胡萝卜ACC合成酶基因DcACS的克隆与表达分析
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摘要
为分析ACS基因在胡萝卜不同组织和逆境胁迫条件下的表达情况,以胡萝卜品种黑田五寸为试验材料,克隆得到编码1-氨基环丙烷-1-羧酸合成酶(ACS)的基因DcACS,采用DNAMAN、NCBI、ExPASy和MEGA 5.1等生物信息学软件对其序列特征进行分析,并利用实时荧光定量PCR检测其在不同胡萝卜组织和非生物胁迫条件下的表达水平。序列分析结果表明,胡萝卜DcACS基因全长1 485 bp,编码494个氨基酸;推测的氨基酸序列含有ACS特有的7个保守结构域和4个不变氨基酸残基,在进化关系上与葫芦科的黄瓜和香瓜亲缘关系最接近。实时荧光定量PCR结果表明,DcACS基因在叶片中的表达量最高,具有明显的组织特异性,并响应高温、低温、干旱和盐胁迫等非生物胁迫。本研究克隆的DcACS基因为研究胡萝卜生长发育及响应非生物胁迫等过程提供了一定的理论依据。
To investigate the expression profiles of DcACS gene in different carrot tissues and abiotic stresses, a gene DcACS that encodes 1-aminocyclopropane-1-carboxylic acid synthase(ACS) was cloned from carrot cultivar ‘Kurodagosun'. Bioinformatic tools including DNAMAN, NCBI, ExPASy and MEGA 5.1 were used to analyze the sequence information, and quantitative real-time PCR(RT-qPCR) was performed to detect the expression levels of DcACS gene in carrot tissues and under abiotic stresses. Sequence analysis showed that the DcACS gene was 1 485 bp in length and encoded 494 amino acids. The deduced amino acid sequence contains 7 conserved domains and four invariant amino acid residues unique to ACS. The evolutionary relationship of DcACS was more close to cucumber and melon from the Cucurbitaceae family. RT-qPCR analysis revealed that DcACS gene had the highest expression in leaves, showing obvious tissue specificity. Moreover, this gene could respond to abiotic stresses, such as high temperature, low temperature, drought, and salt stress. The DcACS gene cloned in this study can shed light on studies about carrot in response to plant growth and abiotic stresses.
To investigate the expression profiles of DcACS gene in different carrot tissues and abiotic stresses, a gene DcACS that encodes 1-aminocyclopropane-1-carboxylic acid synthase(ACS) was cloned from carrot cultivar ‘Kurodagosun'. Bioinformatic tools including DNAMAN, NCBI, ExPASy and MEGA 5.1 were used to analyze the sequence information, and quantitative real-time PCR(RT-qPCR) was performed to detect the expression levels of DcACS gene in carrot tissues and under abiotic stresses. Sequence analysis showed that the DcACS gene was 1 485 bp in length and encoded 494 amino acids. The deduced amino acid sequence contains 7 conserved domains and four invariant amino acid residues unique to ACS. The evolutionary relationship of DcACS was more close to cucumber and melon from the Cucurbitaceae family. RT-qPCR analysis revealed that DcACS gene had the highest expression in leaves, showing obvious tissue specificity. Moreover, this gene could respond to abiotic stresses, such as high temperature, low temperature, drought, and salt stress. The DcACS gene cloned in this study can shed light on studies about carrot in response to plant growth and abiotic stresses.
引文
[1] Lin Y, Yang L, Paul M, Zu Y, Tang Z. Ethylene promotes germination of Arabidopsis seed under salinity by decreasing reactive oxygen species: evidence for the involvement of nitric oxide simulated by sodium nitroprusside [J]. Plant Physiology and Biochemistry, 2013, 73: 211-218
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[11] 孙申申, 温秀萍, 杨菲颖, 李梦思, 李欢, 李科, 陈晓静. ‘云香’水仙ACC合成酶基因NtACS1的克隆及遗传转化 [J]. 西北植物学报, 2017, 37(2): 250-257
[12] 董晨, 李伟才, 张纯, 魏永赞, 王弋, 胡会刚. 香蕉ACS基因家族的系统进化分析 [J]. 分子植物育种, 2017, 15(2): 425-432
[13] 徐士娟, 邝健飞, 陆旺金, 万嗣宝, 陈建业. 龙眼果实ACS和ACO基因克隆及其表达特性 [J]. 果树学报, 2010, 27(1): 39-44
[14] 吴建阳, 李彩琴, 李建国. 荔枝ACS1基因的分离及其与幼果脱落的关系 [J]. 果树学报, 2017, 34(7): 817-827
[15] 张馨月, 王广龙, 黄蔚, 王枫, 倪桢燚, 熊爱生. 胡萝卜抗坏血酸过氧化物酶基因的分离及其对非生物胁迫的响应 [J]. 南京农业大学学报, 2016, 39(1): 55-62
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[18] Geourjon C, Deléage G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments [J]. Bioinformatics, 1995, 11(6): 681-684
[19] Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino T G, Bertoni M, Bordoli L, Schwede T. SWISS-MODEL: modeling protein tertiary and quaternary structure using evolutionary information [J]. Nucleic Acids Research, 2014, 42(W1): 252-258
[20] Tian C, Jiang Q, Wang F, Wang G L, Xu Z S, Xiong A S. Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves [J]. PLoS One, 2015, 10(2): e0117569
[21] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method [J]. Methods, 2001, 25(4): 402-408
[22] 王立红, 张巨松, 李星星, 阿曼古丽·买买提阿力. 外源水杨酸对盐胁迫下棉花幼苗光合作用的影响 [J]. 核农学报, 2016, 30(9): 1864-1871
[23] 汪宝卿, 姜瑶, 解备涛, 张海燕, 董顺旭, 段文学, 王庆美, 张立明. 2个不同耐旱性甘薯品种的苗期根系蛋白组差异分析 [J]. 核农学报, 2017, 31(2): 232-240
[24] 张琪, 周薇, 崔慧萍, 宋丽莉, 郭长虹. 不同类型脱水素在植物低温胁迫应答中的作用 [J]. 核农学报, 2017, 31(4): 689-695
[25] 张弢, 董春海. 乙烯信号转导及其在植物逆境响应中的作用 [J]. 生物技术通报, 2016, 32(10): 11-17
[26] Zarembinski T I, Theologis A. Ethylene biosynthesis and action: a case of conservation [J]. Plant Molecular Biology, 1994, 26(5): 1579-1597
[27] Liu C Y, Lü R H, Li J, Zhao A C, Wang X L, Diane U, Wang X H, Wang C H, Yu Y S, Han S M, Lu C, Yu M D. Characterization and expression profiles of MaACS and MaACO genes from mulberry (Morus alba L.) [J]. Journal of Zhejiang University-SCIENECE B, 2014, 15(7): 611-623
[28] 曾娜霞, 胡姗姗, 周琼, 李刚, 闵丹丹. 罗汉果乙烯合成酶SgACS1基因的克隆与表达分析 [J]. 分子植物育种, 2017, 15(3): 821-832
[29] Salman-Minkov A, Levi A, Wolf S, Trebitsh T. ACC synthase genes are polymorphic in watermelon (Citrullus spp.) and differentially expressed in flowers and in response to auxin and gibberellin [J]. Plant and Cell Physiology, 2008, 49(5): 740-750
[30] Rieu I, Cristescu S M, Harren F J, Huibers W, Voesenek L A, Mariani C, Vriezen W H. RP-ACS1, a flooding-induced 1-aminocyclopropane-1-carboxylate synthase gene of Rumex palustris, is involved in rhythmic ethylene production [J]. Journal of Experimental Botany, 2005, 56(413): 841-849
[31] Yong T E, Meeley R B, Gallie D R. ACC synthase expression regulates leaf performance and drought tolerance in maize [J]. Plant Journal, 2004, 40(5): 813-825
[32] Wi S J, Park K Y. Antisense expression of carnation cDNA encoding ACC synthase or ACC oxidase enhances polyamine content and abiotic stress tolerance in transgenic tobacco plants [J]. Molecular and Cells, 2002, 13(2): 209-220
[2] Liu M, Pirrello J, Chervin C, Roustan J P, Bouzayen M. Ethylene control of fruit ripening: revisiting the complex network of transcriptional regulation [J]. Plant Physiology, 2015, 169(4): 2380-2390
[3] Yin J, Chang X, Kasuga T, Bui M, Reid M S, Jiang C Z. A basic helix-loop-helix transcription factor, PhFBH4, regulates flower senescence by modulating ethylene biosynthesis pathway in petunia [J]. Horticulture Research, 2015, 2: 15059
[4] Mishra A, Khare S, Trivedi P K, Nath P. Ethylene induced cotton leaf abscission is associated with higher expression of cellulase (GhCel1) and increased activities of ethylene biosynthesis enzymes in abscission zone [J]. Plant Physiology and Biochemistry, 2008, 46(1): 54-63
[5] Kazan K. Diverse roles of jasmonates and ethylene in abiotic stress tolerance [J]. Trends in Plant Science, 2015, 20(4): 219-229
[6] Müller M, Munné-Bosch S. Ethylene response factors: a key regulatory hub in hormone and stress signaling [J]. Plant Physiology, 2015, 169(1): 132-141
[7] 孙芝兰, 陈以峰. 乙烯的直接生物合成 [J]. 生物工程学报, 2013, 29(10): 1431-1440
[8] 丁群英, 张瑞, 廖新福, 郭蔼光. 哈密瓜ACC合成酶基因cDNA的克隆及全序列分析 [J]. 园艺学报, 2009, 36(8): 1177-1183
[9] 程立宝, 秦智伟, 刘宏宇, 丁国华, 周秀艳. 黄瓜cs-acs1g基因克隆及不同时空表达的研究 [J]. 园艺学报, 2005, 32(5): 840-843
[10] Takahashih H, Iwasa T, Shinkawa T, Kawahara A, Kurusu T, Inoue Y. Isolation and characterization of the ACC synthase genes from lettuce (Lactuca sativa L.), and the involvement in low pH-induced root hair initiation [J]. Plant and Cell Physiology, 2003, 44(1): 62-69
[11] 孙申申, 温秀萍, 杨菲颖, 李梦思, 李欢, 李科, 陈晓静. ‘云香’水仙ACC合成酶基因NtACS1的克隆及遗传转化 [J]. 西北植物学报, 2017, 37(2): 250-257
[12] 董晨, 李伟才, 张纯, 魏永赞, 王弋, 胡会刚. 香蕉ACS基因家族的系统进化分析 [J]. 分子植物育种, 2017, 15(2): 425-432
[13] 徐士娟, 邝健飞, 陆旺金, 万嗣宝, 陈建业. 龙眼果实ACS和ACO基因克隆及其表达特性 [J]. 果树学报, 2010, 27(1): 39-44
[14] 吴建阳, 李彩琴, 李建国. 荔枝ACS1基因的分离及其与幼果脱落的关系 [J]. 果树学报, 2017, 34(7): 817-827
[15] 张馨月, 王广龙, 黄蔚, 王枫, 倪桢燚, 熊爱生. 胡萝卜抗坏血酸过氧化物酶基因的分离及其对非生物胁迫的响应 [J]. 南京农业大学学报, 2016, 39(1): 55-62
[16] Xu Z S, Tan H W, Wang F, Hou X L, Xiong A S. CarrotDB: a genomic and transcriptomic database for carrot [J]. Database, 2013, 2014(bau096):1229-1245
[17] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods [J]. Molecular Biology and Evolution, 2011, 28(10): 2731-2739
[18] Geourjon C, Deléage G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments [J]. Bioinformatics, 1995, 11(6): 681-684
[19] Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino T G, Bertoni M, Bordoli L, Schwede T. SWISS-MODEL: modeling protein tertiary and quaternary structure using evolutionary information [J]. Nucleic Acids Research, 2014, 42(W1): 252-258
[20] Tian C, Jiang Q, Wang F, Wang G L, Xu Z S, Xiong A S. Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves [J]. PLoS One, 2015, 10(2): e0117569
[21] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method [J]. Methods, 2001, 25(4): 402-408
[22] 王立红, 张巨松, 李星星, 阿曼古丽·买买提阿力. 外源水杨酸对盐胁迫下棉花幼苗光合作用的影响 [J]. 核农学报, 2016, 30(9): 1864-1871
[23] 汪宝卿, 姜瑶, 解备涛, 张海燕, 董顺旭, 段文学, 王庆美, 张立明. 2个不同耐旱性甘薯品种的苗期根系蛋白组差异分析 [J]. 核农学报, 2017, 31(2): 232-240
[24] 张琪, 周薇, 崔慧萍, 宋丽莉, 郭长虹. 不同类型脱水素在植物低温胁迫应答中的作用 [J]. 核农学报, 2017, 31(4): 689-695
[25] 张弢, 董春海. 乙烯信号转导及其在植物逆境响应中的作用 [J]. 生物技术通报, 2016, 32(10): 11-17
[26] Zarembinski T I, Theologis A. Ethylene biosynthesis and action: a case of conservation [J]. Plant Molecular Biology, 1994, 26(5): 1579-1597
[27] Liu C Y, Lü R H, Li J, Zhao A C, Wang X L, Diane U, Wang X H, Wang C H, Yu Y S, Han S M, Lu C, Yu M D. Characterization and expression profiles of MaACS and MaACO genes from mulberry (Morus alba L.) [J]. Journal of Zhejiang University-SCIENECE B, 2014, 15(7): 611-623
[28] 曾娜霞, 胡姗姗, 周琼, 李刚, 闵丹丹. 罗汉果乙烯合成酶SgACS1基因的克隆与表达分析 [J]. 分子植物育种, 2017, 15(3): 821-832
[29] Salman-Minkov A, Levi A, Wolf S, Trebitsh T. ACC synthase genes are polymorphic in watermelon (Citrullus spp.) and differentially expressed in flowers and in response to auxin and gibberellin [J]. Plant and Cell Physiology, 2008, 49(5): 740-750
[30] Rieu I, Cristescu S M, Harren F J, Huibers W, Voesenek L A, Mariani C, Vriezen W H. RP-ACS1, a flooding-induced 1-aminocyclopropane-1-carboxylate synthase gene of Rumex palustris, is involved in rhythmic ethylene production [J]. Journal of Experimental Botany, 2005, 56(413): 841-849
[31] Yong T E, Meeley R B, Gallie D R. ACC synthase expression regulates leaf performance and drought tolerance in maize [J]. Plant Journal, 2004, 40(5): 813-825
[32] Wi S J, Park K Y. Antisense expression of carnation cDNA encoding ACC synthase or ACC oxidase enhances polyamine content and abiotic stress tolerance in transgenic tobacco plants [J]. Molecular and Cells, 2002, 13(2): 209-220