基于RNA-Seq数据筛选的银叶真藓耐干相关基因表达模式研究
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摘要
本文以前期获得的银叶真藓在干旱-复水过程中的转录组数据为基础,选择了9个在干旱-复水条件下基因表达差异明显的相关耐干响应基因,并利用荧光定量PCR技术研究其在干旱-复水、ABA诱导胁迫及盐胁迫下的表达模式。研究结果表明,RT-q PCR实验获得的9个基因在干旱-复水下的表达趋势与RNA-Seq基因表达谱中基因表达规律基本一致,因此较好地验证了RNA-Seq数据的可靠性。在干旱-复水过程中,9个基因均不同程度受到干旱和复水诱导表达,但复水阶段基因诱导表达更为强烈,且大多数基因表达量在复水2 h达到高峰,如ERF、Dehydrin(DHN)和HSF3基因等。在ABA和盐胁迫处理下,9个耐干相关基因中有8个同时受ABA和盐诱导上调表达,其中ERF、DHN和LEA基因受ABA和盐胁迫上调表达最为显著。综上,ERF、DHN和LEA等基因同时受干旱-复水,ABA和高盐强烈诱导表达,可作为将来重点研究的抗逆基因。
Based on our previous dehydration-rehydration transcriptome data of Bryum argenteum,nine desiccation tolerance associated genes were selected and their gene expression patterns under dehydration-rehydration,salt and ABA treatments were investigated using real-time quantitative PCR analysis.The main results were as follows:RT-q PCR results well validated the RNA-seq data,most of genes had similar gene expression trends compared with RNA-seq data under desiccation-rehydration treatment.Nine genes were induced by the dehydration-rehydration treatment at different degrees,and most of genes significantly highly induced at rehydration stage,and their abundance were reached maxima at 2 h,such as ERF,Dehydrin and HSF3 genes.The eight out of nine genes were induced by both ABA and salt treatments,especially the relative expression levels of ERF,DHN and LEA genes were dramatically increased under ABA and salt stresses.In conclusion,genes like ERF,DHN and LEA were intensively induced by dehydration-rehydration,salt and ABA treatments,which should be potential stress-tolerant candidate genes for further gene cloning.
Based on our previous dehydration-rehydration transcriptome data of Bryum argenteum,nine desiccation tolerance associated genes were selected and their gene expression patterns under dehydration-rehydration,salt and ABA treatments were investigated using real-time quantitative PCR analysis.The main results were as follows:RT-q PCR results well validated the RNA-seq data,most of genes had similar gene expression trends compared with RNA-seq data under desiccation-rehydration treatment.Nine genes were induced by the dehydration-rehydration treatment at different degrees,and most of genes significantly highly induced at rehydration stage,and their abundance were reached maxima at 2 h,such as ERF,Dehydrin and HSF3 genes.The eight out of nine genes were induced by both ABA and salt treatments,especially the relative expression levels of ERF,DHN and LEA genes were dramatically increased under ABA and salt stresses.In conclusion,genes like ERF,DHN and LEA were intensively induced by dehydration-rehydration,salt and ABA treatments,which should be potential stress-tolerant candidate genes for further gene cloning.
引文
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Checker VG,Chhibbar AK,Khurana P(2012).Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought,salinity and cold stress.Transgenic Res,21(5):939-957
Duan JL,Cai WM(2012).Os LEA3-2,an abiotic stress induced gene of rice plays a key role in salt and drought tolerance.PLo S One,7(9):e45117
Gao B,Zhang DY,Li XS,Yang HL,Wood AJ(2014).De novo assembly and characterization of the transcriptome in the desiccation-tolerant moss Syntrichia caninervis.BMC Res Notes,7(1):1-12
Gao B,Zhang DY,Li XS,Yang HL,Zhang YM,Wood AJ(2015).De novo transcriptome characterization and gene expression profiling of the desiccation tolerant moss Bryum argenteum,following rehydration.BMC Genomics,16(1):1-14
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Hui R,Li XR,Jia RL,Zhao X,Liu YM,Chen CY(2012).Effects of enhanced UV-B radiation on physiological characteristics of Bryum argenteum.Chin J Ecol,31(1):38-43(in Chinese with English abstract)[回嵘,李新荣,贾荣亮,赵昕,刘艳梅,陈翠云(2012).增强UV-B辐射对真藓结皮生理特性的影响.生态学杂志,31(1):38-43]
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Jin J,Zhang H,Kong L,Gao G,Luo J(2014).Planttfdb 3.0:a portal for the functional and evolutionary study of plant transcription factors.Nucleic Acids Res,42(2):1182-1187
Kong WS,Liu Y,Wang LJ,Li SF,Zhang HR(2016).Research progress of plant family of Sn RK.Plant Physiol J,(4):413-422(in Chinese with English abstract)[孔伟胜,刘言,王林娟,李胜飞,张海荣(2016).植物Sn RK家族的研究进展.植物生理学报,(4):413-422]
Li LB,Zhao JC(2009).Peristome morphology of Bryum hedw,(Musci Bryaceae)and its taxonomic significance.Bull Bota Res,29(6):651-658(in Chinese with English abstract)[李利博,赵建成(2009).真藓属(Bryum hedw.)蒴齿形态特征及其分类学意义.植物研究,29(6):651-658]
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Livak KJ,Schmittgen TD(2001).Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ct,method.Methods,25(4):402-408
Nakano T,Suzuki K,Fujimura T,Shinshi H(2006).Genome-wide analysis of the ERF gene family in Arabidopsis and rice.Plant Physiol,140(2):411-432
Oliver MJ,Dowd SE,Zaragoza J,Mauget SA,Payton PR(2004).The rehydration transcriptome of the desiccation-tolerant bryophyte Tortula ruralis:transcript classification and analysis.BMC Genomics,5(5):1-19
Saavedra L,Svensson J,Carballo V,Izmendi D,Welin B,Vidal S(2006).A dehydrin gene in Physcomitrella patens is required for salt and osmotic stress tolerance.Plant J,45(2):237-249
Sabovljevi M,Bijelovi A,Dragicevic I(2002).Effective and easy way of establishing in vitro culture of mosses,Bryum argenteum hedw.and Bryum capillare hedw.Bryaceae.Arch Biol Sci,54(1-2):7-8
Seo M,Koshiba T(2002).Complex regulation of ABA biosynthesis in plants.Trends Plant Sci,7(1):41-48
Shi Y,Zhao X,Jia R,Li XR(2012).The reactive oxygen species scavenging mechanism of Bryum argenteum and Didymodon vinealis in biological soil crusts under gradual drought stress.JDesert Res,32(3):683-690(in Chinese with English abstract)[石勇,赵昕,贾荣亮,李新荣(2012).逐渐干旱胁迫下生物土壤结皮中真藓和土生对齿藓的活性氧清除机制.中国沙漠,32(3):683-690]
Shinde S,Nurul I,Ng CKY(2012).Dehydration stress-induced oscillations in LEA protein transcripts involves abscisic acid in the moss,Physcomitrella patens.New Phytol,195(2):321-328
Shinde S,Shinde R,Downey F,Ng CKY(2013).Abiotic stress-induced oscillations in steady-state transcript levels of group 3LEA protein genes in the moss,Physcomitrella patens.Plant Signal Behav,8(8):e22535
Werner O,Espín RM.R Bopp M,Atzorn R(1991).Abscisic-acid-induced drought tolerance in Funaria hygrometrica Hedw.Planta,186(1):99-103
Wood AJ,Oliver MJ(1999).Translational control in plantstress:Formation of messenger ribonucleoprotein complexes(m RNPs)in Tortula ruralis in response to desiccation.Plant J,18(4):359-370
Wood AJ(2007).Invited essay:new frontiers in bryology and lichenology:the nature and distribution of vegetative desiccation-tolerance in hornworts,liverworts and mosses.Bryologist,110(2):163-177
Wu N,Zhang YM,Downing A,Zhang J,Yang CH(2012).Membrane stability of the desert moss Syntrichia caninervis mitt.during desiccation and rehydration.J Bryol,34(1):1-8
Zeevaart JAD,Creelman RAD,Zeevaart JAD,Creelman RA(1988).Metabolism and physiology of abscisic acid.Annu Rev Plant Physiol,39(1):439-473
Zheng YP,Zhao JC,Zhang BC,Li L,Zhang YM(2009).Advances on ecological studies of algae and mosses in biological soil crust.Chin Bull Bot,44(3):371-378(in Chinese with English abstract)[郑云普,赵建成,张丙昌,李琳,张元明(2009).荒漠生物结皮中藻类和苔藓植物研究进展.植物学报,44(3):371-378]
Checker VG,Chhibbar AK,Khurana P(2012).Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought,salinity and cold stress.Transgenic Res,21(5):939-957
Duan JL,Cai WM(2012).Os LEA3-2,an abiotic stress induced gene of rice plays a key role in salt and drought tolerance.PLo S One,7(9):e45117
Gao B,Zhang DY,Li XS,Yang HL,Wood AJ(2014).De novo assembly and characterization of the transcriptome in the desiccation-tolerant moss Syntrichia caninervis.BMC Res Notes,7(1):1-12
Gao B,Zhang DY,Li XS,Yang HL,Zhang YM,Wood AJ(2015).De novo transcriptome characterization and gene expression profiling of the desiccation tolerant moss Bryum argenteum,following rehydration.BMC Genomics,16(1):1-14
Hellwege EM,Dietz KJ,Volk OH,Hartung W(1994).Abscisic Acid and the induction of desiccation tolerance in the extremely xerophilic Liverwortexormotheca holstii.Planta,194(4):525-531
Hui R,Li XR,Jia RL,Zhao X,Liu YM,Chen CY(2012).Effects of enhanced UV-B radiation on physiological characteristics of Bryum argenteum.Chin J Ecol,31(1):38-43(in Chinese with English abstract)[回嵘,李新荣,贾荣亮,赵昕,刘艳梅,陈翠云(2012).增强UV-B辐射对真藓结皮生理特性的影响.生态学杂志,31(1):38-43]
Hundertmark M,Hincha DK(2008).LEA(late embryogenesis abundant)proteins and their encoding genes in Arabidopsis thaliana.BMC Genomics,9(9):1-22
Jenks MA,Wood AJ(2008).Plant desiccation tolerance.Ecol Stu,215:51-90
Jin J,Zhang H,Kong L,Gao G,Luo J(2014).Planttfdb 3.0:a portal for the functional and evolutionary study of plant transcription factors.Nucleic Acids Res,42(2):1182-1187
Kong WS,Liu Y,Wang LJ,Li SF,Zhang HR(2016).Research progress of plant family of Sn RK.Plant Physiol J,(4):413-422(in Chinese with English abstract)[孔伟胜,刘言,王林娟,李胜飞,张海荣(2016).植物Sn RK家族的研究进展.植物生理学报,(4):413-422]
Li LB,Zhao JC(2009).Peristome morphology of Bryum hedw,(Musci Bryaceae)and its taxonomic significance.Bull Bota Res,29(6):651-658(in Chinese with English abstract)[李利博,赵建成(2009).真藓属(Bryum hedw.)蒴齿形态特征及其分类学意义.植物研究,29(6):651-658]
Li LB,Zhao JC,Cao N(2009).A taxonomical study on genus Bryum Hedw.of Hebei.Chin J Hebei Normal Univ,29(6):800-809(in Chinese with English abstract)[李利博,赵建成,曹娜(2009).河北省真藓属(Bryum Hedw.)植物分类学研究.河北师范大学学报,29(6):800-809]
Liu Q,Zhao NM,Yamaguchi-Shinozaki K,Shinozaki K(2000).Roles of DREB transcription factor in enhancing stress resistance of plant.Chin Sci Bull,45(1):11-16(in Chinese)[刘强,赵南明,Yamaguchi-Shinozaki K,Shinozaki K(2000).植物DREB转录因子在提高作物抗逆性中的作用.科学通报,45(1):11-16]
Livak KJ,Schmittgen TD(2001).Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ct,method.Methods,25(4):402-408
Nakano T,Suzuki K,Fujimura T,Shinshi H(2006).Genome-wide analysis of the ERF gene family in Arabidopsis and rice.Plant Physiol,140(2):411-432
Oliver MJ,Dowd SE,Zaragoza J,Mauget SA,Payton PR(2004).The rehydration transcriptome of the desiccation-tolerant bryophyte Tortula ruralis:transcript classification and analysis.BMC Genomics,5(5):1-19
Saavedra L,Svensson J,Carballo V,Izmendi D,Welin B,Vidal S(2006).A dehydrin gene in Physcomitrella patens is required for salt and osmotic stress tolerance.Plant J,45(2):237-249
Sabovljevi M,Bijelovi A,Dragicevic I(2002).Effective and easy way of establishing in vitro culture of mosses,Bryum argenteum hedw.and Bryum capillare hedw.Bryaceae.Arch Biol Sci,54(1-2):7-8
Seo M,Koshiba T(2002).Complex regulation of ABA biosynthesis in plants.Trends Plant Sci,7(1):41-48
Shi Y,Zhao X,Jia R,Li XR(2012).The reactive oxygen species scavenging mechanism of Bryum argenteum and Didymodon vinealis in biological soil crusts under gradual drought stress.JDesert Res,32(3):683-690(in Chinese with English abstract)[石勇,赵昕,贾荣亮,李新荣(2012).逐渐干旱胁迫下生物土壤结皮中真藓和土生对齿藓的活性氧清除机制.中国沙漠,32(3):683-690]
Shinde S,Nurul I,Ng CKY(2012).Dehydration stress-induced oscillations in LEA protein transcripts involves abscisic acid in the moss,Physcomitrella patens.New Phytol,195(2):321-328
Shinde S,Shinde R,Downey F,Ng CKY(2013).Abiotic stress-induced oscillations in steady-state transcript levels of group 3LEA protein genes in the moss,Physcomitrella patens.Plant Signal Behav,8(8):e22535
Werner O,Espín RM.R Bopp M,Atzorn R(1991).Abscisic-acid-induced drought tolerance in Funaria hygrometrica Hedw.Planta,186(1):99-103
Wood AJ,Oliver MJ(1999).Translational control in plantstress:Formation of messenger ribonucleoprotein complexes(m RNPs)in Tortula ruralis in response to desiccation.Plant J,18(4):359-370
Wood AJ(2007).Invited essay:new frontiers in bryology and lichenology:the nature and distribution of vegetative desiccation-tolerance in hornworts,liverworts and mosses.Bryologist,110(2):163-177
Wu N,Zhang YM,Downing A,Zhang J,Yang CH(2012).Membrane stability of the desert moss Syntrichia caninervis mitt.during desiccation and rehydration.J Bryol,34(1):1-8
Zeevaart JAD,Creelman RAD,Zeevaart JAD,Creelman RA(1988).Metabolism and physiology of abscisic acid.Annu Rev Plant Physiol,39(1):439-473
Zheng YP,Zhao JC,Zhang BC,Li L,Zhang YM(2009).Advances on ecological studies of algae and mosses in biological soil crust.Chin Bull Bot,44(3):371-378(in Chinese with English abstract)[郑云普,赵建成,张丙昌,李琳,张元明(2009).荒漠生物结皮中藻类和苔藓植物研究进展.植物学报,44(3):371-378]