Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (11): 2285-2297.doi: 10.3864/j.issn.0578-1752.2013.11.013

• HORTICULTURE • Previous Articles     Next Articles

Cloning and Expression Analysis of Lipoxygenase Gene CsLOX2 in Cucumis sativus (Cucumber)

 CHEN  Qiao, CHEN  Shu-Xia, WANG  Cong-Ying, HAO  Li-Ning, MENG  Huan-Wen, WAN  Xu-Hua, SHEN  Xiao-Qing, CHENG  Zhi-Hui   

  1. College of Horticulture, Northwest A&F University/Key Laboratory of Horticultural Plant Biology and Germplasm Resources Utilization in Northwest China, Ministry of Agriculture, Yangling 712100, Shaanxi
  • Received:2012-07-06 Online:2013-06-01 Published:2013-04-26

PDF

1574

Cited

2

Abstract: 【Objective】To investigate the mechanism of cucumber lipoxygenase gene participating in the formation of fruit aldehyde aroma, a full length cDNA named CsLOX2 was cloned from the Northern China germplasm No. 26 and the sequence characteristics of CsLOX2 were analyzed. Furthermore, the expression mode, the relative C6 aldehyde content and the lipoxygenase activity of cucumber fruit during fruit development were studied. 【Method】The full length cDNA of CsLOX2 gene was cloned using the methods of RT-PCR and 3′RACE, and the structure and the coded protein were analyzed. The expression mode of CsLOX2 gene using Real-time PCR and LOX activity were measured during the fruit developing. The head space-solid phase microextraction (HS-SPME) combining with gas chromatography-mass spectrometry (GC-MS) method were used to measure the relative C6 aldehyde content of cucumber fruits. 【Result】The full length cDNA of cucumber lipoxygenase gene named CsLOX2 was 2 878 bp. The open reading frame encompassed 2 640 bp and encoded 879 polypeptides. The calculated protein molecular mass was 99.39 kD and isoelectric point was 6.28. The amino acids sequence of CsLOX2 shared three conserved regions and six highly conserved histidines with other plant LOXs. It showed that the sequence of CsLOX2 had high identity compared with other lipoxygenases by NCBI BLAST. The RT-PCR analysis showed that CsLOX2 gene expressed differently during the fruit development, and highest on the 3 d after anthesis, and then down-regulated gradually and lowest on the 15 d after anthesis. The total LOX activity rose from the 3 d to 12 d after anthesis, and it was up to the highest activity on the 12 d after anthesis, and then went down. The expression peak of CsLOX2 gene preceded that of the LOX activity during the fruit development. The relative C6 aldehyde content was higher at fruit early development stage and decreased gradually during the fruit development and which was the lowest on 12 d after anthesis.【Conclusion】A lipoxygenase gene CsLOX2 was cloned from the Northern China germplasm No. 26 by RT-PCR and RACE. The deduced amino acid sequence of CsLOX2 gene contained the conserved motifs of LOXs family. The expression peak of CsLOX2 gene preceded that of the LOX activity during the fruit development. This study will provide a basis for probing the mechanism of CsLOX2 gene participating in the biosynthesis of aldehydes of cucumber fruits.

Key words: Cucumis sativus(cucumber) , lipoxygenase , gene cloning , sequence analysis , real time-PCR , C6 aldehyde

[1]Pech J C, Bouzayen M, Latche A. Climacteric fruit ripening: Ethylene-dependent and independent regulation of ripening pathways in melon fruit. Plant Science, 2008, 175(1/2): 114-120.

[2]Obando-Ulloa J M, Nicolai B, Lammertyn J, Bueso M C, Monforte A J, Fernandez-Trujillo J P. Aroma volatiles associated with the senescence of climacteric or non-climacteric melon fruit. Postharvest Biology and Technology, 2009, 52(2): 146-155.

[3]Thanh H T, Vergoignan C, Cachon R, Kermasha S, Gervais P, Nguyen T X S, Belin J M,Husson F. Recombinant hydroperoxide lyase for the production of aroma compounds: Effect of substrate on the yeast yarrowia lipolytica. Journal of Molecular Catalysis B-Enzymatic, 2008, 52(3): 146-152.

[4]Blee E. Phytooxylipins and plant defense reactions. Progress in Lipid Research, 1998, 37(1): 33-72.

[5]Melan M A, Dong X N, Endara M E, Davis K R, Ausubel F M, Peterman T K. An arabidopsis-thaliana lipoxygenase gene can be induced by pathogens, abscisic-acid, and methyl jasmonate. Plant Physiology, 1993, 101(2): 441-450.

[6]Ferrie B J, Beaudoin N, Burkhart W, Bowsher C G,Rothstein S J. The cloning of 2 tomato lipoxygenase genes and their differential expression during fruit ripening. Plant Physiology, 1994, 106(1): 109-118.

[7]Kang S G, Hannapel D J. A novel mads-box gene of potato (Solanum tuberosum L) expressed during the early stages of tuberization. Plant Molecular Biology, 1996, 31(2): 379-386.

[8]Matsui K, Nishioka M, Ikeyoshi M, Matsumura Y, Mori T,Kajiwara T. Cucumber root lipoxygenase can act on acyl groups in phosphatidylcholine. Biochimica Et Biophysica Acta-Lipids and Lipid Metabolism, 1998, 1390(1): 8-20.

[9]Liavonchanka A, Feussner N. Lipoxygenases: Occurrence, functions and catalysis. Journal of Plant Physiology, 2006, 163(3): 348-357.

[10]刘春香, 何启伟, 刘扬岷. 黄瓜香气成分的顶空固相微萃取气质联用分析. 园艺学报, 2002, 29(6): 581-583.

Liu C X, He Q W, Liu Y M. Head-space solid phase microextraction and GC-MS analysis of fragrance of cucumber. Acta Horticulturae Sinica, 2002, 29(6): 581-583.

[11]Griffiths A, Barry C, Alpuche-Solis A G, Grierson D. Ethylene and developmental signals regulate expression of lipoxygenase genes during tomato fruit ripening. Journal of Experimental Botany, 1999, 50(335): 793-798.

[12]Heitz T, Bergey D R, Ryan C A. A gene encoding a chloroplast- targeted lipoxygenase in tomato leaves is transiently induced by wounding, systemin, and methyl jasmonate. Plant Physiology, 1997, 114(3): 1085-1093.

[13]Chen G P, Hackett R, Walker D, Taylor A, Lin Z F, Grierson D. Identification of a specific isoform of tomato lipoxygenase (tomloxc) involved in the generation of fatty acid-derived flavor compounds. Plant Physiology, 2004, 136(1): 2641-2651.

[14]Mariutto M, Duby F, Adam A, Bureau C, Fauconnier M L, Ongena M, Thonart P,Dommes J. The elicitation of a systemic resistance by pseudomonas putida btp1 in tomato involves the stimulation of two lipoxygenase isoforms. BMC Plant Biology, 2011, 11(1):16-29.

[15]Liu S Q, Liu X H, Jiang L W. Genome-wide identification, phylogeny and expression analysis of the lipoxygenase gene family in cucumber. Genetics and Molecular Research, 2011, 10(4): 2613-2636.

[16]Matsui K, Hijiya K, Tabuchi Y, Kajiwara T. Cucumber cotyledon lipoxygenase during postgerminative growth. Its expression and action on lipid bodies. Plant Physiology, 1999, 119(4): 1279-1287.

[17]Matsui K, Minami A, Hornung E, Shibata H, Kishimoto K, Ahnert V, Kindl H, Kajiwara T, Feussner I. Biosynthesis of fatty acid derived aldehydes is induced upon mechanical wounding and its products show fungicidal activities in cucumber. Phytochemistry, 2006, 67(7): 649-657.

[18]Santino A, De Paolis A, Gallo A, Quarta A, Casey R,Mita G. Biochemical and molecular characterization of hazelnut (Corylus avellana) seed lipoxygenases. European Journal of Biochemistry, 2003, 270(21): 4365-4375.

[19]Shibata D. Plant lipoxygenases-their structures and functions. Tanpakushitsu kakusan Koso. Protein, Nucleic Acid, Enzyme, 1991, 36(10): 1727-1730.

[20]Galliard T, Phillips D R, Reynolds J. The formation of cis-3-nonenal, trans-2-nonenal and hexanal form linoleic acid hydroperoxide isomers by a hydroperoxide cleavage enzyme system in cucumber (Cucumis sativus) fruits. Biochimica et Biophysica Acta, 1976, 441(2): 181-192.

[21]Ganeko N, Shoda M, Hirohara I, Bhadra A, Ishida T, Matsuda H, Takamura H, Matoba T. Analysis of volatile flavor compounds of sardine (Sardinops melanostica) by solid phase microextraction. Journal of Food Science, 2008, 73(1): S83-S88.

[22]Mao L C, Wang G Z, Zhu C G, Pang H Q. Involvement of phospholipase d and lipoxygenase in response to chilling stress in postharvest cucumber fruits. Plant Science, 2007, 172(2): 400-405.

[23]Huang F C, Studart‐Witkowski C, Schwab W. Overexpression of hydroperoxide lyase gene in nicotiana benthamiana using a viral vector system. Plant Biotechnology Journal, 2010, 8(7): 783-795.

[24]Shibata D, Axelrod B. Plant lipoxygenases. Journal of Lipid Mediators and Cell Signalling, 1995, 12(2/3): 213-228.

[25]Steczko J, Donoho G P, Clemens J C, Dixon J E, Axelrod B. Conserved histidine-residues in soybean lipoxygenase - functional consequences of their replacement. Biochemistry, 1992, 31(16): 4053-4057.

[26]Minor W, Steczko J, Stec B, Otwinowski Z, Bolin J T, Walter R, Axelrod B. Crystal structure of soybean lipoxygenase l-l at 1.4 angstrom resolution. Biochemistry, 1996, 35(33): 10687-10701.

[27]Hornung E, Walther M, Kuhn H, Feussner I. Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(7): 4192-4197.

[28]Marmey P, Jalloul A, Alhamdia M, Assigbetse K, Cacas J L, Voloudakis A E, Champion A, Clerivet A, Montillet J-L,Nicole M. The 9-lipoxygenase ghlox1 gene is associated with the hypersensitive reaction of cotton gossypium hirsutum to xanthomonas campestris pv malvacearum. Plant Physiology and Biochemistry, 2007, 45(8): 596-606.

[29]Porta H, Rocha-Sosa M. Plant lipoxygenases. Physiological and molecular features. Plant Physiology, 2002, 130(1): 15-21.

[30]Feussner I,Wasternack C. The lipoxygenase pathway. Annual Review of Plant Biology, 2002, 53: 275-297.

[31]Yang X Y, Jiang W J, Yu H J. The expression profiling of the lipoxygenase (lOX) family genes during fruit development, abiotic stress and hormonal treatments in cucumber (Cucumis sativus L.). International Journal of Molecular Sciences, 2012, 13(2): 2481-2500.

[32]Wardale D A, Ambert E A. Lipoxygenase from cucumber fruit: Localization and properties. Phytochrmistry, 1980, 19(6): 1013-1016.

[33]徐坤范. 不同季节黄瓜果实芳香物质含量变化及氮对风味品质的影响[D]. 山东泰安：山东农业大学, 2006.

Xu K F. Changes of aromatic compounds content and effect of nitrogen on flavour quality of cucumber fruits at different season[D]. Taian, Shandong: Agriculture University of Shandong, 2006.(in Chinese)

[34]苏雅男. 黄金梨脂氧合酶基因 cdna 片段的克隆及表达分析[D]. 河北保定：河北农业大学, 2012.

Su Y N. Molecular cloning and expression analysis of lipoxygenase gene from Whangkeumbae[D]. Baoding: Agriculture University of Hebei, 2012. (in Chinese)

[35]张莹莹. 苹果脂氧合酶基因克隆及其表达特性研究[D]. 陕西杨凌：西北农林科技大学, 2010.

Zhang Y Y. cloning and expression analysis of lipoxygenase gene of apple(Malus domesdica Brokh) [D]. Yangling, Shaanxi: Northwest A&F University, 2010. (in Chinese)

[36]李靖,马长乐. 植物脂氧合酶研究进展. 生物学杂志, 2007, 24(6): 5-8.

Li J, Ma C L. Recent advances on study of plant lipoxygenases. Journal of Biology, 2007, 24(6): 5-8. (in Chinese)

[37]Siedow J N. Plant lipoxygenase: Structure and function. Annual Review of Plant Biology, 1991, 42(1): 145-188.

[38]刘春香. 黄瓜风味品质的构成因素及部分因素遗传参数的研究[D]. 山东泰安：山东农业大学, 2003.

Liu C X. Study on composed factors of flavor compounds and   some genetic parameters on cucumber (Cucumis sativus L.) fruit[D]. Taian, Shandong: Agriculture University of Shandong, 2003. (in Chinese)
[1] TENG MengXin, XU Ya, HE Jing, WANG Qi, QIAO Fei, LI JingYang, LI XinGuo. Identification and Functional Analysis of Ca2+-ATPase Gene Family in Banana [J]. Scientia Agricultura Sinica, 2025, 58(7): 1418-1433.
[2] ZHENG YaQin, LIU XueQing, WU SiWen, TANG XiaoYan, YANG DanNi, WANG YongKang, AHMAD Aftab, KHAN Afrsyab, WANG ChengGang, CHEN GuoHu. Cloning and Expression of BcDET2 Gene and Functional of Its Regulatory Effect on Bolting and Flowering in Wucai (Brassica campestris L.) [J]. Scientia Agricultura Sinica, 2025, 58(5): 991-1003.
[3] ZOU PeiYi, LIU MeiYan, WANG Ying, LI RanHong. Cloning and Functional Study of AkNAC2 from Actinidia kolomikta [J]. Scientia Agricultura Sinica, 2025, 58(19): 3985-3999.
[4] ZHANG ShuHong, GAO FengJu, WU QiuYing, JI JingXin, ZHANG YunFeng, XU Ke, GU ShouQin, FAN YongShan. Cloning and Expression Analysis of Heat Shock Protein HSP 9/12 Genes in Setosphaeria turcica [J]. Scientia Agricultura Sinica, 2025, 58(18): 3648-3663.
[5] LÜ ShuWei, TANG Xuan, LI Chen. Research Progress on Seed Shattering of Rice [J]. Scientia Agricultura Sinica, 2025, 58(1): 1-9.
[6] WANG Wei, WU ChuanLei, HU XiaoYu, LI JiaJia, BAI PengYu, WANG GuoJi, MIAO Long, WANG XiaoBo. Genome-Wide Identification of Soybean LOX Gene Family and the Effect of GmLOX15A1 Gene Allele on 100-Seed Weight [J]. Scientia Agricultura Sinica, 2025, 58(1): 10-29.
[7] GUAN ZhiLin, JIN FengWei, LIU TingTing, WANG Yi, TAN YingYing, YANG ChunHui, LI RuiTong, WANG Bo, LIU KeDe, DONG Yun. Genetic Analysis and Gene Mapping of Glossy Leaf in Brassica napus [J]. Scientia Agricultura Sinica, 2024, 57(4): 650-662.
[8] ZHANG ShuHong, ZHANG YunFeng, GAO FengJu, WU QiuYing, XU Ke, LI YaZi, LI YanMei, GU ShouQin, FAN YongShan, GONG XiaoDong. Cloning and Expression Analysis of Genes of Small Heat Shock Protein in Setosphaeria turcica [J]. Scientia Agricultura Sinica, 2024, 57(17): 3384-3397.
[9] LIANG LiJuan, CHENG LiXiang, YUAN JianLong, SA Gang, ZHANG Feng. Jasmonic Acid Regulates the Changes of Major Metabolites in Potato Tuber Development in vitro [J]. Scientia Agricultura Sinica, 2024, 57(13): 2525-2538.
[10] HUANG YuXuan, SHEN Chen, JU JiaFei, YANG Lei, LUO GuangHua, FANG JiChao. Identification, Cloning and Expression Profiles of Gustatory Receptor Genes in Chilo suppressalis [J]. Scientia Agricultura Sinica, 2023, 56(13): 2504-2517.
[11] GU LiDan,LIU Yang,LI FangXiang,CHENG WeiNing. Cloning of Small Heat Shock Protein Gene Hsp21.9 in Sitodiplosis mosellana and Its Expression Characteristics During Diapause and Under Temperature Stresses [J]. Scientia Agricultura Sinica, 2023, 56(1): 79-89.
[12] LI YuZe,ZHU JiaWei,LIN Wei,LAN MoYing,XIA LiMing,ZHANG YiLi,LUO Cong,HUANG Gui Xiang,HE XinHua. Cloning and Interaction Protein Screening of RHF2A Gene from Xiangshui Lemon [J]. Scientia Agricultura Sinica, 2022, 55(24): 4912-4926.
[13] QU Cheng,WANG Ran,LI FengQi,LUO Chen. Cloning and Expression Profiling of Gustatory Receptor Genes BtabGR1 and BtabGR2 in Bemisia tabaci [J]. Scientia Agricultura Sinica, 2022, 55(13): 2552-2561.
[14] ZHANG Li,ZHANG Nan,JIANG HuQiang,WU Fan,LI HongLiang. Molecular Cloning and Expression Pattern Analysis of NPC2 Gene Family of Apis cerana cerana [J]. Scientia Agricultura Sinica, 2022, 55(12): 2461-2471.
[15] ZHANG Lu,ZONG YaQi,XU WeiHua,HAN Lei,SUN ZhenYu,CHEN ZhaoHui,CHEN SongLi,ZHANG Kai,CHENG JieShan,TANG MeiLing,ZHANG HongXia,SONG ZhiZhong. Identification, Cloning, and Expression Characteristics Analysis of Fe-S Cluster Assembly Genes in Grape [J]. Scientia Agricultura Sinica, 2021, 54(23): 5068-5082.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd