枸杞抗坏血酸过氧化物酶基因的克隆与表达分析
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摘要
为探索青海枸杞抗坏血酸过氧化物酶(APX)基因信息,并对枸杞APX基因进行生物信息学分析以及基因表达研究,通过RT-PCR和RACE方法克隆枸杞APX基因,利用生物信息学软件预测基因结构,采用实时荧光定量PCR方法分析基因表达的变化。结果显示:枸杞APX基因的全长cDNA序列为1 047bp(Genbank No.KX981601),命名为LcAPX基因。该基因含有1个753bp的完整开放阅读框(ORF),编码250个氨基酸,包含亚铁血红素结合位点、底物结合位点和K~+结合位点。枸杞LcAPX与茄科植物的APX蛋白聚为一类,说明两者的亲缘关系较近。LcAPX基因在枸杞的成熟叶中表达量最高,花中表达量最少。其在聚乙二醇(PEG)、氯化钠(NaCl)胁迫下诱导表达,显示LcAPX在枸杞抗干旱和抗盐逆境过程起一定作用。
The objective of this study was to explore a ascorbate peroxidase(APX)gene in Lycium chinense by the methods of Reverse-Transcription PCR and RACE,and deduce the structure of this gene by bioinformatics,and analyze gene expression by real time PCR.The full-length cDNA of L.chinense APX(designated as LcAPX)was 1 047 bp(Genbank No.KX981601),and contained a complete open reading frame(ORF)of 753 bp,which encoded 250 amino acid residues.Sequence analysis showed that LcAPX protein contains the heme binding site,substrate binding site,and K~+ binding site.Homology analysis indicated that the deduced LcAPX protein was highly homologoud to other APX proteins from different species.LcAPX protein had closer relationship with APXs from Solanaceae plants than from other plants.The expression of LcAPX gene was the strongest in the mature leaves of L.chinense,and the least in flowers.Furthermore,LcAPXtranscription level was induced under the treatment of PEG and NaCl stresses,and LcAPXplayed a role in resistance to drought and salt stress processes.
The objective of this study was to explore a ascorbate peroxidase(APX)gene in Lycium chinense by the methods of Reverse-Transcription PCR and RACE,and deduce the structure of this gene by bioinformatics,and analyze gene expression by real time PCR.The full-length cDNA of L.chinense APX(designated as LcAPX)was 1 047 bp(Genbank No.KX981601),and contained a complete open reading frame(ORF)of 753 bp,which encoded 250 amino acid residues.Sequence analysis showed that LcAPX protein contains the heme binding site,substrate binding site,and K~+ binding site.Homology analysis indicated that the deduced LcAPX protein was highly homologoud to other APX proteins from different species.LcAPX protein had closer relationship with APXs from Solanaceae plants than from other plants.The expression of LcAPX gene was the strongest in the mature leaves of L.chinense,and the least in flowers.Furthermore,LcAPXtranscription level was induced under the treatment of PEG and NaCl stresses,and LcAPXplayed a role in resistance to drought and salt stress processes.
引文
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[6]Carolina W R,Ana P K,Julio A G,Douglas J M,Fabricio E LC,Rachel H V S,Andreia C,Felipe K T,Joaquim A G S.Rice peroxisomal ascorbate peroxidase knockdown affects ROSsignaling and triggers early leaf senescence[J].Plant Science,2017,263:55-65
[7]张馨月,王广龙,黄蔚,王枫,倪桢燚,熊爱生.胡萝卜抗坏血酸过氧化物酶基因的分离及其对非生物胁迫的响应[J].南京农业大学学报,2016,39(1):55-62Zhang X Y,Wang G L,Huang W,Wang F,Ni Z Y,Xiong A S.Cloning of the gene encoding ascorbate peroxidase from carrot and its response to abiotic stress[J].Journal of Nanjing Agricultural University,2016,39(1):55-62(in Chinese)
[8]Hou Y X,Tang H R,Zhang Y.Cloning and expression analysis of ascorbate peroxidase gene during fruit development and ripening in Fragaria×ananassa cv Toyonaka[J].World Journal of Agricultural Sciences,2009,5:675-679
[9]Liu K L,Shen L,Wang J Q,Sheng J P.Rapid inactivation of chloroplastic ascorbate peroxidase is responsible for oxidative modification to Rubisco in tomato(Lycopersicon esculentum)under cadmium stress[J].Journal of Integrative Plant Biology,2008,50(4):415-426
[10]Orvar B L,Ellis B E.Isolation of a cDNA encoding cytosolic ascorbate peroxidase in tobacco[J].Plant Physiology,1995,108(2):839-840
[11]Chen Y P,Wang H Z,Wang X E,Cao A Z,Chen P D.Cloning and expression of peroxisomal ascorbate peroxidase gene from wheat[J].Molecular Biology Reports,2006,33:207-213
[12]曾秀存,孙万仓,方彦,刘自刚,董云,孙佳,武军艳,张鹏飞,史鹏辉,孔德晶,张腾国,何丽,赵彩霞.白菜型冬油菜抗坏血酸过氧化物酶(APX)基因的克隆、表达及其活性分析[J].作物学报,2013,39(8):1400-1408Zeng X C,Sun W C,Fang Y,Liu Z G,Dong Y,Sun J,Wu J Y,Zhang P F,Shi P H,Kong D J,Zhang T G,He L,Zhao C X.Cloning,expression,and activity analysis of ascorbate peroxidase(APX)gene from winter turnip rape(Brassica campestris L)[J].Acta Agronomica Sinica,2013,39(8):1400-1408(in Chinese)
[13]Muhannda K,Ahmad W.A novel L-ascorbate peroxidase 6gene,ScAPX6,plays an important role in the regulation of response to biotic and abiotic stresses in Sugarcane[J].Frontiers in Plant Science,2017,17:2262-2273
[14]Kenneth J K,Thomas D S.Analysis of relative gene expression data using real-time quantitutive PCR and the 2-ΔΔCt method[J].Methods,2001,25(4)402-408
[15]刘娜,吴金华,安亚茹,杨君,张艳,王省芬,马峙英.陆地棉抗坏血酸过氧化物酶基因家族全基因组生物信息学分析[J].棉花学报,2017,29(1):17-28Liu N,Wu J H,An Y R,Yang J,Zhang Y,Wang X F,Ma Z Y.Bioinformatics analysis of the ascorbate peroxidase gene family in the Gossypium hirsutum L Genome[J].Cotton Science,2017,29(1):17-28(in Chinese)
[16]Shigeoka S,Ishikawa T,Tamoi M,Miyagawa Y,Takeda T,Yabuta Y.Regulation and function of ascorbate peroxidase isoenzymes[J].Journal of Experimental Botany,2002,53(372):1305-1319
[17]Cao S,Du X H,Li L H,Liu Y D,Zhang L,Pan X,Li Y,Li H,Lu H.Overexpression of Populus tomentosa cytosolic ascorbate peroxidase enhances abiotic stress tolerance in tobacco plants[J].Russian Journal of Plant Physiology,2017,64(2):224-234
[18]高海伶.枸杞抗坏血酸过氧化物酶基因LmAPX的克隆及表达分析[D].天津:天津大学,2015Gao H L.Cloning and characterization of a cytosolic ascorbate peroxidase gene LmAPX from Lycium chinense Miller[D].Tianjin:Tianjin University,2015(in Chinese)
[19]Li Y J,Hai R L,Du X H,Jiang X N,Lu H.Overexpression of a Populus peroxisomal ascorbate peroxidase(PpAPX)gene in tobacco plants enhances stress tolerance[J].Plant Breeding,2009,128(4):404-410
[20]Wu B M,Li L,Qiu T H,Zhang X,Cui S X.Cytosolic APX2is a pleiotropic protein involved in H2O2 homeostasis,chloroplast protection,plant architecture and fertility maintenance[J].Plant Cell Rep,2018,37(6):833-848
[21]Smirnoff N.Ascorbic acid metabolism and functions:Acomparison of plants and mammals[J].Free Radical Biology Medicine,2018(3):33-46
[22]Liu H C,Tian D Q,Liu J X,Ma G Y,Zou Q C,Zhu Z J.Cloning and functional analysis of a novel ascorbate peroxidase(APX)gene from Anthurium andraeanum[J].Journal of Zhejiang University-Science B:Biomedicine and Biotechnology,2013,14(12):1110-1120
[23]方涛,董艳苹,李亚楠,陈德富,陈喜文.水稻叶绿体抗坏血酸过氧化物酶在干旱和高盐胁迫中的作用[J].植物生理学报,2015,51(12):2207-2213Fang T,Dong Y P,Li Y N,Chen D F,Chen X W.Function of rice chloroplastic ascorbate peroxidases in drought and salt stress conditions[J].Plant Physiology Journal,2015,51(12):2207-2213(in Chinese)
[24]Wang Y J,Michael W,Richaed M.Overexpression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling and salt stress[J].Journal of the American Society for Horticultural Science,2005,130:167-173
[25]陈露,李卓夫,王晓楠,付连双,孙莹璐.低温下冬小麦SOD及APX酶相关基因的表达分析[J].麦类作物学报,2016,36(8):982-988Chen L,Li Z F,Wang X N,Fu L S,Sun Y L.Expression of genes related to SOD and APX enzymes in winter wheat at low temperature[J].Journal of Triticeae Crops,2016,36(8):982-988(in Chinese)
[26]Pandey S,Fartyal D,Agarwal A,Shukla T,James D,Kaul T,Negi Y K,Arora S,Reddy M K.Abiotic stress tolerance in plants:myriad roles of ascorbate peroxidase[J].Frontiers in Plant Science,2017,8:581-587
[27]Chiang C M,Chen C C,Chen S P,Lin K H,Chen L R,Su Y H,Yen H C.Overexpression of the ascorbate peroxidase gene from eggplant and sponge gourd enhances flood tolerance in transgenic Arabidopsis[J].Journal of Plant Research,2017,130(2):373-386
[28]Jiang G H,Yin D D,Zhao J J Y,Chen H L,Guo L Q,Zhu LH,Zhai W X.The rice thylakoid membrane-bound ascorbate peroxidase OsAPX8functions in tolerance to bacterial blight[J].Scientific Reports,2016(6):26104
[2]Shigeoka S,Ishikawa T,Tamoi M,Miyagawa Y,Takeda T,Yoshimura Y.Regulation and function of ascorbate peroxidase isoenzymes[J].Journal of Experimental Botany,2002,53(372):1305-1319
[3]孙卫红,王伟青,孟庆伟.植物抗坏血酸过氧化物酶的作用机制、酶学及分子特性[J].植物生理学通讯,2005,41(2):143-147Sun W H,Wang W Q,Meng Q W.Functional Mechanism and Enzymatic and Molecular Characteristic of ascorbate peroxidase in Plants[J].Plant Physiology Communication,2005,41(2):143-147(in Chinese)
[4]张维静,陆海,杜希华.抗坏血酸过氧化物酶在植物抵抗氧化胁迫中的作用[J].山东师范大学学报:自然科学版,2008,23(4):113-115Zhang W J,Lu H,Du X H.The function of ascorbate peroxidases plant resistance to oxidative stress[J].Journal of Shandong Normal University:Natural Science,2008,23(4):113-115(in Chinese)
[5]Anjum N A,Sharma P,Gill S S,Hasanuzzaman M,Khan E A,Kachhap K,Mohamed A A,Thangavel P,Devi G D,Vasudhevan P,Sofo A,Khan N A,Misra A N,Lukatkin A S,Singh H P,Pereira E,Tuteja N.Catalase and ascorbate peroxidase-representative H2O2-detoxifying heme enzymes in plants[J].Environmental Science and Pollution Research,2016,23(19):19002-19029
[6]Carolina W R,Ana P K,Julio A G,Douglas J M,Fabricio E LC,Rachel H V S,Andreia C,Felipe K T,Joaquim A G S.Rice peroxisomal ascorbate peroxidase knockdown affects ROSsignaling and triggers early leaf senescence[J].Plant Science,2017,263:55-65
[7]张馨月,王广龙,黄蔚,王枫,倪桢燚,熊爱生.胡萝卜抗坏血酸过氧化物酶基因的分离及其对非生物胁迫的响应[J].南京农业大学学报,2016,39(1):55-62Zhang X Y,Wang G L,Huang W,Wang F,Ni Z Y,Xiong A S.Cloning of the gene encoding ascorbate peroxidase from carrot and its response to abiotic stress[J].Journal of Nanjing Agricultural University,2016,39(1):55-62(in Chinese)
[8]Hou Y X,Tang H R,Zhang Y.Cloning and expression analysis of ascorbate peroxidase gene during fruit development and ripening in Fragaria×ananassa cv Toyonaka[J].World Journal of Agricultural Sciences,2009,5:675-679
[9]Liu K L,Shen L,Wang J Q,Sheng J P.Rapid inactivation of chloroplastic ascorbate peroxidase is responsible for oxidative modification to Rubisco in tomato(Lycopersicon esculentum)under cadmium stress[J].Journal of Integrative Plant Biology,2008,50(4):415-426
[10]Orvar B L,Ellis B E.Isolation of a cDNA encoding cytosolic ascorbate peroxidase in tobacco[J].Plant Physiology,1995,108(2):839-840
[11]Chen Y P,Wang H Z,Wang X E,Cao A Z,Chen P D.Cloning and expression of peroxisomal ascorbate peroxidase gene from wheat[J].Molecular Biology Reports,2006,33:207-213
[12]曾秀存,孙万仓,方彦,刘自刚,董云,孙佳,武军艳,张鹏飞,史鹏辉,孔德晶,张腾国,何丽,赵彩霞.白菜型冬油菜抗坏血酸过氧化物酶(APX)基因的克隆、表达及其活性分析[J].作物学报,2013,39(8):1400-1408Zeng X C,Sun W C,Fang Y,Liu Z G,Dong Y,Sun J,Wu J Y,Zhang P F,Shi P H,Kong D J,Zhang T G,He L,Zhao C X.Cloning,expression,and activity analysis of ascorbate peroxidase(APX)gene from winter turnip rape(Brassica campestris L)[J].Acta Agronomica Sinica,2013,39(8):1400-1408(in Chinese)
[13]Muhannda K,Ahmad W.A novel L-ascorbate peroxidase 6gene,ScAPX6,plays an important role in the regulation of response to biotic and abiotic stresses in Sugarcane[J].Frontiers in Plant Science,2017,17:2262-2273
[14]Kenneth J K,Thomas D S.Analysis of relative gene expression data using real-time quantitutive PCR and the 2-ΔΔCt method[J].Methods,2001,25(4)402-408
[15]刘娜,吴金华,安亚茹,杨君,张艳,王省芬,马峙英.陆地棉抗坏血酸过氧化物酶基因家族全基因组生物信息学分析[J].棉花学报,2017,29(1):17-28Liu N,Wu J H,An Y R,Yang J,Zhang Y,Wang X F,Ma Z Y.Bioinformatics analysis of the ascorbate peroxidase gene family in the Gossypium hirsutum L Genome[J].Cotton Science,2017,29(1):17-28(in Chinese)
[16]Shigeoka S,Ishikawa T,Tamoi M,Miyagawa Y,Takeda T,Yabuta Y.Regulation and function of ascorbate peroxidase isoenzymes[J].Journal of Experimental Botany,2002,53(372):1305-1319
[17]Cao S,Du X H,Li L H,Liu Y D,Zhang L,Pan X,Li Y,Li H,Lu H.Overexpression of Populus tomentosa cytosolic ascorbate peroxidase enhances abiotic stress tolerance in tobacco plants[J].Russian Journal of Plant Physiology,2017,64(2):224-234
[18]高海伶.枸杞抗坏血酸过氧化物酶基因LmAPX的克隆及表达分析[D].天津:天津大学,2015Gao H L.Cloning and characterization of a cytosolic ascorbate peroxidase gene LmAPX from Lycium chinense Miller[D].Tianjin:Tianjin University,2015(in Chinese)
[19]Li Y J,Hai R L,Du X H,Jiang X N,Lu H.Overexpression of a Populus peroxisomal ascorbate peroxidase(PpAPX)gene in tobacco plants enhances stress tolerance[J].Plant Breeding,2009,128(4):404-410
[20]Wu B M,Li L,Qiu T H,Zhang X,Cui S X.Cytosolic APX2is a pleiotropic protein involved in H2O2 homeostasis,chloroplast protection,plant architecture and fertility maintenance[J].Plant Cell Rep,2018,37(6):833-848
[21]Smirnoff N.Ascorbic acid metabolism and functions:Acomparison of plants and mammals[J].Free Radical Biology Medicine,2018(3):33-46
[22]Liu H C,Tian D Q,Liu J X,Ma G Y,Zou Q C,Zhu Z J.Cloning and functional analysis of a novel ascorbate peroxidase(APX)gene from Anthurium andraeanum[J].Journal of Zhejiang University-Science B:Biomedicine and Biotechnology,2013,14(12):1110-1120
[23]方涛,董艳苹,李亚楠,陈德富,陈喜文.水稻叶绿体抗坏血酸过氧化物酶在干旱和高盐胁迫中的作用[J].植物生理学报,2015,51(12):2207-2213Fang T,Dong Y P,Li Y N,Chen D F,Chen X W.Function of rice chloroplastic ascorbate peroxidases in drought and salt stress conditions[J].Plant Physiology Journal,2015,51(12):2207-2213(in Chinese)
[24]Wang Y J,Michael W,Richaed M.Overexpression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling and salt stress[J].Journal of the American Society for Horticultural Science,2005,130:167-173
[25]陈露,李卓夫,王晓楠,付连双,孙莹璐.低温下冬小麦SOD及APX酶相关基因的表达分析[J].麦类作物学报,2016,36(8):982-988Chen L,Li Z F,Wang X N,Fu L S,Sun Y L.Expression of genes related to SOD and APX enzymes in winter wheat at low temperature[J].Journal of Triticeae Crops,2016,36(8):982-988(in Chinese)
[26]Pandey S,Fartyal D,Agarwal A,Shukla T,James D,Kaul T,Negi Y K,Arora S,Reddy M K.Abiotic stress tolerance in plants:myriad roles of ascorbate peroxidase[J].Frontiers in Plant Science,2017,8:581-587
[27]Chiang C M,Chen C C,Chen S P,Lin K H,Chen L R,Su Y H,Yen H C.Overexpression of the ascorbate peroxidase gene from eggplant and sponge gourd enhances flood tolerance in transgenic Arabidopsis[J].Journal of Plant Research,2017,130(2):373-386
[28]Jiang G H,Yin D D,Zhao J J Y,Chen H L,Guo L Q,Zhu LH,Zhai W X.The rice thylakoid membrane-bound ascorbate peroxidase OsAPX8functions in tolerance to bacterial blight[J].Scientific Reports,2016(6):26104