苹果细胞分裂素氧化酶基因MdCKX7.2的克隆及抗性鉴定
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摘要
采用同源克隆和PCR技术从‘嘎拉’苹果(Malus×domestica Borkh.)中克隆细胞分裂素氧化酶基因MdCKX7.2(基因序列号:MDP0000279125)。该基因含有1 542 bp的完整开放阅读框,编码513个氨基酸。利用Plant CARE数据库对MdCKX7.2启动子顺式作用元件进行预测分析,发现MdCKX7.2启动子序列中存在光、干旱、脱落酸、水杨酸、茉莉酸甲酯等响应元件。基因表达分析发现,‘嘎拉’幼苗中MdCKX7.2的表达明显受干旱和ABA的诱导。通过农杆菌介导的遗传转化获得MdCKX7.2转基因拟南芥植株。抗性试验表明,异位表达MdCKX7.2基因明显提高了拟南芥对干旱胁迫的抗性。拟南芥种子萌发试验表明,在拟南芥中异位表达MdCKX7.2提高了植株对ABA的敏感性,种子萌发率和幼苗鲜质量明显下降,与种子萌发相关基因的表达量明显上调。以上试验结果表明,MdCKX7.2在植物非生物胁迫响应中发挥重要作用。
MdCKX7.2 gene (MDP0000279125) was cloned from Malus × domestica Borkh‘Gala'using homologous cloning and RT-PCR. The gene contained a 1 542 bp complete open reading frame(ORF)and encoded 513 amino acids. The cis-acting elements of the MdCKX7.2 promoter were analyzed using the Plant CARE database. It was found that there were elements including light,drought,abscisic acid,salicylic acid,and methyl jasmonate in the MdCKX7.2 promoter sequence. Expression analysis revealed that the transcription of MdCKX7.2 in‘Gala'seedlings was significantly induced by drought and ABA treatments. MdCKX7.2 transgenic Arabidopsis plants were obtained by Agrobacterium-mediated genetic transformation. The stress tolerance assays showed that ectopic expression of MdCKX7.2 significantly increased the tolerance of transgenic Arabidopsis to drought stress. Arabidopsis seed germination experiments showed that ectopic expression of MdCKX7.2 in Arabidopsis enhanced the sensitivity of Arabidopsis to ABA,the seed germination rate and seedling fresh weight decreased significantly,and the expression level of genes related to seed germination was significantly up-regulated.The above results indicate that MdCKX7.2 plays an important role in plant abiotic stress response.
MdCKX7.2 gene (MDP0000279125) was cloned from Malus × domestica Borkh‘Gala'using homologous cloning and RT-PCR. The gene contained a 1 542 bp complete open reading frame(ORF)and encoded 513 amino acids. The cis-acting elements of the MdCKX7.2 promoter were analyzed using the Plant CARE database. It was found that there were elements including light,drought,abscisic acid,salicylic acid,and methyl jasmonate in the MdCKX7.2 promoter sequence. Expression analysis revealed that the transcription of MdCKX7.2 in‘Gala'seedlings was significantly induced by drought and ABA treatments. MdCKX7.2 transgenic Arabidopsis plants were obtained by Agrobacterium-mediated genetic transformation. The stress tolerance assays showed that ectopic expression of MdCKX7.2 significantly increased the tolerance of transgenic Arabidopsis to drought stress. Arabidopsis seed germination experiments showed that ectopic expression of MdCKX7.2 in Arabidopsis enhanced the sensitivity of Arabidopsis to ABA,the seed germination rate and seedling fresh weight decreased significantly,and the expression level of genes related to seed germination was significantly up-regulated.The above results indicate that MdCKX7.2 plays an important role in plant abiotic stress response.
引文
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Bohnert H J,Nelson D E,Jensen R G.1995.Adaptations to environmental stresses.The Plant Cell,7(7):1099.
Bray E A.1997.Plant responses to water deficit.Trends Plant Sci,2:48-54.
Brownlee B G,Hall R H,Whitty C D.1975.3-Methyl-2-butenal:an enzymatic degradation product of the cytokinin,N6-(Δ2-Isopentenyl)adenine.Canadian Journal of Biochemistry,53(1):37-41.
Clough S J,Bent A F.1998.Floral dip:a simplifiedmethod for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J,16(6):735-743.
Frébort I,Kowalska M,Hluska T,FrébortováJ,Galuszka P.2011.Evolution of cytokinin biosynthesis and degradation.Journal of Experimental Botany,62(8):2431-2452.
FrébortováJ,Fraaije M W,Galuszka P,?ebela M,Pavel P E?,Hrbá?J,Novák O,Bilyeu K D,English J T,Frébort I.2004.Catalytic reaction of cytokinin dehydrogenase:preference for quinones as electron acceptors.Biochemical Journal,380(1):121-130.
Galuszka P,FrébortováJ,Werner T,Yamada M,Strnad M,Schmülling T,Frébort I.2004.Cytokinin oxidase/dehydrogenase genes in barley and wheat.The FEBS Journal,271(20):3990-4002.
Gómez-Cadenas A,Vives V,I Zandalinas S,Manzi M,M Sanchez-Perez A,M Perez-Clemente R,Arbona V.2015.Abscisic acid:a versatile phytohormone in plant signaling and beyond.Current Protein and Peptide Science,16(5):413-434.
Houba-Hérin N,Pethe C,d’Alayer J,Laloue M.1999.Cytokinin oxidase from Zea mays:purification,c DNA cloning and expression in moss protoplasts.The Plant Journal,17(6):615-626.
Kang N Y,Cho C,Kim N Y,Kim J.2012.Cytokinin receptor-dependent and receptor-independent pathways in the dehydration response of Arabidopsis thaliana.Journal of Plant Physiology,169(14):1382-1391.
Kim H J,Ryu H,Hong S H,Woo H R,Lim P O,Lee I C,Sheen J,Nam H G,Hwang I.2006.Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in Arabidopsis.Proceedings of the National Academy of Sciences of the United States of America,103(3):814-819.
Le D T,Nishiyama R,Watanabe Y,Vankova R,Tanaka M,Seki M,Ham L H,Yamaguchi-Shinozaki K,Shinozaki K,Tran L S P.2012.Identification and expression analysis of cytokinin metabolic genes in soybean under normal and drought conditions in relation to cytokinin levels.PLoS One,7(8),e42411.
Li Jia,Zhou Xue,Li Ting-ting,Wang Zhong-hua,Quan Li.2016.Cloning and functional analysis of a BdCKX gene in Brachypodium distachyon.Agricultural Research in the Arid Areas.34(3),261-266.(in Chinese)李嘉,周雪,李婷婷,王中华,权力.2016.二穗短柄草细胞分裂素氧化酶BdCKX编码基因的克隆及功能分析.干旱地区农业研究,34(3):261-266.
Malito E,Coda A,Bilyeu K D,Fraaije M W,Mattevi A.2004.Structures of Michaelis and product complexes of plant cytokinin dehydrogenase:implications for flavoenzyme catalysis.Journal of Molecular Biology,341(5):1237-1249.
McGaw B A,Horgan R.1983.Cytokinin catabolism and cytokinin oxidase.Phytochemistry,22(5):1103-1105.
Mok D W,Mok M C.2001.Cytokinin metabolism and action.Annu Rev Plant Biol,52:89-118.
Nakashima K,Yamaguchi-Shinozaki K,Shinozaki K.2014.The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought,cold,and heat.Frontiers in Plant Science,5:170.
Nguyen K H,Van Ha C,Nishiyama R,Watanabe Y,Leyva-González M A,Fujita Y,Tran Tu,Li W,Tanaka M,Seki M,Schaller G E,Herrera-Estrella L,Tran Lam-Son P.2016.Arabidopsis type B cytokinin response regulators ARR1,ARR10,and ARR12 negatively regulate plant responses to drought.Proceedings of the National Academy of Sciences,113(11):3090-3095.
Nishiyama R,Watanabe Y,Fujita Y,Le D T,Kojima M,Werner T,Vankova R,Yamaguchi-Shinozaki K,Shinozaki K,Kakimoto T,Sakakibara H,Schmülling T,Tran L S P.2011.Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought,salt and abscisic acid responses,and abscisic acid biosynthesis.The Plant Cell,23(6):2169-2183.
Sakakibara H.2006.Cytokinins:activity,biosynthesis,and translocation.Annu Rev Plant Biol,57:431-449.
Schmülling T,Werner T,Riefler M,KrupkováE,Manns I B.2003.Structure and function of cytokinin oxidase/dehydrogenase genes of maize,rice,Arabidopsis and other species.Journal of Plant Research,116(3):241-252.
To J P,Kieber J J.2008.Cytokinin signaling:two-components and more.Trends in Plant Science,13(2):85-92.
Tuteja N.2007.Abscisic acid and abiotic stress signaling.Plant Signal Behav,2:135-138.
Vyroubalová?,VáclavíkováK,Ture?kováV,Novák O,?mehilováM,Hluska T,OhnoutkováL,Frébort I,Galuszka P.2009.Characterization of new maize genes putatively involved in cytokinin metabolism and their expression during osmotic stress in relation to cytokinin levels.Plant Physiology,151(1):433-447.
Wang Xin-wen.2008.Research progress of jasmonic acid involved in plant stress.Anhui Agricultural Science Bulletin,(6):29-35,24.(in Chinese)汪新文.2008.茉莉酸参与植物逆境胁迫的研究进展.安徽农学通报,(6):29-35,24.
Wang Y,Li L,Ye T,Zhao S,Liu Z,Feng Y Q,Wu Y.2011.Cytokinin antagonizes ABA suppression to seed germination of Arabidopsis by downregulating ABI5 expression.The Plant Journal,68(2):249-261.
Werner T,Motyka V,Laucou V,Smets R,Van Onckelen H,Schmülling T.2003.Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity.The Plant Cell,15(11):2532-2550.
Werner T,Nehnevajova E,K?llmer I,Novák O,Strnad M,Kr?mer U,Schmülling T.2010.Root-specific reduction of cytokinin causes enhanced root growth,drought tolerance,and leaf mineral enrichment in Arabidopsis and tobacco.The Plant Cell,22(12):3905-3920.
Yamaguchi-Shinozaki K,Shinozaki K.2006.Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses.Annu Rev Plant Biol,57:781-803.
Yang S H,Yu H,Goh C J.2003.Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid.Plant Molecular Biology,51(2):237-248.
Yeh S Y,Chen H W,Ng C Y,Lin C Y,Tseng T H,Li W H,Ku M S.2015.Down-regulation of cytokinin oxidase 2 expression increases tiller number and improves rice yield.Rice,8(1):36.
Zhang Fen,Guo De-ping,Lin Ming-li,Yan Miao-miao.2008.Physiological,biochemical and molecular properties of cytokinin oxidase/dehydrogenase.Plant Physiology Communications,44(4):797-803.(in Chinese)张芬,郭得平,林明丽,闫淼淼.2008.细胞分裂素氧化酶/脱氢酶的生理生化和分子特性.植物生理学通讯,44(4):797-803.
Zhao Shi-jie,Xu Chang-cheng,Zou Qi,Meng Qing-wei.1994.Improvements of method for measurement of malondialdehyde in plant tissues.Plant Physiology Communications,30(3):207-210.(in Chinese)赵世杰,许长城,邹琦,孟庆伟.1994.植物组织中丙二醛测定方法的改进.植物生理学通讯,30(3):207-210.
Zhao Yan-xia,Qi Gui-mei,Wang Yong-mei.2017.Research progress of salicylic acid on stress resistance regulation of grape.Shandong Agricultural Sciences,49(11):146-150.赵艳侠,亓桂梅,王咏梅.2017.水杨酸对葡萄抗逆性调控的研究进展.山东农业科学,49(11):146-150.
Zhou Yan-qi,Bu qing-yan.2011.Research progress on the change of endogenous hormone in fruit tress under drought stress.Northern Fruits,(3):1-4.(in Chinese)周晏起,卜庆雁.2011.干旱胁迫下果树内源激素变化规律研究进展.北方果树,(3):1-4.
Bohnert H J,Nelson D E,Jensen R G.1995.Adaptations to environmental stresses.The Plant Cell,7(7):1099.
Bray E A.1997.Plant responses to water deficit.Trends Plant Sci,2:48-54.
Brownlee B G,Hall R H,Whitty C D.1975.3-Methyl-2-butenal:an enzymatic degradation product of the cytokinin,N6-(Δ2-Isopentenyl)adenine.Canadian Journal of Biochemistry,53(1):37-41.
Clough S J,Bent A F.1998.Floral dip:a simplifiedmethod for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant J,16(6):735-743.
Frébort I,Kowalska M,Hluska T,FrébortováJ,Galuszka P.2011.Evolution of cytokinin biosynthesis and degradation.Journal of Experimental Botany,62(8):2431-2452.
FrébortováJ,Fraaije M W,Galuszka P,?ebela M,Pavel P E?,Hrbá?J,Novák O,Bilyeu K D,English J T,Frébort I.2004.Catalytic reaction of cytokinin dehydrogenase:preference for quinones as electron acceptors.Biochemical Journal,380(1):121-130.
Galuszka P,FrébortováJ,Werner T,Yamada M,Strnad M,Schmülling T,Frébort I.2004.Cytokinin oxidase/dehydrogenase genes in barley and wheat.The FEBS Journal,271(20):3990-4002.
Gómez-Cadenas A,Vives V,I Zandalinas S,Manzi M,M Sanchez-Perez A,M Perez-Clemente R,Arbona V.2015.Abscisic acid:a versatile phytohormone in plant signaling and beyond.Current Protein and Peptide Science,16(5):413-434.
Houba-Hérin N,Pethe C,d’Alayer J,Laloue M.1999.Cytokinin oxidase from Zea mays:purification,c DNA cloning and expression in moss protoplasts.The Plant Journal,17(6):615-626.
Kang N Y,Cho C,Kim N Y,Kim J.2012.Cytokinin receptor-dependent and receptor-independent pathways in the dehydration response of Arabidopsis thaliana.Journal of Plant Physiology,169(14):1382-1391.
Kim H J,Ryu H,Hong S H,Woo H R,Lim P O,Lee I C,Sheen J,Nam H G,Hwang I.2006.Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in Arabidopsis.Proceedings of the National Academy of Sciences of the United States of America,103(3):814-819.
Le D T,Nishiyama R,Watanabe Y,Vankova R,Tanaka M,Seki M,Ham L H,Yamaguchi-Shinozaki K,Shinozaki K,Tran L S P.2012.Identification and expression analysis of cytokinin metabolic genes in soybean under normal and drought conditions in relation to cytokinin levels.PLoS One,7(8),e42411.
Li Jia,Zhou Xue,Li Ting-ting,Wang Zhong-hua,Quan Li.2016.Cloning and functional analysis of a BdCKX gene in Brachypodium distachyon.Agricultural Research in the Arid Areas.34(3),261-266.(in Chinese)李嘉,周雪,李婷婷,王中华,权力.2016.二穗短柄草细胞分裂素氧化酶BdCKX编码基因的克隆及功能分析.干旱地区农业研究,34(3):261-266.
Malito E,Coda A,Bilyeu K D,Fraaije M W,Mattevi A.2004.Structures of Michaelis and product complexes of plant cytokinin dehydrogenase:implications for flavoenzyme catalysis.Journal of Molecular Biology,341(5):1237-1249.
McGaw B A,Horgan R.1983.Cytokinin catabolism and cytokinin oxidase.Phytochemistry,22(5):1103-1105.
Mok D W,Mok M C.2001.Cytokinin metabolism and action.Annu Rev Plant Biol,52:89-118.
Nakashima K,Yamaguchi-Shinozaki K,Shinozaki K.2014.The transcriptional regulatory network in the drought response and its crosstalk in abiotic stress responses including drought,cold,and heat.Frontiers in Plant Science,5:170.
Nguyen K H,Van Ha C,Nishiyama R,Watanabe Y,Leyva-González M A,Fujita Y,Tran Tu,Li W,Tanaka M,Seki M,Schaller G E,Herrera-Estrella L,Tran Lam-Son P.2016.Arabidopsis type B cytokinin response regulators ARR1,ARR10,and ARR12 negatively regulate plant responses to drought.Proceedings of the National Academy of Sciences,113(11):3090-3095.
Nishiyama R,Watanabe Y,Fujita Y,Le D T,Kojima M,Werner T,Vankova R,Yamaguchi-Shinozaki K,Shinozaki K,Kakimoto T,Sakakibara H,Schmülling T,Tran L S P.2011.Analysis of cytokinin mutants and regulation of cytokinin metabolic genes reveals important regulatory roles of cytokinins in drought,salt and abscisic acid responses,and abscisic acid biosynthesis.The Plant Cell,23(6):2169-2183.
Sakakibara H.2006.Cytokinins:activity,biosynthesis,and translocation.Annu Rev Plant Biol,57:431-449.
Schmülling T,Werner T,Riefler M,KrupkováE,Manns I B.2003.Structure and function of cytokinin oxidase/dehydrogenase genes of maize,rice,Arabidopsis and other species.Journal of Plant Research,116(3):241-252.
To J P,Kieber J J.2008.Cytokinin signaling:two-components and more.Trends in Plant Science,13(2):85-92.
Tuteja N.2007.Abscisic acid and abiotic stress signaling.Plant Signal Behav,2:135-138.
Vyroubalová?,VáclavíkováK,Ture?kováV,Novák O,?mehilováM,Hluska T,OhnoutkováL,Frébort I,Galuszka P.2009.Characterization of new maize genes putatively involved in cytokinin metabolism and their expression during osmotic stress in relation to cytokinin levels.Plant Physiology,151(1):433-447.
Wang Xin-wen.2008.Research progress of jasmonic acid involved in plant stress.Anhui Agricultural Science Bulletin,(6):29-35,24.(in Chinese)汪新文.2008.茉莉酸参与植物逆境胁迫的研究进展.安徽农学通报,(6):29-35,24.
Wang Y,Li L,Ye T,Zhao S,Liu Z,Feng Y Q,Wu Y.2011.Cytokinin antagonizes ABA suppression to seed germination of Arabidopsis by downregulating ABI5 expression.The Plant Journal,68(2):249-261.
Werner T,Motyka V,Laucou V,Smets R,Van Onckelen H,Schmülling T.2003.Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity.The Plant Cell,15(11):2532-2550.
Werner T,Nehnevajova E,K?llmer I,Novák O,Strnad M,Kr?mer U,Schmülling T.2010.Root-specific reduction of cytokinin causes enhanced root growth,drought tolerance,and leaf mineral enrichment in Arabidopsis and tobacco.The Plant Cell,22(12):3905-3920.
Yamaguchi-Shinozaki K,Shinozaki K.2006.Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses.Annu Rev Plant Biol,57:781-803.
Yang S H,Yu H,Goh C J.2003.Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid.Plant Molecular Biology,51(2):237-248.
Yeh S Y,Chen H W,Ng C Y,Lin C Y,Tseng T H,Li W H,Ku M S.2015.Down-regulation of cytokinin oxidase 2 expression increases tiller number and improves rice yield.Rice,8(1):36.
Zhang Fen,Guo De-ping,Lin Ming-li,Yan Miao-miao.2008.Physiological,biochemical and molecular properties of cytokinin oxidase/dehydrogenase.Plant Physiology Communications,44(4):797-803.(in Chinese)张芬,郭得平,林明丽,闫淼淼.2008.细胞分裂素氧化酶/脱氢酶的生理生化和分子特性.植物生理学通讯,44(4):797-803.
Zhao Shi-jie,Xu Chang-cheng,Zou Qi,Meng Qing-wei.1994.Improvements of method for measurement of malondialdehyde in plant tissues.Plant Physiology Communications,30(3):207-210.(in Chinese)赵世杰,许长城,邹琦,孟庆伟.1994.植物组织中丙二醛测定方法的改进.植物生理学通讯,30(3):207-210.
Zhao Yan-xia,Qi Gui-mei,Wang Yong-mei.2017.Research progress of salicylic acid on stress resistance regulation of grape.Shandong Agricultural Sciences,49(11):146-150.赵艳侠,亓桂梅,王咏梅.2017.水杨酸对葡萄抗逆性调控的研究进展.山东农业科学,49(11):146-150.
Zhou Yan-qi,Bu qing-yan.2011.Research progress on the change of endogenous hormone in fruit tress under drought stress.Northern Fruits,(3):1-4.(in Chinese)周晏起,卜庆雁.2011.干旱胁迫下果树内源激素变化规律研究进展.北方果树,(3):1-4.