干旱和盐胁迫条件下枣树谷胱甘肽过氧化物酶基因（ZjGPX）的差异表达及功能分析

doi:10.3864/j.issn.0578-1752.2015.14.012

中国农业科学 ›› 2015, Vol. 48 ›› Issue (14): 2806-2817.doi: 10.3864/j.issn.0578-1752.2015.14.012

干旱和盐胁迫条件下枣树谷胱甘肽过氧化物酶基因（ZjGPX）的差异表达及功能分析

肖蓉^1，2，罗慧珍³，张小娟³，邓舒^1，2，张春芬^1，2，任莹³，孟玉平^1，2，曹秋芬^{1，2，3，4}，聂园军⁵

¹山西省农业科学院果树研究所，山西太谷 030815
²山西省农业科学院生物技术研究中心，太原 030031
³山西大学生物工程学院，太原 030006
⁴山西省农业科学院农作物品种资源研究所/农业部黄土高原作物基因资源与种质创制重点实验室，太原 030031
⁵山西省农业科学院农业资源与经济研究所，太原 030031

收稿日期:2015-02-06 出版日期:2015-07-16 发布日期:2015-07-16
通讯作者: 曹秋芬，Tel：0351-7369439；E-mail：qiufengcao@163.com
作者简介:肖蓉，E-mail：xiaorongzhujian@126.com
基金资助:
国家科技基础性工作专项重点项目（2012 FY110100-5）、山西省农业科学院院级攻关项目（YGG1432）

Differential Expression and Functional Analysis of Glutathione Peroxidase Gene from jujube (ZjGPX ) Under Drought and Salt Stresses

XIAO Rong^1,2, LUO Hui-zhen³, ZHANG Xiao-juan³, DENG Shu^1,2, ZHANG Chun-fen^1,2, REN Ying³, MENG Yu-ping^1,2, CAO Qiu-fen^1,2,3,4, NIE Yuan-Jun⁵

¹Pomology Institute, Shanxi Academy of Agricultural Sciences, Taigu 030815, Shanxi
²Biotechnolgy Research Center, Shanxi Academy of Agricultural Sciences, Taiyuan 030031
³College of Biological Engineering, Shanxi University, Taiyuan 030006
⁴Key Laboratory of Crop Gene Resources and Germplasm Enhancement on Loess Plateau, Ministry of Agriculture, Taiyuan 030031
⁵Institute of Agricultural Resources and Economy, Shanxi Academy of Agricultural Sciences, Taiyuan 030031

Received:2015-02-06 Online:2015-07-16 Published:2015-07-16

摘要/Abstract

摘要： 【目的】研究枣树谷胱甘肽过氧化物酶基因（ZjGPX）的功能，为其在果树抗逆基因工程改良中的利用奠定基础【方法】以‘壶瓶枣’（Ziziphus jujuba Mill. Hupingzao）结果枝构建的cDNA文库中选取一条与其他植物谷胱甘肽过氧化物酶基因有较高同源性的序列为研究对象进行序列分析，预测其功能。通过实时荧光定量技术分析目的基因在盐胁迫及干旱胁迫条件下的表达特性，并构建植物表达载体PEZR(K)-LNY-ZjGPX，运用农杆菌介导法，将ZjGPX转入拟南芥，对转基因及野生拟南芥植株作盐胁迫及干旱胁迫处理，验证其抗逆功能。【结果】ZjGPX编码序列（CDS）长510 bp，编码169个氨基酸。Blast比对发现该基因与多种植物GPX基因具有高度同源性（>80%）。实时荧光定量分析表明，在辣椒枣组培苗中，ZjGPX能够被一定浓度的干旱胁迫和盐胁迫诱导表达，且反应迅速，仅胁迫15 min，其相对表达量与对照相比即出现大幅升高，暗示该基因可能对枣树抗旱性和耐盐性具有重要的作用。结合50 μg·mL^-1 Kan抗性筛选、PCR验证及激光共聚焦显微镜观察，共获得10株阳性转基因拟南芥株系，干旱及盐胁迫处理结果显示，转基因拟南芥种子萌发率均高于野生型拟南芥；成株在胁迫15 d后，野生型拟南芥出现叶片发黄、萎焉、整株干枯、死亡的迹象，而转ZjGPX拟南芥生长基本正常。表明过表达ZjGPX拟南芥株系具有良好的耐旱性和耐盐性。【结论】ZjGPX在植物的干旱和盐胁迫应答反应机制中起重要作用，过量表达ZjGPX可提高转基因拟南芥的耐旱和耐盐能力。

关键词: 枣树, ZjGPX, 差异表达, 胁迫, 转基因拟南芥

Abstract: 【Objective】As a foundation for further studying on the application of fruit stress tolerance genetic engineering, this study analyzed the function of a cDNA encoding GPX, which was isolated from the fruit-bearing shoot of Ziziphus jujuba Mill. Hupingzao and designated as ZjGPX (GenBank accession No. AB608053). 【Method】 The ZjGPX sequence was analyzed and expression functions were forecasted by using bioinformatics analysis tool first. The differential expressions of ZjGPX on drought and salt stresses were tested by real-time quantitative PCR, and a vector named PEZR(K)-LNY-ZjGPX was constructed and introduced into Arabidopsis by floral dip method. Transgenic and wild Arabidopsis plants were tested under NaCl and drought stresses to verify ZjGPX’s function.【Result】Sequence analysis indicated that ZjGPX was 510 bp in length, encoding 169 amino acids. Blast analysis exhibited that ZjGPX was highly homologous to other GPXs (>80%). Real-time quantitative PCR analysis showed that ZjGPX expression could be rapidly induced by drought and salt stresses just in 15 minutes in Z. jujuba Mill. Lajiaozao test tube seedling. After agrobacterium-mediated transformation, 10 ZjGPX transgenic Arabidopsis lines were obtained. Results of the drought resistance and salt tolerance tests pointed out that in the stress treatments, the transgenic plants exhibited higher seed germination rates than the untransformed control plants. After 15 days of stress treatment, the untransformed control plants showed signs of death, but the transgenic plants maintained normal growth.【Conclusion】The results showed that ZjGPX was likely related to drought and salt resistances and over-expression of ZjGPX gene increased the drought resistance and salt tolerance of the transgenic Arabidopsis.

Key words: Ziziphus jujube, ZjGPX gene, differential expression, stress, transgenic Arabidopsis

肖蓉，罗慧珍，张小娟，邓舒，张春芬，任莹，孟玉平，曹秋芬，聂园军. 干旱和盐胁迫条件下枣树谷胱甘肽过氧化物酶基因（ZjGPX）的差异表达及功能分析[J]. 中国农业科学, 2015, 48(14): 2806-2817.

XIAO Rong, LUO Hui-zhen, ZHANG Xiao-juan, DENG Shu, ZHANG Chun-fen, REN Ying, MENG Yu-ping, CAO Qiu-fen, NIE Yuan-Jun. Differential Expression and Functional Analysis of Glutathione Peroxidase Gene from jujube (ZjGPX ) Under Drought and Salt Stresses[J]. Scientia Agricultura Sinica, 2015, 48(14): 2806-2817.

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.0578-1752.2015.14.012

https://www.chinaagrisci.com/CN/Y2015/V48/I14/2806

参考文献

[1] Smirnoff N. Environment and plant metabolism. Oxford: Bios Scientific Publishers, 1995: 217-243.

[2] Sugimoto M, Sakamoto W. Putative phospholipid hydroperoxide glutathione peroxidase gene from Arabidopsis thaliana induced by oxidative stress. Genes & Genetic Systems, 1997, 72(5): 311-316.

[3] Criqui M C, Jamet E, Parmentier Y, Marbach J, Durr A, Fleck J. Isolation and characterization of a plant cDNA showing homology to animal glutathione peroxidases. Plant Molecular Biology, 1992, 18: 623-627.

[4] Li W J, Feng H, Fan J H, Zhang R Q, Zhao N M, Liu J Y. Molecular cloning and expression of a phospholipid hydroperoxide glutathione peroxidase homolog in Oryza sativa. Biochim Biophys Acta, 2000, 1493(1/2): 225-230.

[5] 马亭亭, 周宜君, 高飞, 赵竹, 隋欣, 刘楠, 刘冉. 盐芥谷胱甘肽过氧化物酶基因（THGPX6）的克隆及表达分析. 植物遗传资源学报, 2012, 13(2): 252-258.

Ma T T, Zhou Y J, Gao F, Zhao Z, Sui X, Liu N, Liu R. Cloning and expression analysis of glutathione peroxidase 6 (ThGPX6) in Thellungiella halophile. Journal of Plant Genetic Resources, 2012, 13(2): 252-258. (in Chinese)

[6] 王菲菲, 丁明全, 邓澍荣, 王美娟, 孙健, 侯佩臣, 马旭君, 张玉红, 赵楠, 沈昕, 陈少良. 胡杨谷胱甘肽过氧化物酶 PeGPX基因的克隆及转化植株耐盐性分析. 基因组学与应用生物学, 2012, 31(3): 231-239.

Wang F F, Ding M Q, Deng S R, Wang M J, Sun J, Hou P C, Ma X J, Zhang Y H, Zho N, Shen X, Chen S L. Cloning of glutathione peroxidase gene PeGPX from Populus euphratica and the salt tolerance of the transformed plants. Genomics and Applied Biology, 2012, 31(3): 231-239. (in Chinese)

[7] 李文君, 刘进元, 赵南明. 苦瓜谷胱甘肽磷脂氢过氧化物酶 cDNA 的克隆及其特征分析. 生物化学与生物物理进展, 2001, 28(6): 908-911.

Li W J, Liu J Y, Zhao N M. Cloning and characterization of a phospholipid hydroperoxide glutathione peroxidase gene from Momordica charantia. Progress in Biochemistry and Biophysics, 2001, 28(6): 908-911 . (in Chinese)

[8] Depege N, Drevet J, Boyer N. Molecular cloning and characterization of tomato cDNAs encoding glutathione peroxidase-like proteins. European Journal of Biochemistry, 1998, 253(2): 445-451.

[9] 韩立敏. 丹参APX和GPX基因克隆及其表达分析[D]. 西安: 陕西师范大学, 2007.

Han L M. Study on cloning and expression of ascorbate peroxidase and glutathione peroxidase gene of Salvia miltiorrhiza Bge.[D]. Xi’an: Shaanxi Normal University, 2007. (in Chinese)

[10] Sugimoto M, Furui S, Suzuki Y. Molecular cloning and characterization of a cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase from spinach. Biosciences Biotechnology and Biochemistry, 1997, 61(8): 1379-1381.

[11] Avsian-Kretchmer O, Eshdat Y, Gueta-Dahan Y, Ben-Hayyim G. Regulation of stress-induced phospholipid hydroperoxide glutathione peroxidase expression in citrus. Planta, 1999, 209(4): 469-477.

[12] 陈义挺, 赖钟雄, 方智振, 蔡英卿, 林玉玲, 李焕苓, 陈登云. 龙眼GPX基因的克隆, 原核表达, 及其在体胚发生过程中的表达分析.热带作物学报, 2013, 34(8): 1483-1489.

Chen Y T, Lai Z X, Fang Z Z, Cai Y Q, Lin Y L, Li H L, Chen D Y. Cloning of GPX, prokaryotic expression and its expression during longan somatic embryogenesis of Dimocarpus longan Lour. Chinese Jurnal of Tropical Crops, 2013, 34(8): 1483-1489. (in Chinese)

[13] 王卫. 猕猴桃遗传多样性的SRAP分析及GPX和cAPX基因片段的克隆[D]. 福建: 福建师范大学, 2013.

Wang W. Using SRAP markers to analyze genetic diversity of Kiwifruit and cloning the gene of GPX and cAPX [D]. Fujian: Fujian Normal University, 2013. (in Chinese)

[14] 陈义挺, 王卫, 陈婷. 福建省地方梨种质资源莆田豆梨GPX基因克隆与生物信息学分析. 福建省农业科学院青年科技人才创新基金学术论文集. 2011: 200-207.

Chen Y T, Wang W, Chen T. Cloning and bioinformatics analysis of GPX gene of local pear germplasm resources Putian douli in Fujian. Proceedings of the young talents of science and technology innovation fund of Fujian institute of agricultural science, 2011: 200-207. (in Chinese)

[15] 曹秋芬, 孟玉平, 孙毅. 苹果属植物总RNA有效, 快速提取方法简介. 农业生物技术学报, 2003, 11(4): 428-429.

Cao Q F, Meng Y P, Sun Y. An effective quick method for total RNA extraction in Malus. Journal of Agricultural Biotechnology, 2003, 11(4): 428-429. (in Chinese)

[16] 孟玉平, 曹秋芬, 孙海峰. 枣树内参基因ZjH3的克隆与筛选. 生物技术通报, 2010, (11): 101-107.

Meng Y P, Cao Q F, Sun H F. Cloning and selection of housekeeping gene ZjH3 for Ziziphus jujuba. Biotechnology Bulletin, 2010, (11): 101-107. (in Chinese)

[17] 张小娟. 枣树ZjGPX, ZjPIP2和ZjMT基因的功能分析[D]. 山西: 山西大学, 2012.

Zhang X J. Functional analysis of ZjGPX, ZjPIP2 and ZjMT gene of Ziziphus jujuba [D]. Shanxi: Shanxi University, 2012. (in Chinese)

[18] 张洁, 杨大威, 孟玉平, 赵玉云, 曹秋芬. 枣树水通道蛋白基因生物信息学分析及原核表达载体的构建. 山西农业科学, 2010, 38(1): 6-10．

Zhang J, Yang D W, Meng Y P, Zhao Y Y, Cao Q F. The biological information analysis construction of the recombinant prokaryotic expression plasmidd of Ziziphus jujuba Aquaporin(AQP). Journal of Shanxi Agricultural Sciences, 2010, 38(1): 6-10. (in Chinese)

[19] Andrews G K. Regulation of metallothionein gene expression. Progress in Food & Nutrition Science, 1990, 14(2/3): 193-258.

[20] Zhang X, Henriques R, Lin S S, Niu Q W, Chua N H. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocols, 2006, 1: 641-646.

[21] F.M.奥斯伯, R. 布伦特. 精编分子生物学实验指南. 北京: 科学出版社, 1999: 37.

Ausbury F M, Brunt R. Short Protocols in Molecular Biology. Beijing: Science Press, 1999: 37. (in Chinese)

[22] Gaber A, Ogata T, Maruta T, Yoshimura K, Tamoi M, Shigeoka s. The Involvement of Arabidopsis glutathione peroxidase 8 in the suppression of oxidative damage in the nucleus and cytosol. Plant and Cell Physiology,2012, 53(9): 1596-1606.

[23] Milla M, Maurer A, Huete A R, Gustafson J P. Glutathione peroxidase genes in Arabidopsis are ubiquitous and regulated by abiotic stresses though diverse signaling pathways. The Plant Journal, 2003, 36(5): 602-615.

[24] 苗雨晨, 白玲, 苗琛, 陈珈, 宋纯鹏. 植物谷胱甘肽过氧化物酶研究进展.植物学通报, 2005, 22(3): 350-356.

Miao Y C, Bai L, Miao C, Chen J, Song C P. Progress in plant glutathione peroxidase. Chinese Bulletin of Botany, 2005, 22(3): 350-356. (in Chinese)

[25] 周立敬, 周宜君, 高飞, 赵竹. 拟南芥谷胱甘肽过氧化物酶的生物信息学分析. 中央民族大学学报: 自然科学版, 2010, 19(2): 11-17.

Zhou L J, Zhou Y J, Gao F, Zhao Z. Bioinformatics analysis of glutathione peroxidase in Arabidopsis. Journal of Minzu University of China: Natural Science Edition, 2010, 19(2): 11-17. (in Chinese)

[26] Herbette S, Labrouhe D T, Drevet J R, Roeckel-Drevet P. Transgenic tomatoes showing higher glutathione peroxydase antioxidant activity are more resistant to an abiotic stress but more susceptible to biotic stresses. Plant Science, 2011, 180(3): 548-553.

[27] Gong M, Chen B, Li Z G, Guo L H, Heat-shock-induced cross adaptation to heat, chilling,droughe and salt stress in maize seedlings and involvement of H₂O₂. Plant Physiology, 2001, 158: 1125-1130.

[28] Dietz K J. Redox signaling integration: from stimulus to networks and genes, Plant Physiology, 2008, 133: 459-468.

[29] Mehdy M C. Active oxygen species in plant defense against pathogens. Plant Physiology, 1994, 105: 467-472.

[30] 朱竹君, 陈海敏, 骆其君, 杨锐, 严小军, 王秀娟, 何山. 琼胶寡糖诱导坛紫菜活性氧爆发. 水产学报, 2012, 36(6): 969-973.

Zhu Z J, Chen H M, Luo Q J, Yang R, Yan X J, Wang X J, He S. Oxidative burst in Porphyra haitanensis (Rhodophyta). Journal of Fisheries of China, 2012, 36(6): 969-973. (in Chinese)

[31] 彭媛媛, 龚凌燕, 曹丹琴, 杨健, 朱立武, 张水明. 石榴PgGPX基因克隆及盐胁迫下的表达分析. 西北植物学报, 2014, 34(9): 1735-1741.

Peng Y Y, Gong L Y, Cao D Q, Yang J, Zhu L W, Zhang S M. Cloning of PgGPX Geng in pomegranate and its expression analysis under salt stress. Acta Botanica Boreali-Occidentalia Sinica, 2014, 34(9): 1735-1741. (in Chinese)

[32] 张丽丽, 徐碧玉, 刘菊华, 贾彩红, 张建斌, 王甲水, 金志强. 香蕉谷胱甘肽过氧化物酶基因MaGPX的克隆和表达分析. 园艺学报, 2012, 39(8): 1471-1481.

Zhang L L, Xu B Y, Liu J H, Jia C H, Zhang J B, Wang J S, Jin Z Q. Isolation and expression analysis of a cDNA encoding glutathione peroxidase from banana. Acta Horticulturae Sinica, 2012, 39(8): 1471-1481. (in Chinese)

[33] 安秀红, 徐锴, 厉恩茂, 李壮, 李敏, 刘志, 程存刚. 苹果抗性相关的谷胱甘肽转移酶基因MdGSTU1的生物信息学和表达分析. 中国农业科学, 2014, 47(24): 4868-4877.

An X H, Xu K, Li E M, Li Z, Li M, Liu Z, Cheng C G. Bioinformatics and expression analysis of MdGSTU1 gene encoding a resistance- related glutathione transferase from apple. Scientia Agricultura Sinica, 2014, 47(24): 4868-4877. (in Chinese)

干旱和盐胁迫条件下枣树谷胱甘肽过氧化物酶基因（ZjGPX）的差异表达及功能分析

Differential Expression and Functional Analysis of Glutathione Peroxidase Gene from jujube (ZjGPX ) Under Drought and Salt Stresses

RichHTML

PDF

赞

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	古丽旦,刘洋,李方向,成卫宁. 小麦吸浆虫小热激蛋白基因Hsp21.9的克隆及在滞育过程与温度胁迫下的表达特性[J]. 中国农业科学, 2023, 56(1): 79-89.
[2]	胡盛,李阳阳,唐章林,李加纳,曲存民,刘列钊. 干旱胁迫下甘蓝型油菜籽粒含油量和蛋白质含量变化的全基因组关联分析[J]. 中国农业科学, 2023, 56(1): 17-30.
[3]	莫文静,朱嘉伟,何新华,余海霞,江海玲,覃柳菲,张艺粒,李雨泽,罗聪. 芒果MiZAT10A和MiZAT10B功能分析[J]. 中国农业科学, 2023, 56(1): 193-202.
[4]	隋心意,赵小刚,陈鹏宇,李亚灵,温祥珍. 生菜LsPHYB可变剪接体的克隆与高温诱导表达模式[J]. 中国农业科学, 2022, 55(9): 1822-1830.
[5]	王俊娟,陆许可,王延琴,王帅,阴祖军,付小琼,王德龙,陈修贵,郭丽雪,陈超,赵兰杰,韩迎春,孙亮庆,韩明格,张悦新,范亚朋,叶武威. 陆地棉遗传标准系TM-1的特性及其耐冷性[J]. 中国农业科学, 2022, 55(8): 1503-1517.
[6]	李青林,张文涛,徐慧,孙京京. 低磷胁迫下黄瓜木质部与韧皮部汁液的代谢物变化[J]. 中国农业科学, 2022, 55(8): 1617-1629.
[7]	董桑婕,姜小春,王羚羽,林锐,齐振宇,喻景权,周艳虹. 远红光补光对辣椒幼苗生长和非生物胁迫抗性的影响[J]. 中国农业科学, 2022, 55(6): 1189-1198.
[8]	胡雪华,刘宁宁,陶慧敏,彭可佳,夏晓剑,胡文海. 低温胁迫对番茄幼苗不同叶龄叶片叶绿素荧光成像特性的影响[J]. 中国农业科学, 2022, 55(24): 4969-4980.
[9]	李宁,柳坤,刘彤彤,史雨刚,王曙光,杨进文,孙黛珍. 小麦响应干旱胁迫环状RNA的鉴定[J]. 中国农业科学, 2022, 55(23): 4583-4599.
[10]	刘浩,庞婕,李欢欢,强小嫚,张莹莹,宋嘉雯. 叶面喷施硒与土壤水分耦合对番茄产量和品质的影响[J]. 中国农业科学, 2022, 55(22): 4433-4444.
[11]	李刚,白阳,贾子颖,马正阳,张祥池,李春艳,李诚. 两种磷素水平下小麦苗期对干旱胁迫的离子组和代谢组响应[J]. 中国农业科学, 2022, 55(2): 280-294.
[12]	崔鹏,赵逸人,姚志鹏,庞林江,陆国权. 低温对甘薯淀粉理化特性及代谢关键基因表达量的影响[J]. 中国农业科学, 2022, 55(19): 3831-3840.
[13]	苏倩,杜文宣,马琳,夏亚迎,李雪,祁智,庞永珍. 紫花苜蓿MsCIPK2的克隆及功能分析[J]. 中国农业科学, 2022, 55(19): 3697-3709.
[14]	张川,刘栋,王洪章,任昊,赵斌,张吉旺,任佰朝,刘存辉,刘鹏. 不同时期高温胁迫对夏玉米物质生产性能及籽粒产量的影响[J]. 中国农业科学, 2022, 55(19): 3710-3722.
[15]	李小凡,邵靖宜,于维祯,刘鹏,赵斌,张吉旺,任佰朝. 高温干旱复合胁迫对夏玉米产量及光合特性的影响[J]. 中国农业科学, 2022, 55(18): 3516-3529.