龙眼咖啡酰辅酶A-O-甲基转移酶（DLCCoAOMT）基因的克隆和表达分析

doi:10.3864/j.issn.0578-1752.2012.01.014

摘要/Abstract

摘要： 【目的】克隆龙眼咖啡酰辅酶A-O-甲基转移酶(DLCCoAOMT)基因，分析其序列特征和在低温胁迫下不同组织中的表达情况并进行原核表达研究。【方法】采用RT-PCR 和RACE 技术从龙眼叶片中克隆DLCCoAOMT基因，用生物信息学方法对获得的氨基酸序列进行分析，利用荧光定量PCR 研究DLCCoAOMT 基因在不同组织中的表达。【结果】克隆得到DLCCoAOMT 基因，GenBank 登录号为JN093023。该cDNA 全长993 bp，具有1 个744 bp 的完整开放阅读框（ORF），编码247 个氨基酸。序列分析表明，DLCCoAOMT 编码的氨基酸序列与其它植物的 CCoAOMT 蛋白有很高的相似性。系统进化树分析显示，龙眼 DLCCoAOMT 与桦木属的CCoAOMT 蛋白亲缘关系较近。利用荧光定量技术进行组织表达模式分析发现，DLCCoAOMT 基因在根、茎、叶中均有表达。总体上表达量在根和茎中较多，叶中较少，随着低温胁迫时间延长，DLCCoAOMT 基因在各组织的表达量也发生变化。原核表达结果表明，DLCCoAOMT 基因在大肠杆菌中获得表达。【结论】从龙眼中克隆到咖啡酰辅酶A-O-甲基转移酶基因，该基因可能参与调控低温胁迫。

关键词: 龙眼, 咖啡酰辅酶A-O-甲基转移酶, 基因克隆, 表达分析, 原核表达

Abstract: 【Objective】The aim of this study was to clone full-length cDNA of a key enzyme gene DLCCoAOMT related to lignin metabolism in longan, investigate its sequence characteristics and analyze its expression in different organs under low temperature stress, as well as expression in the prokaryote. 【Method】 The DLCCoAOMT gene cDNA sequence was cloned from longan leaf using RT-PCR and RACE techniques. The bioinformatics methods were used to analyze putative amino acid sequence and real-time PCR method were used to study the expression of DLCCoAOMT gene in different tissues. 【Result】 The full-length cDNA of DLCCoAOMT (GenBank accession number: JN093023) in longan was cloned. The sequence consists of 993 bp with an open reading frame of 744 bp, encoding a polypeptide of 247 amino acids. Homology analysis showed that the deduced DLCCoAOMT protein was highly homologous to other CCoAOMT proteins from different species. Phylogenetic analysis also indicated that DLCCoAOMT was very closely related to DLCCoAOMT of Betula. Real-time PCR results showed that the DLCCoAOMT expressed in root, stem and leaf, and its expression was different among three organs. The mRNA of DLCCoAOMT was abundant in root and stem, but less in leaf. Furthermore, DLCCoAOMT transcription level was significantly different among root, stem and leaf with time courses of chilling treatment. Prokaryotic expression showed that recombinant plasmid was efficiently expressed in Escherichia coli BL21. 【Conclusion】 Gene DLCCoAOMT was firstly isolated and characterized from longan, which may be involved in chilling stress.

Key words: Dimocarpus longan Lour., caffeoyl CoA 3-O-methyltransferase, gene cloning, expression analysis, prokaryotic expression

陈虎, 何新华, 罗聪, 杨丽涛, 张保青, 宋修鹏. 龙眼咖啡酰辅酶A-O-甲基转移酶（DLCCoAOMT）基因的克隆和表达分析[J]. 中国农业科学, 2012, 45(1): 118-126.

CHEN Hu, HE Xin-Hua, LUO Cong, YANG Li-Tao, ZHANG Bao-Qing, SONG Xiu-Peng. Molecular Cloning of Longan Caffeoyl-CoA O-methyltransferase (DLCCoAOMT) and Its Expression Analysis[J]. Scientia Agricultura Sinica, 2012, 45(1): 118-126.

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参考文献

[1]Ye Z H. Association of caffeoyl Coenzyme A 3-O-methyltransferase expression with lignifying tissues in several dicot plants. Plant Physiology, 1997, 115: 1341-1350.

[2]Costa P, Bahrman N, Frigerio J M, Kremer A, Plomion C. Water-deficit-responsive proteins in Maritime pine. Plant Molecular Biology, 1998, 38: 587-596.

[3]Vincent D, Lapierre C, Pollet B, Cornic G, Negroni L, Zivy M. Water deficits affect caffeate O-methyltransferase, lignification, and related enzymes in maize leaves. a proteomic investigation. Plant Physiology, 2005, 137: 949-960.

[4]Yoshimura K, Masuda A, Kuwano M, Yokota A, Akashi K. Programmed proteome response for drought avoidance/tolerance in the root of a C3 xerophyte (wild watermelon) under water deficits. Plant Cell Physiology, 2008, 49(2): 226-241.

[5]Riccardi F, Gazeau P, de Vienne D, Zivy M. Protein changes in response to progressive water deficit in maize: Quantitative variation and polypeptide identification. Plant Physiology, 1998, 117(4): 1253-1263.

[6]Salekdeh G H, Siopongco J, Wade L J, Ghareyazie B, Bennett J. A proteomic approach to analyzing drought-and salt-responsiveness in rice. Field Crops Research, 2002, 76: 199-219.

[7]Kim J K, Bamba T, Harada K, Fukusaki E, Kobayashi A. Time-course metabolic profiling in Arabidopsis thaliana cell cultures after salt stress treatment. Journal of Experimental Botany, 2007, 58(3): 415-424.

[8]倪志勇, 吕萌, 范玲. 棉花咖啡酰辅酶A-O-甲基转移酶基因的克隆及特征分析. 西北植物学报, 2010, 30(6): 1083-1091.

Ni Z Y, Lu M, Fan L. Cloning and characterization of CCoAOMT gene from Gossypium hirsuturm L. Acta Botanica Boreali -Occidentalia Sinica, 2010, 30(6): 1083-1091. (in Chinese)

[9]胡轶. 玉米木质素合成相关基因在干旱胁迫下的差异表达[D]. 四川雅安: 四川农业大学, 2008.

Hu Y. Differential expression of candidate genes for lignin biosynthesis under drought stress in maize[D]. Yaan, Sichuan: Sichuan Agricultural University, 2008. (in Chinese)

[10]陈建荣, 郭清泉, 张学文. 苎麻CCoAOMT 基因全长cDNA 克隆与序列分析. 中国农业科学, 2006, 39(5):1058-1063.

Chen J R, Guo Q Q, Zhang X W. Cloning full Length of ramie caffeoyl-CoA 3-O- methyltransferase cDNA and sequence analysis. Scientia Agricultura Sinica, 2006, 39(5): 1058-1063. (in Chinese)

[11]蒋向辉, 佘朝文, 许　栋, 张青桦, 赵　旺. 杉木CCoAOMT 基因部分cDNA 克隆与序列分析. 中南林业科技大学学报, 2009, 29(6): 25-29.

Jiang X H, She C W, Xu D, Zhang Q H, Zhao W. Partial cDNA cloning and sequence analysis of CCoAOMT gene of Cunninghamia lanceolata (Lamb.). Journal of Central South University of Forestry & Technology, 2009, 29(6): 25-29. (in Chinese)

[12]陈肃. 白桦4CL与CCoAOMT基因表达分析及蛋白预测[D]. 哈尔滨:东北林业大学, 2008.

Chen S. Expression and forecast of 4CL and CCoAOMT of Birch[D]. Harbin: Northeast Forestry University, 2008. (in Chinese)

[13]Yang L T, Hong L, Takahashi Y, Chen F, Walkerd M A, Cinerolo L E. Proteomic analysis of grapevine stem in response to Xylella fastidiosa inoculation. Physiological and Molecular Plant Pathology, 2011, 75: 90-99.

[14]游向荣.龙眼成花转变与成花逆转的差异蛋白质组学研究[D]. 福建农林大学, 2009.

You X R. Analysis of differential proteomice on longan(Dimocarpus longan Lour.) floral reversion and normal flowering [D]. Fujian: Fujian Agriculture and Forestry University, 2009. (in Chinese)

[15]罗聪, 何新华, 陈虎, 韦泳丽, 李明娟. 一种高效获取基因5′末端的RACE 方法. 植物生理学报, 2011, 47(4): 409-414.

Luo C, He X H, Chen H, Wei Y L, Li M J. A high-efficient method of RACE technique for obtaining the gene 5′end. Plant Physiology Journal, 2011, 47(4): 409-414. (in Chinese)

[16]黄春琼, 刘国道, 郭安平. 反义CCoAOMT 基因调控烟草木质素的生物合成. 安徽农业科学, 2008, 36(19) :8026-8027.

Huang C Q, Liu G D, Guo A P. Lignin biosynthesis regulated by antisense CCoAOMT gene in tobacco. Journal of Anhui Agriculture Science, 2008, 36(19): 8026-8027. (in Chinese)

[17]Marina L D, Ingrid G, Viviana E. Allele-specific expression of a weeping lovegrass gene from the lignin biosynthetic pathway, caffeoyl-coenzyme A 3-O-methyltransferase. Molecular Breeding, 2010, 26: 627-637.

[18]Martz F, Manury S, Pincon G, Legrand M. cDNA cloning substrate specificity and expression study of tobacco caffeoyl-CoA 3-O- methyltransferase, a lignin biosynthetic enzyme. Plant Molecular Biology, 1998, 36: 427-437.

[19]Pagadala N S, Arha M, Reddy P S, Kumar R, Sirisha V L, Prashant S, Reddy K J, Khan B, Rawal S K, Kishor P B K. Phylogenetic analysis, homology modelling, molecular dynamics and docking studies of caffeoyl-CoA-O-methyl transferase (CCoAOMT 1 and 2) isoforms isolated from subabul (Leucaena leucocephala). Journal of Molecular Modeling, 2009, 15: 203-221.

[20]Wei J H, Wang Y Z, Wang H Z, Li R F, Lin N, Ma R C, Qu L Q, Song Y R. Pulping performance of transgenic poplar with depressed Caffeoyl-CoA O-methyltransferase. Chinese Science Bulletin, 2008, 53(22): 3553-3558.

[21]Ye Z H, Zhong R Q, Morrison W H, Himmelsbach D S. Caffeoyl coenzyme A-O-methyltransferase and lignin biosynthesis. Phytochemistry, 2001, 57: 1177-1185.

[22]孙静文. 构树DREB转录因子及木质素合成代谢相关基因的克隆及功能分析[D]. 北京: 中国科学院, 2006.

Sun J W. Isolation and functional characterization of DREB transcription factor and Genes involved in lignin biosynthetic pathway from Broussonetia papyrifera L[D]. Beijing: Chinese Academy of Sciences, 2006. (in Chinese)

[23]Zhao H Y, Sheng Q X, Lu S Y, Wang T, Song Y R. Characterization of three rice CCoAOMT genes. Chinese Science Bulletin, 2004, (49)15: 1602-1606.

[24]崔光红, 王学勇, 冯华, 赵静雪, 黄璐琦. 丹参乙酰CoA 酰基转移酶基因全长克隆和SNP分析. 药学学报, 2010, 45(6): 785-790.

Cui G H, Wang X Y, Feng H, Zhao J X, Huang L Q. Molecular cloning and SNP analysis of a acetyl-CoA C-acetyltransferase gene (SmAACT) from Salvia miltiorrhiza. Acta Pharmaceutica Sinica, 2010, 45(6): 785−790. (in Chinese)

[25]Jeroen R, Antje R, Jorgen H C, Yves V D P, Wout B. Genome-wide characterization of the lignification toolbox in arabidopsis. Plant Physiology, 2003, 133: 1051-1071.

[26]Ma Q H, Xu Y. Characterization of a caffeic acid 3-Omethyltransferase from wheat and its function in lignin biosynthesis. Biochimie, 2008, 90: 515-524.