烟草过氧化氢酶基因CAT2克隆与表达特征分析
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摘要
基于已有的植物Catalase 2基因(CAT2)序列设计烟草Nicotiana tabacum CAT2的特异性引物,通过RT-PCR扩增克隆获得Nicotiana tabacum var. NC89 CAT2全长mRNA序列,包含1479 bp完整的读码框(ORF),可编码492个氨基酸残基。遗传进化分析显示,烟草NC89 CAT2与N. benthamiana CAT2基因亲缘关系最近。利用Quantitative Real-time PCR技术分析了CAT2基因在NC89烟草中的组织特异性表达和其应对生物和非生物胁迫的表达差异。结果表明,CAT2基因在烟草NC89的根、茎、叶、花瓣、花萼、种子中均有表达,其中在花中相对表达量最高,其次为叶、茎、种子和根。生物和非生物胁迫处理烟株,其CAT2诱导表达分析显示,机械损伤、渗透压、低温和高温、干旱和感染PVY时CAT2表达量均上调。上述研究表明烟草CAT2基因可能参与了应对非生物和生物胁迫的过程。
The full-length cDNA of Catalase 2(CAT2) from Nicotiana tabacum var. NC89 was cloned usingspecific primers designed according to CAT2 mRNA sequences in other plants. Amino acid sequence alignment indicated that CAT2 contains a complete reading frame(ORF) of 1479 bp, which encodes 492 amino acid residues. Phylogenetic analysis showed that NC89 CAT2 shares high similarity with the CAT2 gene from N. benthamiana. Quantitative real-time PCR(qPCR) analysis revealed that CAT2 is highly expressed in petal and calyx, while less expressed in leaves, stems, seeds, and roots. The expression of NC89 CAT2 was enhanced by abiotic and biotic stressors, such as mechanical damage, osmotic pressure, low temperature, high temperature, drought and PVY infection. It was suggested that CAT2 plays an important role in growth, development, and interplay of abiotic and biotic stresses in plant.
The full-length cDNA of Catalase 2(CAT2) from Nicotiana tabacum var. NC89 was cloned usingspecific primers designed according to CAT2 mRNA sequences in other plants. Amino acid sequence alignment indicated that CAT2 contains a complete reading frame(ORF) of 1479 bp, which encodes 492 amino acid residues. Phylogenetic analysis showed that NC89 CAT2 shares high similarity with the CAT2 gene from N. benthamiana. Quantitative real-time PCR(qPCR) analysis revealed that CAT2 is highly expressed in petal and calyx, while less expressed in leaves, stems, seeds, and roots. The expression of NC89 CAT2 was enhanced by abiotic and biotic stressors, such as mechanical damage, osmotic pressure, low temperature, high temperature, drought and PVY infection. It was suggested that CAT2 plays an important role in growth, development, and interplay of abiotic and biotic stresses in plant.
引文
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[20]J KIM J Y,PARK S J,JANG B,et al.Functional characterization of a glycine-rich RNA-binding protein 2in Arabidopsis thaliana under abiotic stress conditions[J].Plant Journal,2010,50(3):439-451.
[21]FARIDUDDIN Q,MIR B A,AHMAD A.Physiological and biochemical traits as tools to screen sensitive and resistant varieties of tomatoes exposed to salt stress[J].Brazilian Journal of Plant Physiology,2012,24(4):281-292.
[22]胡义,胡庭兴,陈洪,等.干旱胁迫及复水对香樟幼树生理特性及生长的影响[J].西北植物学报,2015,35(2):294-301.HU Y,HU T X,CHEN H,et al.Physiological properties and growth of cinnamomum camphora saplings under drought stress and rewatering[J].Acta Botanica Boreali-Occidentalia Sinica,2015,35(2):294-301.
[23]MASOUMI H,MASOUMI M,DARVISH F,et al.Change in several antioxidant enzymes activity and seed yield by water deficit stress in soybean(Glycine max L.)cultivars[J].Notulae Botanicae Horti Agrobotanici Cluj-Napoca,2010,38(3):86-94.
[24]HE L,JIA X,GAO Z,et al.Genotype-dependent responses of wheat(Triticum aestivum L.)seedlings to drought,UV-B radiation and their combined stresses[J].African Journal of Biotechnology,2011,1020(20):4046-4056.
[25]BOLDT R,SCANDALIOS J G.Influence of UV-light on the expression of the Cat2 and Cat3 catalase genes in maize[J].Free Radic Biol Med,1997,23(3):505-514.
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[27]KWON S I,LEE H,AN C S.Differential expression of three catalase genes in the small radish(Rhaphanus sativus L.var.sativus)[J].Molecules and Cells,2007,24(1):37-44.
[28]RIZHSKY L,HALLAK-HERR E,VAN BREUSEGEMF,et al.Double antisense plants lacking ascorbate peroxidase and catalase are less sensitive to oxidative stress than single antisense plants lacking ascorbate peroxidase or catalase[J].The Plant Journal,2002,32(3):329-342.
[29]杨丽烨,吴元华,赵秀香.烤烟品种K326抗PVYN突变株SN02的筛选及其抗病生理生化特性[J].烟草科技,2010(9):61-64.YANG L Y,WU Y H,ZHAO X X.Screening of tobacco mutant plant SN02 against PVYN from K326 and physiological and biochemical properties of the same[J].Tobacco Science&Technology,2010(9):61-64.
[30]陈学平,姚忠达,郭家明,等.不同烟草品种感染TMV病程过程中CAT,PAL活力变化研究[J].安徽农业大学学报,2002,29(2):103-107.CHEN X P,YAO Z D,GUO J M,et al.Studies on the CAT and PAL vigors of various tobacco varieties response to TMV[J].Journal of Anhui Agricultural University,2002,29(2):103-107.
[31]吴元华,文才艺,李浩戈,等.烟草感染马铃薯Y病毒脉坏死株系后六种酶活性变化的研究[J].中国烟草学报,1999,5(2):30-34.WU Y H,WEN C Y,LI H G,et al.Changes on the activities of six enzymes of tobacco leaves infected with potato virus Y-vein necrosis strain[J].Acta Tabacaria Sinica,1999,5(2):30-34.
[2]陈卫国,刘勇,周冀衡.低温胁迫对烟草膜保护酶系统的影响[J].安徽农业科学,2011,39(7),3978-3980.CHEN W G,LIU Y,ZHOU J H.Influence of low temperature stress on the membrane-protected enzyme system in tobacco[J].Journal of Anhui Agricultural Sciences,2011,39(7):3978-3980.
[3]贾晨晨.葡萄过氧化氢酶基因在毕赤酵母中的表达及酶学特性的初级研究[D].南京:南京农业大学,2012.JIA C C.Expression,characterization and the primaryresearch of catalase from vitis vinifera in pichia pastoris[D].Nanjing:Nanjing Agricultural University,2012.
[4]SAXENA I,SRIKANTH S,CHEN Z.Cross talk betweenH2O2 and interacting signal molecules under plant stress response[J].Frontiers in Plant Science,2016,7:570.https://www.frontiersin.org/article/10.3389/fpls.2016.00570.
[5]MAILLOUX R J,MCBRIDE S L,HARPER M E.Unearthing the secrets of mitochondrial ROS and glutathione in bioenergetics[J].Trends in Biochemical Sciences,2013,38(12):592-602.
[6]宋新华,赵凤云.植物体内过氧化氢酶的研究进展[J].安徽农业科学,2007,35(31):7-10.SONG X H,ZHAO F Y.Research progress on catalase in plants[J].Journal of Anhui Agricultural Sciences,2007,35(31):7-10.
[7]MARSHALL O J.PerlPrimer:cross-platform,graphical primer design for standard,bisulphite and real-time PCR[J].Bioinformatics,2004,20(15):2471-2472.
[8]REILLY K,HAN Y,TOHME J,et al.Isolation and characterisation of a cassava catalase expressed during post-harvest physiological deterioration 1[J].Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,2001,1518(3):317-323.
[9]AZOOZ M M,Ismail A M,Elhamd M F A.Growth,lipid peroxidation and antioxidan enzyme activities as a selection criterion for the salt tolerance of maize cultivars grown under salinity stress[J].International Journal of Agriculture&Biology,2009,11(1):572-577.
[10]GILL S S,KHAN N A,TUTEJA N.Differential cadmium stress tolerance in five indian mustard(Brassica juncea L.)cultivars:an evaluation of the role of antioxidant machinery[J].Plant Signaling&Behavior,2011,6(2):293-300.
[11]HAFEZ Y M.A pivotal role of reactive oxygen species and antioxidants to attenuate Tobacco mosaic virus[J].Egyptian Journal of Pest Control,2013,23(2):277-285.
[12]WADSWORTH G J,SCANDALIOS J G.Differential expression of the maize catalase genes during kernel development:the role of steady-state m RNA levels[J].Genesis,2010,10(4):304-310.
[13]REDINBAUGH M G,WADSWORTH G J,SCANDALIOS J G.Characterization of catalase transcripts and their differential expression in maize[J].Biochimica Et Biophysica Acta(bba)-gene Structure and Expression,1988,951(1):104-116.
[14]高翔.拟南芥cat2突变体中叶片偏下性生长机理的研究[D].武汉:武汉大学,2013.GAO X.Mutation of Arabidopsis CATALASE2 results in hyponastic leaves by changes of auxin levels[D].Wuhan:Wuhan University,2013.
[15]SYTYKIEWICZ H.Transcriptional responses of catalase genes in maize seedlings exposed to cereal aphids'herbivory[J].Biochemical Systematics&Ecology,2015,60:131-142.
[16]JUNG S,CHON S U,KUK Y I.Differential antioxidant responses in catalase-deficient maize mutants exposed to norflurazon[J].Biologia Plantarum,2006,50(3):383-388.
[17]BO?OVáB,HUTTOVáJ,Liptáková?,et al.Impact of short-term cadmium treatment on catalase and ascorbate peroxidase activities in barley root tips[J].Biologia Plantarum,2012,56(4):724-728.
[18]MUTLU S,ATICI?.Alleviation of high salt toxicity-induced oxidative damage by salicylic acid pretreatment in two wheat cultivars[J].Toxicology&Industrial Health,2013,29(1):89-96.
[19]El-MONEM A,SHARAF M.Improvement growth,and yield of wheat plants grown under salinity stress by using silicon[J].Journal of American Science,2010,66:559-566.
[20]J KIM J Y,PARK S J,JANG B,et al.Functional characterization of a glycine-rich RNA-binding protein 2in Arabidopsis thaliana under abiotic stress conditions[J].Plant Journal,2010,50(3):439-451.
[21]FARIDUDDIN Q,MIR B A,AHMAD A.Physiological and biochemical traits as tools to screen sensitive and resistant varieties of tomatoes exposed to salt stress[J].Brazilian Journal of Plant Physiology,2012,24(4):281-292.
[22]胡义,胡庭兴,陈洪,等.干旱胁迫及复水对香樟幼树生理特性及生长的影响[J].西北植物学报,2015,35(2):294-301.HU Y,HU T X,CHEN H,et al.Physiological properties and growth of cinnamomum camphora saplings under drought stress and rewatering[J].Acta Botanica Boreali-Occidentalia Sinica,2015,35(2):294-301.
[23]MASOUMI H,MASOUMI M,DARVISH F,et al.Change in several antioxidant enzymes activity and seed yield by water deficit stress in soybean(Glycine max L.)cultivars[J].Notulae Botanicae Horti Agrobotanici Cluj-Napoca,2010,38(3):86-94.
[24]HE L,JIA X,GAO Z,et al.Genotype-dependent responses of wheat(Triticum aestivum L.)seedlings to drought,UV-B radiation and their combined stresses[J].African Journal of Biotechnology,2011,1020(20):4046-4056.
[25]BOLDT R,SCANDALIOS J G.Influence of UV-light on the expression of the Cat2 and Cat3 catalase genes in maize[J].Free Radic Biol Med,1997,23(3):505-514.
[26]BOLDT R,SCANDALIOS J G.Circadian regulation of the Cat3,catalase gene in maize(Zea mays,L.):entrainment of the circadian rhythm of Cat3,by different light treatments[J].Plant Journal,2010,7(6):989-999.
[27]KWON S I,LEE H,AN C S.Differential expression of three catalase genes in the small radish(Rhaphanus sativus L.var.sativus)[J].Molecules and Cells,2007,24(1):37-44.
[28]RIZHSKY L,HALLAK-HERR E,VAN BREUSEGEMF,et al.Double antisense plants lacking ascorbate peroxidase and catalase are less sensitive to oxidative stress than single antisense plants lacking ascorbate peroxidase or catalase[J].The Plant Journal,2002,32(3):329-342.
[29]杨丽烨,吴元华,赵秀香.烤烟品种K326抗PVYN突变株SN02的筛选及其抗病生理生化特性[J].烟草科技,2010(9):61-64.YANG L Y,WU Y H,ZHAO X X.Screening of tobacco mutant plant SN02 against PVYN from K326 and physiological and biochemical properties of the same[J].Tobacco Science&Technology,2010(9):61-64.
[30]陈学平,姚忠达,郭家明,等.不同烟草品种感染TMV病程过程中CAT,PAL活力变化研究[J].安徽农业大学学报,2002,29(2):103-107.CHEN X P,YAO Z D,GUO J M,et al.Studies on the CAT and PAL vigors of various tobacco varieties response to TMV[J].Journal of Anhui Agricultural University,2002,29(2):103-107.
[31]吴元华,文才艺,李浩戈,等.烟草感染马铃薯Y病毒脉坏死株系后六种酶活性变化的研究[J].中国烟草学报,1999,5(2):30-34.WU Y H,WEN C Y,LI H G,et al.Changes on the activities of six enzymes of tobacco leaves infected with potato virus Y-vein necrosis strain[J].Acta Tabacaria Sinica,1999,5(2):30-34.