铁皮石斛2个P型H~+-ATPase基因的克隆、表达及生物信息学分析
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摘要
利用RACE进行铁皮石斛质膜PMA(plasma membrane H~+-ATPase)基因的全长克隆,采用生物信息学方法、DNASTAR 7.0和MEGA 7.0对其编码蛋白结构、序列和分子进化等进行分析,并借助实时荧光定量PCR技术检测基因表达模式。结果表明,从铁皮石斛叶片cDNA中分离到2个P型H~+-ATPase基因 DoPMA1和 DoPMA2(GenBank注册号KU160470和KU160471),各编码1条由973和951个氨基酸组成的肽链;2个基因编码蛋白均含有2个保守的P型ATPase结构域和5个H~+运输ATPase结合位点,不含信号肽,定位在质膜。2个蛋白与植物H~+-ATPases蛋白相似性大于72%,聚在拟南芥、水稻等植物H~+-ATPase分子进化树的Ⅴ和Ⅱ分支。实时荧光定量PCR分析表明, DoPMA1在根和茎中上调,分别为叶中的2.98、1.95倍, DoPMA2在根与叶中表达无显著差异,茎中下调为叶中的0.86倍。研究结果为进一步揭示 DoPMA1和 DoPMA2分子作用奠定基础。
RACE was used for full-length cloning of Dendrobium officinale Kimura et Migo membrane H~+-ATPase gene. Bioinformatics method,DNASTAR 7.0 and MEGA 7.0 were used to analyze the structure,sequence and molecular evolution of the encoded protein,and real-time quantitative PCR technology was used to detect the expression pattern of gene organs. The results showed that two full length P-type H~+-ATPase genes, DoPMA1 and DoPMA2(GenBank accessions KU160470 and KU160471,respectively) were isolated from the leaves cDNAs of D. Officinale. DoPMA1 was deduced to a 973 aa(amino acid) protein,while DoPMA2 encoded a 951 aa protein. Both deduced DoPMA1 and DoPMA2 proteins had two conserved P-type H~+-ATPase domains and five H~+-transporting ATPase signature. The proteins did not contain any signal peptide,but located in plasma membrane. The two proteins were highly identical to the plant H~+-ATPases by more than 72%,similarity,and were grouped to the Ⅴ and Ⅱ clades of the evolutionary tree of Arabidopsis,rice,and other plants H~+-ATPase members. DoPMA1 and DoPMA2 showed deferentially expression profiles among the three organs. DoPMA1 transcripts were higher in root and stem,2.98 and 1.95 fold than that in leaf; DoPMA2 did not display significant difference between root and leaf,while it reduced in stem by 0.86 fold. The studies lay a foundation for further exploring the function of DoPMA1 and DoPMA2.
RACE was used for full-length cloning of Dendrobium officinale Kimura et Migo membrane H~+-ATPase gene. Bioinformatics method,DNASTAR 7.0 and MEGA 7.0 were used to analyze the structure,sequence and molecular evolution of the encoded protein,and real-time quantitative PCR technology was used to detect the expression pattern of gene organs. The results showed that two full length P-type H~+-ATPase genes, DoPMA1 and DoPMA2(GenBank accessions KU160470 and KU160471,respectively) were isolated from the leaves cDNAs of D. Officinale. DoPMA1 was deduced to a 973 aa(amino acid) protein,while DoPMA2 encoded a 951 aa protein. Both deduced DoPMA1 and DoPMA2 proteins had two conserved P-type H~+-ATPase domains and five H~+-transporting ATPase signature. The proteins did not contain any signal peptide,but located in plasma membrane. The two proteins were highly identical to the plant H~+-ATPases by more than 72%,similarity,and were grouped to the Ⅴ and Ⅱ clades of the evolutionary tree of Arabidopsis,rice,and other plants H~+-ATPase members. DoPMA1 and DoPMA2 showed deferentially expression profiles among the three organs. DoPMA1 transcripts were higher in root and stem,2.98 and 1.95 fold than that in leaf; DoPMA2 did not display significant difference between root and leaf,while it reduced in stem by 0.86 fold. The studies lay a foundation for further exploring the function of DoPMA1 and DoPMA2.
引文
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[15] CHEN W,SI G Y,ZHAO G,et al.Genomic comparison of the P-ATPase gene family in four cotton species and their expression patterns in Gossypium hirsutum [J].Molecules,2018,23(5):1092.
[16] ARANGO M,BOUTRY M.The plasma membrane proton pump ATPase:the significance of gene subfamilies [J].Planta,2003,216(3):355-365.
[17] OUFATTOLE M,ARANGO M,BOUTRY M.Identification and expression of three new Nicotiana plumbaginifolia genes which encode isoforms of a plasma-membrane H+-ATPase,and one of which is induced by mechanical stress [J].Planta,2000,210(5):715-722.
[18] LIU J J,LIU J L,JI M J,et al.Idetification and expression analysis of tomato plasma membrane H+-ATPase family genes [J].Journal of Zhejiang A&F University,2016,33(5):734-741.
[19] MORIAU L,MICHELET B,BOGAERTS P,et al.Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme [J].The Plant Journal,1999,19(1):31-41.
[20] ZHU Y,DI T,XU G,et al.Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition [J].Plant Cell and Environment,2010,32(10):1428-1440.
[21] KRAJINSKI F,HAUSE B,GIANINAZZI-PEARSON V,et al.Mtha1,a plasma membrane H+-ATPase gene from Medicago truncatula,shows arbuscule-specific induced expression in mycorrhizal tissue [J].Plant Biology,2002,4:754-761.
[2] 杨颖丽,杨宁,安黎哲,等.植物质膜H+-ATPase的研究进展[J].西北植物学报,2006,26(11):2388-2396.YANG Y L,YANG N ,AN L ZH,et al.Research advances about plasmalemma H+ -ATPase in plants[J].Acta Botanica Boreali-Occidentalia Sinica,2006,26(11):2388-2396.
[3] FALHOF J,PEDERSEN J T,FUGLSANG A T,et al.Plasma membrane H+-ATPase regulation in the center of plant physiology [J].Molecular Plant,2016,9(3):323-337.
[4] ZHAO R,DIELEN V,KINET J M,et al.Cosuppression of a plasma membrane H+-ATPase isoform impairs sucrose translocation,stomatal opening,plant growth,and male fertility [J].The Plant Cell Online,2000,12(4):535-546.
[5] YUAN W,ZHANG D,SONG T,et al.Arabidopsis plasma membrane H+-ATPase genes AHA2 and AHA7 have distinct and overlapping roles in the modulation of root tip H+ efflux in response to low-phosphorus stress [J].Journal of Experimental Botany,2017,68(7):1731-1741.
[6] WANG Y,NOGUCHI K,ONO N,et al.Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth[J].Proceedings of the National Academy of Sciences of the United States of America,2014,111(1):533-538.
[7] GéVANUDANT F,DUBY G,STEDINGK E V,et al.Expression of a constitutively activated plasma membrane H+-ATPase alters plant development and increases salt tolerance [J].Plant Physiology,2007,144(4):1763.
[8] WANG E,YU N,BANO S A,et al.A H+-ATPase that energizes nutrient uptake during mycorrhizal symbioses in rice and Medicago truncatula [J].The Plant Cell,2014,26(4):1818-1830.
[9] 何铁光,杨丽涛,李扬瑞,等.铁皮石斛原球茎多糖提取最佳工艺研究[J].西北农业学报,2006,15(5):240-243.HE T G,YANG L T,LI Y R,et al.Studies on the optimum technology for extraction of polysaccharides from suspension-cultured protocorms of Dendrobium candidum [J].Acta Agriculturae Boreali-occidentalis Sinica,2006,15(5):240-243.
[10] 陈晓梅,王春兰,杨峻山,等.铁皮石斛化学成分及其分析的研究进展[J].中国药学杂志,2013,48(19):1634-1640.CHEN X M,WANG CH L,YANG J SH,et al.Research progress on chemical composition and chemical analysis of Dendrobium officinale[J].Chinese Pharmaceutical Journal,2013,48(19):1634-1640.
[11] ZHAO M M,ZHANG G,ZHANG D W,et al.ESTs analysis reveals putative genes involved in symbiotic seed germination in Dendrobium officinale [J].Public Library of Science One,2013,8(8):e72705.
[12] PFAEEL M W.A new mathematical model for relative quantification in real-time RT-PCR [J].Nucleic Acids Research,2001,29(9):e45.
[13] KOZAK M W.An analysis of 50-noncoding sequences from 699 vertebrate messenger RNAs [J].Nucleic Acids Research,1987,15(20):8125-8132.
[14] ZHANG G Q,XU Q,CHAO B,et al.The Dendrobium catenatum Lindl.genome sequence provides insights into polysaccharide synthase,floral development and adaptive evolution [J].Scientific Reports,2016,6(19029):19029.
[15] CHEN W,SI G Y,ZHAO G,et al.Genomic comparison of the P-ATPase gene family in four cotton species and their expression patterns in Gossypium hirsutum [J].Molecules,2018,23(5):1092.
[16] ARANGO M,BOUTRY M.The plasma membrane proton pump ATPase:the significance of gene subfamilies [J].Planta,2003,216(3):355-365.
[17] OUFATTOLE M,ARANGO M,BOUTRY M.Identification and expression of three new Nicotiana plumbaginifolia genes which encode isoforms of a plasma-membrane H+-ATPase,and one of which is induced by mechanical stress [J].Planta,2000,210(5):715-722.
[18] LIU J J,LIU J L,JI M J,et al.Idetification and expression analysis of tomato plasma membrane H+-ATPase family genes [J].Journal of Zhejiang A&F University,2016,33(5):734-741.
[19] MORIAU L,MICHELET B,BOGAERTS P,et al.Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in Nicotiana plumbaginifolia reveals the major transport functions of this enzyme [J].The Plant Journal,1999,19(1):31-41.
[20] ZHU Y,DI T,XU G,et al.Adaptation of plasma membrane H+-ATPase of rice roots to low pH as related to ammonium nutrition [J].Plant Cell and Environment,2010,32(10):1428-1440.
[21] KRAJINSKI F,HAUSE B,GIANINAZZI-PEARSON V,et al.Mtha1,a plasma membrane H+-ATPase gene from Medicago truncatula,shows arbuscule-specific induced expression in mycorrhizal tissue [J].Plant Biology,2002,4:754-761.