核桃CBF基因在低温胁迫中表达
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摘要
研究果树抗寒机理及其抗寒生理机制对于抗寒基础理论研究,以及解决实际生产上的问题十分重要。为了了解核桃相关抗寒基因的表达机制,本研究采用2、4、6、8℃几个低温进行处理,利用实时荧光定量PCR技术研究了在不同温度下‘阳光’‘云新’核桃叶片内CBF、(GenBank序列号:JX875914.1)随时间表达量的变化。本课题从处理后核桃叶片中提取出了c-DNA,并成功得到了实时荧光定量PCR数据并进一步进行比较分析。为了了解核桃相关抗氧化生理机制,采用分光光度比色法研究了核桃叶片的多酚、黄酮成分对低温胁迫响应的规律,并得到了相关抗寒物质的变化规律,旨在为以后选育核桃优良抗寒品种等提供更多的参考依据。1.本课题以核桃18S基因为内参,对‘阳光’和‘云新’核桃内CBF基因在2、4、6、8℃胁迫下的表达进行了初步的研究,表明当受到冷胁迫时这些基因在核桃叶片内均有表达量的变化,其中‘阳光’核桃叶片受到冷胁迫时这种变化更为明显,表现为‘阳光’核桃在6℃胁迫下出现了先升高后下降再升高下降的趋势,在8℃胁迫下出现了先升高后下降的明显趋势,推测CBF基因在核桃对低温胁迫的响应中发挥重要功能。本研究同时表明‘阳光’核桃对冷胁迫更敏感,更能在寒冷胁迫迅速响应。
Studying the molecular mechanism of cold resistance in fruit trees will contribute to the understanding of plant stress response and also the agriculture industry.In order to understand the effect of polyphenols and flavone in the response to cold stress under various conditions(at the temperature 2、4、6、8℃)RT-PCR was used to examine the expression of CBF gene(GenBank:JX875914.1)of two different species Juglans leaves.Moreover,we studied the response of polyphenols and flavonoids to the low temperature stress by spectrophotometer.Using Juglans 18 S gene as a reference,we observed that the expression of CBF gene were changed in Walnut Leaves under cold stress,and the expression fluctuation is more dramatic in the‘Yangguang'Walnut Leaves under cold stress.All of the three genes in the‘Yangguang'Walnut leaf at 6℃first increased and then decreased,followed by a trend of increasing and then decreasing,whereas the expression of the genes rises at first and then decrease at 8℃.Thus we propose that the three genes play important roles in the response of Juglans to low temperature stress.
Studying the molecular mechanism of cold resistance in fruit trees will contribute to the understanding of plant stress response and also the agriculture industry.In order to understand the effect of polyphenols and flavone in the response to cold stress under various conditions(at the temperature 2、4、6、8℃)RT-PCR was used to examine the expression of CBF gene(GenBank:JX875914.1)of two different species Juglans leaves.Moreover,we studied the response of polyphenols and flavonoids to the low temperature stress by spectrophotometer.Using Juglans 18 S gene as a reference,we observed that the expression of CBF gene were changed in Walnut Leaves under cold stress,and the expression fluctuation is more dramatic in the‘Yangguang'Walnut Leaves under cold stress.All of the three genes in the‘Yangguang'Walnut leaf at 6℃first increased and then decreased,followed by a trend of increasing and then decreasing,whereas the expression of the genes rises at first and then decrease at 8℃.Thus we propose that the three genes play important roles in the response of Juglans to low temperature stress.
引文
[1]李静,王永,杨耀东,等.植物CBF转录因子抗寒作用机制研究进展[J].江西农业学报,2014(01):59-63.
[2]包永霞,满达,肖波,等.CBF/DREB转录因子与植物矮化的相关性研究进展[J].生物技术通报,2011(03):7-12.
[3]Yamaguchi Shinozaki K,Shinozaki K.A novel cis-acting element in an Arabidopsisgene is involved in responsiveness to drought,low temperature or high salt stress[J].Plant Cell,1994,6:251-264.
[4]Ouellet F,Vazquea-Tello A,Sarhan F.The wheat wcs120promoter is cold-inducible in both monocotyledonous and dicotyledonous species[J].FEBS Letter,1998,423(3):324-328.
[5]Haake V,Cook D,Riechman JL,et al.Transcription factor CBF4is a regulator of drought adapta tion in Arabidopsis[J].Plant Physiol,2002,130(2):639-648.
[6]Thomashow M F.So what’s new in the field of plant cold acclimation?Lots![J].Plant Physiology,2001,125(1):89-93.
[7]Gilmour SJ,Zarka DG,Stockinger EJ,et al.Low temperature regulation of the Arabidopsis CBF family of AF2transcriptional activators as an early step in cold-induced COR gene expression[J].Plant J,1998,16(4):433-442.
[8]Chinnusamy V.ICE1:a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.[J].Genes&Development,2003,17:1043-1054.
[9]Shinwari Z K,Nakashima K,Miura S,et al.An Arabidopsisgene family encoding DRE/CRT binding proteins involved in low-temperature-re-sponsive gene expression[J].Biochem.Biophys.Res.Commun.,1998,250:161-170.
[10]Guo Y,Xiong L,Ishitani M,et al.AnArabidopsis mutation in translation elongation factor 2causes superinduction of CBF/DREB1transcription factor genes but blocks the induction of their downstream targets under low temperature[J].Proc.Natl.Acad.Sci.,2002,99:7786-7791.
[11]Wisniewski M,Norelli J,Bassett C,et al.Ectopic expression of a novel peach(Prunus persica)CBF transcription factor in apple(Malus×domestica)results in short-day induced dormancy and increased cold hardiness[J].Planta,2011,233:971-983.
[12]Gong Z Z,Dong C H,Lee H,et al.A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis[J].The Plant Cell January,2005,17(1):256-267.
[13]Lee H,Xiong L,Gong Z,et al.The Arabidopsis HOS1genenegatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo-cytoplasmic partitioning[J].Genes&Development,2001,15:912-924.
[14]Liu D C,He L G,Wang H L,et al.Molecular cloning,characterization and expression analysis of PtrHOS1,a novel gene of cold responses from trifoliate orange[Poncirus trifoliata(L.)Raf.][J].Acta Physiologiae Plantarum,2010,32:271-279.
[15]Wang Y,Hua J.A moderate decrease in temperature induces COR15aexpression through the CBF signaling cascade and enhances freezing tolerance[J].The Plant Journal,2009,60(2):340-349.
[16]Boyce J M,Knight H,Deyholos M,et al.The sfr6mutant ofArabidopsis is defective in transcriptional activation via CBF/DREB1and DREB2and shows sensitivity to osmotic stress[J].The Plant Journal,2003,34(4):395-406.
[17]Knight H,Mugford S G,lker B,et al.Identification of SFR6,a key component in cold acclimation acting post-translationally on CBF function[J].The Plant Journal,2009,58(1):97-108.
[18]Novillo F,Alonso JM,Ecker JR,et al.CBF2/DREB1Cis a negative regulator of CBF1/DREB1Band CBF3/DREB1Aexpression and plays a central role in stress tolerance in Arabidopsis[J].Proc Natl Acad Sci,2004,101(11):3985-3990.
[19]钟克亚,叶妙水,胡新文,等.转录因子CBF在植物抗寒中的重要作用[J].遗传,2006,28(2):249-254.
[20]Fowler S,Thomashow MF.Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway[J].Plant Cell,2002,14:1675-1690.
[21]李科友,朱海兰.植物非生物逆境胁迫DREB/CBF转录因子的研究进展[J].林业科学,2011,47(1):124-134.
[22]Artus NN,Uemura M,Steponkus PL,et al.Constitutive expression of the cold regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance[J].Proc Natl Acad Sci,1996,93(23):13404-13409.
[23]李勇鹏.香樟CBF类似基因的克隆及其在植物转基因抗寒育种中的潜在应用[C].南阳:南阳师范学院,2015:17-19.
[24]吴纯倩.CBF3和COR15a基因对烟草抗寒性的影响[C].重庆:西南大学,2006:26-35.
[25]雷恒久,苏淑钗,马履一,等.平欧杂交榛CBF/DREB1转录因子ChaCBF1基因的克隆与功能分析[J].北京林业大学学报,2016,38(10):69-79.
[26]冯勋伟,才宏伟.结缕草CBF基因的同源克隆及其转基因拟南芥的抗寒性验证[J].作物学报,2014,40(9):1572-1578.
[27]张建朋,张士刚,陈新,等.麻核桃CBF基因的克隆与生物信息学分析[J].山东农业科学2013,45(11):7-11.
[28]陈暄,房婉萍,邹中伟,等.茶树冷胁迫诱导抗寒基因CBF的克隆与表达分析[J].茶叶科学,2009,29(1):53-59.
[29]王京京.巨桉低温胁迫SSHEST文库构建及CBF基因的克隆和表达分析[C].浙江:浙江农林大学,2012:20-33.
[30]张亮,张智俊,李疆,等.扁桃CBF1基因的克隆与生物信息学分析[J].中国农学通报,2014,30(34):16-23.
[31]孟平红,李桂莲,李苍山,等.双价抗寒基因CBF3/COR15A转化大白菜的初步研究[J].中国农业大学学报,2016(6):41-51.
[32]张哲敏,孙萍,王旺田,等.三种不同抗冻性葡萄中CBF2基因的生物信息学分析及植物表达载体构建[J].广西植物Guihaia Jan,2013,33(1):82-88.
[33]徐丽,陈新,张力思,等.核桃JrCBF基因的克隆与表达和单核苷酸多态性分析[J].植物遗传资源学报,2014(02):320-326.
[2]包永霞,满达,肖波,等.CBF/DREB转录因子与植物矮化的相关性研究进展[J].生物技术通报,2011(03):7-12.
[3]Yamaguchi Shinozaki K,Shinozaki K.A novel cis-acting element in an Arabidopsisgene is involved in responsiveness to drought,low temperature or high salt stress[J].Plant Cell,1994,6:251-264.
[4]Ouellet F,Vazquea-Tello A,Sarhan F.The wheat wcs120promoter is cold-inducible in both monocotyledonous and dicotyledonous species[J].FEBS Letter,1998,423(3):324-328.
[5]Haake V,Cook D,Riechman JL,et al.Transcription factor CBF4is a regulator of drought adapta tion in Arabidopsis[J].Plant Physiol,2002,130(2):639-648.
[6]Thomashow M F.So what’s new in the field of plant cold acclimation?Lots![J].Plant Physiology,2001,125(1):89-93.
[7]Gilmour SJ,Zarka DG,Stockinger EJ,et al.Low temperature regulation of the Arabidopsis CBF family of AF2transcriptional activators as an early step in cold-induced COR gene expression[J].Plant J,1998,16(4):433-442.
[8]Chinnusamy V.ICE1:a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.[J].Genes&Development,2003,17:1043-1054.
[9]Shinwari Z K,Nakashima K,Miura S,et al.An Arabidopsisgene family encoding DRE/CRT binding proteins involved in low-temperature-re-sponsive gene expression[J].Biochem.Biophys.Res.Commun.,1998,250:161-170.
[10]Guo Y,Xiong L,Ishitani M,et al.AnArabidopsis mutation in translation elongation factor 2causes superinduction of CBF/DREB1transcription factor genes but blocks the induction of their downstream targets under low temperature[J].Proc.Natl.Acad.Sci.,2002,99:7786-7791.
[11]Wisniewski M,Norelli J,Bassett C,et al.Ectopic expression of a novel peach(Prunus persica)CBF transcription factor in apple(Malus×domestica)results in short-day induced dormancy and increased cold hardiness[J].Planta,2011,233:971-983.
[12]Gong Z Z,Dong C H,Lee H,et al.A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis[J].The Plant Cell January,2005,17(1):256-267.
[13]Lee H,Xiong L,Gong Z,et al.The Arabidopsis HOS1genenegatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo-cytoplasmic partitioning[J].Genes&Development,2001,15:912-924.
[14]Liu D C,He L G,Wang H L,et al.Molecular cloning,characterization and expression analysis of PtrHOS1,a novel gene of cold responses from trifoliate orange[Poncirus trifoliata(L.)Raf.][J].Acta Physiologiae Plantarum,2010,32:271-279.
[15]Wang Y,Hua J.A moderate decrease in temperature induces COR15aexpression through the CBF signaling cascade and enhances freezing tolerance[J].The Plant Journal,2009,60(2):340-349.
[16]Boyce J M,Knight H,Deyholos M,et al.The sfr6mutant ofArabidopsis is defective in transcriptional activation via CBF/DREB1and DREB2and shows sensitivity to osmotic stress[J].The Plant Journal,2003,34(4):395-406.
[17]Knight H,Mugford S G,lker B,et al.Identification of SFR6,a key component in cold acclimation acting post-translationally on CBF function[J].The Plant Journal,2009,58(1):97-108.
[18]Novillo F,Alonso JM,Ecker JR,et al.CBF2/DREB1Cis a negative regulator of CBF1/DREB1Band CBF3/DREB1Aexpression and plays a central role in stress tolerance in Arabidopsis[J].Proc Natl Acad Sci,2004,101(11):3985-3990.
[19]钟克亚,叶妙水,胡新文,等.转录因子CBF在植物抗寒中的重要作用[J].遗传,2006,28(2):249-254.
[20]Fowler S,Thomashow MF.Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway[J].Plant Cell,2002,14:1675-1690.
[21]李科友,朱海兰.植物非生物逆境胁迫DREB/CBF转录因子的研究进展[J].林业科学,2011,47(1):124-134.
[22]Artus NN,Uemura M,Steponkus PL,et al.Constitutive expression of the cold regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance[J].Proc Natl Acad Sci,1996,93(23):13404-13409.
[23]李勇鹏.香樟CBF类似基因的克隆及其在植物转基因抗寒育种中的潜在应用[C].南阳:南阳师范学院,2015:17-19.
[24]吴纯倩.CBF3和COR15a基因对烟草抗寒性的影响[C].重庆:西南大学,2006:26-35.
[25]雷恒久,苏淑钗,马履一,等.平欧杂交榛CBF/DREB1转录因子ChaCBF1基因的克隆与功能分析[J].北京林业大学学报,2016,38(10):69-79.
[26]冯勋伟,才宏伟.结缕草CBF基因的同源克隆及其转基因拟南芥的抗寒性验证[J].作物学报,2014,40(9):1572-1578.
[27]张建朋,张士刚,陈新,等.麻核桃CBF基因的克隆与生物信息学分析[J].山东农业科学2013,45(11):7-11.
[28]陈暄,房婉萍,邹中伟,等.茶树冷胁迫诱导抗寒基因CBF的克隆与表达分析[J].茶叶科学,2009,29(1):53-59.
[29]王京京.巨桉低温胁迫SSHEST文库构建及CBF基因的克隆和表达分析[C].浙江:浙江农林大学,2012:20-33.
[30]张亮,张智俊,李疆,等.扁桃CBF1基因的克隆与生物信息学分析[J].中国农学通报,2014,30(34):16-23.
[31]孟平红,李桂莲,李苍山,等.双价抗寒基因CBF3/COR15A转化大白菜的初步研究[J].中国农业大学学报,2016(6):41-51.
[32]张哲敏,孙萍,王旺田,等.三种不同抗冻性葡萄中CBF2基因的生物信息学分析及植物表达载体构建[J].广西植物Guihaia Jan,2013,33(1):82-88.
[33]徐丽,陈新,张力思,等.核桃JrCBF基因的克隆与表达和单核苷酸多态性分析[J].植物遗传资源学报,2014(02):320-326.