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作物学报 ›› 2013, Vol. 39 ›› Issue (09): 1602-1611.doi: 10.3724/SP.J.1006.2013.01602

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

林烟草钾离子通道基因NKT6的克隆与表达定位分析

靳义荣1,2,白岩1,2,宋毓峰1,2,张良1,2,董连红1,2,刘朝科3,冯祥国3,胡晓明3,王倩,*,刘好宝1,*   

  1. 1中国农业科学院烟草研究所 / 农业部烟草生物学与加工重点实验室, 青岛266101; 2中国农业科学院研究生院, 北京100081; 3川渝中烟工业有限责任公司, 成都610000
  • 收稿日期:2012-12-31 修回日期:2013-04-22 出版日期:2013-09-12 网络出版日期:2013-07-09
  • 通讯作者: 王倩, E-mail: wangqian2000_zb@163.com, Tel: 0532-88701031; 刘好宝, E-mail: l88702236@163.com, Tel: 0532-88701829
  • 基金资助:

    本研究由中央级公益性科研院所基本科研业务费专项(2012ZL058)和国家自然科学基金青年基金(31201489)资助。

Molecular Cloning and Expression Analysis of Potassium Channel Gene NKT6 in Nicotiana sylvestris

JIN Yi-Rong1,2,BAI Yan1,2,SONG Yu-Feng1,2,ZHANG Liang1,2,DONG Lian-Hong1,2,LIU Chao-Ke3,FENG Xiang-Guo3,HU Xiao-Ming3,WANG Qian1,*,LIU Hao-Bao1,*   

  1. 1 Key Laboratory of Tobacco Biology and Processing, Ministry of Agriculture, Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, China; 2 Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081, China; 3 China Tobacco Chuanyu Industrial Co. Ltd., Chengdu 610000, China
  • Received:2012-12-31 Revised:2013-04-22 Published:2013-09-12 Published online:2013-07-09
  • Contact: 王倩, E-mail: wangqian2000_zb@163.com, Tel: 0532-88701031; 刘好宝, E-mail: l88702236@163.com, Tel: 0532-88701829

摘要:

Shaker家族钾离子通道在植物钾的吸收转运及其他生命过程中发挥重要作用。本研究利用同源克隆的策略从林烟草中获得一个Shaker家族钾离子通道基因,命名为NKT6 (GenBank登录号为KC310448)。该基因cDNA序列全长2 317 bp,编码由681个氨基酸组成的蛋白,该蛋白与Shaker家族其他成员具有较高同源性。NKT6基因组CDS序列共含有11个外显子、10个内含子。系统进化树分析表明,NKT6蛋白是Shaker家族Group II的成员之一。荧光定量PCR分析发现,NKT6的表达量在林烟草的茎和腋芽中最高,在萼片、叶、花中其次,在根中最低。亚细胞定位结果表明,NKT6主要定位于细胞膜和核膜附近的内质网上。干旱与外源ABA胁迫处理下,NKT6的表达量均呈下降趋势。推测NKT6可能在林烟草气孔开放中发挥作用。

关键词: 钾离子通道, 林烟草, NKT6, 克隆表达, 亚细胞定位

Abstract:

Shaker family potassium channels play a crucial role in absorption and transportation of potassium in plants. A new potassium channel gene, designated as NKT6 (GenBank accession number KC310448), was isolated from N. sylvestris using homologous cloning strategy. The full length sequence of NKT6 cDNA was 2137 bp. The deduced protein sequence contained 618 amid acids and exhibited high homology with Shaker family potassium channels. The genomic sequence of NKT6 contained 11 exons and 10 introns. The phylogenetic tree showed that NKT6 belonged to Group II of Shaker family. Real-time quantitative PCR assay indicated that the expression level of NKT6 in stem was the highest at full-bloom stage of Nicotiana sylvestris. Subcellular localization assay showed NKT6 was localized on plasma membrane and endoplasmic reticulum of tobacco epidermal cells. The expression level of NKT6 declined under drought and ABA treatment. These results demonstrated that NKT6 might play a role in stomatal movement.

Key words: Potassium channel, Nicotiana sylvestris, NKT6, Cloing and expression, Subcellular localization

[1]Mäser P, Thomine S, Schroeder J I, Ward J M, Hirschi K, Sze H, Talke I N, Amtmann A, Maathuis F J M, Sanders D, Phylogenetic relationships within cation transporter families of Arabidopsis. Plant Physiol, 2001, 126: 1646–1667



[2]Amtmann A, Blatt M R. Regulation of macronutrient transport. New Phytol, 2008, 181: 35–52



[3]Spalding E P, Hirsch R E, Lewis D R, Qi Z, Sussman M R, Lewis B D. Potassium uptake supporting plant growth in the absence of AKT1 channel activity inhibition by ammonium and stimulation by sodium. J General Physiol, 1999, 113: 909–918



[4]Wang Y(王毅), Wu W-H(武维华). Molecular genetic mechanism of high efficient potassium uptake in plants. Chin Bull Bot (植物学报), 2009, 44(1): 27–36 (in Chinese)



[5]Bezanilla F. The voltage sensor in voltage-dependent ion channels. Physiol Rev, 2000, 80: 555–592



[6]Doyle D A, Cabral J M, Pfuetzner R A, Kuo A, Gulbis J M, Cohen S L, Chait B T, MacKinnon R. The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science, 1998, 280: 69–77



[7]Jan L Y, Jan Y N. Cloned potassium channels from eukaryotes and prokaryotes. Ann Rev Neurosci, 1997, 20: 91–123



[8]Gambale F, Uozumi N. Properties of shaker-type potassium channels in higher plants. J Membrane Bioly, 2006, 210: 1–19



[9]Qu P-Z(曲平治), Liu G-S(刘贯山), Liu H-B(刘好宝), Si C-C(司丛丛), Liu C-K(刘朝科), Hu X-M(胡晓明), Feng X-G(冯祥国), Zhang S-H(张守厚), Zhao J(赵静). Research advances in tobacco potassium ion channel. Chin Tob Sci (中国烟草科学), 2009, 30(2): 74–80 (in Chinese with English abstract)



[10]Sano T, Becker D, Ivashikina N, Wegner L H, Zimmermann U, Roelfsema M R G, Nagata T, Hedrich R. Plant cells must pass a K+ threshold to re-enter the cell cycle. Plant J, 2007, 50: 401–413



[11]Guo Z-K(郭兆奎), Yang Q(杨谦), Yan P-Q(颜培强), Wan X-Q(万秀清). Cloning and homology modeling of a potassium channel gene NKC1 from Nicotiana rustica. Acta Tob Sin (中国烟草学报), 2008, 14(5): 63–68 (in Chinese with English abstract)



[12]Liu H B, Qu P Z, Liu G S, Sun Y H, Wang Q, Jin Y R, Song Y F, Zhao M. Cloning and expression analysis of potassium channel gene NKT3 from Nicotiana tabacum. Afr J Biotechnol, 2012, 11: 10824–10830



[13]Qu P-Z(曲平治), Liu G-S(刘贯山), Liu H-B(刘好宝), Si C-C(司丛丛), Hu X-M(胡晓明), Feng X-G(冯祥国), Liu C-K(刘朝科). Cloning and sequence and expression analysis of potassium channel gene NKT4 in Nicotiana tabacum. J Plant Genet Resourc (植物遗传资源学报), 2009, 10(3): 354–359 (in Chinese with English abstract)



[14]Si C-C(司丛丛), Liu G-S(刘贯山), Liu H-B(刘好宝), Jiao R(焦蓉), Wang S-L(王树林), Qu P-Z(曲平治), Liu C-K(刘朝科), Hu X-M(胡晓明), Feng X-G(冯祥国). Cloning and sequence analysis of potassium ion channel gene NKT5 in Nicotiana tabacum. Chin Tob Sci (中国烟草科学) 2010, 31(4): 8–12 (in Chinese with English abstract)



[15]Goodin M M, Dietzgen R G, Schichnes D, Ruzin S, Jackson A O. pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves. Plant J, 2002, 31: 375–383



[16]Wang Q, Tao T, Zhang Y, Wu W, Li D, Yu J, Han C. Rice black-streaked dwarf virus P6 self-interacts to form punctate, viroplasm-like structures in the cytoplasm and recruits viroplasm-associated protein P9-1. Virol J, 2011, 8: 8–24



[17]Müller-Röbe B, Ellenberg J, Provart N, Willmitzer L, Busch H, Becker D, Dietrich P, Hoth S, Hedrich R. Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells. EMBO J, 1995, 14: 2409–2416



[18]Su Y H, North H, Grignon C, Thibaud J B, Sentenac H, Véry A A. Regulation by external K+ in a maize inward Shaker channel targets transport activity in the high concentration range. Plant Cell, 2005, 17: 1532–1548



[19]Hoffman M. New role found for a common protein. Science, 1991, 253: 742



[20]Pilot G, Pratelli R, Gaymard F, Meyer Y, Sentenac H. Five-group distribution of the Shaker-like K+ channel family in higher  plants. J Mol Evol, 2003, 56: 418–434



[21]Comai L, Henikoff S. TILLING: practical single-nucleotide mutation discovery. Plant J, 2006, 45: 684–694



[22]Hedrich R, Becker D, Geiger D, Marten I, Roelfsema M R G. Role of Ion Channels in Plants. Springer Protocols Handbooks, 2012. 295–322



[23]Pilot G, Gaymard F, Mouline K, Chérel I, Sentenac H. Regulated expression of Arabidopsis Shaker K+ channel genes involved in K+ uptake and distribution in the plant. Plant Mol Biol, 2003, 51: 773–787



[24]Mouline K, Véry A A, Gaymard F, Boucherez J, Pilot G, Devic M, Bouchez D, Thibaud J B, Sentenac H. Pollen tube development and competitive ability are impaired by disruption of a Shaker K+ channel in Arabidopsis. Gene Dev, 2002, 16: 339–350



[25]Mansfield T, Hetherington A, Atkinson C. Some current aspects of stomatal physiology. Annu Rev Plant Biol, 1990, 41: 55–75



[26]Pilot G, Lacombe B, Gaymard F, Chérel I, Boucherez J, Thibaud J B, Sentenac H. Guard cell inward K+ channel activity in Arabidopsis involves expression of the twin channel subunits KAT1 and KAT2. J Biol Chem, 2001, 276: 3215–3221



[27]Kang J Y, Choi H I, Im M Y, Kim S Y. Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling. Plant Cell Online, 2002, 14: 343–357



[28]Xu J, Li H D, Chen L Q, Wang Y, Liu L L, He L, Wu W H. A protein kinase, interacting with two calcineurin B-like proteins, regulates K+ transporter AKT1 in Arabidopsis. Cell, 2006, 125: 1347–1360



[29]Mori I C, Uozumi N, Muto S. Phosphorylation of the inward-rectifying potassium channel KAT1 by ABR kinase in Vicia guard cells. Plant Cell Physiol, 2000, 41: 850–856

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