黄瓜脂氢过氧化物裂解酶基因13-CsHPL的克隆及其表达分析
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摘要
为了研究黄瓜脂氢过氧化物裂解酶基因13-Cs HPL在不同生物胁迫和非生物胁迫条件下的表达模式,本研究以黄瓜种质26号为材料,通过同源克隆的方法克隆了黄瓜氢过氧化物裂解酶基因13-CsHPL的cDNA全长并分析其序列特征;通过Real-time PCR技术检测了13-CsHPL在华北型26号黄瓜幼苗受到不同逆境胁迫条件下的表达模式。结果表明:13-Cs HPL基因的cDNA序列全长为1 478 bp,编码481个氨基酸,分子量为54.34 kD。黄瓜种质26号幼苗的13-Cs HPL基因在受到非生物胁迫如干旱、NaCl、甲基紫精、H2O2、损伤与外源激素MeJA、SA和灰霉侵染处理后其表达水平均显著升高。干旱、Na Cl、H2O2胁迫处理12 h时13-CsHPL的表达水平升高,其中H2O2处理12 h时13-CsHPL的升高最为明显,比对照显著增加了216.79%;在各个处理中,13-CsHPL对甲基紫精及损伤胁迫处理的响应较早,与对照相比,分别在6 h和8 h后显著升高,分别比对照显著增加了52.96%和146.67%。本研究可为黄瓜13-CsHPL参与抗逆性调控的分子机理提供帮助。
In order to study the expression patterns of hydroperoxide lyase gene 13-CsHPL in cucumber under different biotic and abiotic stresses, the cDNA full length of cucumber hydroperoxide lyase gene 13-CsHPL was cloned by homologous cloning and its sequence characteristics were analyzed with cucumber germplasm No.26 as the material. The Real-time PCR was used to detect the expression pattern of 13-CsHPL in the seedling of cucumber germplasm Huabei No.26 under different stress conditions. The results showed that the cDNA full length of 13-CsHPL was 1 478 bp, encoding 481 amino acids, and the molecular weight was 54.34 kD. The expression of 13-CsHPL in seedlings of cucumber germplasm 26 was significantly increased under abiotic stresses such as drought, NaCl, methyl violet, H_2O_2, wounding and exogenous hormones Me JA, SA and Botrytis cinerea infection. The expression of 13-CsHPL increased in 12 hours with drought, NaCl and H2 O2 treatment, and the increase of 13-Cs HPL was the most significant in H2 O2 treatment for 12 h, which was significantly increased by216.79% compared with the control. In each treatment, the response of 13-CsHPL to methyl viologenine and injury stress was earlier than that of the control, which increased significantly after 6 h and 8 h, and increased by 52.96% and 146.67%, respectively. These results might be helpful for the molecular mechanism of 13-CsHPL involved in the regulation of stress resistance of cucumber.
In order to study the expression patterns of hydroperoxide lyase gene 13-CsHPL in cucumber under different biotic and abiotic stresses, the cDNA full length of cucumber hydroperoxide lyase gene 13-CsHPL was cloned by homologous cloning and its sequence characteristics were analyzed with cucumber germplasm No.26 as the material. The Real-time PCR was used to detect the expression pattern of 13-CsHPL in the seedling of cucumber germplasm Huabei No.26 under different stress conditions. The results showed that the cDNA full length of 13-CsHPL was 1 478 bp, encoding 481 amino acids, and the molecular weight was 54.34 kD. The expression of 13-CsHPL in seedlings of cucumber germplasm 26 was significantly increased under abiotic stresses such as drought, NaCl, methyl violet, H_2O_2, wounding and exogenous hormones Me JA, SA and Botrytis cinerea infection. The expression of 13-CsHPL increased in 12 hours with drought, NaCl and H2 O2 treatment, and the increase of 13-Cs HPL was the most significant in H2 O2 treatment for 12 h, which was significantly increased by216.79% compared with the control. In each treatment, the response of 13-CsHPL to methyl viologenine and injury stress was earlier than that of the control, which increased significantly after 6 h and 8 h, and increased by 52.96% and 146.67%, respectively. These results might be helpful for the molecular mechanism of 13-CsHPL involved in the regulation of stress resistance of cucumber.
引文
Blokhina O.,Virolainen E.,and Frgerstedt K.V.,2003,Antioxidants,oxidative damage and oxygen deprivation stress:a review,Ann.Bot.,91(2):179-194
Bruce T.J.A.,Wadhams L.J.,and Woodcock C.M.,2005,Insect host location:a volatile situation,Trends Plant Sci.,10(6):269-274
Croft K.P.C.,Juttner F.,and Slusarenko A.J.,1993,Volatile products of the lipoxygenase athway evolved from Phaseolus vulgaris(L.)leaves inoculated with Pseudomonas syringae pv phaseolicola,Plant Physiol.,101(1):13-24
Grant G.G.,Ryall K.L.,Lyons D.B.,and Abou-Zaid M.M.,2010,Differential response of male and female emerald ash borers(Col.,Buprestidae)to(Z)-3-hexenol and manuka oil,J.Appl.Entomol.,134:26-33
Guo F.Z.,Jiang C.C.,Feng H.,Chen G.X.,Xia H.W.,She W.Q.,and Pan D.M.,2007,Cloning and sequencing of cDNA encoding conservative regions of hydroperoxide lyase in petals of Murraya paniculata,Citrus maxima,Yarendai Nongye Yanjiu(Subtropical Agriculture Research),3(1):73-76(郭凤芝,姜翠翠,冯会,陈桂信,夏海武,佘文琴,潘东明,2007,九里香,柚花瓣HPL基因保守序列的克隆与分析,亚热带农业研究,3(1):73-76)
Howe G.A.,Lee G.I.,Itoh A.,Li L.,and Derocher A.E.,2000,Cytochrome P450-dependent metabolism of oxylipins in tomato.Cloning and expression of allene oxide synthase and fatty acid hydroperoxide lyase,Plant Physiol.,123(2):711-724
Jia G.L.,Dai H.P.,Feng B.L.,Zhang S.Q.,and Zhang S.W.,2008,Biochemical characteristics in broomcorn millet(Panicum miliaceum L.)seedlings under PEG simulated drought stress,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),28(10):2073-2079(贾根良,代惠萍,冯佰利,张社奇,张嵩午,2008,PEG模拟干旱胁迫对糜子幼苗生理特性的影响,西北植物学报,28(10):2073-2079)
Li C.F.,Zhao L.Y.,Chen Y.T.,Yue P.,Gu W.,Wang Y.Y.,Teng X.Y.,and Wang N.B.,2010,Research advances on higher plant lipoxygenase,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),41(10):143-149(李彩凤,赵丽影,陈业婷,越鹏,谷维,王园园,滕祥勇,王楠博,2010,高等植物脂氧合酶研究进展,东北农业大学学报,41(10):143-149)
Li L.Z.,Li H.P.,and Liang E.N.,2008,Effect on salicylic acid to germination on cucumber seed and its seedling growth,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),36(10):3983-3984(李灵芝,李海平,梁二妮,2008,水杨酸对黄瓜种子萌发和幼苗生长的影响,安徽农业科学,36(10):3983-3984)
Matsui K.,Wilkinson J.,Hiatt B.,Knauf V.,and Kajiwara T.,1999,Molecular cloning and expression of Arabidopsis fatty acid hydroperoxide lyase,Plant Cell Physiol.,40(5):477-481
Mita G.,Quarta A.,Fasano P.,Paolis A.,Sansebastiano G.,Perrotta C.,Iannacone R.,Belfield E.,Hughes R.,Tsesmetzis N.,Casey R.,and Santino A.,2005,Molecular cloning and characterization of an almond 9-hydroperoxide lyase,a new CYP74 targeted to lipid bodies,J.Exp.Bot.,56(419):2321-2333
Noordermeer M.A.,Dijken A.J.H.,Smeekens S.C.,Veldink G.A.,and Vliegenthart J.F.,2001,Characterization of three cloned and expressed 13-hydroperoxide lyase isoenzymes from alfalfa with unusual N-terminal sequences and different enzyme kinetics,Eur.J.Biochem.,267(9):2473-2482
Shiojiri K.,Kishimoto K.,Ozawa R.,Kugimiya S.,Urashimo S.,Arimura G.,Horiuchi J.,Nishioka T.,Matsui K.,and Takabayashi J.,2006,Changing green leaf volatile biosynthesis in plants:an approach for improving plant resistance against both herbivores and pathogens,Proc.Natl.Acad.Sci.USA,103(45):16672-16676
Stefania D.D.,Stefania B.,Ralf H.,Vict oria P.,Marco T.,Palmiro P.,Muhammad I.,Gunter K.,Victor F.,Peter W.,and Angelo S.,2012,Transcriptomic analysis of oxylipin biosynthesis genes and chemical profiling reveal an early induction of jasmonates in chickpea roots under drought stress,Plant Physiol.Biochem.,61(4):115-122
Uematsu S.,and Fujimoto K.,2010,The innate immune system in the intestine,Microbiol.Immunol.,54(11):645-657
Vancanneyt G.,Sanz C.,Farmaki T.,Paneque M.,Ortego F.,Casta觡era P.,and Sánchez-Serrano J.J.,2001,Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance,Proc.Natl.Acad.Sci.USA,98(14):8139-8144
Wang L.Y.,Wang Y.,Meng X.,and Meng Q.W.,2012,Overexpression of tomato GDP-L-galactose phosphorylase gene enhanced tolerance of transgenic tobacco to methyl viologen-mediated oxidative stress,Zhiwu Shengli Xuebao(Plant Physiology Communications),48(7):689-698(王丽燕,王玉,孟夏,孟庆伟,2012,过表达番茄GDP-L-半乳糖磷酸酶基因提高烟草抗MV诱导的氧化胁迫能力,植物生理学报,48(7):689-698)
Weichert H.,Kolbe A.,Kraus A.,Wasternack C.,and Feussner I.,2002,Metabolic profiling of oxylipins in germinating cucumber seedlings lipoxygenase-dependant degradation of triacylglycerols and biosynthesis of volatile aldehydes,Planta,215(4):612-619
Xin Z.J.,Sun X.L.,Zhang Z.Q.,and Chen Z.M.,2013,Cloning and expression analysis of tea hydroperoxide lyase gene CsiHPL1,Zhiwu Yanjiu(Bulletin of Botanical Research),33(1):66-72(辛肇军,孙晓玲,张正群,陈宗懋,2013,茶树脂氢过氧化物裂解酶基因CsiHPL1的克隆及表达,植物研究,33(1):66-72)
Yang X.Y.,Jiang W.J.,and Yu H.J.,2012,The expression profiling of the lipoxygenase(LOX)family genes during fruit development,abiotic stress and hormonal treatments in cucumber(Cucumis sativus L.),Int.J.Mol.Sci.,13(2):2481-2500
Yao P.F.,Zhao X.R.,Li M.F.,Wang A.H.,and Wu Q.,2016,Cloning and expression analysis of transcription factor gene FtbHLH3 from Fagopyrum tataricum under abiotic stress,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(2):429-435(姚攀锋,赵学荣,李茂菲,王安虎,吴琦,2016,苦荞转录因子基因FtbHLH3的克隆及其非生物胁迫下的表达分析,基因组学与应用生物学,35(2):429-435)
Zhang Y.P.,2011,Effects of choline chloride on osmotic adjustment substances and reactive oxygen species metabolism of cucumber seedlings under salt stress,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),31(1):137-143(张永平,2011,氯化胆碱对盐胁迫黄瓜幼苗渗透调节物质及活性氧代谢系统的影响,西北植物学报,31(1):137-143)
Bruce T.J.A.,Wadhams L.J.,and Woodcock C.M.,2005,Insect host location:a volatile situation,Trends Plant Sci.,10(6):269-274
Croft K.P.C.,Juttner F.,and Slusarenko A.J.,1993,Volatile products of the lipoxygenase athway evolved from Phaseolus vulgaris(L.)leaves inoculated with Pseudomonas syringae pv phaseolicola,Plant Physiol.,101(1):13-24
Grant G.G.,Ryall K.L.,Lyons D.B.,and Abou-Zaid M.M.,2010,Differential response of male and female emerald ash borers(Col.,Buprestidae)to(Z)-3-hexenol and manuka oil,J.Appl.Entomol.,134:26-33
Guo F.Z.,Jiang C.C.,Feng H.,Chen G.X.,Xia H.W.,She W.Q.,and Pan D.M.,2007,Cloning and sequencing of cDNA encoding conservative regions of hydroperoxide lyase in petals of Murraya paniculata,Citrus maxima,Yarendai Nongye Yanjiu(Subtropical Agriculture Research),3(1):73-76(郭凤芝,姜翠翠,冯会,陈桂信,夏海武,佘文琴,潘东明,2007,九里香,柚花瓣HPL基因保守序列的克隆与分析,亚热带农业研究,3(1):73-76)
Howe G.A.,Lee G.I.,Itoh A.,Li L.,and Derocher A.E.,2000,Cytochrome P450-dependent metabolism of oxylipins in tomato.Cloning and expression of allene oxide synthase and fatty acid hydroperoxide lyase,Plant Physiol.,123(2):711-724
Jia G.L.,Dai H.P.,Feng B.L.,Zhang S.Q.,and Zhang S.W.,2008,Biochemical characteristics in broomcorn millet(Panicum miliaceum L.)seedlings under PEG simulated drought stress,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),28(10):2073-2079(贾根良,代惠萍,冯佰利,张社奇,张嵩午,2008,PEG模拟干旱胁迫对糜子幼苗生理特性的影响,西北植物学报,28(10):2073-2079)
Li C.F.,Zhao L.Y.,Chen Y.T.,Yue P.,Gu W.,Wang Y.Y.,Teng X.Y.,and Wang N.B.,2010,Research advances on higher plant lipoxygenase,Dongbei Nongye Daxue Xuebao(Journal of Northeast Agricultural University),41(10):143-149(李彩凤,赵丽影,陈业婷,越鹏,谷维,王园园,滕祥勇,王楠博,2010,高等植物脂氧合酶研究进展,东北农业大学学报,41(10):143-149)
Li L.Z.,Li H.P.,and Liang E.N.,2008,Effect on salicylic acid to germination on cucumber seed and its seedling growth,Anhui Nongye Kexue(Journal of Anhui Agricultural Sciences),36(10):3983-3984(李灵芝,李海平,梁二妮,2008,水杨酸对黄瓜种子萌发和幼苗生长的影响,安徽农业科学,36(10):3983-3984)
Matsui K.,Wilkinson J.,Hiatt B.,Knauf V.,and Kajiwara T.,1999,Molecular cloning and expression of Arabidopsis fatty acid hydroperoxide lyase,Plant Cell Physiol.,40(5):477-481
Mita G.,Quarta A.,Fasano P.,Paolis A.,Sansebastiano G.,Perrotta C.,Iannacone R.,Belfield E.,Hughes R.,Tsesmetzis N.,Casey R.,and Santino A.,2005,Molecular cloning and characterization of an almond 9-hydroperoxide lyase,a new CYP74 targeted to lipid bodies,J.Exp.Bot.,56(419):2321-2333
Noordermeer M.A.,Dijken A.J.H.,Smeekens S.C.,Veldink G.A.,and Vliegenthart J.F.,2001,Characterization of three cloned and expressed 13-hydroperoxide lyase isoenzymes from alfalfa with unusual N-terminal sequences and different enzyme kinetics,Eur.J.Biochem.,267(9):2473-2482
Shiojiri K.,Kishimoto K.,Ozawa R.,Kugimiya S.,Urashimo S.,Arimura G.,Horiuchi J.,Nishioka T.,Matsui K.,and Takabayashi J.,2006,Changing green leaf volatile biosynthesis in plants:an approach for improving plant resistance against both herbivores and pathogens,Proc.Natl.Acad.Sci.USA,103(45):16672-16676
Stefania D.D.,Stefania B.,Ralf H.,Vict oria P.,Marco T.,Palmiro P.,Muhammad I.,Gunter K.,Victor F.,Peter W.,and Angelo S.,2012,Transcriptomic analysis of oxylipin biosynthesis genes and chemical profiling reveal an early induction of jasmonates in chickpea roots under drought stress,Plant Physiol.Biochem.,61(4):115-122
Uematsu S.,and Fujimoto K.,2010,The innate immune system in the intestine,Microbiol.Immunol.,54(11):645-657
Vancanneyt G.,Sanz C.,Farmaki T.,Paneque M.,Ortego F.,Casta觡era P.,and Sánchez-Serrano J.J.,2001,Hydroperoxide lyase depletion in transgenic potato plants leads to an increase in aphid performance,Proc.Natl.Acad.Sci.USA,98(14):8139-8144
Wang L.Y.,Wang Y.,Meng X.,and Meng Q.W.,2012,Overexpression of tomato GDP-L-galactose phosphorylase gene enhanced tolerance of transgenic tobacco to methyl viologen-mediated oxidative stress,Zhiwu Shengli Xuebao(Plant Physiology Communications),48(7):689-698(王丽燕,王玉,孟夏,孟庆伟,2012,过表达番茄GDP-L-半乳糖磷酸酶基因提高烟草抗MV诱导的氧化胁迫能力,植物生理学报,48(7):689-698)
Weichert H.,Kolbe A.,Kraus A.,Wasternack C.,and Feussner I.,2002,Metabolic profiling of oxylipins in germinating cucumber seedlings lipoxygenase-dependant degradation of triacylglycerols and biosynthesis of volatile aldehydes,Planta,215(4):612-619
Xin Z.J.,Sun X.L.,Zhang Z.Q.,and Chen Z.M.,2013,Cloning and expression analysis of tea hydroperoxide lyase gene CsiHPL1,Zhiwu Yanjiu(Bulletin of Botanical Research),33(1):66-72(辛肇军,孙晓玲,张正群,陈宗懋,2013,茶树脂氢过氧化物裂解酶基因CsiHPL1的克隆及表达,植物研究,33(1):66-72)
Yang X.Y.,Jiang W.J.,and Yu H.J.,2012,The expression profiling of the lipoxygenase(LOX)family genes during fruit development,abiotic stress and hormonal treatments in cucumber(Cucumis sativus L.),Int.J.Mol.Sci.,13(2):2481-2500
Yao P.F.,Zhao X.R.,Li M.F.,Wang A.H.,and Wu Q.,2016,Cloning and expression analysis of transcription factor gene FtbHLH3 from Fagopyrum tataricum under abiotic stress,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),35(2):429-435(姚攀锋,赵学荣,李茂菲,王安虎,吴琦,2016,苦荞转录因子基因FtbHLH3的克隆及其非生物胁迫下的表达分析,基因组学与应用生物学,35(2):429-435)
Zhang Y.P.,2011,Effects of choline chloride on osmotic adjustment substances and reactive oxygen species metabolism of cucumber seedlings under salt stress,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),31(1):137-143(张永平,2011,氯化胆碱对盐胁迫黄瓜幼苗渗透调节物质及活性氧代谢系统的影响,西北植物学报,31(1):137-143)