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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).

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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
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