茶树CsLHTs亚家族基因的克隆与表达分析
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摘要
氨基酸是茶叶的重要品质成分和氮素贮存形式,因此开展茶树体内氨基酸转运蛋白的研究尤为重要。本研究从茶树转录组中筛选得到5条茶树LHTs(Lysine histidine transporters,赖氨酸/组氨酸转运蛋白)家族基因序列,并从龙井43中成功克隆获得4个茶树LHTs基因,分别命名为CsLHT1、CsLHT6、CsLHT8.1和CsLHT8.2。对该亚家族编码的氨基酸序列的理化性质和功能结构进行预测,结果显示CsLHTs亚家族含有9～11个跨膜区;且含有与氨基酸转运相关的结构域。为了明确在不同茶树品种和氮素水平间CsLHTs基因的表达差异,本研究选用3个茶树品种的扦插苗为试验材料,水培条件下氮饥饿两周后,分别用0.2、2、10 mmol·L~(-1)的NH_4NO_3进行处理。利用qRT-PCR分析了CsLHTs在不同组织间的表达情况,结果表明4个基因在茶树营养组织中均有表达。经氮素处理后,CsLHTs基因在3个氮素水平和3个品种中呈现出不同的变化规律。CsLHT1、CsLHT6和CsLHT8.2在品种间的表达差异程度高于不同氮素水平间的基因表达差异。CsLHT8.1对氮素处理有明显的响应,尤其经0.2 mmol·L~(-1)和10 mmol·L~(-1)的NH_4NO_3处理72 h后,在氮高效品种中茶302根中呈上调表达,表明CsLHT8.1可能参与氨基酸由根部向地上部的转运过程。
Amino acids are important quality components and forms of nitrogen storage in tea plants(Camellia sinensis).Therefore,it is of great significance to study the function of amino acid transporters.In present study,five LHTs(Lysine histidine transporters,CsLHTs)were found from transcriptomes of tea plants.Among them,four CsLHTs were successfully cloned from the cultivar‘Longjing 43’by RT-PCR,and named CsLHT1,CsLHT6,CsLHT8.1 and CsLHT8.2,respectively.The prediction results from amino acid sequences showed that each of CsLHT contained 9-11 transmembrane domains and one domain of amino acid transporter.To investigate the expression patterns of these CsLHT genes under different nitrogen levels,cutting seedlings of three tea cultivars were fed with NH_4NO_3 of three levels(0.2 mmol·L~(-1),2 mmol·L~(-1) and 10 mmol·L~(-1))after two weeks of nitrogen starvation.The qRT-PCR results showed that four CsLHTs were expressed in all vegetative tissues,and exhibited diverse expression patterns.For the genes of CsLHT1,CsLHT6 and CsLHT8.2,the gene expression were affected more highly by tea cultivars rather than by nitrogen levels.However,the gene expression of CsLHT8.1 changed greatly by nitrogen treatments.Especially,after 72 h treatments of 0.2 mmol·L~(-1) and 10 mmol·L~(-1) NH_4NO_3,the CsLHT8.1expressions were significantly up-regulated in the root of‘Zhongcha 302’,which was identified as a high nitrogen efficient cultivar.These results implied that CsLHT8.1 might participate in the transport of amino acids from root to aerial parts in tea plants.
Amino acids are important quality components and forms of nitrogen storage in tea plants(Camellia sinensis).Therefore,it is of great significance to study the function of amino acid transporters.In present study,five LHTs(Lysine histidine transporters,CsLHTs)were found from transcriptomes of tea plants.Among them,four CsLHTs were successfully cloned from the cultivar‘Longjing 43’by RT-PCR,and named CsLHT1,CsLHT6,CsLHT8.1 and CsLHT8.2,respectively.The prediction results from amino acid sequences showed that each of CsLHT contained 9-11 transmembrane domains and one domain of amino acid transporter.To investigate the expression patterns of these CsLHT genes under different nitrogen levels,cutting seedlings of three tea cultivars were fed with NH_4NO_3 of three levels(0.2 mmol·L~(-1),2 mmol·L~(-1) and 10 mmol·L~(-1))after two weeks of nitrogen starvation.The qRT-PCR results showed that four CsLHTs were expressed in all vegetative tissues,and exhibited diverse expression patterns.For the genes of CsLHT1,CsLHT6 and CsLHT8.2,the gene expression were affected more highly by tea cultivars rather than by nitrogen levels.However,the gene expression of CsLHT8.1 changed greatly by nitrogen treatments.Especially,after 72 h treatments of 0.2 mmol·L~(-1) and 10 mmol·L~(-1) NH_4NO_3,the CsLHT8.1expressions were significantly up-regulated in the root of‘Zhongcha 302’,which was identified as a high nitrogen efficient cultivar.These results implied that CsLHT8.1 might participate in the transport of amino acids from root to aerial parts in tea plants.
引文
[1]宋奇超,曹凤秋,巩元勇,等.高等植物氨基酸吸收与转运及生物学功能的研究进展[J].植物营养与肥料学报,2012,18(6):1507-1517.
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[4]Chen L,Bush D R.LHT1,a lysine-and histidine-specific amino acid transporter in Arabidopsis[J].Plant Physiol,1997,115(3):1127-1134.
[5]Hirner A,Ladwig F,Stransky H,et al.Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll[J].Plant Cell,2006,18(8):1931-1946.
[6]Foster J,Lee Y H,Tegeder M.Distinct expression of members of the LHT amino acid transporter family in flowers indicates specific roles in plant reproduction[J].Sexual Plant Reproduction,2008,21(2):143-152.
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[9]Ruan J,Gerendas J,Hardter R,et al.Effect of nitrogen form and root-zone pH on growth and nitrogen uptake of tea(Camellia Sinensis)plants[J].Annals of Botany,2006,99(2):301-310.
[10]Xia E H,Zhang H B,Sheng J,et al.The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis[J].Molecular Plant,2017,10(6):866-877.
[11]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the TC-2(35)(35)Method[J].Methods,2001,25(4):402-408.
[12]Tegeder M,Rentsch D.Uptake and partitioning of amino acids and peptides[J].Molecular Plant,2010,3(6):997-1011.
[13]Tegeder M.Transporters for amino acids in plant cells:some functions and many unknowns[J].Current Opinion in Plant Biology,2012,15(3):315-321.
[14]Tegeder M.Transporters involved in source to sink partitioning of amino acids and ureides:opportunities for crop improvement[J].Journal of Experimental Botany,2014,65(7):1865-1878.
[15]Feng L,Yang T,Zhang Z,et al.Identification and characterization of cationic amino acid transporters(CATs)in tea plant(Camellia sinensis)[J].Plant Growth Regulation,2018,84(1):57-69.
[16]王新超.不同品种茶树氮素营养差异及其机制的研究[D].北京:中国农业科学院,2003.
[17]刘圆.不同氮效率茶树品种氮素吸收利用相关基因表达模式探究[D].北京:中国农业科学院,2003.
[2]Rentsch D,Schmidt S,Tegeder M.Transporters for uptake and allocation of organic nitrogen compounds in plants[J].Febs Letters,2007,581(12):2281-2289.
[3]Lee Y H,Tegeder M.Selective expression of a novel high-affinity transport system for acidic and neutral amino acids in the tapetum cells of Arabidopsis flowers[J].Plant Journal for Cell&Molecular Biology,2004,40(1):60-74.
[4]Chen L,Bush D R.LHT1,a lysine-and histidine-specific amino acid transporter in Arabidopsis[J].Plant Physiol,1997,115(3):1127-1134.
[5]Hirner A,Ladwig F,Stransky H,et al.Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll[J].Plant Cell,2006,18(8):1931-1946.
[6]Foster J,Lee Y H,Tegeder M.Distinct expression of members of the LHT amino acid transporter family in flowers indicates specific roles in plant reproduction[J].Sexual Plant Reproduction,2008,21(2):143-152.
[7]宛晓春.茶叶生物化学[M].3版.北京:中国农业出版社,2003.
[8]郭桂义,胡孔峰,袁丁.信阳毛尖茶的化学成分[J].食品科技,2006,31(9):298-301.
[9]Ruan J,Gerendas J,Hardter R,et al.Effect of nitrogen form and root-zone pH on growth and nitrogen uptake of tea(Camellia Sinensis)plants[J].Annals of Botany,2006,99(2):301-310.
[10]Xia E H,Zhang H B,Sheng J,et al.The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis[J].Molecular Plant,2017,10(6):866-877.
[11]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the TC-2(35)(35)Method[J].Methods,2001,25(4):402-408.
[12]Tegeder M,Rentsch D.Uptake and partitioning of amino acids and peptides[J].Molecular Plant,2010,3(6):997-1011.
[13]Tegeder M.Transporters for amino acids in plant cells:some functions and many unknowns[J].Current Opinion in Plant Biology,2012,15(3):315-321.
[14]Tegeder M.Transporters involved in source to sink partitioning of amino acids and ureides:opportunities for crop improvement[J].Journal of Experimental Botany,2014,65(7):1865-1878.
[15]Feng L,Yang T,Zhang Z,et al.Identification and characterization of cationic amino acid transporters(CATs)in tea plant(Camellia sinensis)[J].Plant Growth Regulation,2018,84(1):57-69.
[16]王新超.不同品种茶树氮素营养差异及其机制的研究[D].北京:中国农业科学院,2003.
[17]刘圆.不同氮效率茶树品种氮素吸收利用相关基因表达模式探究[D].北京:中国农业科学院,2003.