欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2016, Vol. 42 ›› Issue (09): 1282-1290.doi: 10.3724/SP.J.1006.2016.01282

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦蛋白磷酸酶2A 基因TaPP2AbB″-α 启动子的克隆及表达分析

扆珩1, 2,李昂2,刘惠民1,景蕊莲2,*   

  1. 1 山西大学生物工程学院, 山西太原 030006; 2 农作物基因资源与基因改良国家重大科学工程 / 中国农业科学院作物科学研究所,北京 100081
  • 收稿日期:2016-02-05 修回日期:2016-05-09 出版日期:2016-09-12 网络出版日期:2016-06-06
  • 通讯作者: 景蕊莲, E-mail: jingruilian@caas.cn, Tel: 010-82105829
  • 基金资助:

    本研究由国家自然科学基金项目(31201206)和中国农业科学院创新工程项目资助。

Cloning and Expression Analysis of Protein Phosphatase 2A Gene TaPP2AbB″-α Promoter in Wheat

YI Heng1,2,LI Ang2,LIU Hui-Min1,JING Rui-Lian2,*   

  1. 1 College of Bioengineering, Shanxi University, Taiyuan 030006, China; 2 National Key Facility for Crop Gene Resources and Genetic Improvement /Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2016-02-05 Revised:2016-05-09 Published:2016-09-12 Published online:2016-06-06
  • Contact: 景蕊莲, E-mail: jingruilian@caas.cn, Tel: 010-82105829
  • Supported by:

    This study was supported by the National Natural Science Foundation of China (31201206) and the Agricultural Science and Technology Innovation Program (CAAS).

RichHTML

PDF

1656

被引次数

2

摘要：

植物蛋白磷酸酶2A (protein phosphatase 2A, PP2A)由结构亚基A、调节亚基B 和催化亚基C 组成, 在应答逆境胁迫途径中发挥着重要作用。小麦基因TaPP2AbB″-α 是调节亚基亚家族B″的成员, 过量表达该基因可以促进拟南芥的根系生长及侧根发育, 在盐胁迫和渗透胁迫条件下的作用更显著。本研究从小麦(Triticum aestivum L.)抗旱品种“旱选10 号”基因组中克隆了TaPP2AbB″-α 基因的启动子PB″α, 序列长度为1899 bp, 含有TATA-box 和CAAT-box,以及响应干旱和渗透胁迫的顺式作用元件EECCRCAH1-(?1058 bp 至?1052 bp)、GCCCORE (?1073 bp 至?1068 bp)和MYCCONSE (?1179 bp 至?1174 bp)。将启动子PB″α 和5 种5′端缺失启动子片段与报告基因GUS (β-glucuronidase)连接后转化拟南芥, 组织化学染色结果显示PB″α 在植株的叶片和根中均有表达。5′端缺失分析表明缺失片段PB″α-1545 和PB″α-1389 具有启动子活性, 活性区域位于?1389 bp 和?946 bp 之间。GUS 定量分析结果显示, 在盐和渗透胁迫条件下, PB″α、PB″α-1545 和PB″α-1389 的活性显著上升。本研究表明PB″α 具有较强的启动子基本活性, 并且在盐胁迫及渗透胁迫条件下活性显著上升, 该结果为合理选用启动子改良作物提供了依据。

关键词: 小麦, 蛋白磷酸酶2A, 启动子, 顺式作用元件, GUS 组织化学染色, GUS 定量分析

Abstract:

Protein phosphatase 2A (PP2A) is a heterotrimeric protein, consisting of a scaffolding subunit (A), a catalytic subunit (C), and a member of four families of regulatory subunits (B). PP2A plays significant roles in the pathway responding to abiotic stresses in plants. TaPP2AbB″-α, a member of regulatory subunit B″ in wheat (Triticum aestivum L.), enhanced root development and could develop more lateral roots in the gene overexpressed Arabidopsis, especially under the osmotic stresses of mannitol and NaCl. In order to elucidate transcriptional regulatory mechanism of the promoter PB″α of TaPP2AbB″-α, we isolated an 1899 bp full-length sequence of promoter PB″α from a drought-tolerant wheat cultivar Hanxuan 10. The PB″α sequence contained TATA-box, CAAT-box and a series of cis-acting elements responding to drought and osmotic stresses, such as elements of EECCRCAH1 (from ?1058 to ?1052 bp), GCCCORE (from ?1073 to ?1068 bp) and MYCCONSE (from ?1179 to ?1174 bp). The full-length promoter PB″α and five 5′-end truncated PB″α promoters in different lengths fused with the reporter gene β-glucuronidas (GUS) were transformed into Arabidopsis, respectively. The histochemical staining results showed that the full-length promoter PB″α, and the deletion fragments of PB″α-1545 and PB″α-1389 could drive GUS gene expression in shoots and roots of transgenic Arabidopsis seedlings. As the result of quantitative fluorometric GUS assay, only the expression of PB″α, PB″α-1545 and PB″α-1389 could be up-regulated by salt and osmotic stresses in transgenic Arabidopsis lines, and the active region of PB″α promoter was located in the interval between ?1389 bp and ?946 bp. In conclusion, PB″α has strong basic promoter activity which is up-regulated significantly by salt and osmotic stresses. These findings contribute to the selection of a suitable promoter for crop improvement.

Key words: Wheat, PP2A, Promoter, cis-acting Element, GUS histochemical staining, Quantitative fluorometric GUS assay

[1]Shinozaki K, Yamaguchi-Shinozaki K. Gene networks involved in drought stress response and tolerance. J Exp Bot, 2007, 58: 221–227
[2]Janssens V, Goris J. Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J, 2001, 353: 417–439
[3]Høyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, Bianchi K, Fehrenbacher N, Elling F, Rizzuto R, Mathiasen I S, Jäättelä M. Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2. Mol Cell, 2007, 25: 193–205
[4]Ballesteros I, Dominguez T, Sauer M, Paredes P, Duprat A, Rojo E, Sanmartin M, Sanchez-Serrano J J. Specialized functions of the PP2A subfamily II catalytic subunits PP2A-C3 and PP2A-C4 in the distribution of auxin fluxes and development in Arabidopsis. Plant J, 2013, 73: 862–872
[5]Tang W, Yuan M, Wang R, Yang Y, Wang C, Oses-Prieto J A, Kim T W, Zhou H W, Deng Z, Gampala S S, Gendron J M, Jonassen E M, Lillo C, DeLong A, Burlingame A L, Sun Y, Wang Z Y. PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1. Nat Cell Biol, 2011, 13: 124–131
[6]Michniewicz M, Zago M K, Abas L, Weijers D, Schweighofer A, Meskiene I, Heisler M G, Ohno C, Zhang J, Huang F, Schwab R, Weigel D, Meyerowitz E M, Luschnig C, Offringa R, Friml J. Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux. Cell, 2007, 130: 1044–1056
[7]Chavez-Aviles M N, Andrade-Perez C L, de la Cruz H R. PP2A mediates lateral root development under NaCl-induced osmotic stress throughout auxin redistribution in Arabidopsis thaliana. Plant & Soil, 2013, 368: 591–602
[8]Xu C, Jing R, Mao X, Jia X, Chang X. A wheat (Triticum aestivum) protein phosphatase 2A catalytic subunit gene provides enhanced drought tolerance in tobacco. Ann Bot, 2007, 99: 439–450
[9]Shi Y. Serine/threonine phosphatases: mechanism through structure. Cell, 2009, 193: 468–484
[10]Slupe A M, Merrill R A, Strack S. Determinants for substrate specificity of protein phosphatase 2A. Enzyme Res, 2011, 2011: 398751
[11]Pandey S, Mahato P K, Bhattacharyya S. Metabotropic glu-tamate receptor 1 recycles to the cell surface in protein phosphatase 2A-dependent manner in non-neuronal and neuronal cell lines. J Neurochem, 2014, 131: 602–614
[12]Uhrig R G, Labandera A M, Moorhead G B. Arabidopsis PPP family of serine/threonine protein phosphatases: many targets but few engines. Trends Plant Sci, 2013, 18: 505–513
[13]Farkas I, Dombradi V, Miskei M, Szabados L, Koncz C. Arabidopsis PPP family of serine/threonine phosphatases. Trends Plant Sci, 2007, 12: 169–176
[14]Harris D M, Myrick T L, Rundle S J. The Arabidopsis homolog of yeast TAP42 and mammalian alpha4 binds to the catalytic subunit of protein phosphatase 2A and is induced by chilling. Plant Physiol, 1999, 121: 609–617
[15]Liu D, Li A, Mao X, Jing R. Cloning and characterization of TaPP2AbB"-alpha, a member of the PP2A regulatory subunit in wheat. PLoS One, 2014, 9: e94430
[16]Benfey P N, Chua N H. The Cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science, 1990, 250: 959–966
[17]Estrada-Melo A C, Ma C, Reid M S, Jiang C Z. Overexpression of an ABA biosynthesis gene using a stress-inducible promoter enhances drought resistance in petunia. Hort Res, 2015, 2: 15013
[18]Nakashima K, Jan A, Todaka D, Maruyama K, Goto S, Shi-nozaki K, Yamaguchi-Shinozaki K. Comparative functional analysis of six drought-responsive promoters in transgenic rice. Planta, 2014, 239: 47–60
[19]Li M, Wang X, Cao Y, Liu X, Lin Y, Ou Y, Zhang H, Liu J. Strength comparison between cold-inducible promoters of Arabidopsis cor15a and cor15b genes in potato and tobacco. Plant Physiol Biochem, 2013, 71: 77–86
[20]Hernandez-Garcia C M, Finer J J. A novel cis-acting element in the GmERF3 promoter contributes to inducible gene expres-sion in soybean and tobacco after wounding. Plant Cell Rep, 2015, 35: 1–14
[21]Murray M G, Thompson W F. Rapid isolation of high mo-lecular weight plant DNA. Nucl Acids Res, 1980, 8: 4321–4325
[22]Sieburth L E, Meyerowitz E M. Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell, 1997, 9: 355–365
[23]Vasil I K, Vasil V. Transformation of wheat via particle bombardment. Methods Mol Biol, 2006, 318: 273–283
[24]Bradford M M. A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem, 1976, 72: 248–254
[25]Luo K, Zhang G, Deng W, Luo F, Qiu K, Pei Y. Functional characterization of a cotton late embryogenesis-abundant D113 gene promoter in transgenic tobacco. Plant Cell Rep, 2008, 27: 707–717
[26]Singh H, Sen R, Baltimore D, Sharp P A. A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature, 1986, 319: 154–158
[27]Li J J, Herskowitz I. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science, 1993, 262: 1870–1874
[28]Chakravarthy S, Tuori R P, D’Ascenzo M D, Fobert P R, Despres C, Martin G B. The tomato transcription factor Pti4 regulates defense-related gene expression via GCC box and non-GCC box cis elements. Plant Cell, 2003, 15: 3033–3050
[29]Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-                 Shinozaki K. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell, 2003, 15: 63–78
[30]Kim H J, Kim Y K, Park J Y, Kim J. Light signalling mediated by phytochrome plays an important role in cold-induced gene expression through the C-repeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana. Plant J, 2002, 29: 693–704
[31]Bihmidine S, Lin J, Stone J M, Awada T, Specht J E, Clemente T E. Activity of the Arabidopsis RD29A and RD29B promoter elements in soybean under water stress. Planta, 2013, 237: 55–64
[32]Kovalchuk N, Jia W, Eini O, Morran S, Pyvovarenko T, Fletcher S, Bazanova N, Harris J, Beck-Oldach K, Shavrukov Y, Langridge P, Lopato S. Optimization of TaDREB3 gene expression in transgenic barley using cold-inducible promoters. Plant Biotechnol J, 2013, 11: 659–670
[33]Yi N, Oh S J, Kim Y S, Jang H J, Park S H, Jeong J S, Song S I, Choi Y D, Kim J K. Analysis of the Wsi18, a stress-inducible promoter that is active in the whole grain of transgenic rice. Transgenic Res, 2011, 20: 153–163
[1] 胡文静, 李东升, 裔新, 张春梅, 张勇. 小麦穗部性状和株高的QTL定位及育种标记开发和验证[J]. 作物学报, 2022, 48(6): 1346-1356.
[2] 郭星宇, 刘朋召, 王瑞, 王小利, 李军. 旱地冬小麦产量、氮肥利用率及土壤氮素平衡对降水年型与施氮量的响应[J]. 作物学报, 2022, 48(5): 1262-1272.
[3] 付美玉, 熊宏春, 周春云, 郭会君, 谢永盾, 赵林姝, 古佳玉, 赵世荣, 丁玉萍, 徐延浩, 刘录祥. 小麦矮秆突变体je0098的遗传分析与其矮秆基因定位[J]. 作物学报, 2022, 48(3): 580-589.
[4] 周悦, 赵志华, 张宏宁, 孔佑宾. 大豆紫色酸性磷酸酶基因GmPAP14启动子克隆与功能分析[J]. 作物学报, 2022, 48(3): 590-596.
[5] 冯健超, 许倍铭, 江薛丽, 胡海洲, 马英, 王晨阳, 王永华, 马冬云. 小麦籽粒不同层次酚类物质与抗氧化活性差异及氮肥调控效应[J]. 作物学报, 2022, 48(3): 704-715.
[6] 刘运景, 郑飞娜, 张秀, 初金鹏, 于海涛, 代兴龙, 贺明荣. 宽幅播种对强筋小麦籽粒产量、品质和氮素吸收利用的影响[J]. 作物学报, 2022, 48(3): 716-725.
[7] 马红勃, 刘东涛, 冯国华, 王静, 朱雪成, 张会云, 刘静, 刘立伟, 易媛. 黄淮麦区Fhb1基因的育种应用[J]. 作物学报, 2022, 48(3): 747-758.
[8] 徐龙龙, 殷文, 胡发龙, 范虹, 樊志龙, 赵财, 于爱忠, 柴强. 水氮减量对地膜玉米免耕轮作小麦主要光合生理参数的影响[J]. 作物学报, 2022, 48(2): 437-447.
[9] 王洋洋, 贺利, 任德超, 段剑钊, 胡新, 刘万代, 郭天财, 王永华, 冯伟. 基于主成分-聚类分析的不同水分冬小麦晚霜冻害评价[J]. 作物学报, 2022, 48(2): 448-462.
[10] 陈新宜, 宋宇航, 张孟寒, 李小艳, 李华, 汪月霞, 齐学礼. 干旱对不同品种小麦幼苗的生理生化胁迫以及外源5-氨基乙酰丙酸的缓解作用[J]. 作物学报, 2022, 48(2): 478-487.
[11] 马博闻, 李庆, 蔡剑, 周琴, 黄梅, 戴廷波, 王笑, 姜东. 花前渍水锻炼调控花后小麦耐渍性的生理机制研究[J]. 作物学报, 2022, 48(1): 151-164.
[12] 孟颖, 邢蕾蕾, 曹晓红, 郭光艳, 柴建芳, 秘彩莉. 小麦Ta4CL1基因的克隆及其在促进转基因拟南芥生长和木质素沉积中的功能[J]. 作物学报, 2022, 48(1): 63-75.
[13] 韦一昊, 于美琴, 张晓娇, 王露露, 张志勇, 马新明, 李会强, 王小纯. 小麦谷氨酰胺合成酶基因可变剪接分析[J]. 作物学报, 2022, 48(1): 40-47.
[14] 李玲红, 张哲, 陈永明, 尤明山, 倪中福, 邢界文. 普通小麦颖壳蜡质缺失突变体glossy1的转录组分析[J]. 作物学报, 2022, 48(1): 48-62.
[15] 石磊, 苗利娟, 黄冰艳, 高伟, 张忠信, 齐飞艳, 刘娟, 董文召, 张新友. 花生AhFAD2-1基因启动子及5'-UTR内含子功能验证及其低温胁迫应答[J]. 作物学报, 2021, 47(9): 1703-1711.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2