丝瓜铜锌超氧化物歧化酶Cu/Zn-SOD基因家族的克隆与表达分析

doi:10.3864/j.issn.0578-1752.2017.17.013

摘要/Abstract

摘要： 【目的】克隆丝瓜铜/锌超氧化物歧化酶Cu/Zn-SOD基因家族，并分析其序列特征、表达情况及其在丝瓜褐变中的作用，为进一步揭示丝瓜褐变的发生机理提供科学依据，为丝瓜品种遗传改良奠定基础。【方法】通过转录组测序和RT-PCR方法获得丝瓜Cu/Zn-SOD基因家族的cDNA序列，采用生物信息学方法分析氨基酸序列，利用荧光定量PCR技术研究Cu/Zn-SOD基因家族在不同组织及褐变条件下的表达情况。采用氮蓝四唑（NBT）光还原法测定总超氧化物歧化酶（SOD）活性，采用福林-酚比色测定总酚含量。【结果】获得了3个丝瓜Cu/Zn-SOD基因家族cDNA序列，依次命名为LcCu/Zn-SOD1（GenBank登录号：KP178922）、LcCu/Zn-SOD2 （GenBank登录号：KX092445）和LcCu/Zn-SOD3（GenBank登录号：KX092446）。LcCu/Zn-SOD1全长758 bp，包含一个456 bp的开放读码框（ORF），编码152个氨基酸；LcCu/Zn-SOD2全长799 bp，ORF为471 bp，编码157个氨基酸；LcCu/Zn-SOD3全长1 011 bp，ORF为663 bp，编码221个氨基酸；编码的蛋白与甜瓜、南瓜、黄瓜同源蛋白的相似性均在90%以上。生物信息学分析表明3个基因编码的酶蛋白均无信号肽，无跨膜结构域，为亲水性稳定蛋白，Wolf Psort预测其亚细胞定位于细胞质。Cu/Zn-SOD基因家族在根中的表达量最高，在花中表达量最低。在丝瓜采后储藏期间，LcCu/Zn-SOD1和LcCu/Zn-SOD3在丝瓜储藏初期表达量较采后当时（0 d）上调，后期表达量受到抑制；在丝瓜鲜切条件下，3个基因表达量在鲜切后较采后当时（0 h）总体呈下降趋势。相关性分析显示，在采后储藏和鲜切条件下，LcCu/Zn-SOD1表达量均与SOD酶活性呈极显著性正相关，LcCu/Zn-SOD3表达量均与SOD酶活性呈显著性正相关，SOD酶活性在鲜切条件下与总酚含量呈显著负相关，LcCu/Zn-SOD1和LcCu/Zn-SOD3在调控SOD酶活性方面起作重要作用，LcCu/Zn-SOD1和LcCu/Zn-SOD3的表达影响了SOD酶活性，并影响了丝瓜褐变进程。【结论】从丝瓜果肉中获得Cu/Zn-SOD基因家族的3个，其中LcCu/Zn-SOD1和LcCu/Zn-SOD3在普通丝瓜果肉褐变过程中可能发挥着重要作用。

关键词: 丝瓜, 褐变, Cu/Zn-SOD, 表达分析, SOD活性

Abstract: 【Objective】The aim of this study was to clone the Cu/Zn-SOD gene family from Luffa cylindrical, investigate their sequence characteristics and analyze their expression in luffa browning. These findings will provide a scientific basis for further revealing the mechanism of luffa browning and lay a practical foundation for the genetic improvement of luffa. 【Method】 The cDNA sequences of Cu/Zn-SOD gene family were obtained by transcriptome sequencing and RT-PCR. The bioinformatics methods were used to analyze the putative amino acid sequence, and quantitative real-time PCR (qRT-PCR) method was used to study the expression of Cu/Zn-SOD gene family in different tissues and browning conditions. The superoxide dismutase enzyme activity was measured by NBT deoxidization method. The total phenols was measured by folin-cioncaleuc method. 【Result】Three cDNAs of Cu/Zn-SOD were cloned from luffa fruit, in turn being named LcCu/Zn-SOD1, LcCu/Zn-SOD2 and LcCu/Zn-SOD3. The cDNA sequence of LcCu/Zn-SOD1 was 758 bp in length, containing a 456 bp opening reading frame(ORF), encoded a polypeptide of 152 amino acids. The cDNA sequence of LcCu/Zn-SOD2 was 799 bp in length, containing a 471 bp ORF, encoded a polypeptide of 157 amino acids. The cDNA sequence of LcCu/Zn-SOD3 was 1 011 bp in length, containing a 663 bp ORF, encoded a polypeptide of 221 amino acids. They shared over 90% identity with the homologous proteins from Cucumis melo, Cucurbita pepo and Cucumis sativus. The bioinformatics analysis showed that three proteins were hydrophilic protein without signal-peptide and transmembrane region, and the Wolf Psort protection indicated that they were located in the cytoplasm. The expression of LcCu/Zn-SODgene familywas the highest in root and the lowest in flower. During post-harvest storage, the expression of LcCu/Zn-SOD1 and LcCu/Zn-SOD3 was up-regulated in the early, and then decreased. The expression levels of three genes were overall down-regulated in fresh-cut luffa fruit. Correlation analysis showed that the expression level of LcCu/Zn-SOD1 showed a extremely significant positive correlation with SOD activity, and the expression level of LcCu/Zn-SOD3 showed a significant positive correlation with SOD activity during fresh-cut and post-harvest storage. SOD activity was significantly and negatively correlated with total phenols content during fresh-cut conditions. LcCu/Zn-SOD1 and LcCu/Zn-SOD3 play an important role in regulating the activity of SOD, and the expression of LcCu/Zn-SOD1 and LcCu/Zn-SOD3 may influence the activity of SOD and the process of luffa browning.【Conclusion】Three cDNAs of Cu/Zn-SOD were firstly obtained and characterized from luffa fruit, LcCu/Zn-SOD1 and LcCu/Zn-SOD3 may play an important role in luffa browning process.

Key words: Luffa cylindrical, browning, Cu/Zn-SOD, expression analysis, activity of SOD

朱海生，刘建汀，陈敏氡，李永平，王彬，张前荣，叶新如，林珲，温庆放. 丝瓜铜锌超氧化物歧化酶Cu/Zn-SOD基因家族的克隆与表达分析[J]. 中国农业科学, 2017, 50(17): 3386-3399.

ZHU HaiSheng, LIU JianTing, CHEN MinDong, LI YongPing, WANG Bin, ZHANG QianRong, YE XinRu, LIN Hui, WEN QingFang. Cloning and Expression Analysis of Copper and Zinc Superoxide Dismutase Cu/Zn-SOD Gene Family from Luffa cylindrical[J]. Scientia Agricultura Sinica, 2017, 50(17): 3386-3399.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.17.013

https://www.chinaagrisci.com/CN/Y2017/V50/I17/3386

参考文献

[1] 李程斌, 李恩, 郑艳, 施媚, 吴琼. 丝瓜藤和叶营养成分分析. 安徽师范大学学报(自然科学版), 2009, 32(1): 69-71.

LI C B, LI E, ZHENG Y, SHI M, WU Q. Towel gourd stem and leaf nutrient analysis. Journal of Anhui Normal University (Natural Science Edition), 2009, 32(1): 69-71. (in Chinese)

[2] 颜国纲, 郑振佳, 时新刚, 葛长军. 丝瓜的营养价值及其综合利用研究进展. 中国果菜, 2011(7): 35-36.

YAN G G, ZHENG Z J, SHI X G, GE C J. Towel gourd nutrition value and its comprehensive utilization. China Fruit Vegetable, 2011(7): 35-36. (in Chinese)

[3] 娄丽娜, 刘哲, 苏小俊. 普通丝瓜耐褐变研究进展. 长江蔬菜, 2015(4): 1-4.

LOU L N, LIU Z, SU X J. Research progress of flesh browning resistance in luffa (Luffa cylindrical L.). Journal of Changjiang Vegetables, 2015(4):1-4. (in Chinese)

[4] 王成, 王辉, 娄丽娜, 苏小俊. 普通丝瓜果肉褐变的鉴定方法. 江苏农业科学, 2012, 40(11): 137-138.

WANG C, WANG H, LOU L N，SU X J. Identification of luffa browning. Jiangsu Agricultural Sciences, 2012, 40(11): 137-138. (in Chinese)

[5] 黄树苹, 徐长城, 谈杰, 张敏, 王春丽, 陈霞, 甘莉, 谈太明. 不同影响因素下普通丝瓜果肉褐变度、多酚氧化酶活性及多酚质量比变化研究. 西南大学学报, 2015, 37(9): 31-36.

HUANG S P, XU C C, TAN J, ZHANG M, WANG C L, CHEN X, GAN L, TAN T M. Studies on the browning degree, polyphenol oxidase (PPO) activity and polyphenol content in fruit flesh of Luffa (Luffa cylindrica) influenced by different factors. Journal of Southwest Agricultural University, 2015, 37(9): 31-36. (in Chinese)

[6] BUSTOS M C, MAZZOBRE M F, BUERA M P. Stabilization of refrigerated avocado pulp: effect of Allium and Brassica extracts on enzymatic browning. Food Science and Technology, 2015, 61(1): 89-97.

[7] 曾秀存, 孙万仓, 孙佳, 许耀照, 方彦, 史鹏辉, 杨刚, 孔德晶, 武军艳, 刘自刚. 白菜型冬油菜铁超氧化物歧化酶（Fe-SOD）基因的克隆及表达分析. 中国农业科学, 2013, 46(21): 4603-4611.

ZENG X C, SUN W C, SUN J, XU Y Z, FANG Y, SHI P H, YANG G, KONG D J, WU J Y, LIU Z G. Cloning and expression analysis of Fe superoxide dismutase (Fe-SOD) gene from winter turnip rape (Brassica rapa L.). Scientia Agricultura Sinica, 2013, 46(21): 4603-4611. (in Chinese)

[8] LEE S H, AHSAN N, LEE K W, KIM D H, LEE D G, KWAK S S, KWON S Y, KIM T H, LEE B. Simultaneous overexpression of both CuZn-superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses. Journal of Plant Physiology, 2007(164): 1626-1638.

[9] ALLEN R D, WEBB R P, SCHAKE S A. Use of transgenic plants to study antioxidant defenses. Free Radical Biology Medicine, 1997, 23(3): 473-479.

[10] ALSCHER R G, ERTURKN, HEATH L S. Role of superoxide dismutases (SODs) in controlling oxidative stress in plant. Journal of Experimental Botany, 2002, 53: 1331-1341.

[11] LAWRENCE V G, NICOLE T B, WU G H, LUO X M, LIU X J, MICHAEL L G, ALAN H. Superoxide dismutase: An all-purpose gene for agri-biotechnology. Molecular Breeding, 2009, 24: 103-115.

[12] 臧洁, 余梅, 王先磊, 王荣富, 李娟, 吉虎, 徐凯, 鲁茂龙. 盐角草Cu/Zn-SOD基因的克隆及耐盐性分析. 农业生物技术学报, 2013, 21(7): 847-854.

ZANG J,YU M, WANG X L, WANG R F, LI J, JI H, XI K, LU M L. Cloning and salt-tolerance analysis of the salicornia europaea gene Cu/Zn-SOD. Journal of Agricultural Biotechnology, 2013, 21(7): 847-854. (in Chinese)

[13] 严伶俐, 甘小迎, 韩冬梅, 吴振先. 荔枝果皮LcMn-SOD、LcCu/Zn-SOD-1、LcCu/Zn-SOD-2、LcPRDX5的表达特性分析. 安徽农学通报, 2016, 22(7): 114-118.

YAN L L, GAN X Y, HAN D M, WU Z X. The expression of LcMn-SOD, LcCu/Zn-SOD-1, LcCu/Zn-SOD-2, LcPRDX5 genes in Litchi pericarp. Anhui Agricultural Science Bulletin, 2016, 22(7): 114-118. (in Chinese)

[14] BUBLIY O A, LOESEHCHE V. Correlated responses to selection for stress resistance and longevity in a laboratory population of Drosophila melanogaste. Journal of Evolutionary Biology, 2005, 18(4): 789-803.

[15] 汤燕姗, 赖钟雄, 赖呈纯, 林玉玲. “台农甜蜜桃”胚性愈伤组织SOD基因家族的克隆与序列分析. 热带作物学报, 2013, 34(10): 1925-1934.

TANG Y S, LAI Z X, LAI C C, LIN Y L. Cloning and sequence analysis of SOD gene family from the peach cultivar ‘Premier’ honey peach. Chinese Journal of Tropical Crop, 2013, 34(10): 1925-1934. (in Chinese)

[16] 王星, 刘肖飞, 周宜君, 张根发. 植物SOD基因表达调控的分子机制. 中国油料作物学报, 2014, 36(2): 275-280.

WANG X, LIU X F, ZHOU Y J, ZHANG G F.Molecular mechanism of expression and regulation of SOD gene in plant. Chinese Journal of Oil Crop Sciences, 2014, 36(2): 275-280. (in Chinese)

[17] DU J F, ZENG Y Q, CHEN W, CUI J X, CHEN Q. Cloning, expression and functional analysis of CuZnSOD gene in swine. Hereditas, 2010, 32(10): 1037.

[18] 纪砚耘, 化文平, 王喆之. 丹参铜锌超氧化物歧化酶(Cu/Zn-SOD)基因的克隆与生物信息学分析. 陕西师范大学学报(自然科学版), 2011, 39(3): 56-60.

JI Y Y, HUA W P, WANG Z Z. Cloning and bioinformatics analysis of Cu/Zn/SOD gene from Salvia miltiorrhiza Bunge. Journal of Shaanxi Normal University(Natural Science Edition), 2011, 39(3): 56-60. (in Chinese)

[19] 陆燕元, 邓西平. 转入Cu/Zn-SOD和APX基因对甘薯旱后复水的恢复作用. 西北农林科技大学学报, 2010, 38(1):67-74.

LU Y Y, DENG X P. Recovery effects of transferring both Cu/Zn-SOD and APX genes in sweet potato under water stress. Journal of Northwest A&F University (in Chinese), 2010, 38(1): 67-74.

[20] 史艳梅, 王燃, 武明珠, 陈千思, 李锋, 魏春阳, 罗朝鹏, 杨军, 屈凌波, 林福呈. 烟草Cu/Zn-SOD基因克隆及在低温弱光胁迫条件下的表达模式分析. 烟草科技, 2013(11): 73-77.

SHI Y M, WANG R, WU M Z, CHEN Q S, LI F, WEI C Y, LUO Z P, YANG J, QU L B, LIN F C. Cloning of Cu/Zn-SOD gene in tobacco and analysis of expression pattern of stresses induced by low temperature and weak light exposure. Tobacco Science＆Technology, 2013(11): 73-77. (in Chinese)

[21] SONG F N, YANG C P, LIU M, LI G B. Effect of salt stress on activity of superoxide dismutase (SOD) in Ulmus pumila L. Journal of Forestry Research, 2006(17): 13-16.

[22] HERNFINDEZ-NISTAL J, DOPICO B, LABRADOR E. Cold and salt stress regulates the expression and activity of a chickpea cytosolic Cu/Zn superoxide dismutase. Plant Science, 2002, 163(3): 507-514.

[23] KAYIHAN C, EYIDOGAN F, AFSAR N. Cu/Zn superoxide dismutase activity and respective gene expression during cold acclimation and freezing stress in barley cultivars. Biologia Plantarum, 2012, 56(4): 693-698.

[24] 蒋娟. 鲜切莲藕褐变的生理生化机制及蛋白表达差异研究[D]. 南京: 南京农业大学, 2011.

JIANG J. The physio-chemical mechanism and differential expressions of proteins for the browning of fresh-cut lotus root [D]. Nanjing: Nanjing Agriculture University, 2011. (in Chinese)

[25] 李文香, 周莎莎, 刘洁, 王丽娇, 刘树堂. 微真空贮藏条件下莱阳梨果实褐变的生理机制. 食品科学, 2013, 34(6): 266-270.

LI W X, ZHOU S S, LIU J, WANG L J, LIU S T. Browning mechanism of laiyang pears during micro-vacuum storage. Food Science, 2013, 34(6): 266-270. (in Chinese)

[26] FAN M, Li W, HU X. Effect of micro-vacuum storage on active oxygen metabolism, internal browning and related enzyme activities in Laiyang pear (Pyrus bretschneideri Rehd). LWT-Food Science and Technology, 2016, 72: 467-474.

[27] 胡根海, 喻树迅, 范术丽, 宋美珍. 编码棉花胞质铜锌超氧物歧化酶基因的克隆与表达分析. 中国农业科学, 2007, 40(8): 1602-1609.

HU G H, YU S X, FAN S L, SONG M Z. Cloning and expressing of a gene encoding cytosolic coppe/zinc superoxide dismutase in the upland cotton. Scientia Agricultura Sinica, 2007, 40(8): 1602-1609. (in Chinese)

[28] SUMIO K, KOZI A. Characteristic of amino acid sequences of chloroplast and cytoplast isozymes of Cu/Zn superoxide dismutase in spinach, rice and horsetail. Plant Cell Physiology, 1990, 31: 99-112.

[29] Kernodle S, Scandalios J G. A comparison of the structure and function of the highly homologous maize antioxidant Cu/Zn superoxide dismutase genes SOD4 and SOD4A. Genetics, 1996, 144: 317-328.

[30] 王盛, 张保青, 黄杏, 樊艳姣, 杨丽涛, 李杨瑞. 甘蔗Cu/Zn-SOD的克隆和表达分析. 中国农业科学 2013, 46(15): 3277-3284.

WANG S, ZHANG B q, HUANG X, FAN Y J, YANG L T, LI Y R. Molecular cloning of sugarcane Cu/Zn superoxide dismutase (Cu/Zn-SOD) and its expression analysis. Scientia Agricultura Sinica, 2013, 46(15): 3277-3284. (in Chinese)

[31] CUI L J, HUANG Q, YAN B, WANG Y, QIAN Z Y, PAN J X, KAI G Y. Molecular cloning and expression analysis of a Cu/Zn SOD gene (BcCSD1) from Brassica campestris ssp. Chinese Food Chemistry, 2015, 186(1): 306-311.

[32] ZHU X J, LI Q HQ, HU J Y, WANG M L, LI X H. Molecular cloning and characterization of spermine synthesis gene associated with cold tolerance in tea plant (Camellia sinensis). Applied Biochemistry and Biotechnology, 2015, 177(5): 1055-1068.

[33] 朱海生, 陈敏氡, 温庆放, 蓝新隆, 李永平, 王彬, 张前荣, 吴卫大化东. 丝瓜18S rRNA基因克隆及其作为内参基因的应用. 核农学报, 2016, 30(1): 35-41.

ZHU H S, CHEN M D, WEN Q F, LAN X L, LI Y P, WANG B, ZHANG Q R, WU W D. Cloning of 18S rRNA gene from Luffa cylindrical and its application as an internal standard. Journal of Nuclear Agricultural Sciences, 2016, 30(1): 35-41. (in Chinese)

[34] LIU J, WU Y, KANG J. Changes in reactive oxygen species production and antioxidant enzyme activity of Agaricus bisporus harvested at different stages of maturity. Journal of the Science of Food and Agriculture, 2013, 93(9): 2201-2206.

[35] 朱海生, 庄尹宏, 刘建汀, 王彬，陈敏氡, 张前荣, 温庆放, 李大忠, 薛珠政. 丝瓜总酚提取和测定方法的优化. 福建农业学报, 2016, 31(11): 1204-1209.

ZHU H S, ZHUANG Y H, LIU J T, WANG B, CHEN M D, ZHANG Q R, WEN Q F, LI D Z, XUE Z Z. Extraction and determination of total phenol in luffa (Luffa cylindrical Roem.). Fujian Journal of Agricultural Sciences, 2016, 31(11): 1204-1209. (in Chinese)

[36] 温文旭, 朱海生, 温庆放, 陈敏氡, 林碧英, 薛珠政. 超高效液相色谱法分析丝瓜酚类物质组分及其含量. 园艺学报, 2016, 43(7): 1391-1401.

WEN W X, ZHU H S, WEN Q F, CHEN M D, LIN B Y, XUE Z Z. Determination of polyphenols in luffa by ultra performance liquid chromatography.Acta Horticulturae Sinica, 2016, 43(7): 1391-1401. (in Chinese)

[37] LIN Y L, LAI Z X. Superoxide dismutase multigene family in longan somatic embryos: a comparison of CuZn-SOD, Fe-SOD, and Mn-SOD gene structure, splicing, phylogeny, and expression. Molecular Breeding, 2013, 32(3): 595-615.

[38] KLIEBENSTEIN D J, GERSHENZON J, MITCHELLOLDS T. Comparative quantitative trait loci mapping of aliphatic, indolic and benzylic glucosinolate production in Arabidopsis thaliana leaves and seeds. Genetics, 2001, 159: 359-370.

[39] LIU J J, GOH C J, LOH C S, TAY E B H, PUA E C. Cloning of two cDNAs encoding Cu/Zn-superoxide dismutase (accession No. X95726, X95728) of mustard (Brassicajuncea L. Czern & Coss). Plant Physiology, 1998, 116: 867.

[40] 章慧慧, 励勤荣. 超氧化物歧化酶的研究和应用现状. 农产品加工(学刊), 2007(8): 28-32.

ZHANG H H, LI Q R. Study on superoxide dismutase. The processing of Agricultural Products (in Chinese), 2007(8): 28-32.

[41] 杨鸯鸯, 李云, 丁勇, 徐春雷, 张成桂, 刘英, 甘莉. 甘蓝型油菜Cu/Zn-SOD和FeSOD基因的克隆及菌核病菌诱导表达. 作物学报, 2009, 35(1): 7l-78.

YANG Y Y, LI Y, DING Y, XU C L, ZHANG C G, LIU Y, GAN L. Cloning of Cu/Zn-Superoxide dismutase of Brassica napus and its induced expression by Sclerotinia sclerotiorum. Acta Agronomica Sinica, 2009, 35(1): 7l-78. (in Chinese)

[42] QUE Y X, LIU J X, XU L P, GUO J R, CHEN R K. Molecular cloning and expression analysis of an Mn superoxide dismutase gene in sugarcane. African Journal of Biotechnology, 2012, 11(3): 552-560.

[43] RUBIO M C, BUSTOS-SANNMAMED P, CLEMENTE M R, BECANA M. Effects of salt stress on the expression of antioxidant genes and proteins in the model legume Lotus japonicus. New Phytologist, 2009, 181(4): 851-859.

[44] ALSCHER R G, DONAHUE J L, CRAMER C L. Reactive oxygen species and antioxidants relationships in green cells. Plant Physiology, l997, 100: 224-233.

[45] 陈艺晖, 林河通, 林艺芬, 张居念, 赵云峰. 拟茎点霉侵染对龙眼果实采后果皮褐变和活性氧代谢的影响. 中国农业科学, 2011, 44(23): 4858-4866.

CHEN Y H, LIN H T, LIN Y F, ZHANG J N , ZHAO Y F. Effects of phomopsis longanae Scientia Agricultura Sinica, 2011, 44(23): 4858-4866. (in Chinese)Chi infection on browning and active oxygen metabolism in pericarp of harvested longan fruits.