油用牡丹脂肪酸脱氢酶基因FAD3的克隆与表达分析

doi:10.3864/j.issn.0578-1752.2017.10.016

摘要/Abstract

摘要： 【目的】ω-3脂肪酸脱氢酶(ω-3 FAD)为植物脂肪酸生物合成途径中的关键酶，通过对油用牡丹‘凤丹’ω-3 FAD基因结构特征及其在不同组织中的表达模式进行分析，为研究FAD3在油用牡丹‘凤丹’脂肪酸形成过程中的调控提供理论基础。【方法】采用RACE和RT-PCR的方法克隆油用牡丹‘凤丹’ω-3 FAD基因，用Vector NTI Advance 11软件进行序列分析，BLAST进行同源性比较，并用MEGA7.0的邻接法（neighbor-joining，NJ）构建系统进化树，利用在线蛋白质分析工具ExPASy预测FAD3蛋白的基本参数和特性，利用Phyre2预测蛋白质二级和三级结构，并通过实时荧光定量PCR检测该基因在油用牡丹‘凤丹’不同发育期的种子以及不同组织中的特异性表达情况。【结果】获得油用牡丹‘凤丹’脂肪酸脱氢酶FAD3，命名为FAD3（GenBank登录号：KX906966）。序列分析表明该基因cDNA序列全长1 723 bp，其中，开放阅读框1 308 bp，编码435个氨基酸，3′端非编码区长287 bp，5′末端非编码区长99 bp，预测成熟蛋白分子量为49.9 kD，理论等电点（pI）为7.42，N端无信号肽，脂肪系数为83.08，不稳定指数35.67，总水平亲水性为-0.222。经FAD3蛋白的二级结构预测，FAD3以α-螺旋、随机卷曲为主，其次是延伸链、β-转角含量较少；多序列比对结果表明，油用牡丹FAD3氨基酸序列含有2个保守结构域，系统发育分析结果显示‘凤丹’与芍药处于同一分支，其亲缘关系最近。TMHMM和TargetP亚细胞定位分析得知，FAD3蛋白有3个跨膜区域，可能定位于内质网中发挥功能。组织特异性结果分析表明，FAD3在‘凤丹’的根、茎、叶、花瓣、雌蕊、雄蕊、种子中均有表达，其中，在叶中表达量最高，雌蕊次之，在雄蕊中表达量最低；不同时期种子中，10 d表达量最高，20 d次之，在60 d中表达量最低。【结论】成功从油用牡丹‘凤丹’中克隆获得FAD3全长cDNA序列，其在‘凤丹’不同组织中呈现出多种表达模式。

关键词: 油用牡丹, 脂肪酸脱氢酶, 克隆, 实时荧光定量PCR

Abstract: 【Objective】 The Omega -3 fatty acid desaturase (ω-3 FAD) is a key enzyme in the fatty acid biosynthesis pathway of plant. Through analysis of omega-3 fatty acid desaturase gene structure and its expression in different tissues from Paeonia ostii, this study will lay a foundation for the research of regulating effects of FAD3 gene on fatty acid biosynthesis and provide a theoretical basis for the formation in the process of regulation. 【Method】 The FAD gene from Paeonia ostii (FAD3) was cloned using RT-PCR and RACE-PCR strategies. Nucleotide sequence was analyzed using Vector NTI Advance 11 software. Homology was analyzed using BLAST. A phylogenetic tree was constructed using neighbor-joining of MEGA 7.0. The secondary structure and three-dimensional model of FAD3 were predicted using ExPASy and Phyre2, respectively. Expression profiles of FAD3 at different developmental stages and in different tissues of the P. ostii were assayed using real-time quantitative PCR. 【Result】 The FAD gene in P. ostii was cloned and named as FAD3 (GenBank accession number: KX906966). The full length of FAD3 cDNA is 1 723 bp, contains a 1 308 bp open reading frame (ORF) encoding a putative protein of 435 amino acids with a molecular mass of 49.9 kD and an isoelectric point (pI) of 7.42. The 3′-untranslated region of 287 bp and 5′-untranslated region of 99 bp were obtained from the FAD3 gene of P. ostii. N end without signal peptide, fat coefficient is 83.08, instability index 35.67, the grand average of hydrophobicity value of -0.222. By FAD3 protein secondary structure prediction, FAD3 mainly in the alpha helix and random coil, followed by less extended strand, beta turn content; multiple sequence alignment results showed that it contains two conserved domains of FAD3 gene. Phylogenetic tree analysis showed that the P. ostii has the closest evolutionary relationship with P. lactiflora. Subcellular localization analysis of TMHMM and Target P indicated that it might be targeted to the endoplasmic reticulum with three transmembrane regions. FAD3 gene was expressed in the root, stem, leaf, petal, pistil, stamen and seed of P. ostii by the tissue-specificity expression. The expressive content of FAD3 gene in different tissues of P. ostii was different: The highest was leaf, the next was pistil, and the lowest level was in stamen; In different periods of seeds, the highest expression was seeds of 10d, the next was 20d, in 60d expression was the lowest. 【Conclusion】 The full length cDNA sequence of FAD3 gene was successfully cloned from P. ostii, It shows a variety of expression patterns in different tissues, which laid a foundation for the further study on the function and expression regulation mechanism of FAD3 gene in the process of unsaturated fatty acid biosynthesis.

Key words: Paeonia suffruticosa Andr., fatty acid desaturase, cloning, real-time PCR

黄兴琳，陆俊杏，廖冰楠，白辉扬，管丽，张涛. 油用牡丹脂肪酸脱氢酶基因FAD3的克隆与表达分析[J]. 中国农业科学, 2017, 50(10): 1914-1921.

HUANG XingLin, LU JunXing, LIAO BingNan, BAI HuiYang, GUAN Li, ZHANG Tao. Cloning and Expression Analysis of Fatty Acid Desaturase Gene FAD3 from Oil Peony[J]. Scientia Agricultura Sinica, 2017, 50(10): 1914-1921.

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参考文献

[1] RUIZ-LOPEZ N, HASLAM R P, USHER S, NAPIER J A, SAYANOVA1 O. An alternative pathway for the effective production of the omega-3 long-chain polyunsaturates EPA and ETA in transgenic oil seeds. Plant Biotechnology Journal, 2015, 13(9): 1264-1275.

[2] KÖNIGS A, KILIAAN A J. Critical appraisal of omega-3 fatty acids in attention-deficit/hyperactivity disorder treatment. Neuropsychiatric Disease & Treatment, 2016, 12(1): 1869-1882.

[3] MAZZA M, POMPONI M, JANIRI L, BRIA P, MAZZA S. Omega-3 fatty acids and antioxidants in neurological and psychiatric diseases: An overview. Progress in Neuro-Psychopharmacology and Biological Psychiatry, 2007, 31(1): 12-26.

[4] SIMOPOULOS A P. n-3 fatty acids and human health: Defining strategies for public policy. Lipids, 2001, 36(1): S83-S89.

[5] HU F B, STAMPFER M J, MANSON J E, RIMM E B, WOLK A, COLDITZ G A, HENNEKENS C H, WILLETT W C. Dietary intake of alpha-linolenic acid and risk of fatal ischemic heart disease among women. American Journal of Clinical Nutrition, 1999, 69(5): 890-897.

[6] MCLENNAN P L, ABEYWARDENA M Y, CHARNOCK J S. Dietary fish oil prevents ventricular fibrillation following coronary artery occlusion and reperfusion. American Heart Journal, 1988, 116(3): 709-717.

[7] WHELAN J, BROUGHTON K S, KINSELLA J E. The comparative effects of dietary alpha-linolenic acid and fish oil on 4-and 5-series leukotriene formation in vivo. Lipids, 1991, 26(2): 119-126.

[8] PITTAWAY J K, CHUANG L T, AHUJA K D K, BECKETT J M, GLEW R H, BALL M J. Omega-3 dietary fatty acid status of healthy older adults in Tasmania, Australia: An observational study. Journal of Nutrition Health & Aging, 2015, 19(5): 505-510.

[9] BERUTO M, CURIR P. In vitro culture of tree peony through axillary budding. Protocols for Micropropagation of Woody Trees and Fruits, 2007: 477-497.

[10] LI S S, WANG L S, SHU Q Y, WU J, CHEN L G, SHAO S, YIN D D. Fatty acid composition of developing tree peony (Paeonia section Moutan DC.) seeds and transcriptome analysis during seed development. BMC Genomics, 2015, 16(1): 1-14.

[11] 陈景震, 李培旺, 张良波, 肖志红, 吴苏喜, 吴红, 赵志伟. 湖南油用牡丹籽油的理化性质及脂肪酸组分分析. 经济林研究, 2015, 4(33): 119-122.

CHEN J Z, LI P W, ZHANG L B, XIAO Z H, WU S X, WU H, ZHAO Z W. Analysis of physicochemical properties and fatty acid composition of peony seed oil in Hunan. Nonwood Forest Research, 2015, 4(33): 119-122. (in Chinese)

[12] 张涛, 高天姝, 白瑞英, 黄兴琳, 郭楠, 陆俊杏, 罗安才. 油用牡丹利用与研究进展. 重庆师范大学学报(自然科学版), 2015, 2(32): 143-149.

ZHANG T, GAO T S, BAI R Y, HUANG X L, GUO N, LU J X, LUO A C. Utilization and research progress of oil tree peony. Journal of Chongqing Normal University (Natural Science), 2015, 2(32): 143-149. (in Chinese)

[13] 林萍, 姚小华, 曹永庆, 薛亮, 王开良. 油用牡丹‘凤丹’果实性状及其脂肪酸组分的变异分析. 经济林研究, 2015, 1(33): 67-72.

LIN P, YAO X H, CAO Y Q, XUE L, WANG K L. Variation analysis of fruit characteristics and fatty acid components in Paeonia ostii ‘Phoenix White’. Nonwood Forest Research, 2015, 1(33): 67-72. (in Chinese)

[14] 周志钦, 潘开玉, 洪德元. 牡丹组野生种间亲缘关系和栽培牡丹起源研究进展. 园艺学报, 2003, 30(6): 751-757.

ZHOU Z Q, PAN K Y, HONG D Y. Advance in studies on relationships among wild tree peony species and the origin of cultivated tree peonies, Acta Horticulturae Sinica, 2003, 30(6): 751-757. (in Chinese)

[15] VRINTEN P, HU Z, MUNCHINSKY M A, ROWLAND G, QIU X. Two FAD3 desaturase genes control the level of linolenic acid in flax seed. Plant Physiology, 2005, 139(1): 79-87.

[16] PUTTICK D, DAUK M, LOZINSKY S, SMITH M A. Overexpression of a FAD3 desaturase increases synthesis of a polymethylene- interrupted dienoic fatty acid in seeds of Arabidopsis thaliana L.. Lipids, 2009, 44(8): 753-757.

[17] DAMUDE H G, ZHANG H, FARRALL L, RIPP K G, TOMB J F, HOLLERBACH D, YADAV N S. Identification of bifunctional Δ12/ω-3 fatty acid desaturases for improving the ratio of ω-3 to ω-6 fatty acid in microbes and plants. Proceedings of the National Academy of Sciences of the USA, 2006, 103(25): 9446-9451.

[18] ECKERT H, LAVALLEE B, SCHWEIGER B J, KINNEY A J, CAHOON E B, CLEMENTE T. Co-expression of the borage Δ6 desaturase and the Arabidopsis Δ15 desaturase results in high accumulation of stearidonic acid in the seeds of transgenic soybean. Planta, 2006, 224(5): 1050-1057.

[19] BHUNIA R K, ANIRBAN C, RANJEET K, MRINAL K M, SOUMITRA K S. Enhancement of a-linolenic acid content in transgenic tobacco seeds by targeting a plastidial x-3 fatty acid desaturase (fad7) gene of Sesamum indicum to ER. Plant Cell Report, 2016, 35(1): 213-226.

[20] LEE K R, LEE Y, KIM E H, LEE S B, ROh K H, KIM J B, KANG H C, KIM H U. Functional identification of oleate 12-desaturase and ω-3 fatty acid desaturase genes from Perilla frutescens var. frutescens. Plant Cell Report, 2016, 12(35): 2523-2537.

[21] MURAKAMI Y, TSUYANA M, KOBAYASHI Y. Trienoic fatty acids and plant tolerance of high temperature. Science, 2000, 287: 476-479.

[22] ZHOU Z, WANG M J, HU J J, LU M Z, WANG J H. Improve freezing tolerance in transgenic poplar by overexpressing a ω-3 fatty acid desaturase gene. Molecular Breeding, 2010, 25(4): 571-579.

[23] 刘洪峰, 高乐旋, 胡永红. 牡丹不同发育阶段种子和花瓣组织实时荧光定量PCR中内参基因的挖掘与筛选. 农业生物技术学报, 2015, 23(12): 1639-1648.

LIU H F, GAO L X, HU Y H. Reference genes discovery and selection for quantitative real-time PCR in tree peony seed and petal tissue of different development stages. Journal of Agricultural Biotechnology, 2015, 23(12): 1639-1648. (in Chinese)

[24] 宋淑香, 郭先锋, 马燕, 李俊杰, 韩璐璐. 凤丹(Paeonia ostii)脂肪酸去饱和酶基因PoFAD2的克隆及表达分析. 园艺学报, 2016, 43(2): 347-355.

SONG S X, GUO X F, MA Y, LI J J, HAN L L. Isolation and expression analysis of PoFAD2 gene in Paeonia ostii. Acta Horticulturae Sinica,2016, 43(2): 347-355. (in Chinese)

[25] 王俊娟, 穆敏, 王帅, 陆许可, 陈修贵, 王德龙, 樊伟丽, 阴祖军, 郭丽雪, 叶武威, 喻树迅. 棉花脱水素GhDHN1的克隆及其表达. 中国农业科学, 2016, 49(15): 2867-2878.

WANG J J, MU M, WANG S, LU X K, CHEN X G, WANG D L, FAN W L, YIN Z J, GUO L X, YE W W, YU S X. Molecular clone and expression of GhDHN1 gene in cotton (Gossypium hirsutum L.). Scientia Agricultura Sinica, 2016, 49(15): 2867-2878. (in Chinese)

[26] 曹新文, 王秀珍, 李永梅, 赵李婧, 马海霞, 闫洁. 橡胶草法尼基焦磷酸合酶基因的克隆与功能分析. 中国农业科学, 2016, 49(6): 1034-1046.

CAO X W, WANG X Z, LI Y M, ZHAO L J, MA H X, YAN J. Molecular cloning and functional analysis of farnesyl pyrophosphate synthase genes from Taraxacum kok-saghyz Rodin. Scientia Agricultura Sinica, 2016, 49(6): 1034-1046. (in Chinese)

[27] 王慧飞, 孙艳香, 冯雪, 张一名, 杨江涛, 马梅芳, 陈光. 棉花精氨琥珀酸合成酶基因GhASS1的克隆及表达分析. 中国农业科学, 2016, 49(7): 24-35.

WANG H F, SUN Y X, FENG X, ZHANG Y M, YANG J T, MA M F, CHEN G. Cloning and expression analysis of argininosuccinate synthetase gene GhASS1from Gossypium hirsutum. Scientia Agricultura Sinica, 2016, 49(7): 24-35. (in Chinese)

[28] 梁文裕, 杨佳, 吴诗杰, 焦广飞, 王淑萍, 徐婷婷. 发菜耐旱相关蛋白NXL-01的基因克隆与表达分析. 西北植物学报, 2015, 35(1): 44-49.

LIANG W Y, YANG J, WU S J, JIAO G F, WANG S P, XU T T. Clone and expression of HypOthetical protein NXL-01 related to drought- tolerant in Nostoc flagelliforme. Acta Botanica Boreali- Occidentalia Sinica, 2015, 35(1): 44-49. (in Chinese)

[29] 官玲亮. 红花(Carthamus tinctor ius L.)不同组织多不饱和脂肪酸积累模式及调控机制[D]. 成都: 四川农业大学, 2011.

GUAN L L. Accumulation pattern and regulatory mechanisms of fatty acid in different safflower (Carthamus tinctorius L.) tissues[D]. Chengdu: Sichuan Agricultural University. 2011. (in Chinese)

[30] 白瑞英, 黄兴琳, 陆俊杏, 袁中厚, 高天姝, 郭楠, 张涛. 紫苏ω-3脂肪酸脱氢酶基因FAD3的克隆与表达分析. 分子植物育种, 2015, 13(12): 2728-2735.

BAI R Y, HUANG X L, LU J X, YUAN Z H, GAO T S, GUO N, ZHANG T.Cloning and expression analysis of ω-3 fatty acid desaturase gene FAD3 from Perilla frutescens. Molecular Plant Breeding, 2015, 13(12): 2728-2735. (in Chinese)

[31] MARTÍNEZ-RIVAS J M, SPERLING P, LÜHS W, SPERLING P, LÜHS W, HEINZ E. Spatial and temporal regulation of three different microsomal oleate desaturase genes (FAD2) from normal-type and high-oleic varieties of sunflower (Helianthus annuus L.). Molecular Breeding, 2001, 8(2): 159-168.