目的 分离珍稀濒危药用铁皮石斛过氧化氢酶基因DoCAT1cDNA全长,并进行生物信息和表达分析。方法 采用RACE技术获基因全长;利用生物信息软件预测基因编码蛋白的理化性质、结构域和亚细胞定位等分子特性;用DNASTAR 6.0和MEGA 6.0 分别进行氨基酸多序列比对和进化树构建分析;借助实时荧光定量PCR检测基因的表达模式。结果 克隆到DoCAT1(GenBank获得注册号 KF876840),cDNA全长1 796 bp,ORF长1 479 bp,编码蛋白含492个氨基酸,相对分子质量56.90×103,等电点6.7。DoCAT1蛋白不含跨膜域和信号肽,包含过氧化氢酶核心结构域(18~401)、活性位点(54~70)、血红素结合位点(344~352)和多个保守基序;DoCAT1与植物CATs蛋白一致性为83.3%~88.3%,所在分支隶属于CATs分子进化的Ⅲ分支,与单子叶植物CATs亲缘关系近;DoCAT1基因的表达呈器官特异性,根中相对表达量较高,为叶中的4.27倍,叶中次之,茎中表达量很低。结论 DoCAT1基因鉴定与表达特征为石斛生长发育和抗逆生理提供分子基础。
Abstract
OBJECTIVE To isolate and characterize a catalase gene DoCAT1, the full length cDNA was cloned, in a rare endangered medicinal orchid species Dendrobium officinale, followed by bioinformatics analysis and expression pattern detection. METHODS RACE Technology was used for gene clone. Characteristics of physiochemical properties, conserved domains and subcellular localization of the protein were determined using a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 6.0 softwares, respectively. Real time quantitative PCR was used for gene expression analysis. RESULTS The full length cDNA of DoCAT1 was 1 796 bp in length, encoding a 492 amino acid protein with a molecular weight of 56.90×103 and an isoelectric point of 6.7. The deduced DoCAT1 protein, without transmembrane or signal peptide residues, contains catalase core domain (18-401), active site (54-70), heme binding site (344-352) and multiple conserved motifs. DoCAT1 had high identities (83.3%-88.3%) with CATs proteins from various plants; DoCAT1 belonged to class Ⅲ of the CATs evolutionary tree . DoCAT1 gene was differentially and organs specifically expressed. The transcripts were more abundant in the roots, with 4.27 fold, then in the leaves and the lowest in the stems. CONCLUSION Molecular characterization of DoCAT1 provides important basis for the growth and development and stress adaptation in D. officinale.
关键词
铁皮石斛 /
过氧化氢酶 /
基因克隆 /
序列分析 /
定量PCR
{{custom_keyword}} /
Key words
Dendrobium officinale /
catalase /
gene cloning /
sequence analysis /
quantitative PCR
{{custom_keyword}} /
中图分类号：
R915
R931.5
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] NEILL S J, DESIKAN R, HANCOCK J T. Hydrogen peroxide signaling. Curr Opin Plant Biol, 2002, 5(5):388-395.
[2] DU Y Y, WANG P C, CHEN J, et al. Comprehensive functional analysis of the catalase gene family in Arabidopsis thaliana. J Integrat Plant Biol, 2008, 50(10):1318-1326.
[3] NASSER S, KEMAL K, SCHENK P M. Global plant stress signaling:reactive oxygen species at the cross-road. Front Plant Sci, 2016, 7(187):1-21.
[4] PEI Z M, MURATA Y, BENNING G, et al. Calcium channels activated by hydrogen peroxide mediate abscisic acid signalling in guard cells. Nature, 2000, 406(6797):731-734.
[5] MANDHANIA S, MADAN S, SAWHNEY V. Antioxidant defense mechanism under salt stress in wheat seedlings. Biol Plantarum, 2006, 50(2):227-231.
[6] JING P, WANG X L, SUN X N, et al. Effects of high temperature stress on antioxidant enzymes in Xanthoceras sorbifolia. Acta Agric Univ Jiangxi (江西农业大学学报), 2008, 30(2):220-225.
[7] CHEN J F, WANG G N, CHENG S M. Progress about catalase function in plant stress reactions. Acta Bot Boreal Occident Sin (西北植物学报), 2008, 28(1):188-193.
[8] LI J, LIU J, WANG G, et al. A chaperone function of NO CATALASE ACTIVITY1 is required to maintain catalase activity and for multiple stress responses in Arabidopsis . Plant Cell, 2015, 27(3):908-925.
[9] YE N, ZHU G, LIU Y, et al. ABA controls H2O2 accumulation through the induction of OsCATB in rice leaves under water stress. Plant Cell Physiol, 2011, 52(4):689-698.
[10] LI Q, YANG A, ZHANG W H. Comparative studies on tolerance of rice genotypes differing in their tolerance to moderate salt stress. BMC Plant Biol, 2017, 17(1):1-13.
[11] LIN X G, KONG D C, PANG X M, et al. Cloning and tissue expression analysis of catalase gene in Dongzao (Ziziphus jujuba). Acta Bot Boreal Occident Sin (西北植物学报), 2012, 32(6):1086-1092.
[12] XING Z B, LIU Y, XIU L S, et al. Cloning and expression analysis of catalase gene in Eleutherococcus senticosu. Nonwood Forest Res (经济林研究), 2014, 2(2):24-27.
[13] ZHANG G, LIU S S, PENG L, et al. Molecular cloning and characterization of a transcription factor gene DoWRKY3 in Dendrobium officinale. Chin Tradit Herb Drugs (中草药), 2017, 48(14):2930-2936.
[14] CHEN X M, WANG C L, YANG J S, et al. Research progress on chemical composition and chemical analysis of Dendrobium officinale. Chin Pham J (中国药学杂志), 2013, 48(19):1634-1640.
[15] ZHAO M M, ZHANG G, ZHANG D W, et al. ESTs analysis reveals putative genes involved in symbiotic seed germination in Dendrobium officinale . PLoS One, 2013, 8(8):e72705.
[16] PFAFFL M W. A new mathematical model for relative quantification in Real-time RT-PCR. Nucleic Acids Res, 2001, 29(9):e45.
[17] KOZAK M. An analysis of 50-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res, 1987, 15(20):8125-8132.
[18] LOEWEN P C, VILLANUEVA J, SWITALA J, et al. Unprecedented access of phenolic substrates to the heme active site of a catalase: substrate binding and peroxidase-like reactivity of Bacillus pumilus catalase monitored by X-ray crystallography and EPR spectroscopy. Proteins, 2015, 83(5):853-866.
[19] ZAMOCKY M, FURTMÜLLER P G, OBINGER C. Evolution of catalases from bacteria to humans. Antioxid Redox Sign, 2008, 10(9):1527-1548.
[20] WANG S P, YANG J G, ZAHN H X, et al. Cloning of catalase gene (CAT1) and its expression patterns in Nicotiana tabacum L.. Acta Tabacaria Sin (中国烟草学报), 2014, 20(5):103-109.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
国家自然科学基金项目资助(31101608);陕西省普通高校青年杰出人才计划项目资助;咸阳市中青年科技领军人才项目资助;中国医学科学院医学与健康科学创新工程资助(2017-IZM-3-013)
{{custom_fund}}
PDF(2989 KB)
