桃冷处理响应基因PdCIbHLH的克隆和功能鉴定
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摘要
以山桃(Prunus davidiana Franch)为试材,克隆获得冷处理响应基因PdCIbHLH(cold-induced b HLH),其ORF为1 617 bp,编码含539个氨基酸的蛋白质,含有典型的bHLH结构域。系统发生分析表明,Pd CIbHLH与苹果MdICE1的同源性最高。PdCIbHLH在山桃不同器官中均可表达,在叶片中表达最高,在枝条中表达较弱。叶片中PdCIbHLH受4℃处理诱导表达。在拟南芥中超量表达PdCIbHLH,其低温抗性较野生型显著升高。酵母杂交试验结果显示,PdCIbHLH可与AtCBF3的启动子相互作用。烟草瞬时表达试验结果表明,PdCIbHLH可激活AtCBF3的表达。推测PdCIbHLH通过直接激活CBF的表达正调控植株对低温处理的响应。
This study is aiming at cloning PdICbHLH from Prunus davidiana Franch,to analyze its expression pattern and reveal biological functions involved in cold stress. PdCIbHLH gene was obtained,and the open reading frame(ORF)contained 1 617 bp,encoding a protein of 539 amino acid residues. Sequence analysis indicated that PdCIbHLH exhibited the structures of bHLH domain. Phylogenetic relationship analysis showed that the homology of PdCIbHLH was close to the MdICE1. The qRT-PCR analysis indicated that PdCIbHLH was expressed in different organs. The expression showed high level in leaves,and the lowest in stems,and then expression of PdCIbHLH in leaves was induced by 4 ℃ treatment. The PdCIbHLH-overexpressing Arabidopsis exhibited increased tolerance to cold stress. Yeast one-hybrid assay showed that PdCIbHLH interacted with promoter of AtCBF3. The transient expression assays showed that PdCIbHLH could induce the expression of AtCBF3. It is speculated that PdCIbHLH was an important regulatory in response to cold treatment by directly activating the transcript of CBF.
This study is aiming at cloning PdICbHLH from Prunus davidiana Franch,to analyze its expression pattern and reveal biological functions involved in cold stress. PdCIbHLH gene was obtained,and the open reading frame(ORF)contained 1 617 bp,encoding a protein of 539 amino acid residues. Sequence analysis indicated that PdCIbHLH exhibited the structures of bHLH domain. Phylogenetic relationship analysis showed that the homology of PdCIbHLH was close to the MdICE1. The qRT-PCR analysis indicated that PdCIbHLH was expressed in different organs. The expression showed high level in leaves,and the lowest in stems,and then expression of PdCIbHLH in leaves was induced by 4 ℃ treatment. The PdCIbHLH-overexpressing Arabidopsis exhibited increased tolerance to cold stress. Yeast one-hybrid assay showed that PdCIbHLH interacted with promoter of AtCBF3. The transient expression assays showed that PdCIbHLH could induce the expression of AtCBF3. It is speculated that PdCIbHLH was an important regulatory in response to cold treatment by directly activating the transcript of CBF.
引文
Abe H,Hrao T,Ito T,Seki M,Shinozaki K,Yamaguchi-shinozaki K.2003.Arabidopsis At MYC2(bHLH)and AtMYB2(MYB)function as transcriptional activators in abscisic acid signaling.The Plant Cell,15:63-78.
An X H,Hao Y J,Li N M,Xu K,Cheng C G.2017.Functional identification of apple MdJAZ2 in Arabidopsis with reduced JA-sensitivity and increased stress tolerance.Plant Cell Reports,36:255-265.
Badawi M,Reddy Y V,Agharbaoui Z,Tominaga Y,Danyluk J,Sarhan F,Houde M.2008.Structure and functional analysis of wheat ICE(inducer of CBF expression)genes.Plant Cell Physiology,49(8):1237-1249.
Bailey P C,Martin C,Toledo-Ortiz G,Quail P H,Huq E,Heim M A,Jakoby M,Werber M,Weisshaar B.2003.Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana.The Plant Cell,15:2497-2502.
Budhagatapalli N,Narasimhan R,Rajaraman J,Viswanathan C,Nataraja K N.2016.Ectopic expression of At ICE1 and Os ICE1,transcription factor delays stress-induced senescence and improves tolerance to abiotic stresses in tobacco.Journal of Plant Biochemistry and Biotechnology,25(3):258-293.
Chinnusamy V,Ohta M,Kanrar S,Lee B,Hong X,Agarwal M,Zhu J K.2003.ICE1:a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.Genes Dev,17:1043-1054.
Farichild C D,Schumaker M A,Quail P H.2000.HFR1 encodes an atypical bHLH protein that acts in phytochrome A signal transduction.Genes Dev,14:2377-2391.
Feng X M,Zhao Q,Zhao L L,Xie X B,Li H F,Yao Y X,You C X,Hao Y J.2012.The cold-induced basic helix-loop-helix transcription factor gene MdCIbHLH1 encodes an ICE-like protein in apple.BMC Plant Biology,12:22.
Friedrichsen D M,Nemhauser J,Muramitsu T,Maloof J N,Alonso J,Ecker J R,Furuya M,Chory J.2002.Three redundant brassinosteroid early response genes encode putative bHLH transcription factors required for normal growth.Genetics,162:1445-1456.
Heim M A,Jakoby M,Werber M,Martin C,Weisshaar B,Baildy P C.2003.The basic helix-loop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity.Molecular Biology and Evolution,20:735-747.
Heisler M G,Atlomspm A,Bylstra Y H,Walsh R,Smyth D R.2001.SPATULA,a gene that controls development of carpel margin tissues in Arabidopsis,encodes a bHLH protein.Development,128:1089-1098.
Huq E,Quaio P H.2002.PIF4,a phytochrome-interacting bHLH factor,functions as a negative regulator of phytochrome B signaling in Arabidopsis.EMBO Journal,21:2441-2450.
Jefferson R A,Kavanagh T A,Bevan M W.1987.GUS fusions:?-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO Journal,6(13):3901-3907.
Lee B H,Henderson D A,Zhu J K.2005.The Arabidopsis cold-responsive transcriptome and its regulation by ICE1.The Plant Cell,17:3155-3175.
Li J T,Wang L N,Zhu W,Wang N,Xin H P,Li S H.2014.Characterization of two VvICE1 genes isolated from‘Muscat Hamburg’grapevine and their effect on the tolerance to abiotic stresses.Scientia Horticulturae,165:266-273.
Li X X,Duan X P,Jiang H X,Sun Y J,Tang Y P,Yuan Z,Guo J K,Liang W Q,Chen L,Yin J Y,Ma H,Wang J,Zhang D B.2006.Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis.Plant Physiology,141:1167-1184.
Lill R E,O’Donoghue E M,King G A.1989.Postharvest physiology of peaches and nectarines.Horticultural Reviews,11:413-418.
Liu L,Duan L,Zhang Z,Mi G,Ren H.2010.Cucumber(Cucumis sativus L.)over-expressing cold-induced transcriptome regulator ICE1 exhibits changed morphological characters and enhances chilling tolerance.Scientia Horticulturae,124:29-33.
Mao Lin-chun,Zhang Shang-long.2000.Relationship between pectolytic enzymes and chilling injury in peach.Plant Physiology Journal,36(3):266-271.(in Chinese)茅林春,张上隆.2000.果胶酶与桃果实冷害的关系.植物生理学通讯,36(3):266-271.
Nesi N,Dereaujon I,Jond C,PelletierR G,Caboche M,Lepiniec L.2000.The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques.The Plant Cell,12:1863-1878.
Ni M,Tepperman J M,Quail P H.1998.PIF3,a phytochrome-interacting factor necessary for normal photoinduced signal transduction,is a novel basic helix-loop-helix protein.Cell,95:657-667.
Rajani S,Sundaresan V.2001.The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence.Current Biology,11:1914-1922.
Shinozaki K,Yamaguchi-Shinozaki K.2000.Molecular response to dehydration and low temperature:differences and cross-talk between two stress signaling pathways.Current Opinion in Plant Biology,3:217-223.
Sorensen A M,Krober S,Unte U S,Huijser P,Dekker K,Saedler H.2003.The Arabidopsis ABORTED MICROSPORES(AMS)gene encodes a MYC class transcription factor.The Plant Journal,33:413-423.
Thomashow M F.1994.Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance.New York:Cold Spring Harbor Laboratory Press:807-834.
Thomashow M F.2001.So what’s new in the field of plant clod acclimation?Lots!Plant Physiology,125:89-93.
Tong Z G,Gao Z H,Wang F,Zhou J,Zhang Z.2009.Selection of reliable reference genes for gene expression studies in peach using real-time PCR.BMC Molecular Biology,10:71.
Xiang D,Hu X,Zhang Y,Yin K.2008.Over-expression of ICE1 gene in transgenic rice improves cold tolerance.Rice Science,15:173-178.
Yang Chun-xiang,Li Xian-li,Gao Dong-sheng,Shi Zuo-an.2005.Effects of chilling stress on membrane lipid peroxidation and activities of protective enzymes in nectarine flower organs.Journal of Fruit Science,22(1):69-71.(in Chinese)杨春祥,李宪利,高东升,史作安.2005.低温胁迫对油桃花器官膜脂过氧化和保护酶活性的影响.果树学报,22(1):69-71.
Yin X R,Allan A C,Chen K S,Ferguson I B.2010.Kiwifruit EIL and ERF genes involved in regulating fruit ripening.Plant Physiology,153:1280-1292.
Zhang Tengguo,Chang Yan,Wang Juan,Wang Ning,Wang Yuanyuan,Chen Qiongqiong,Sun Wancang.2013.Cloning and expression analysis of a BnICE1 from Brassica napus L.Scientia Agricultura Sinica,46(1):205-214.(in Chinese)张腾国,常燕,王娟,王宁,王圆圆,陈琼琼,孙万仓.2013.油菜BnICE1的克隆及表达分析.中国农业科学,46(1):205-214.
An X H,Hao Y J,Li N M,Xu K,Cheng C G.2017.Functional identification of apple MdJAZ2 in Arabidopsis with reduced JA-sensitivity and increased stress tolerance.Plant Cell Reports,36:255-265.
Badawi M,Reddy Y V,Agharbaoui Z,Tominaga Y,Danyluk J,Sarhan F,Houde M.2008.Structure and functional analysis of wheat ICE(inducer of CBF expression)genes.Plant Cell Physiology,49(8):1237-1249.
Bailey P C,Martin C,Toledo-Ortiz G,Quail P H,Huq E,Heim M A,Jakoby M,Werber M,Weisshaar B.2003.Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana.The Plant Cell,15:2497-2502.
Budhagatapalli N,Narasimhan R,Rajaraman J,Viswanathan C,Nataraja K N.2016.Ectopic expression of At ICE1 and Os ICE1,transcription factor delays stress-induced senescence and improves tolerance to abiotic stresses in tobacco.Journal of Plant Biochemistry and Biotechnology,25(3):258-293.
Chinnusamy V,Ohta M,Kanrar S,Lee B,Hong X,Agarwal M,Zhu J K.2003.ICE1:a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.Genes Dev,17:1043-1054.
Farichild C D,Schumaker M A,Quail P H.2000.HFR1 encodes an atypical bHLH protein that acts in phytochrome A signal transduction.Genes Dev,14:2377-2391.
Feng X M,Zhao Q,Zhao L L,Xie X B,Li H F,Yao Y X,You C X,Hao Y J.2012.The cold-induced basic helix-loop-helix transcription factor gene MdCIbHLH1 encodes an ICE-like protein in apple.BMC Plant Biology,12:22.
Friedrichsen D M,Nemhauser J,Muramitsu T,Maloof J N,Alonso J,Ecker J R,Furuya M,Chory J.2002.Three redundant brassinosteroid early response genes encode putative bHLH transcription factors required for normal growth.Genetics,162:1445-1456.
Heim M A,Jakoby M,Werber M,Martin C,Weisshaar B,Baildy P C.2003.The basic helix-loop-helix transcription factor family in plants:a genome-wide study of protein structure and functional diversity.Molecular Biology and Evolution,20:735-747.
Heisler M G,Atlomspm A,Bylstra Y H,Walsh R,Smyth D R.2001.SPATULA,a gene that controls development of carpel margin tissues in Arabidopsis,encodes a bHLH protein.Development,128:1089-1098.
Huq E,Quaio P H.2002.PIF4,a phytochrome-interacting bHLH factor,functions as a negative regulator of phytochrome B signaling in Arabidopsis.EMBO Journal,21:2441-2450.
Jefferson R A,Kavanagh T A,Bevan M W.1987.GUS fusions:?-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO Journal,6(13):3901-3907.
Lee B H,Henderson D A,Zhu J K.2005.The Arabidopsis cold-responsive transcriptome and its regulation by ICE1.The Plant Cell,17:3155-3175.
Li J T,Wang L N,Zhu W,Wang N,Xin H P,Li S H.2014.Characterization of two VvICE1 genes isolated from‘Muscat Hamburg’grapevine and their effect on the tolerance to abiotic stresses.Scientia Horticulturae,165:266-273.
Li X X,Duan X P,Jiang H X,Sun Y J,Tang Y P,Yuan Z,Guo J K,Liang W Q,Chen L,Yin J Y,Ma H,Wang J,Zhang D B.2006.Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis.Plant Physiology,141:1167-1184.
Lill R E,O’Donoghue E M,King G A.1989.Postharvest physiology of peaches and nectarines.Horticultural Reviews,11:413-418.
Liu L,Duan L,Zhang Z,Mi G,Ren H.2010.Cucumber(Cucumis sativus L.)over-expressing cold-induced transcriptome regulator ICE1 exhibits changed morphological characters and enhances chilling tolerance.Scientia Horticulturae,124:29-33.
Mao Lin-chun,Zhang Shang-long.2000.Relationship between pectolytic enzymes and chilling injury in peach.Plant Physiology Journal,36(3):266-271.(in Chinese)茅林春,张上隆.2000.果胶酶与桃果实冷害的关系.植物生理学通讯,36(3):266-271.
Nesi N,Dereaujon I,Jond C,PelletierR G,Caboche M,Lepiniec L.2000.The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques.The Plant Cell,12:1863-1878.
Ni M,Tepperman J M,Quail P H.1998.PIF3,a phytochrome-interacting factor necessary for normal photoinduced signal transduction,is a novel basic helix-loop-helix protein.Cell,95:657-667.
Rajani S,Sundaresan V.2001.The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence.Current Biology,11:1914-1922.
Shinozaki K,Yamaguchi-Shinozaki K.2000.Molecular response to dehydration and low temperature:differences and cross-talk between two stress signaling pathways.Current Opinion in Plant Biology,3:217-223.
Sorensen A M,Krober S,Unte U S,Huijser P,Dekker K,Saedler H.2003.The Arabidopsis ABORTED MICROSPORES(AMS)gene encodes a MYC class transcription factor.The Plant Journal,33:413-423.
Thomashow M F.1994.Arabidopsis thaliana as a model for studying mechanisms of plant cold tolerance.New York:Cold Spring Harbor Laboratory Press:807-834.
Thomashow M F.2001.So what’s new in the field of plant clod acclimation?Lots!Plant Physiology,125:89-93.
Tong Z G,Gao Z H,Wang F,Zhou J,Zhang Z.2009.Selection of reliable reference genes for gene expression studies in peach using real-time PCR.BMC Molecular Biology,10:71.
Xiang D,Hu X,Zhang Y,Yin K.2008.Over-expression of ICE1 gene in transgenic rice improves cold tolerance.Rice Science,15:173-178.
Yang Chun-xiang,Li Xian-li,Gao Dong-sheng,Shi Zuo-an.2005.Effects of chilling stress on membrane lipid peroxidation and activities of protective enzymes in nectarine flower organs.Journal of Fruit Science,22(1):69-71.(in Chinese)杨春祥,李宪利,高东升,史作安.2005.低温胁迫对油桃花器官膜脂过氧化和保护酶活性的影响.果树学报,22(1):69-71.
Yin X R,Allan A C,Chen K S,Ferguson I B.2010.Kiwifruit EIL and ERF genes involved in regulating fruit ripening.Plant Physiology,153:1280-1292.
Zhang Tengguo,Chang Yan,Wang Juan,Wang Ning,Wang Yuanyuan,Chen Qiongqiong,Sun Wancang.2013.Cloning and expression analysis of a BnICE1 from Brassica napus L.Scientia Agricultura Sinica,46(1):205-214.(in Chinese)张腾国,常燕,王娟,王宁,王圆圆,陈琼琼,孙万仓.2013.油菜BnICE1的克隆及表达分析.中国农业科学,46(1):205-214.