厚藤脱水素基因IpDHN启动子IpDHN-Pro的克隆和调控转录活性分析
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摘要
为了解厚藤(Ipomoea pes-caprae)脱水素基因IpDHN (GenBank登录号:KX426069)启动子的转录活性和对非生物胁迫和植物激素ABA的响应,通过染色体步移法克隆了IpDHN的上游启动子序列IpDHN-Pro,长度为974 bp。构建IpDHN-Pro调控下GUS转基因载体,转化拟南芥(Arabidopsis thaliana)植株获得IpDHN-Pro::GUS转基因植株并进行GUS染色,验证IpDHN-Pro启动转录活性以及在氯化钠、甘露醇、ABA处理后拟南芥GUS基因表达变化。结果表明,扩增获得的IpDHN-Pro序列包含多个顺式作用元件,包括1个ABRE、3个Myb转录因子结合位点、富含TC的重复序列以及Skn-1基序等。转基因拟南芥GUS染色及qRT-PCR表明该序列可驱动GUS基因在拟南芥稳定表达,且表达受高盐、渗透压及ABA的诱导。这表明IpDHN-Pro是一个盐旱、ABA诱导的启动子序列,可应用于相关的植物抗逆遗传工程研究。
Based on the previous research about Ipomoea pes-caprae cDNA library screening, a full length cDNA encoding dehydrin was characterized and named as IpDHN(GenBank accession: No. KX426069). In order to explore the transcriptional activity of the Ip DHN promoter and the characteristics of this promoter region responding to abiotic stresses and ABA, the 5' upstream sequence of Ip DHN(974 bp), named as IpDHN-Pro was cloned by genomic walking method, and then was subcloned into plant expression vector, in which GUS is under control of Ip DHN-Pro. The transgenic Arabidopsis plants harboring Ip DHN-Pro::GUS were checked by GUS staining assay, then the expression of GUS was further detected by qRT-PCR after the transgenic plants were challenged by NaCl, mannitol and ABA. The results showed that the Ip DHN-Pro contained several cis-acting elements, including an ABRE, three Myb transcription factor binding sites, a TC-rich repeat, and a Skn-1 motif.The GUS staining and q RT-PCR showed that Ip DHN-Pro could drive the stable expression of GUS gene in transgenic Arabidopsis, and this promoter was up-regulated by high salinity, osmotic stress and ABA treatment.Therefore, it was indicated that IpDHN-Pro was a salt/dehydration and ABA induced promoter sequence, and can be applied in plant genetic engineering research aiming at abiotic stress tolerance.
Based on the previous research about Ipomoea pes-caprae cDNA library screening, a full length cDNA encoding dehydrin was characterized and named as IpDHN(GenBank accession: No. KX426069). In order to explore the transcriptional activity of the Ip DHN promoter and the characteristics of this promoter region responding to abiotic stresses and ABA, the 5' upstream sequence of Ip DHN(974 bp), named as IpDHN-Pro was cloned by genomic walking method, and then was subcloned into plant expression vector, in which GUS is under control of Ip DHN-Pro. The transgenic Arabidopsis plants harboring Ip DHN-Pro::GUS were checked by GUS staining assay, then the expression of GUS was further detected by qRT-PCR after the transgenic plants were challenged by NaCl, mannitol and ABA. The results showed that the Ip DHN-Pro contained several cis-acting elements, including an ABRE, three Myb transcription factor binding sites, a TC-rich repeat, and a Skn-1 motif.The GUS staining and q RT-PCR showed that Ip DHN-Pro could drive the stable expression of GUS gene in transgenic Arabidopsis, and this promoter was up-regulated by high salinity, osmotic stress and ABA treatment.Therefore, it was indicated that IpDHN-Pro was a salt/dehydration and ABA induced promoter sequence, and can be applied in plant genetic engineering research aiming at abiotic stress tolerance.
引文
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[17]MISHRA A,TANNA B.Halophytes:Potential resources for salt stress tolerance genes and promoters[J].Front Plant Sci,2017,8:829.doi:10.3389/fpls.2017.00829.
[18]HOBO T,ASADA M,KOWYAMA Y,et al.ACGT-containing abscisic acid response element(ABRE)and coupling element 3(CE3)are functionally equivalent[J].Plant J,1999,19(6):679-689.doi:10.1046/j.1365-313x.1999.00565.x.
[19]MISHRA S,SHUKLA A,UPADHYAY S,et al.Identification,occurrence,and validation of DRE and ABRE cis-regulatory motifs in the promoter regions of genes of Arabidopsis thaliana[J].J Integr Plant Biol,2014,56(4):388-399.doi:10.1111/jipb.12149.
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[21]URAO T,YAMAGUCHI-SHINOZAKI K,URAO S,et al.An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence[J].Plant Cell,1993,5(11):1529-1539.doi:10.1105/tpc.5.11.1529.
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[23]GERMAIN H,LACHANCE D,PELLETIER G,et al.The expression pattern of the Picea glauca Defensin 1 promoter is maintained in Arabidopsis thaliana,indicating the conservation of signalling pathways between angiosperms and gymnosperms[J].J Exp Bot,2012,63(2):785-795.doi:10.1093/jxb/err303.
[24]FAUTEUX F,STR?MVIK M V.Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae,Fabaceae and Poaceae[J].BMC Plant Biol,2009,9:126.doi:10.1186/1471-2229-9-126.
[2]OUYANG P Y,LIU N,ZHANG W W,et al.Biological and ecophysiological characteristics of a beach plant Ipomoea pescaprae[J].JHunan Univ Sci Technol(Nat Sci),2011,26(4):117-121.doi:10.3969/j.issn.1672-9102.2011.04.025.欧阳蒲月,刘楠,张伟伟,等.海滩植物厚藤(Ipomoea pescaprae)的生物学及生理生态特性[J].湖南科技大学学报(自然科学版),2011,26(4):117-121.doi:10.3969/j.issn.1672-9102.2011.04.025.
[3]GRAETHER S P,BODDINGTON K F.Disorder and function:Areview of the dehydrin protein family[J].Front Plant Sci,2014,5:576.doi:10.3389/fpls.2014.00576.
[4]HAND S C,MENZE M A,TONER M,et al.LEA proteins during water stress:Not just for plants anymore[J].Annu Rev Physiol,2011,73:115-134.doi:10.1146/annurev-physiol-012110-142203.
[5]BIES-ETHèVE N,GAUBIER-COMELLA P,DEBURES A,et al.Inventory,evolution and expression profiling diversity of the LEA(late embryogenesis abundant)protein gene family in Arabidopsis thaliana[J].Plant Mol Biol,2008,67(1/2):107-124.doi:10.1007/s11103-008-9304-x.
[6]CLOSE T J.Dehydrins:Emergence of a biochemical role of a family of plant dehydration proteins[J].Physiol Plant,1996,97(4):795-803.doi:10.1111/j.1399-3054.1996.tb00546.x.
[7]OUELLET F,HOUDE M,SARHAN F.Purification,characterization and cDNA cloning of the 200 kDa protein induced by cold acclimation in wheat[J].Plant Cell Physiol,1993,34(1):59-65.doi:10.1093/oxfordjournals.pcp.a078400.
[8]HANIN M,BRINI F,EBEL C,et al.Plant dehydrins and stress tolerance:Versatile proteins for complex mechanisms[J].Plant Signal Behav,2011,6(10):1503-1509.doi:10.4161/psb.6.10.17088.
[9]ZHOU Y,HU L F,XU S Y,et al.Identification and transcriptional analysis of dehydrin gene family in cucumber(Cucumis sativus)[J].Acta Physiol Plant,2018,40(8):144.doi:10.1007/s11738-018-2715-7.
[10]YANG Y Z,HE M Y,ZHU Z G,et al.Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress[J].BMC Plant Biol,2012,12:140.doi:10.1186/1471-2229-12-140.
[11]VAZQUEZ-HERNANDEZ M,ROMERO I,ESCRIBANO M I,et al.Deciphering the role of CBF/DREB transcription factors and dehydrins in maintaining the quality of table grapes cv.autumn royal treated with high CO2 levels and stored at 0℃[J].Front Plant Sci,2017,8:1591.doi:10.3389/fpls.2017.01591.
[12]ZHU W N,ZHANG L S,LüH,et al.The dehydrin wzy2 promoter from wheat defines its contribution to stress tolerance[J].Funct Integr Genomics,2014,14(1):111-125.doi:10.1007/s10142-013-0354-z.
[13]ABEDINI R,GHANEGOLMOHAMMADI F,PISHKAMRAD R,et al.Plant dehydrins:Shedding light on structure and expression patterns of dehydrin gene family in barley[J].J Plant Res,2017,130(4):747-763.doi:10.1007/s10265-017-0941-5.
[14]ZHANG H,ZHENG J X,SU H X,et al.Molecular cloning and functional characterization of the dehydrin(IpDHN)gene from Ipomoea pes-caprae[J].Front Plant Sci,2018,9:1454.doi:10.3389/fpls.2018.01454.
[15]HARA M.The multifunctionality of dehydrins:An overview[J].Plant Signal Behav,2010,5(5):503-508.doi:10.4161/psb.11085.
[16]KOSOVáK,VíTáMVáS P,PRá?IL I T.Wheat and barley dehydrins under cold,drought,and salinity:What can LEA-II proteins tell us about plant stress response?[J].Front Plant Sci,2014,5:343.doi:10.3389/fpls.2014.00343.
[17]MISHRA A,TANNA B.Halophytes:Potential resources for salt stress tolerance genes and promoters[J].Front Plant Sci,2017,8:829.doi:10.3389/fpls.2017.00829.
[18]HOBO T,ASADA M,KOWYAMA Y,et al.ACGT-containing abscisic acid response element(ABRE)and coupling element 3(CE3)are functionally equivalent[J].Plant J,1999,19(6):679-689.doi:10.1046/j.1365-313x.1999.00565.x.
[19]MISHRA S,SHUKLA A,UPADHYAY S,et al.Identification,occurrence,and validation of DRE and ABRE cis-regulatory motifs in the promoter regions of genes of Arabidopsis thaliana[J].J Integr Plant Biol,2014,56(4):388-399.doi:10.1111/jipb.12149.
[20]WATANABE K A,HOMAYOUNI A,GU L K,et al.Transcriptomic analysis of rice aleurone cells identified a novel abscisic acid response element[J].Plant Cell Environ,2017,40(9):2004-2016.doi:10.1111/pce.13006.
[21]URAO T,YAMAGUCHI-SHINOZAKI K,URAO S,et al.An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence[J].Plant Cell,1993,5(11):1529-1539.doi:10.1105/tpc.5.11.1529.
[22]ABE H,URAO T,ITO T,et al.Arabidopsis AtMYC2(bHLH)and AtMYB2(MYB)function as transcriptional activators in abscisic acid signaling[J].Plant Cell,2003,15(1):63-78.doi:10.1105/tpc.06130.
[23]GERMAIN H,LACHANCE D,PELLETIER G,et al.The expression pattern of the Picea glauca Defensin 1 promoter is maintained in Arabidopsis thaliana,indicating the conservation of signalling pathways between angiosperms and gymnosperms[J].J Exp Bot,2012,63(2):785-795.doi:10.1093/jxb/err303.
[24]FAUTEUX F,STR?MVIK M V.Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae,Fabaceae and Poaceae[J].BMC Plant Biol,2009,9:126.doi:10.1186/1471-2229-9-126.