEA2属于Group I,只含有4个Motif基序,而FveKEA3～FveKEA5属于Group II,含有7个Motif基序;系统进化树表明草莓FveKEA2和FveKEA4分别与葡萄VvKEA2和苹果MdoKEA6紧密聚在一起,草莓FveKEA1、FveKEA3和FveKEA5分别与葡萄VvKEA1、白杨PtrKEA4、桃PpeKEA4等相应成员在遗传距离上较近;草莓KEA家族蛋白主要定位于细胞质膜,均含有12～14个跨膜区,除FveKEA3外,均为稳定蛋白,且只有FveKEA5含有信号肽;转录表达谱分析结果揭示草莓KEA家族基因在多种组织或器官中均有表达,实时荧光定量PCR结果表明FveKEA1在5AF8草莓不同组织中的整体表达水平最高,在花瓣和未成熟果实中的表达量最为突出,其次是FveKEA4,而其他3个基因的整体表达水平相对较低。此外,在草莓KEA基因启动子区域鉴定到至少16种顺式作用元件,且均含有光感应、胚乳表达和脱落酸(ABA)响应的作用元件。
In plants, K~+ efflux antiporters(KEAs) mediate the cellular K~+/H~+ homeostasis and play key role in the ion balance, growth and development and signal transduction. However, the relevant studies mainly focused on model plant Arabidopsis, the functions of KEA family genes in fruit trees were rarely known. Using Yellow Wonder 5 AF8 strawberry as material, KEA family genes bioinformatics identification and gene expression characteristics were analyzed to provide gene resources and theoretical foundation for the study of K~+/H~+ balance and K~+ homeostasis mechanisms in fruit trees. Results showed that five KEA family genes were isolated from strawberry, named FveKEA1-FveKEA5, which belonged to the typical plant K~+/H~+ antiporter gene. The encoded proteins shared an overall identity of 25.00% at the amino acid level with the KEA family proteins of seven different families, and could be divided into two subgroups, Group I and Group II. FveKEA1 and FveKEA2 belonged to Group I, and contained four Motif motifs. FveKEA3-FveKEA5 belonged to Group II, and contained seven Motif motifs. Phylogenetic tree analysis showed that FveKEA2 and FveKEA4 of strawberry were closely related to VvKEA2 of grape and MdoKEA6 of apple, respectively, and FveKEA1, FveKEA3 and FveKEA5 were closely related to VvKEA1 of grape, PtrKEA4 of polar and PpeKEA4 of peach, correspondingly. All FveKEA proteins were majorly localized in plasma membrane and contained 12-14 transmembrane domains(TMs). All strawberry KEA proteins were stable protein with the exception of FveKEA3, and only FveKEA5 possessed the signal peptide. The results of transcriptional profiling revealed that FveKEA genes could be detected in various tissues or organs of strawberry. qRT-PCR analysis results indicated that FveKEA1 had the highest expression level in different tissues of 5 AF8 strawberry on the whole and the most prominent expression level in petals and immature fruits, followed by FveKEA4, while the overall expression levels of the other three genes were relatively low. Moreover, at least 16 cis-acting elements were identified in the promoter region of strawberry KEA family genes, and all of them contained elements of light sensing, endosperm expression and abscisic acid response.
In plants, K~+ efflux antiporters(KEAs) mediate the cellular K~+/H~+ homeostasis and play key role in the ion balance, growth and development and signal transduction. However, the relevant studies mainly focused on model plant Arabidopsis, the functions of KEA family genes in fruit trees were rarely known. Using Yellow Wonder 5 AF8 strawberry as material, KEA family genes bioinformatics identification and gene expression characteristics were analyzed to provide gene resources and theoretical foundation for the study of K~+/H~+ balance and K~+ homeostasis mechanisms in fruit trees. Results showed that five KEA family genes were isolated from strawberry, named FveKEA1-FveKEA5, which belonged to the typical plant K~+/H~+ antiporter gene. The encoded proteins shared an overall identity of 25.00% at the amino acid level with the KEA family proteins of seven different families, and could be divided into two subgroups, Group I and Group II. FveKEA1 and FveKEA2 belonged to Group I, and contained four Motif motifs. FveKEA3-FveKEA5 belonged to Group II, and contained seven Motif motifs. Phylogenetic tree analysis showed that FveKEA2 and FveKEA4 of strawberry were closely related to VvKEA2 of grape and MdoKEA6 of apple, respectively, and FveKEA1, FveKEA3 and FveKEA5 were closely related to VvKEA1 of grape, PtrKEA4 of polar and PpeKEA4 of peach, correspondingly. All FveKEA proteins were majorly localized in plasma membrane and contained 12-14 transmembrane domains(TMs). All strawberry KEA proteins were stable protein with the exception of FveKEA3, and only FveKEA5 possessed the signal peptide. The results of transcriptional profiling revealed that FveKEA genes could be detected in various tissues or organs of strawberry. qRT-PCR analysis results indicated that FveKEA1 had the highest expression level in different tissues of 5 AF8 strawberry on the whole and the most prominent expression level in petals and immature fruits, followed by FveKEA4, while the overall expression levels of the other three genes were relatively low. Moreover, at least 16 cis-acting elements were identified in the promoter region of strawberry KEA family genes, and all of them contained elements of light sensing, endosperm expression and abscisic acid response.
引文
[1] VéRY A A,SENTENAC H.Molecular mechanisms and regulation of K+ transport in higher plants [J].Annual Review of Plant Biology,2003,54:575-603.
[2] GRABOV A.Plant KT/KUP/HAK potassium transporters:Single family-multiple functions [J].Annals of Botany,2007,99:1035-1041.
[3] DEMIRAL M A,K?SEOGLU A T.Effect of potassium on yield,fruit quality,and chemical composition of green house-grown aalia melon [J].Journal of Plant Nutrition,2005,28:93-100.
[4] YURTSEVEN E,KESMEZ G D,üNLüKARA A.The effects of water salinity and potassium levels on yield,fruit quality and water consumption of a native central anatolian tomato species (Lycopersicon esculantum) [J].Agricultural Water Management,2005,78:128-135.
[5] HARTZ T K,JOHNSTONE P R,FRANCIS D M,et al.Processing tomato yield and fruit quality improved with potassium fertigation [J].Hort Science,2005,40:1862-1867.
[6] 宋志忠,郭绍雷,马瑞娟,等.KT/HAK/KUP家族基因在桃开花期的表达及对钾肥施放的响应分析 J].中国农业科学,2015,48(6):1177-1185.
[7] 宋志忠,许建兰,张斌斌,等.叶面喷施钾肥对霞脆桃果实品质及KUP基因表达的影响[J].江苏农业学报,2018,34(5):1107-1112.
[8] CHANROJ S,LU Y,PADMANABAN S,et al.Plant-specific cation/H+ exchanger 17 and its homologs are endomembrane K+ transporters with roles in protein sorting [J].Journal of Biology Chemistry,2011,286(39):33931-33941.
[9] CHANROJ S,WANG G,VENEMA K,et al.Conserved and diversified gene families of monovalent cation/H+ antiporters from algae to flowering plants [J].Frontiers in Plant Science,2012,3:25.
[10] BASSIL E,BLUMWALD E.The ins and outs of intracellular ion homeostasis:NHX-type cation/H+ transporters [J].Current Opinion in Plant Biology,2014,22:1-6.
[11] MASER P,THOMINE S,SCHROEDER J I,et al.Phylogenetic relationships within cation transporter families of Arabidopsis [J].Plant Physiology,2001,126:1646-1667.
[12] HAN L,LI J L,WANG L,et al.Identification and localized expression of putative K+/H+ antiporter genes in Arabidopsis [J].Acta Physiologiae Plantarum,2015,37(5):1-14.
[13] ARANDA-SICILIA M N,CAGNAC O,CHANROJ S,et al.Arabidopsis KEA2,a homolog of bacterial KefC,encodes a K+/H+ antiporter with a chloroplast transit peptide [J].BBA-Biomembranes,2012,1818(9):2362-2371.
[14] ZHENG S,PAN T,FAN L G,et al.A novel AtKEA gene family,homolog of bacterial K+/H+ antiporters,plays potential roles in K+ homeostasis and osmotic adjustment in Arabidopsis [J].PLoS ONE,2013,8(11):e81463.
[15] ARMBRUSTER U,CARRILLO L R,VENEMA K,et al.Ion antiport accelerates photosynthetic acclimation in fluctuating light environments [J].Nature Communications,2014,5:5439.
[16] KUNZ H H,GIERTH M,HERDEAN A,et al.Plastidial transporters KEA1,-2,and -3 are essential for chloroplast osmoregulation,integrity,and pH regulation in Arabidopsis [J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(20):7480-7485.
[17] ZHOU H,QI K,LIU X,et al.Genome-wide identification and comparative analysis of the cation proton antiporters family in pear and four other Rosaceae species [J].Molecular Genetics and Genomics,2016,291:1727-1742.
[18] KANG C,DARWISH O,GERETZ,et al.Genome-scale transcriptomic insights into early-stage fruit development in woodland strawberry Fragaria vesca [J].Plant Cell,2013,25:1960-1978.
[19] FAIT A,HANHINEVA K,BELEGGIA R,et al.Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development [J].Plant Physiology,2008,148 (2):730-750.
[20] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ct method [J].Methods,2001,25(4):402-408.
[21] WANG Y,WU W H.Genetic approaches for improvement of the crop potassium acquisition and utilization efficiency [J].Current Opinion in Plant Biology,2015,25:46-52.
[22] CHEN H T,CHEN X,WU B Y,et al.Whole-genome identification and expression analysis of K+ efflux antiporter (KEA) and Na+/H+ antiporter (NHX) families under abiotic stress in soybean [J].Journal of Intergrative Agriculture,2015,14(6):1171-1183.
[23] SONG Z Z,GUO S L,ZHANG C H,et al.KT/HAK/KUP potassium transporter genes differentially expressed during fruit devcelopment,ripening,and postharvest shelf-life of ‘Xiahui6’ peaches [J].Acta Physiologiae Plantarum,2015,37:131.
[2] GRABOV A.Plant KT/KUP/HAK potassium transporters:Single family-multiple functions [J].Annals of Botany,2007,99:1035-1041.
[3] DEMIRAL M A,K?SEOGLU A T.Effect of potassium on yield,fruit quality,and chemical composition of green house-grown aalia melon [J].Journal of Plant Nutrition,2005,28:93-100.
[4] YURTSEVEN E,KESMEZ G D,üNLüKARA A.The effects of water salinity and potassium levels on yield,fruit quality and water consumption of a native central anatolian tomato species (Lycopersicon esculantum) [J].Agricultural Water Management,2005,78:128-135.
[5] HARTZ T K,JOHNSTONE P R,FRANCIS D M,et al.Processing tomato yield and fruit quality improved with potassium fertigation [J].Hort Science,2005,40:1862-1867.
[6] 宋志忠,郭绍雷,马瑞娟,等.KT/HAK/KUP家族基因在桃开花期的表达及对钾肥施放的响应分析 J].中国农业科学,2015,48(6):1177-1185.
[7] 宋志忠,许建兰,张斌斌,等.叶面喷施钾肥对霞脆桃果实品质及KUP基因表达的影响[J].江苏农业学报,2018,34(5):1107-1112.
[8] CHANROJ S,LU Y,PADMANABAN S,et al.Plant-specific cation/H+ exchanger 17 and its homologs are endomembrane K+ transporters with roles in protein sorting [J].Journal of Biology Chemistry,2011,286(39):33931-33941.
[9] CHANROJ S,WANG G,VENEMA K,et al.Conserved and diversified gene families of monovalent cation/H+ antiporters from algae to flowering plants [J].Frontiers in Plant Science,2012,3:25.
[10] BASSIL E,BLUMWALD E.The ins and outs of intracellular ion homeostasis:NHX-type cation/H+ transporters [J].Current Opinion in Plant Biology,2014,22:1-6.
[11] MASER P,THOMINE S,SCHROEDER J I,et al.Phylogenetic relationships within cation transporter families of Arabidopsis [J].Plant Physiology,2001,126:1646-1667.
[12] HAN L,LI J L,WANG L,et al.Identification and localized expression of putative K+/H+ antiporter genes in Arabidopsis [J].Acta Physiologiae Plantarum,2015,37(5):1-14.
[13] ARANDA-SICILIA M N,CAGNAC O,CHANROJ S,et al.Arabidopsis KEA2,a homolog of bacterial KefC,encodes a K+/H+ antiporter with a chloroplast transit peptide [J].BBA-Biomembranes,2012,1818(9):2362-2371.
[14] ZHENG S,PAN T,FAN L G,et al.A novel AtKEA gene family,homolog of bacterial K+/H+ antiporters,plays potential roles in K+ homeostasis and osmotic adjustment in Arabidopsis [J].PLoS ONE,2013,8(11):e81463.
[15] ARMBRUSTER U,CARRILLO L R,VENEMA K,et al.Ion antiport accelerates photosynthetic acclimation in fluctuating light environments [J].Nature Communications,2014,5:5439.
[16] KUNZ H H,GIERTH M,HERDEAN A,et al.Plastidial transporters KEA1,-2,and -3 are essential for chloroplast osmoregulation,integrity,and pH regulation in Arabidopsis [J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(20):7480-7485.
[17] ZHOU H,QI K,LIU X,et al.Genome-wide identification and comparative analysis of the cation proton antiporters family in pear and four other Rosaceae species [J].Molecular Genetics and Genomics,2016,291:1727-1742.
[18] KANG C,DARWISH O,GERETZ,et al.Genome-scale transcriptomic insights into early-stage fruit development in woodland strawberry Fragaria vesca [J].Plant Cell,2013,25:1960-1978.
[19] FAIT A,HANHINEVA K,BELEGGIA R,et al.Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development [J].Plant Physiology,2008,148 (2):730-750.
[20] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ct method [J].Methods,2001,25(4):402-408.
[21] WANG Y,WU W H.Genetic approaches for improvement of the crop potassium acquisition and utilization efficiency [J].Current Opinion in Plant Biology,2015,25:46-52.
[22] CHEN H T,CHEN X,WU B Y,et al.Whole-genome identification and expression analysis of K+ efflux antiporter (KEA) and Na+/H+ antiporter (NHX) families under abiotic stress in soybean [J].Journal of Intergrative Agriculture,2015,14(6):1171-1183.
[23] SONG Z Z,GUO S L,ZHANG C H,et al.KT/HAK/KUP potassium transporter genes differentially expressed during fruit devcelopment,ripening,and postharvest shelf-life of ‘Xiahui6’ peaches [J].Acta Physiologiae Plantarum,2015,37:131.