甘蔗抗坏血酸过氧化物酶基因(ScAPX)的克隆及表达分析

摘要
图/表
参考文献
相关文章 (15)

全文: PDF (3309 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要抗坏血酸过氧化物酶(ascorbate peroxidase, APX)是清除活性氧(reactive oxygen species, ROS)的重要酶类。本研究对针刺接种黑粉菌(Sporisorium scitamineum)后的抗、感基因型甘蔗(Saccharum officinarum)进行APX活性测定。结果表明，甘蔗接种黑穗病菌48 h内，抗病品种(崖城05-179) APX活性显著高于感病品种(柳城03-182) (P＜0.05)。借助电子克隆技术，结合RT-PCR方法，从甘蔗中分离到一个APX基因，并命名为ScAPX(GenBank登录号: KJ7565501)。生物信息学分析显示，ScAPX基因全长1 171 bp，包含一个1 038 bp的完整开放阅读框，编码345个氨基酸。ScAPX编码的蛋白不含信号肽，推测其为非分泌蛋白，定位于线粒体基质和叶绿体基质中的概率分别为91.1%和88.7%。实时荧光定量PCR检测结果显示，ScAPX在甘蔗根、芽、叶、蔗皮和蔗髓中均有表达，为组成型表达基因，表达量在蔗皮中最高，叶中最低，前者是后者的19.7倍；在外源因子处理后0~48 h，ScAPX基因的表达受水杨酸(salicylic acid, SA)、茉莉酸甲酯(methyl jasmonate, MeJA)、过氧化氢(hydrogen peroxide, H2O2)、脱落酸(abscisic acid, ABA)、氯化钠(sodium chloride, NaCl)和聚乙二醇(polyethylene glycol, PEG)诱导，SA、MeJA和H2O2处理后的ScAPX转录本积累量峰值较外源激素(ABA)和环境(NaCl和PEG)胁迫早。前者的ScAPX转录本变化呈现出胁迫初期积累量增加，达到峰值后又逐步下降的特点；在同样的测试时间(处理后24 h)内，ABA、NaCl和PEG处理后ScAPX转录本在达到峰值后未见下降。虽然在外源胁迫下，ScAPX表达量变化存在明显差异，但其表达均表现出正响应上述外源因子对甘蔗的胁迫。本研究为后续深入鉴定该基因的功能与进一步应用提供基础资料。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	王竹青
	陈云
	杨玉婷
	苏亚春
	陈珊珊
	吴期滨
	许莉萍

关键词 ：甘蔗, 甘蔗黑穗病菌, 抗坏血酸过氧化物酶基因(ScAPX), 非生物胁迫, qRT-PCR

Abstract：Ascorbate peroxidase (APX) is one of the important enzymes which can remove the reactive oxygen species (ROS) in the plant. In this study, APX activity in sugarcane (Saccharum officinarum) smut resistant variety (Yacheng05-179) was significantly higher (P＜0.05) than that in the susceptible one (Liucheng03-182) after inoculated with Sporisorium scitamineum within 48 h. ScAPX (GenBank accession No. KJ7565501) was separated from sugarcane with cloning and RT-PCR technologies. The bioinformatic analysis showed that the total length of ScAPX gene was 1 171 bp, which contained a complete open reading frame (1 038 bp) and encoded 345 amino acids. The ScAPX contained no signal peptide and belonged to nonsecretory protein which was likely located in the matrix of mitochondrial (91.1%) and chloroplast (88.7%). The results of tissue specificity analysis showed that the highest expression level of ScAPX was in stem skin which was 19.7 times of that in leaves. The transcript of ScAPX increased under the stresses of salicylic acid (SA), methyl jasmonate (MeJA), hydrogen peroxide (H2O2), abscisic acid (ABA), sodium chloride (NaCl) and polyethylene glycol (PEG). The peak of the ScAPX transcript under the stresses of SA, MeJA and H2O2 was accumulated during the initial period, which was earlierly found than that under ABA, NaCl and PEG stresses, and then it declined gradually. The expression pattern of ScAPX in response to ABA, NaCl and PEG treatments was not clear after 24 h, while the transcripts were kept in the peak at 24 h. Though the gene expression under the exogenous stresses were different, it was undoubted that ScAPX was positive response to the external stress. This study provides the basis for further functional analysis and application of this gene in the future.

Key words： Sugarcane Sporisorium scitamineum Ascorbate peroxidase gene(ScAPX) Abiotic stresses qRT-PCR

收稿日期: 2014-06-03 出版日期: 2015-01-13

基金资助:现代农业产业技术体系建设专项资金

通讯作者: 许莉萍 E-mail: xlpmail@126.com

引用本文:

王竹青陈云杨玉婷苏亚春陈珊珊吴期滨许莉萍. 甘蔗抗坏血酸过氧化物酶基因(ScAPX)的克隆及表达分析[J]. , 2015, 23(2): 170-180.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I2/170

陈国强, 孟鹏, 刘李黎. 2011. 高粱抗坏血酸过氧化物酶基因的电子克隆及序列分析[J]. 生物信息学, 9(2): 124-125. (Chen G Q, Meng P, Liu L S. 2011. In silico cloning and characterization of Sorghum APX Gene[J]. China Journal of Bioinformatics, 9(2): 124-125.)
关西贞, 张卫东, 田纪春. 2000. 小麦近等基因系与白粉病菌互作的生理指标研究[J]. 华北农学报, 25(1): 217-2291. (Guan X Z, Zhang W D, Tian J C. 2000. Physiological indicators of near-isogenic Wheat lines in interaction with Powdery Mildew[J]. Acta Agriculturae Boreali-Sinica, 25(1): 217-2291.)
龚德明, 陈如凯. 1995. 甘蔗对黑穗病抗病机制及遗传的研究进展[J]. 福建省农科院学报, 10(1): 33-36. (Gong D M, Chen R K. 1995. Advances in the study on resistance mechanism and heritance to sugarcane smut caused by Usilago scitaminea[J]. Journal of Fujian Academy of Agricultural Sciences.)
韩立敏. 2007. 丹参APX和GPX基因克隆及其表达分析[D]. 硕士学位论文, 陕西师范大学, 导师: 王喆之. (Han L M. 2007. Study on cloning and expression of ascorbate peroxidase and glutathione peroxidase gene of Salvia Miltiorrhiza Bge[D]. Thesis for M.S., Shaanxi Normal University, Supervisor: Wang Z Z.)
蒋明, 张志仙, 袁菱婧. 2012. 青花菜抗坏血酸过氧化物酶基因BoAPX2的克隆与表达分析[J]. 植物病理学报, 42(4): 374-380. (Jiang M, Zhang Z X, Yuan L J. 2012. Cloning and expression analysis of an ascorbate peroxidase gene BoAPX2 from Brassica oleracea var.italica[J]. Acta Phytopathologica Sinica, 42(4): 374-380.)
蒋明义. 1999. 水分胁迫下植物体内HO?的产生与细胞的氧化损伤[J]. 植物学报, 41(3): 229-234. (Jiang M Y. 1999. Generation of hydroxyl radicals and it’s relation to cellular oxidative damage in plants subjected to water stress[J]. Bulletin of Botany, 41(3): 229-234.)
李泽琴, 李静晓, 张根发. 2013. 植物抗坏血酸过氧化物酶的表达调控以及对非生物胁迫的耐受作用[J]. 遗传, 35(1): 45-54. (Li Z Q, Li X J, Zhang G F. 2013. Expression regulation of pant ascorbate peroxidase and its tolerance to abiotic stress[J]. Hereditas, 35(1): 45-54.)
刘家忠, 龚明. 1999. 植物抗氧化系统研究进展[J]. 云南师范大学学报, 19(6): 1-11. (Liu J Z, Gong M. 1999. The research progress of antiodant plant systems[J]. Journal of Yunnan Normal University, 19(6): 1-11.)
刘稳. 2002. 植物过氧化物酶超家族的分子结构[J]. 生命科学, 14(4): 212-215. (Liu W. 2002. Molecular structure of plant peroxidase superfamily[J]. Chinese Bulletin of Life Sciences, 14(4): 212-215.)
刘稳, 李杨, 高培基, 等. 2000. 过氧化物酶研究进展[J]. 纤维素科学与技术, 8(2): 50-64. (Liu W, Li Y, Gao P J, et al. 2000. The research progress of ascorbate peroxidase[J]. Journal of Cellulose Science and Technology, 8(2): 50-64. )
马长乐, 王萍萍, 曹子谊, 等. 2002. 盐地碱蓬(Suaeda salsa) APX基因的克隆及盐胁迫下的表达[J]. 植物生理与分子生物学学报, 28(4): 261-266. (Ma C L, Wang P P, Cao Z Y, et al. 2002. cDNA cloning and gene expression of APX in Suaeda salsa in response to salt stress[J]. Journal of Plant Physiology and Molecular Biology, 28(4): 261-266.)
苏亚春, 李国印, 阙友雄, 等. 2011. 甘蔗几丁质酶基因的电子克隆与生物信息学分析[J]. 生物信息学, 9(4): 322-326. (Su Y C, Li G Y, Que Y X, et al. 2011. In silico cloning and characterization of chitinase gene from sugarcane using bioinformatics tools[J]. China Journal of Bioinformatics, 9(4): 322-326.)
孙云, 江春柳, 赖钟雄, 等. 2008. 茶树鲜叶抗坏血酸过氧化物酶活性[J]. 热带作物学报, 29(5): 562-566. (Sun Y, Jiang C L, Lai Z X, et al. 2008. Determination and observation of the changes of the ascorbate peroxidase activities in the fresh leaves of tea plants[J]. Chinese Journal of Tropical Crops, 29(5): 562-566.)
王伯辉. 2007. 我国甘蔗病害的发生现状与研究进展[J]. 中国糖料, 2007(3): 48-51. (Wang B H. 2007. The occurrence status and the research progress of sugarcane disease in China[J]. Sugar Crops of China, 2007(3): 48-51.)
王利军, 黄卫东, 战吉成. 2003. 水杨酸和高温锻炼与葡萄抗热性及抗氧化的关系[J]. 园艺学报, 30(4): 452-454. (Wang L J, Huang W D, Zhan J C. 2003. Thermotolerance related to antioxidation induced by SA and heat acclimation in grape seedlings[J]. Acta Horticulturae Sinica, 30(4): 452-454.)
王媛. 2007. 杨树与溃疡病菌(Botryosphaeria dothidea)互作中的细胞生物学、活性氧代谢及细胞过敏性反应[D]. 博士学位论文, 中国林业科学研究院, 导师: 张星耀, 梁军. (Wang Y. 2007. Cell biology, reactive oxygen metabolism and cell hypersensitive response of Poplar-canker pathogen interaction[D]. Thesis for DOC., State Forestry Administration, P. R. China. Supervisor: Zhang X Y, Liang J.)
谢小群, 高山林. 2002. 黄芩过氧化物酶同工酶电泳和抗环血酸过氧化物酶活性分析[J]. 植物资源与环境学报, 11(1): 5-8. (Xie X Q, Gao S L. 2002. The analysis of peroxidase isoenzyme electrophoresis and activity of ascorbate peroxidase(ASP) in Scutellaria baicalensis Georgi[J]. Journal of Plant Resources and Environment, 11(1): 5-8.)
许传俊, 孙叙卓, 李玲, 等. 2012. 蝴蝶兰抗坏血酸过氧化物酶基因克隆及其表达研究[J]. 园艺学报, 39(4): 769-776. (Xu C J, Sun Z J, Li L, et al. 2012. Molecular cloning and expression analysis of homological gene APX from phalaenopsis[J]. Acta Horticulturae Sinica, 39(4): 769-776.)
许莉萍, 陈如凯. 2000. 甘蔗黑穗病及其抗病育种的现状与展望[J]. 福建农业学报. 15(2): 26-31. (Xu L P, Chen R K. 2000. Current status and prospects of smut and smut resistance breeding in sugarcane[J]. Fujian Journal of Agricultural of Sciences, 15(2): 26-31.)
曾秀存, 孙万仓, 方彦. 2013. 白菜型冬油菜抗坏血酸过氧化物酶(APX)基因的克隆、表达及其活性分析[J]. 作物学报, 39(8): 1400-1408. (Zeng X C, Sun W C, Fang Y. 2013. Cloning, expression and activity analysis of ascorbate peroxidase (APX) gene from winter turnip rape (Brassica campestris L.)[J]. Acta Agronomica Sinica, 39(8): 1400-1408.)
Agarwal S, Sairam R K, Srivastava G C, et al. 2005. Role of salicylic acid, calcium and hydrogen peroxide on antioxidant enzymes induction in wheat seedlings[J]. Plant Science, 169: 559-570.
Chao C P, Hoy J W, Saxton A M, et al. 1990. Heritability of resistance and repeatability of clone reactions to sugarcane smut in Louisiana [J]. Phytopathology, 80(7): 622-626.
Dat J, Vandenabeele S, Vranova E, et al. 2000. Dual action of the active oxygen species during plant stress responses[J]. Cellular and Molecular Life Sciences CMLS, 57(5): 779-795.
De-Leonardis S, Dipierro N, Dipierro S. 2000. Purification and characterization of an ascorbate peroxidase from potato tuber mitochondria[J]. Plant Physiology and Biochemistry, 38: 773-779.
Elia M R, Borraccino G, Dipierro S. 1992. Soluble ascorbate peroxidase from potato tuber[J]. Plant Science, 85(1): 17-21.
Narendra S, Venkataramani S, Shen G X, et al. 2006. The Arabidopsis ascorbate peroxidase 3 is a peroxisomal membrane-bound antioxidant enzyme and is dispensable for Arabidopsis growth and development. Journal of Experimental Botany, 57(12): 3033-3042.
Padmanaban P, Alexander K, Shanmugan N. 1988. Effect of smut on growth and yield parameters of sugarcane[J]. Indian Phytopathology, 41(3): 367-369.
Su Y C, Guo J L, Ling H, et al. 2014. Isolation of a novel peroxisomal catalase gene from sugarcane, which is responsive to biotic and abiotic stresses[J]. PLoS ONE, 9(1): e84426.
Su Y C, Xu L P, Xue B T, et al. 2013. Molecular cloning and characterization of two pathogenesis-related β-1,3-Glucanase genes ScGluA1 and ScGluD1 from sugarcane infected by Sporisorium sitamineum[J]. Plant Cell Reports, 32: 1503-1519.
Wang, H Z. 2005. Reactive oxygen species (ROS) acts as activating cells death and defense genes expression in plant[A]. Journal of Mountain Agriculture and Biology, 24(1): 170-175.
Willekens H, Langebartels C, Tire C, et al. 1994. Differential expression of catalase genes in Mieotiana ginifolia[J]. Proceedings of the National Academy of Sciences, 91: 10450-10454.