烟草质膜ATPase4基因的克隆、表达载体构建及表达分析

doi:10.3969/j.issn.1004-1524.2019.02.01

浙江农业学报 ›› 2019, Vol. 31 ›› Issue (2): 173-181.DOI: 10.3969/j.issn.1004-1524.2019.02.01

烟草质膜ATPase4基因的克隆、表达载体构建及表达分析

王静^{1, 2}, 彭双^{1, 2}, 胡圣^{1, 2}, 卓维^{1, 2}, 陈倩^{1, 2}, 李立芹^{1, 2, *}

1.四川农业大学农学院,四川成都 611130;
2.作物科学国家级实验教学示范中心,四川成都 611130

收稿日期:2018-03-19 出版日期:2019-02-25 发布日期:2019-03-06
通讯作者: 李立芹,E-mail: liliqin88@163.com
作者简介:王静(1994—),女,河南濮阳人,硕士研究生,主要从事植物分子生物学研究。E-mail: 1015229363@qq.com
基金资助:
植物生理学与生物化学国家重点实验室开放课题(SKLPPBKF 1505,SKLPPBKF 1506)

Cloning expression vector construction and expression analysis of tobacco ATPase4 gene

WANG Jing^{1, 2}, PENG Shuang^{1, 2}, HU Sheng^{1, 2}, ZHUO Wei^{1, 2}, CHEN Qian^{1, 2}, LI Liqin^{1, 2, *}

1.College of Agronomy, Sichuan Agricultural University, Chengdu 611130, China;
2. National Demonstration Center for Experimental Crop Science Education, Chengdu 611130, China

Received:2018-03-19 Online:2019-02-25 Published:2019-03-06

摘要/Abstract

摘要： 植物细胞质膜ATPase基因表达可以引起细胞向外界环境释放H⁺,从而形成细胞膜内外的电化学式梯度,促进各种离子的运输,抵抗外界的各种胁迫。为研究ATPase基因在烟草中的非生物胁迫调控机制及相关生物学功能,本研究采用同源克隆的方法,从普通烟草K326中克隆到了1个ATPase4同源性基因,成功构建ATPase4-pcambia1300过表达载体。利用生物信息学分析ATPase4基因编码蛋白的疏水性、理化性质、蛋白结构预测、信号肽预测、磷酸位点预测及同源性分析,结果显示,该基因与绒毛状烟草(Nicotiana tomentosiformis)质膜ATPase4具有99%同源性,故命名为质膜ATPase4基因。利用荧光定量PCR(qRT-PCR)测定该基因在烟草组织中的表达量及其在非生物逆境胁迫中的表达模式,结果表明,该基因在根中表达量最高,且能快速响应低钾、高盐、PEG和冷胁迫诱导,说明ATPase4基因在烟草逆境胁迫中发挥着重要的调节作用。

关键词: 烟草, ATPase, 克隆, 表达

Abstract: Expression of cytoplasmic membrane ATPase gene in plants can cause cells to release H⁺ to the external environment, thus form the membrane inside and outside electrical chemical formula of the gradient, promote all sorts of ion transport, various stress resistance to the outside world. In order to study the regulatory mechanism of ATPase in tobacco and its related biological functions, this article adopted homologous cloning method to clone a ATPase4 homologous gene from common tobacco K326 and construct ATPase4-pcambia1300 overexpression vector. The bioinformatics software was used to predict the hydrophobic, physical and chemical properties, protein structure, signal peptide, phosphate site and origin of ATPase4. The homology analysis showed that the modified gene had 99% homology with plasma membrane ATPase4 of Nicotiana tomentosiformis, so it was named as plasma membrane ATPase4 gene. Fluorescence quantitative real time PCR (qRT-PCR) was used to determine the relative expression pattern in different tissues under different stresses. The results showed that the length of gene sequence was 2 982 bp, encoding 963 amino acid residues, the gene had the highest expression level in the roots, and it could respond quickly to low potassium, high salt, PEG and cold stress. It was shown that ATPase4 played an important regulatory role in tobacco stress.

Key words: tobacco, ATPase, cloning, expression

中图分类号:

S572

王静, 彭双, 胡圣, 卓维, 陈倩, 李立芹. 烟草质膜ATPase4基因的克隆、表达载体构建及表达分析[J]. 浙江农业学报, 2019, 31(2): 173-181.

WANG Jing, PENG Shuang, HU Sheng, ZHUO Wei, CHEN Qian, LI Liqin. Cloning expression vector construction and expression analysis of tobacco ATPase4 gene[J]. , 2019, 31(2): 173-181.

参考文献

[1] GAXIOIA R A, PALMGREN M G, SCHUMACHER K.Plant proton pumps[J]. FEBS Letters, 2007, 581(12):2204-2214.
[2] FALHOF J, FUGLSANG A T, PALMGREN M.Plasma membrane H+-ATPase regulation in the center of plant physiology[J]. Molecular Plant, 2016, 9(3):323-337.
[3] BRISKIN D P.The plasma membrane H+-ATPase of higher plant cells: biochemistry and transport function[J]. Biochimica et Biophysica Acta (BBA) Bioenergetics, 1990, 1019(2):95-109.
[4] MUNIZ M N, PAIS S M, TELLEZ M T, et al.Characterization of StPPI1, aproton pump interactor from Solanum tubeosum L. that is up regulated during tuber development and by abiotic stress[J]. Planta, 2011, 233(4):661-674.
[5] MORSOMME P, BOUTRY M.The plant plasma membrane H+-ATPase: structure, function and regulation[J]. Biochimica et Biophysica Acta Biomembranes, 2000, 1465(1/2):1-16.
[6] AYALA F, O'LEARY W, SCHUMAKER K S. Increased vacuolar and plasma membrane H+-ATPase activities in Salicornia bigelovii Torr. in response to NaCl[J]. Journal of Experimental Botany, 1996, 47(294):25-32.
[7] BINZEL M L.NaCl-induced accumulation of tonoplast and plasma membrane H+-ATPase message in tomato[J]. Physiologia Plantarum, 1995, 94(4):722-728.
[8] 赵昕, 谭会娟, 丁楠. NaCl胁迫对盐芥质膜和液泡膜ATPase活性的影响[J]. 西北植物学报, 2009, 29(4):717-723.
ZHAO X, TAN H J, DING N.Changes of PM and TP ATPase activities in Thellungiella seedlings under salt stress[J]. Acta Botanica Boreali-Occidentalia Sinica, 2009, 29(4):717-723. (in Chinese with English abstract)
[9] 张敏, 黄利斌, 季永华,等. NaCl胁迫对构树质膜H+-ATPase活性和表达的影响[J]. 北京林业大学学报, 2011, 33(6):21-26.
ZHANG M, HUANG L B, JI Y H, et al.Effects of NaCl stress on activity and expression of plasma membrane H+-ATPase in Broussonetia papyrifera[J]. Journal of Beijing Forestry University, 2011, 33(6):21-26. (in Chinese with English abstract)
[10] 司丛丛, 刘好宝, 曲平治. 烟草钾离子通道及转基因烟草抗逆性的研究进展[J]. 中国农学通报, 2010, 26(2):45-49.
SI C C, LIU H B, QU P Z.Research advances in potassium ion channel of tobacco and stress resistance of transgenic tobacco[J]. Chinese Agricultural Science Bulletin, 2010, 26(2):45-49. (in Chinese with English abstract)
[11] HODGES T K, LEONARD R T, BRACKER C E, et al.Purification of an ion-stimulated adenosine triphosphatase from plant roots: association with plasma membranes[J]. Proceedings of the National Academy of Sciences of the United States of America, 1972, 69(11):3307-3311.
[12] SHEN H, HE L F, SASAKI T, et al.Citrate secretion coupled with the modulation of soybean root tip under aluminum stress. Up-regulation of transcription, and threonine-oriented phosphorylation of plasma membrane ATPase[J]. Plant Physiology, 2005, 139(1):287-296.
[13] JUNGMIN O, HYUNSUNG K, HYUNJONG B.Jatropha is vulnerable to cold injury due to impaired activity and expression of plasma membrane H+-ATPase[J]. Acta Physiologiae Plantarum, 2014, 36(1):231-241.
[14] 刘健健, 刘俊丽, 季敏杰, 等. 番茄质膜ATPase家族基因的鉴定和表达分析[J]. 浙江农林大学学报, 2016, 33(5):734-741.
LIU J J, LIU J L, JI M J, et al.Identification and expression analysis of tomato plasma membrane H+-ATPase family genes[J]. Journal of Zhejiang A & F University, 2016, 33(5):734-741. (in Chinese with English abstract)
[15] MITO N, WIMMERS L E, BENNETT A B.Sugar regulates mRNA abundance of H+-ATPase gene family members in tomato[J]. Plant Physiology, 1996, 112(3):1229-1236.
[16] 武维华. 植物生理学[M].北京:科学出版社,2003.
[17] 刘建新, 胡浩斌, 赵国林. NaCI胁迫对骆驼蓬幼苗液泡膜ATPase和ATPase活性的影响[J]. 中国沙漠, 2008, 28(2):275-279.
LIU J X, HU H B, ZHAO G L.Effects of NaCI stress on the activity of tonoplast ATPase and ATPase in Peganum harmala seedlings[J]. Journal of Desert Research, 2008, 28(2):275-279. (in Chinese with English abstract)
[18] LI X, YANG Y Q, ZHANG M, et al.Effect of PEG priming on plasma membrane H+-ATPase activities and mitochondriu-m function in soybean seeds[J]. Seed Science & Technology, 2010,38: 49-60.
[19] YANG Y Q, WANG X F.Changes of plasma membrane H+-ATPase activities of Glycine max seeds by PEG treatment[J]. Forest Studies in China, 2005, 7(2):7-11.
[20] 刘尼歌, 王占义, 莫丙波, 等. 质膜ATPase与环境胁迫[J]. 热带亚热带植物学报, 2006, 14(3):263-268.
LIU N G, WANG Z Y, MO B B, et al.Plasma membrane H+-ATPase and environmental stress[J]. Journal of Tropical and Subtropical Botany, 2006, 14(3):263-268. (in Chinese with English abstract)
[21] BALLESTEROS E, KERKEB B, DONAIRE J P, et al.Effects of salt stress on H+-ATPase activity of plasma membrane-enriched vesicles isolated from sunflower roots[J]. Plant Science, 1998, 134(2):181-190.
[22] PONS R, CORNEIO M J, SANZ A.Differential salinity-induced variations in the activity of H+-pumps and Na+/H+ antiporters that are involved in cytoplasm ion homeostasis as a function of genotype and tolerance level in rice cell lines[J].Plant Physiology and Biochemistry, 2011, 49(12):1399-1409.
[23] JUNG S, HUTSCH B W, SCHUBERT S.Salt stress reduces kernel number of corn by inhibiting plasma membrane H+-ATPase activity[J]. Plant Physiology and Biochemistry, 2017, 113: 198-207.
[24] 简令成, 董合铸, 孙龙华. 番茄子叶细胞内三磷酸腺苷酶活性的超微结构定位及其在冷害中的变化[J]. 植物学报, 1981, 23(4):257-261.
JIAN L C, DONG H Z, SUN L H.Ultrastructural localization of adenosine triphosphatase activity in cotyledon cells of tomato and its changes during chilling stress[J]. Acta Botanica Sinica, 1981, 23(4):257-261. (in Chinese with English abstract)

烟草质膜ATPase4基因的克隆、表达载体构建及表达分析

Cloning expression vector construction and expression analysis of tobacco ATPase4 gene

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	赵秀平, 王双, 闫星伊, 段强, 张帅, 陈永胜, 李国瑞. 稻瘟病菌MGG-01005的表达纯化与生物信息学分析[J]. 浙江农业学报, 2021, 33(3): 470-478.
[2]	吴俊静, 乔木, 周佳伟, 梅书棋, 彭先文. 猪长链非编码RNA lnc-000649在PRRSV感染增殖中的作用[J]. 浙江农业学报, 2021, 33(2): 223-229.
[3]	黄咏明, 宋放, 王策, 姚京磊, 王志静, 何利刚, 吴黎明, 蒋迎春. 根系修剪对枳生长及相关基因表达的影响[J]. 浙江农业学报, 2021, 33(2): 270-277.
[4]	何佳琦, 翟莹, 张军, 邱爽, 李铭杨, 赵艳, 张梅娟, 马天意. 大豆转录因子GmDof1.5的克隆与非生物胁迫诱导表达[J]. 浙江农业学报, 2021, 33(1): 1-7.
[5]	苏学思, 张玉宝, 王若愚, 王亚军, 唐国亮, 金卫杰. 车前草花叶病毒衣壳蛋白的原核表达和多克隆抗体制备[J]. 浙江农业学报, 2021, 33(1): 104-111.
[6]	刘金玉, 黄鹰. 粟酒裂殖酵母SpTrz2蛋白全长和N端的原核表达与多克隆抗体制备[J]. 浙江农业学报, 2021, 33(1): 34-42.
[7]	王伟科, 陆娜, 闫静, 宋吉玲, 袁卫东, 周祖法. 秀珍菇S-腺苷甲硫氨酸合成酶基因(PpSAMS)的克隆与表达分析[J]. 浙江农业学报, 2021, 33(1): 62-68.
[8]	徐秀红, 刘金亮, 李栋成, 刘仁祥. 不同类型烟草种质的烟碱含量变化与相关基因表达水平[J]. 浙江农业学报, 2020, 32(9): 1555-1563.
[9]	王伟, 滚双宝, 王鹏飞, 黄晓宇, 谢开会, 雒瑞瑞, 高小莉, 张博, 闫尊强, 杨巧丽, 马艳萍. 猪miR-204组织表达与重要靶基因筛选[J]. 浙江农业学报, 2020, 32(9): 1564-1573.
[10]	刘坤举, 张小辉, 庞有志, 赵淑娟, 祁艳霞, 王乾昆. 朝鲜鹌鹑GNAS基因表达、克隆及其多态性与羽色的相关性[J]. 浙江农业学报, 2020, 32(8): 1369-1377.
[11]	杜炎斌, 张港琛, 王瑜欣, 刘宝宝, 宫胜龙, 东笑, 汪洋. 猪链球菌rpoE基因克隆及生物信息学分析[J]. 浙江农业学报, 2020, 32(7): 1149-1154.
[12]	盛中伟, 姬改革, 刘一帆, 巨晓军, 单艳菊, 邹剑敏, 章明, 屠云洁, 束婧婷. 鸡生长发育早期骨骼肌IGF1R mRNA表达规律研究[J]. 浙江农业学报, 2020, 32(7): 1160-1165.
[13]	黄小珍, 乔中全, 曾慧杰, 李永欣, 何钢, 王晓明. 紫薇花器官发育调控基因LiFUL1的分离与表达分析[J]. 浙江农业学报, 2020, 32(7): 1206-1214.
[14]	孙瑞萍, 王峰, 晁哲, 刘海隆, 邢漫萍, 刘圈炜, 黄丽丽, 郑心力, 魏立民. 屯昌猪PDK4基因克隆及其组织表达分析[J]. 浙江农业学报, 2020, 32(6): 978-985.
[15]	李秋玲, 齐颖, 王琛, 张一名, 王新妤, 尚校兰, 贾永红, 李美茹, 储明星. 热应激对中国荷斯坦牛乳腺组织基因表达及信号通路的影响[J]. 浙江农业学报, 2020, 32(5): 770-778.