过表达香樟CcCBFs提高转基因拟南芥抗寒能力
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摘要
为系统性研究香樟CcCBFs(CcCBFa、CcCBFb、CcCBFc和CcCBFd)基因在增强抗寒性方面的功能,以香樟无性系EL_6组培苗为试验材料,采用荧光定量PCR技术,分析其在低温(4℃)下的表达情况,结果表明:CcCBFs均可持续且强烈地响应低温信号,且CcCBFa和CcCBFc对低温诱导的响应更加显著;通过农杆菌介导的花序侵染法转化哥伦比亚野生型拟南芥,获得转CcCBFs基因拟南芥种子,并对野生型和T_2代转基因拟南芥种子在4℃条件下的发芽率进行统计,结果显示:转基因拟南芥种子发芽率均显著高于野生型拟南芥,可见过表达香樟CcCBFs能够增强拟南芥种子对低温的抵御能力;此外,对T_2代转基因拟南芥和野生型拟南芥幼苗在低温胁迫下的生理指标进行测定,结果发现,未胁迫处理前转基因与野生型拟南芥SOD活性以及脯氨酸质量分数、丙二醛质量摩尔浓度均无显著差异,低温胁迫处理一定时间后转基因拟南芥SOD活性和总游离脯氨酸质量分数均显著高于野生型,而丙二醛质量摩尔浓度均显著低于野生型拟南芥,且在相同的胁迫条件下转CcCBFa、CcCBFc拟南芥的抗寒生理数据变化更为显著。结合基因表达分析、种子发芽率及抗寒生理指标可知,过表达香樟CcCBFs均能提高转基因拟南芥抗寒能力,其中CcCBFa和CcCBFc在香樟抵御低温胁迫时可能发挥更为重要的调控作用。
In order to systematically study the cold resistance of Cc CBFs(Cc CBFa,Cc CBFb,Cc CBFc and Cc CBFd) genes from Cinnamomum camphora,with EL6 tissue culture seedling as experimental material,fluorescence quantitative PCR technique was used to analyze its expression at low temperature(4 ℃).The results showed that Cc CBFs could respond to the low temperature signal continuously and strongly,Cc CBFa and Cc CBFc were more sensitive to low temperature induction.The transgenic Arabidopsis thaliana seeds with Cc CBFs were obtained by Agrobacterium tumefaciens mediated inflorescence soaking,and the germination rates of wild type and T_2 generation transgenic A.thaliana seeds at 4 ℃ were analyzed.The germination rate of T_2 generation positive transgenic A.thaliana seeds was significantly higher than that of wild type A.thaliana seeds.In other words,overexpression of Cc CBFs could enhance the resistance of A.thaliana seeds to low temperature.In addition,physiological indexes of transgenic A.thaliana seedlings and wild type A.thaliana seedlings under low temperature stress were determined.There were no significant differences in proline,malondialdehyde molarity and SOD activity between transgenic A.thaliana and wild type A.thaliana before stress.The total free proline mass fraction and SOD activity of transgenic A.thaliana were significantly higher than those of wild type A.thaliana,but the malondialdehyde molarity of transgenic A.thaliana was significantly lower than that of wild type A.thaliana.And under the same stress conditions,the change of cold resistance physiological data of Cc CBFa and Cc CBFc were more significant than that of Cc CBFb and Cc CBFd.Combined with gene expression analysis,seed germination rate and cold resistance physiological index,overexpression of camphor Cc CBFs can improve the cold resistance of transgenic A.thaliana,and Cc CBFa and Cc CBFc may play a more important role in controlling camphor to resist cold stress.
In order to systematically study the cold resistance of Cc CBFs(Cc CBFa,Cc CBFb,Cc CBFc and Cc CBFd) genes from Cinnamomum camphora,with EL6 tissue culture seedling as experimental material,fluorescence quantitative PCR technique was used to analyze its expression at low temperature(4 ℃).The results showed that Cc CBFs could respond to the low temperature signal continuously and strongly,Cc CBFa and Cc CBFc were more sensitive to low temperature induction.The transgenic Arabidopsis thaliana seeds with Cc CBFs were obtained by Agrobacterium tumefaciens mediated inflorescence soaking,and the germination rates of wild type and T_2 generation transgenic A.thaliana seeds at 4 ℃ were analyzed.The germination rate of T_2 generation positive transgenic A.thaliana seeds was significantly higher than that of wild type A.thaliana seeds.In other words,overexpression of Cc CBFs could enhance the resistance of A.thaliana seeds to low temperature.In addition,physiological indexes of transgenic A.thaliana seedlings and wild type A.thaliana seedlings under low temperature stress were determined.There were no significant differences in proline,malondialdehyde molarity and SOD activity between transgenic A.thaliana and wild type A.thaliana before stress.The total free proline mass fraction and SOD activity of transgenic A.thaliana were significantly higher than those of wild type A.thaliana,but the malondialdehyde molarity of transgenic A.thaliana was significantly lower than that of wild type A.thaliana.And under the same stress conditions,the change of cold resistance physiological data of Cc CBFa and Cc CBFc were more significant than that of Cc CBFb and Cc CBFd.Combined with gene expression analysis,seed germination rate and cold resistance physiological index,overexpression of camphor Cc CBFs can improve the cold resistance of transgenic A.thaliana,and Cc CBFa and Cc CBFc may play a more important role in controlling camphor to resist cold stress.
引文
[1] 刘辉,李德军,邓治.植物应答低温胁迫的转录调控网络研究进展[J].中国农业科学,2014,47(18):3523-3533.
[2] 董亚茹,王应民,王向誉.DREB/CBF转录因子植物抗逆性研究进展[J].山东农业科学,2017,49(10):139-142.
[3] LEE C M, THOMASHOW M F. Photoperiodic regulation of the C-repeat binding factor (CBF) cold acclimation pathway and freezing tolerance in Arabidopsis thaliana[J]. Proceedings of the National Academy of Sciences of the United States of America,2012,109(37):15054-15059.
[4] KELLER G, CAO P B, SAN C H, et al. Transcript profiling combined with functional annotation of 2,662 ESTs provides a molecular picture of Eucalyptus gunnii cold acclimation[J]. Trees,2013,27(6):1713-1735.
[5] XUE Y, WANG Y Y, PENG R H, et al. Transcription factor MdCBF1 gene increases freezing stress tolerance in transgenic Arabidopsis thaliana[J]. Biologia Plantarum,2014,58(3):499-506.
[6] 柏星轩, 闫雪,要宇晨,等.DREB/CBF转录因子在植物非生物胁迫中的作用及研究进展[J].生物学杂志,2017,34(4):88-93.
[7] 乌凤章,王贺新,徐国辉,等.木本植物低温胁迫生理及分子机制研究进展[J].林业科学,2015,51(7):116-128.
[8] 庄倩倩,陈少鹏,刘洪章.低温胁迫对紫萼玉簪根系3种生理指标的影响[J].东北林业大学学报,2018,46(3):33-36.
[9] 任军,黄志霖,曾立雄,等.低温胁迫下植物生理反应机理研究进展[J].世界林业研究,2013,26(6):15-20.
[10] DU L, LI Y P, YAO Y, et al. An efficient protocol for plantlet regeneration via direct organogenesis by using nodal segments from embryo-cultured seedlings of Cinnamomum camphora L[J]. PLoS One,2015,10(5).doi:10.1371/journal.pone.0127215.
[11] 李勇鹏,张佳佳,张力维,等.香樟两个DREB1转录因子基因的分离及其对不同胁迫的响应分析[J].园艺学报,2016,43(4):743-751.
[12] 李勇鹏,张力维,张佳佳,等.香樟CcCBFs基因的克隆及表达模式[J].东北林业大学学报,2016,44(8):34-40.
[13] YAO P, SUN Z, LI C, et al. Overexpression of Fagopyrum tataricum FtbHLH2 enhances tolerance to cold stress in transgenic Arabidopsis.[J]. Plant Physiol Biochem,2018,125:85-94.
[14] WU D, SUN Y, WANG H, et al. The SlNAC8 gene of the halophyte Suaeda liaotungensis enhances drought and salt stress tolerance in transgenic Arabidopsis thaliana[J]. Gene,2018,662(7):10-20.
[15] 杜丽.香樟子叶胚培养的研究[J].北方园艺,2014(14):89-92.
[16] 张佳佳,朱逢玲,焦晓琳,等.过表达香樟CcCBFb的烟草非生物胁迫抗性增强[J].园艺学报,2017,44(7):1397-1404.
[17] 张力维,李勇鹏,姚瑶,等.香樟延伸因子EF1α基因片段的克隆和表达分析[J].中南林业科技大学学报,2015,35(5):122-128.
[18] 张佳佳,张力维,李勇鹏,等.不同非生物胁迫下香樟实时定量PCR内参基因的选择[J].生物技术通报,2016,32(10):205-211.
[19] HARRISON S J, MOTT E K, PARSLEY K, et al. A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation[J]. Plant Methods,2006,2(1):19-25.
[20] 张瑛,罗志祥,周俊峰.植物脯氨酸测定方法优化及其应用[J].分子植物育种,2013,11(30):1219-1226.
[21] GUPTA K, JHA B, AGARWAL P K. A dehydration-responsive element binding (DREB) transcription factor from the succulent halophyte Salicorniabrachiataenhances abiotic stress tolerancein transgenic tobacco[J]. Mar Biotechnol,2014,16(6):657-673.
[22] GUO R G, ZHAO J, WANG X H, et al. Constitutive expression of a grape aspartic protease genein transgenic Arabidopsis confersosmotic stress tolerance[J]. Plant Cell Tiss Organ Cult,2015,121(2):275-287.
[23] 刘海妹,陈韵琳,李成浩.PtrDREB28转录因子的克隆及抗逆性[J].东北林业大学学报,2018,46(2):12-16.
[24] 刘晨旭,刘彧,刘杰,等.过量表达甘菊CBF1基因提高拟南芥抗旱耐盐能力[J].草业科学,2018,35(6):1400-1408.
[25] ZHAO J, ZHANG X, GUO R, et al. Over-expression of a grape WRKY transcription factor gene, VlWRKY48, in Arabidopsis thaliana, increases disease resistance and drought stress tolerance[J]. Plant Cell Tissue & Organ Culture,2018,132(2):359-370.
[26] 李文明,辛建攀,魏驰宇,等.植物抗寒性研究进展[J].江苏农业科学,2017,45(12):6-11.
[27] 王宁.植物抗寒生理性研究进展[J].北方园艺,2014(4):174-177.
[28] 安飞飞,李庚虎,陈霆,等.植物耐寒生理及蛋白质组学研究进展[J].中国农学通报,2015,31(14):96-101.
[2] 董亚茹,王应民,王向誉.DREB/CBF转录因子植物抗逆性研究进展[J].山东农业科学,2017,49(10):139-142.
[3] LEE C M, THOMASHOW M F. Photoperiodic regulation of the C-repeat binding factor (CBF) cold acclimation pathway and freezing tolerance in Arabidopsis thaliana[J]. Proceedings of the National Academy of Sciences of the United States of America,2012,109(37):15054-15059.
[4] KELLER G, CAO P B, SAN C H, et al. Transcript profiling combined with functional annotation of 2,662 ESTs provides a molecular picture of Eucalyptus gunnii cold acclimation[J]. Trees,2013,27(6):1713-1735.
[5] XUE Y, WANG Y Y, PENG R H, et al. Transcription factor MdCBF1 gene increases freezing stress tolerance in transgenic Arabidopsis thaliana[J]. Biologia Plantarum,2014,58(3):499-506.
[6] 柏星轩, 闫雪,要宇晨,等.DREB/CBF转录因子在植物非生物胁迫中的作用及研究进展[J].生物学杂志,2017,34(4):88-93.
[7] 乌凤章,王贺新,徐国辉,等.木本植物低温胁迫生理及分子机制研究进展[J].林业科学,2015,51(7):116-128.
[8] 庄倩倩,陈少鹏,刘洪章.低温胁迫对紫萼玉簪根系3种生理指标的影响[J].东北林业大学学报,2018,46(3):33-36.
[9] 任军,黄志霖,曾立雄,等.低温胁迫下植物生理反应机理研究进展[J].世界林业研究,2013,26(6):15-20.
[10] DU L, LI Y P, YAO Y, et al. An efficient protocol for plantlet regeneration via direct organogenesis by using nodal segments from embryo-cultured seedlings of Cinnamomum camphora L[J]. PLoS One,2015,10(5).doi:10.1371/journal.pone.0127215.
[11] 李勇鹏,张佳佳,张力维,等.香樟两个DREB1转录因子基因的分离及其对不同胁迫的响应分析[J].园艺学报,2016,43(4):743-751.
[12] 李勇鹏,张力维,张佳佳,等.香樟CcCBFs基因的克隆及表达模式[J].东北林业大学学报,2016,44(8):34-40.
[13] YAO P, SUN Z, LI C, et al. Overexpression of Fagopyrum tataricum FtbHLH2 enhances tolerance to cold stress in transgenic Arabidopsis.[J]. Plant Physiol Biochem,2018,125:85-94.
[14] WU D, SUN Y, WANG H, et al. The SlNAC8 gene of the halophyte Suaeda liaotungensis enhances drought and salt stress tolerance in transgenic Arabidopsis thaliana[J]. Gene,2018,662(7):10-20.
[15] 杜丽.香樟子叶胚培养的研究[J].北方园艺,2014(14):89-92.
[16] 张佳佳,朱逢玲,焦晓琳,等.过表达香樟CcCBFb的烟草非生物胁迫抗性增强[J].园艺学报,2017,44(7):1397-1404.
[17] 张力维,李勇鹏,姚瑶,等.香樟延伸因子EF1α基因片段的克隆和表达分析[J].中南林业科技大学学报,2015,35(5):122-128.
[18] 张佳佳,张力维,李勇鹏,等.不同非生物胁迫下香樟实时定量PCR内参基因的选择[J].生物技术通报,2016,32(10):205-211.
[19] HARRISON S J, MOTT E K, PARSLEY K, et al. A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation[J]. Plant Methods,2006,2(1):19-25.
[20] 张瑛,罗志祥,周俊峰.植物脯氨酸测定方法优化及其应用[J].分子植物育种,2013,11(30):1219-1226.
[21] GUPTA K, JHA B, AGARWAL P K. A dehydration-responsive element binding (DREB) transcription factor from the succulent halophyte Salicorniabrachiataenhances abiotic stress tolerancein transgenic tobacco[J]. Mar Biotechnol,2014,16(6):657-673.
[22] GUO R G, ZHAO J, WANG X H, et al. Constitutive expression of a grape aspartic protease genein transgenic Arabidopsis confersosmotic stress tolerance[J]. Plant Cell Tiss Organ Cult,2015,121(2):275-287.
[23] 刘海妹,陈韵琳,李成浩.PtrDREB28转录因子的克隆及抗逆性[J].东北林业大学学报,2018,46(2):12-16.
[24] 刘晨旭,刘彧,刘杰,等.过量表达甘菊CBF1基因提高拟南芥抗旱耐盐能力[J].草业科学,2018,35(6):1400-1408.
[25] ZHAO J, ZHANG X, GUO R, et al. Over-expression of a grape WRKY transcription factor gene, VlWRKY48, in Arabidopsis thaliana, increases disease resistance and drought stress tolerance[J]. Plant Cell Tissue & Organ Culture,2018,132(2):359-370.
[26] 李文明,辛建攀,魏驰宇,等.植物抗寒性研究进展[J].江苏农业科学,2017,45(12):6-11.
[27] 王宁.植物抗寒生理性研究进展[J].北方园艺,2014(4):174-177.
[28] 安飞飞,李庚虎,陈霆,等.植物耐寒生理及蛋白质组学研究进展[J].中国农学通报,2015,31(14):96-101.