雷公藤AP2/ERF转录因子基因的克隆与分析
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摘要
该研究以雷公藤发状根为材料,根据雷公藤根转录组数据设计引物,采用RT-PCR方法克隆得到2个雷公藤AP2/ERF转录因子,分别命名为TwAP2/ERF1基因(GenBank登录号:GAVZ01042389.1)和TwAP2/ERF2基因(GenBank登录号:GAVZ01016765.1)。TwAP2/ERF1基因含有一个525bp开放阅读框(ORF),编码186个氨基酸;TwAP2/ERF2基因的ORF为789bp,编码262个氨基酸;2个基因编码的蛋白质均为亲水性蛋白质。系统进化分析表明,TwAP2/ERF1与油桐(Vernicia fordii)AP2/ERF(APQ47444.1)和木油桐(Vernicia montana)AP2/ERF(APQ47365.1)相似性较高,TwAP2/ERF2与毛果杨(Populus trichocarpa)AP2/ERF(XP_002304640.1)和樱桃(Prunus pseudocerasus)AP2/ERF(ALD84477.1)相似性较高。雷公藤发状根经MeJA诱导后,TwAP2/ERF1基因的相对表达量明显提高,并于处理后9h达到最高值,为对照表达量的16.77倍;而MeJA处理对TwAP2/ERF2基因的表达表现出抑制作用,但于处理后48h相对表达量有所提高。研究表明,雷公藤TwAP2/ERF1转录因子响应MeJA早期诱导正调控,推测其可能参与调控雷公藤植物次生代谢产物的生物合成,该研究结果为阐明雷公藤次生代谢物质的生物合成调控与利用现代生物技术提高雷公藤植物细胞中次生代谢物质的含量奠定了基础。
In this paper,the hairy roots of T.wilfordii were used as the experimental material.Primers were designed based on the transcriptomic data of T.wilfordii roots.We cloned the two AP2/ERF transcription factors fromT.wilfordii hairy roots using reverse transcription polymerase chain reaction(RTPCR),separately named TwAP2/ERF1(GenBank accession GAVZ01042389.1)and TwAP2/ERF2(GenBank accession GAVZ01016765.1).The TwAP2/ERF1 gene contained 525 bp open reading frame(ORF)which encoding 186 amino acids.The ORF of the TwAP2/ERF2 gene was 789 bp,which encoding 262 amino acids.Both genes encode proteins that were hydrophilic proteins.The phylogenetic analysis showed that TwAP2/ERF1 was highly similar with VerniciafordiiAP2/ERF(APQ47444.1)and Vernicia montana AP2/ERF(APQ47365.1).TwAP2/ERF2 was closely related to Populus trichocarpa AP2/ERF(XP_002304640.1)and Prunus pseudocerasus AP2/ERF(ALD84477.1).After methyl jasmonate(MeJA)treatment in T.wilfordii hairy roots,the relative expression of TwAP2/ERF1 gene was significantly increased and reached the highest level at 9 h,which was 16.77 times than the control expression.In the early stage MeJA inhibited the expression of TwAP2/ERF2 gene,but after 48 hthe relative expression level increased.These results showed that TwAP2/ERF1 response positively MeJA induction in early stage and probably involve the regulation of secondary metabolites biosynthesis in T.wilfordii.These will provide the basis for elucidating the regulation of secondary metabolites biosynthesis and for using biotechnology to enhance the content of secondary metabolites in T.wilfordii.
In this paper,the hairy roots of T.wilfordii were used as the experimental material.Primers were designed based on the transcriptomic data of T.wilfordii roots.We cloned the two AP2/ERF transcription factors fromT.wilfordii hairy roots using reverse transcription polymerase chain reaction(RTPCR),separately named TwAP2/ERF1(GenBank accession GAVZ01042389.1)and TwAP2/ERF2(GenBank accession GAVZ01016765.1).The TwAP2/ERF1 gene contained 525 bp open reading frame(ORF)which encoding 186 amino acids.The ORF of the TwAP2/ERF2 gene was 789 bp,which encoding 262 amino acids.Both genes encode proteins that were hydrophilic proteins.The phylogenetic analysis showed that TwAP2/ERF1 was highly similar with VerniciafordiiAP2/ERF(APQ47444.1)and Vernicia montana AP2/ERF(APQ47365.1).TwAP2/ERF2 was closely related to Populus trichocarpa AP2/ERF(XP_002304640.1)and Prunus pseudocerasus AP2/ERF(ALD84477.1).After methyl jasmonate(MeJA)treatment in T.wilfordii hairy roots,the relative expression of TwAP2/ERF1 gene was significantly increased and reached the highest level at 9 h,which was 16.77 times than the control expression.In the early stage MeJA inhibited the expression of TwAP2/ERF2 gene,but after 48 hthe relative expression level increased.These results showed that TwAP2/ERF1 response positively MeJA induction in early stage and probably involve the regulation of secondary metabolites biosynthesis in T.wilfordii.These will provide the basis for elucidating the regulation of secondary metabolites biosynthesis and for using biotechnology to enhance the content of secondary metabolites in T.wilfordii.
引文
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[14]LU X,ZHANG L,ZHANG F,et al.AaORA,a trichomespecific AP2/ERF transcription factor of Artemisia annua,is apositive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea[J].New Phytologist,2013,198(4):1 191-1 202.
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[16]DAI Y,QIN Q,DAI D,et al.Isolation and characterization of a novel cDNA encoding methyl jasmonate-responsive transcription factor TcAP2from Taxus cuspidata[J].Biotechnology Letters,2009,31(11):1 801-1 809.
[17]MENKE F L,CHAMPION A,KIJNE J W,et al.A novel jasmonate-and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate-and elicitor-inducible AP2-domain transcription factor,ORCA2[J].Embo Journal,1999,18:4 455-4 463.
[18]VAN D F L,MEMELINK J.ORCA3,ajasmonate-responsive transcriptional regulator of plant primary and secondary metabolism[J].Science,2000,289(5 477):295-297.
[19]ZHU C S,MIAO G P,GUO J,et al.Establishment of Tripterygium wilfordii Hook.f.hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine[J].Journal Microbiology Biotechnology,2014,24(6):823-834.
[20]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408.
[21]ALLEN M D,YAMASAKI K,OHME-TAKAGI M,et al.A novel mode of DNA recognition by aβ-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[J].Embo Journal,1998,17(18):5 484-5 496.
[22]GEYTER D,NATHAN,GHOLAMI,et al.Transcriptional machineries in jasmonate-elicited plant secondary metabolism[J].Trends in Plant Science,2012,17(6):349-359.
[2]刘强,张贵友.植物转录因子的结构与调控作[J].科学通报,2000,45(14):1 465-1 474.LIU Q,ZHANG G Y.The structure and regulation function of plant transcription factors[J].Chinese Science Bulletin,2000,45(14):1 465-1 474.
[3]RIECHMANN J L,MEYROWITZ E M.The AP2/EREBP family of plant transcription factors[J].Biological Chemistry,1998,379:633-646.
[4]TOSHITSUGU N,KAORU S,TATSUHITO F,et al.Genome-wide analysis of the ERF gene family in Arabidopsis and rice[J].Plant Physiology,2006,140(2):411-432.
[5]ZHANG G Y,CHEN M,CHEN X P,et al.Phylogeny,gene structures,and expression patterns of the ERF gene family in soybean(Glycine max L.)[J].Journal of Experimental Botany,2008,59(15):4 095-4 107.
[6]SHARONI A M,NURUZZAMAN M,SATOH K,et al.Gene structures,classification and expression models of the AP2/EREBP transcription factor family in rice[J].Plant and Cell Physiology,2011,52(2):344-360.
[7]LICAUSI F,GIORGI F M,ZENONI S,et al.Genomic and transcriptomic analysis of the AP2/ERF superfamily in Vitis vinifera[J].BMC Genomics,2010,11(1):719.
[8]SEARS M T,ZHANG H,RUSHTON P J,et al.NtERF32:a non-NIC2locus AP2/ERF transcription factor required in jasmonate-inducible nicotine biosynthesis in tobacco[J].Plant Molecular Biology,2014,84(1/2):49-66.
[9]XU Z S,CHEN M,LI L C,et al.Functions and application of the AP2/ERF transcription factor family in crop improvement[J].Journal of Integrative Plant Biology,2011,53(7):570-585.
[10]MIZOI J,SHINOZAKI K,YAMAGUCHI-SHINOZAKI K.AP2/ERF family transcription factors in plant abiotic stress responses[J].Biochimica et Biophysica Acta(BBA)-Gene Regulatory Mechanisms,2012,1 819(2):86-96.
[11]LICAUSI F,OHME-TAKAGI M,PERATA P.APETALA2/Ethylene Responsive Factor(AP2/ERF)transcription factors:Mediators of stress responses and developmental programs[J].New Phytologist,2013,199(3):639-649.
[12]LEE D K,JUNG H,JANG G,et al.Overexpression of the OsERF71transcription factor alters rice root structure and drought resistance[J].Plant Physiology,2016,172(1):575-588.
[13]YU Z X,LI J X,YANG C Q,et al.The jasmonate-responsive AP2/ERF transcription factors AaERF1and AaERF2positively regulate artemisinin biosynthesis in Artemisia annua L.[J].Molecular Plant,2012,5:353-365.
[14]LU X,ZHANG L,ZHANG F,et al.AaORA,a trichomespecific AP2/ERF transcription factor of Artemisia annua,is apositive regulator in the artemisinin biosynthetic pathway and in disease resistance to Botrytis cinerea[J].New Phytologist,2013,198(4):1 191-1 202.
[15]TAN H,XIAO L,GAO S,et al.Trichome and artemisinin regulator 1is required for trichome development and artemisinin biosynthesis in Artemisia annua L.[J].Molecular Plant,2015,8(9):1 396-1 411.
[16]DAI Y,QIN Q,DAI D,et al.Isolation and characterization of a novel cDNA encoding methyl jasmonate-responsive transcription factor TcAP2from Taxus cuspidata[J].Biotechnology Letters,2009,31(11):1 801-1 809.
[17]MENKE F L,CHAMPION A,KIJNE J W,et al.A novel jasmonate-and elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate-and elicitor-inducible AP2-domain transcription factor,ORCA2[J].Embo Journal,1999,18:4 455-4 463.
[18]VAN D F L,MEMELINK J.ORCA3,ajasmonate-responsive transcriptional regulator of plant primary and secondary metabolism[J].Science,2000,289(5 477):295-297.
[19]ZHU C S,MIAO G P,GUO J,et al.Establishment of Tripterygium wilfordii Hook.f.hairy root culture and optimization of its culture conditions for the production of triptolide and wilforine[J].Journal Microbiology Biotechnology,2014,24(6):823-834.
[20]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408.
[21]ALLEN M D,YAMASAKI K,OHME-TAKAGI M,et al.A novel mode of DNA recognition by aβ-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[J].Embo Journal,1998,17(18):5 484-5 496.
[22]GEYTER D,NATHAN,GHOLAMI,et al.Transcriptional machineries in jasmonate-elicited plant secondary metabolism[J].Trends in Plant Science,2012,17(6):349-359.
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