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作物学报 ›› 2018, Vol. 44 ›› Issue (02): 236-244.doi: 10.3724/SP.J.1006.2018.00236

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陆地棉钾转运体基因GhHAK5的序列特征及表达分析

晁毛妮1, 温青玉2, 张志勇1, 胡根海1, 张金宝1, 王果1, 王清连1,*()   

  1. 1河南科技学院 / 现代生物育种河南省协同创新中心 / 河南省棉麦分子生态与种质创新重点实验室, 河南新乡 453003
    2河南省农业科学院, 河南郑州 450002
  • 收稿日期:2017-04-25 接受日期:2017-09-10 出版日期:2018-02-12 网络出版日期:2017-10-27
  • 通讯作者: 王清连
  • 基金资助:
    本研究由国家自然科学基金项目(31601347, 31571600), 河南省科技攻关计划项目(172102110022), 河南省高等学校重点科研计划项目(15A210001)和河南科技学院高层次人才计划项目(2014004)资助

Sequence Characteristics and Expression Analysis of Potassium Transporter Gene GhHAK5 in Upland Cotton (Gossypium hirsutum L.)

Mao-Ni CHAO1, Qing-Yu WEN2, Zhi-Yong ZHANG1, Gen-Hai HU1, Jin-Bao ZHANG1, Guo WANG1, Qing-Lian WANG1,*()   

  1. 1 Henan Institute of Science and Technology / Henan Collaborative Innovation Center of Modern Biological Breeding / Henan Key Laboratory for Molecular Ecology and Germplasm Innovation of Cotton and Wheat, Xinxiang 453003, Henan, China
    2 Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
  • Received:2017-04-25 Accepted:2017-09-10 Published:2018-02-12 Published online:2017-10-27
  • Contact: Qing-Lian WANG
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31601347, 31571600), the Henan Scientific and Technological Research Program (172102110022), Key Research Projects of Henan Higher Education Institutions (15A210001), and High Level Talent Program of Henan Institute of Science and Technology (2014004).

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摘要：

KUP/HAK/KT钾转运体基因家族对植物吸收钾离子发挥重要作用, 鉴定和克隆棉花的钾转运体基因, 对于改良棉花的钾吸收特性, 提高棉花的产量和品质具有重要意义。基于已测序的陆地棉基因组序列, 本研究通过同源克隆的方法鉴定到陆地棉钾转运体基因GhHAK5, 并以陆地棉品种百棉1号为材料对其CDS序列进行扩增。结果表明, GhHAK5基因的CDS全长为2451 bp, 编码816个氨基酸, 分子量和等电点分别为91.23 kD和8.15。GhHAK5蛋白具有KUP/HAK/KT家族基因的保守结构域“K-trans”(Pfam02705)和标志性序列GXXXGDXXXSPLY, 并具有11个跨膜区。在进化上, GhHAK5蛋白与拟南芥AtHAK5亲缘关系最近, 其次是与水稻的OsHAK5, 它们同属Cluster I进化簇。亚细胞定位结果显示, GhHAK5是一个定位于质膜的蛋白, 这与其主要作为钾转运子参与K+吸收的功能是一致的。GhHAK5基因在根中表达量最高, 在茎、叶、花瓣、纤维和花萼中表达量很低, 且其表达受外界低钾环境诱导。本研究结果为进一步了解GhHAK5基因的功能及培育钾高效棉花品种奠定了基础。

关键词: 陆地棉, 钾转运体基因, 亚细胞定位, 低钾, 基因表达

Abstract:

KUP/HAK/KT potassium transporter gene family plays an important role in the absorption of K+ in plants. Identification and cloning of potassium transporter genes in cotton are helpful to improve potassium absorption, and facilitate the increase of yield and quality in cotton. Based on the genome sequence of upland cotton, we identified the potassium transporter gene GhHAK5 by homologous cloning, and amplified its CDS sequence from upland cotton variety Baimian 1. The CDS of GhHAK5 was 2451 bp, encoding a protein with 816 amino acids. The molecular weight and isoelectric point of this protein were 91.23 kD and 8.15, respectively. The GhHAK5 protein contained a conserved domain “K-trans” (Pfam02705) and a symbolic amino acid sequence GXXXGDXXXSPLY of KUP/HAK/KT family gene, and had 11 transmembrane domains. Phylogenetic tree analysis demonstrated that GhHAK5 had a close relationship with AtHAK5, followed by OsHAK5, and they all belong to Cluster I evolutionary cluster. The subcellular localization experiment indicated that GhHAK5 was located in plasma membrane, which is consistent with its function as a potassium transporter involved in K+ uptake. The expression of GhHAK5 was high in roots, but low in leaf, stem, petal, fiber and sepal, and induced by low potassium environment. These results provide a foundation for understanding the function of GhHAK5 and breeding varieties with high potassium efficiency in cotton.

Key words: upland cotton, potassium transporter gene, subcellular localization, low potassium, gene expression

图1

陆地棉GhHAK5蛋白的序列分析 A: GhHAK5的保守域分析; B: GhHAK5中含有的钾转运体标志性序列(用横线表出); C: GhHAK5的跨膜区预测。"

图2

陆地棉GhHAK5蛋白与拟南芥和水稻KUP/HAK/KT家族成员的进化关系分析空心和实心分别代表水稻和拟南芥KUP/HAK/KT家族成员。"

图3

陆地棉GhHAK5蛋白的亚细胞定位 a和d: 荧光下照片; b和e: 明场下照片; c和f: 荧光和明场叠加图。"

图4

陆地棉GhHAK5基因表达水平 A: GhHAK5在陆地棉不同组织中的表达分析(误差线代表3个独立重复的标准差); B: 低钾处理下GhHAK5在根中的表达分析(CK: normal potassium; LK: low potassium; **: P < 0.01; 误差线代表3个独立重复的标准差)。"

[1] Véry A A, Sentenac H.Molecular mechanisms and regulation of K+ transport in higher plants.Annu Rev Plant Biol, 2003, 54: 575-603
[2] Grabov A.Plant KT/KUP/HAK potassium transporters: single family-multiple functions.Ann Bot, 2007, 99: 1035-1041
[3] 张炎, 李宁, 胡伟, 高媛, 姚银坤, 汤明尧. 钾肥用量对棉花蕾铃脱落及产量品质的影响. 新疆农业科学, 2012, 11: 2088-2095
Zhang Y, Li N, Hu W, Gao Y, Yao Y K, Tang M Y.Bud & boll falling, yield and quality of cotton affected by rates of potassium.Xinjiang Agric Sci, 2012, 11: 2088-2095 (in Chinese with English abstract)
[4] Mian A, Oomen R J, Isayenkov S, Sentenac H, Maathuis F J, Véry A A.Over-expression of an Na+- and K+-permeable HKT transporter in barley improves salt tolerance.Plant J, 2011, 68: 468-479
[5] 高祥照, 马文奇. 我国耕地土壤养分变化与肥料投入状况. 植物营养与肥料学报, 2000, 6: 363-369
Gao X Z, Ma W Q.Changes of soil nutrient contents and input of nutrientsin arable of China.Plant Nutr Fert Sci, 2000, 6: 363-369 (in Chinese with English abstract)
[6] 张志勇, 王清连, 李召虎, 段留生, 田晓莉. 缺钾对棉花幼苗根系生长的影响及其生理机制. 作物学报, 2009, 35: 718-723
Zhang Z Y, Wang Q L, Li Z H, Duan L S, Tian X L.Effect of potassium deficiency on root growth of cotton (Gossypium hirsutum L.) seedlings and its physiological mechanisms involved. Acta Agron Sin, 2009, 35: 718-723 (in Chinese with English abstract)
[7] 孔祥强, 罗振, 李存东, 董合忠. 棉花早衰的分子机理研究进展. 棉花学报, 2015, 27: 71-79
Kong X Q, Luo Z, Li C D, Dong H Z.Molecular mechanisms of premature senescence in cotton.Cott Sci, 2015, 27: 71-79 (in Chinese with English abstract)
[8] 刘冬青, 刘锐. 转基因抗虫棉早衰与土壤肥力的相关性分析. 土壤肥料, 2002, (6): 41-42
Liu D Q, Liu R.Correlation analysis between soil fertility and premature senescence of transgenic cotton.Soils & Fert, 2002, (6): 41-42 (in Chinese with English abstract)
[9] Pettigrew W, Meredith Jr W.Dry matter production, nutrient uptake, and growth of cotton as affected by potassium fertilization.J Plant Nutr, 1997, 20: 531-548
[10] 李书田, 邢素丽, 张炎, 崔荣宗. 钾肥用量和施用时期对棉花产量品质和棉田钾素平衡的影响. 植物营养与肥料学报, 2016, 22: 111-121
Li S T, Xing S L, Zhang Y, Cui R Z.Application rate and time of potash for high cotton yield, quality and balance of soil potassium.Plant Nutr Fert Sci, 2016, 22: 111-121 (in Chinese with English abstract)
[11] 宋美珍, 毛树春, 邢金松, 杨惠元. 钾素对棉花光合产物的积累及产量形成的影响. 棉花学报, 1994, 6(增刊): 52-57
Song M Z, Mao S C, Xing J S, Yang H Y.Effects of potassium on photosynthetic matter accumulation and yield.Acta Gossipii Sin, 1994, 6(suppl): 52-57 (in Chinese with English abstract)
[12] Ahn S J, Shin R, Schachtman D P.Expression ofKT/KUP genes in Arabidopsis and the role of root hairs in K+ uptake. Plant Physiol, 2004, 134: 1135-1145
[13] Véry A A, Nieves-Cordones M, Daly M, Khan I, Fizames C, Sentenac H.Molecular biology of K+ transport across the plant cell membrane: what do we learn from comparison between plant species?J Plant Physiol, 2014, 171: 748-769
[14] Mäser P, Thomine S, Schroeder J I, Ward J M, Hirschi K, Sze H, Talke I N, Amtmann A, Maathuis F J, Sanders D, Harper J F, Tchieu J, Gribskov M, Persans M W, Salt D E, Kim S A, Guerinot M L.Phylogenetic relationships within cation transporter families ofArabidopsis. Plant Physiol, 2001, 126: 1646-1667
[15] Yang Z, Gao Q, Sun C, Li W, Gu S, Xu C.Molecular evolution and functional divergence of HAK potassium transporter gene family in rice (Oryza sativa L.). J Genet Genomics, 2009, 36: 161-172
[16] Zhang Z, Zhang J, Chen Y, Li R, Wang H, Wei J.Genome-wide analysis and identification of HAK potassium transporter gene family in maize (Zea mays L.). Mol Biol Rep, 2012, 39: 8465-8473
[17] 晁毛妮, 温青玉, 张晋玉, 张志勇, 董洁, 于亚鑫. 大豆KUP/HAK/KT钾转运体基因家族的鉴定与表达分析. 西北植物学报, 2017, 37: 239-249
Chao M N, Wen Q Y, Zhang J Y, Zhang Z Y, Dong J, Yu Y X.Identification and expression analysis of KUP/HAK/KT potassium transporter gene family in soybean [Glycine max (>L.) Merr.]. Acta Bot Boreal-Occident Sin, 2017, 37: 239-249 (in Chinese with Englishabstract)
[18] Shin R, Schachtman D P.Hydrogen peroxide mediates plant root cell response to nutrient deprivation.Proc Natl Acad Sci USA, 2004, 101: 8827-8832
[19] Gierth M, Mäser P, Schroeder J I.The potassium transporter AtHAK5 functions in K+ deprivation-induced high-affinity K+ uptake and AKT1 K+ channel contribution to K+ uptake kinetics in Arabidopsis roots. Plant Physiol, 2005, 137: 1105-1114
[20] Zhao S, Zhang M L, Ma T L, Wang Y.Phosphorylation ofARF2 relieves its repression of transcription of the K+ transporter gene HAK5 in response to low potassium stress. Plant Cell, 2016, 28: 3005-3019
[21] Wang Y H, Garvin D F, Kochian L V.Rapid induction of regulatory and transporter genes in response to phosphorus, potassium, and iron deficiencies in tomato roots. Evidence for cross talk and root/rhizosphere-mediated signals.Plant Physiol, 2002, 130: 1361-1370
[22] Santa-María G E, Rubio F, Dubcovsky J, Rodríguez-Navarro A. The HAK1 gene of barley is a member of a large gene family and encodes a high-affinity potassium transporter. Plant Cell, 1997, 9: 2281-2289
[23] 徐娟. 棉花钾离子通道基因GhAKT1和转运体基因GhKT2的克隆及功能分析. 中国农业大学博士学位论文, 北京, 2014
Xu J.Cloning and Functional Characterization of Potassium Channel Gene GhAKT1 and Potassium Transporter Gene GhKT2 from Cotton (Gossypium hirsutum L.). PhD Dissertation of China Agricultural University, Beijing, China, 2014 (in Chinese with English abstract)
[24] 穆春, 安静, 李婷婷, 王逸茹, 张明才, 田晓莉. 应用病毒诱导基因沉默技术研究棉花GhAKT1和GhKT2基因的功能. 棉花学报, 2017, 29: 40-49
Mu C, An J, Li T T, Wang Y R, Zhang M C, Tian X L.Functional characterization ofGhAKT1 and GhKT2 in cotton(Gossypium hirsutum L.) using virus-induced gene silencing. Cott Sci, 2017, 29: 40-49 (in Chinese with English abstract)
[25] 郭兆奎, 杨谦, 姚泉洪, 万秀清, 颜培强. 转拟南芥AtKup1基因高含钾量烟草获得. 中国生物工程杂志, 2005, 25: 24-28
Guo Z K, Yang Q, Yao Q H, Wan X Q, Yan P Q.Transgenic tobacco withArabidopsis thaliana AtKup1 gene has high potassium content in leaves. J Chin Biotechnol, 2005, 25: 24-28 (in Chinese with English abstract)
[26] Zhang T, Hu Y, Jiang W, Fang L, Guan X, Chen J, Zhang J, Saski C A, Scheffler B E, Stelly D M, Hulse-Kemp A M, Wan Q, Liu B, Liu C, Wang S, Pan M, Wang Y, Wang D, Ye W, Chang L, Zhang W, Song Q, Kirkbride R C, Chen X, Dennis E, Llewellyn D J, Peterson D G, Thaxton P, Jones D C, Wang Q, Xu X, Zhang H, Wu H, Zhou L, Mei G, Chen S, Tian Y, Xiang D, Li X, Ding J, Zuo Q, Tao L, Liu Y, Li J, Lin Y, Hui Y, Cao Z, Cai C, Zhu X, Jiang Z, Zhou B, Guo W, Li R, Chen Z J. Sequencing of allotetraploid cotton (Gossypium hirsutum L. acc. TM-1) provides a resource for fiber improvement. Nat Biotechnol, 2015, 33: 531-537
[27] Zhang Z, Chao M, Wang S, Bu J, Tang J, Li F, Wang Q, Zhang B.Proteome quantification of cotton xylem sap suggests the mechanisms of potassium-deficiency-induced changes in plant resistance to environmental stresses.Sci Rep, 2016, 6: 21060
[28] Kumar S, Nei M, Dudley J, Tamura K.MEGA: a biologist- centric software for evolutionary analysis of DNA and protein sequences.Brief Bioinform, 2008, 9: 299-306
[29] Engler C, Kandzia R, Marillonnet S.A one pot, one step, precision cloning method with high throughput capability.PLoS One, 2008, 3: e3647
[30] Chen S, Tao L, Zeng L, Vega-Sanchez M E, Umemura K, Wang G L. A highly efficient transient protoplast system for analyzing defence gene expression and protein-protein interactions in rice.Mol Plant Pathol, 2006, 7: 417-427
[31] 宋毓峰, 张良, 董连红, 靳义荣, 史素娟, 白岩, 刘朝科, 冯广林, 冯祥国, 王倩, 刘好宝. 植物KUP/HAK/KT家族钾转运体研究进展. 中国农业科技导报, 2013, 15: 92-98
Song Y F, Zhang L, Dong L H, Jin Y R, Shi S J, Bai Y, Liu C K, Feng G L, Feng X G, Wang Q, Liu H B.Research progress on KUP/HAK/KT potassium transporter family in plant.J Agric Sci Tech China, 2013, 15: 92-98 (in Chinese with English abstract)
[32] Ashley M K, Grant M, Grabov A.Plant responses to potassium deficiencies: a role for potassium transport proteins.J Exp Bot, 2006, 57: 425-436
[33] Rodrı́guez-Navarro A. Potassium transport in fungi and plants.Biochim Biophys Acta, 2000, 1469: 1-30
[34] Rubio F, Santamaría G E, Rodrígueznavarro A.Cloning ofArabidopsis and barley cDNAs encoding HAK potassium transporters in root and shoot cells. Physiol Plant, 2000, 109: 34-43
[35] Véry A A, Sentenac H.Molecular mechanisms and regulation of K+ transport in higher plants.Annu Rev Plant Biol, 2003, 54: 575-603
[36] Sato Y, Nanatani K, Hamamoto S, Shimizu M, Takahashi M, Tabuchi-Kobayashi M, Mizutani A, Schroeder J I, Souma S, Uozumi N.Defining membrane spanning domains and crucial membrane-localized acidic amino acid residues for K+ transport of a Kup/HAK/KT-typeEscherichia coli potassium transporter. J Biochem, 2014, 155: 315-323
[37] Kim E J, Kwak J M, Uozumi N, Schroeder J I. AtKUP1: an Arabidopsis gene encoding high-affinity potassium transport activity. Plant Cell, 1998, 10:51-62
[38] Maathuis F J.The role of monovalent cation transporters in plant responses to salinity.J Exp Bot, 2006, 57: 1137-1147
[39] Takahashi R, Nishio T, Ichizen N, Takano T.High-affinity K+ transporterPhaHAK5 is expressed only in salt-sensitive reed plants and shows Na+ permeability under NaCl stress. Plant Cell Rep, 2007, 26: 1673-1679
[40] Grossman A, Takahashi H.Macronutrient utilization by photosynthetic eukaryotes and the fabric of interactions.Annu Rev Plant Physiol Plant Mol Biol, 2001, 52: 163-210
[41] Coello P, Polacco J C.ARR6, a response regulator from Arabidopsis, is differentially regulated by plant nutritional status. Plant Sci, 1999, 143: 211-220
[42] 晁毛妮, 张志勇, 宋海娜, 李成奇, 张新, 胡根海, 张金宝, 王清连. 陆地棉Pht1家族成员的全基因组鉴定及表达分析. 棉花学报, 2017, 29: 59-69
Chao M N, Zhang Z Y, Song H N, Li C Q, Zhang X, Hu G H, Zhang J B, Wang Q L.Genome-wide identification and expression analysis of Pht1 family genes in cotton (Gossypium hirsutum L.). Cott Sci, 2017, 29: 59-69 (in Chinese with English abstract)
[43] 韩璐. 棉花氮高效基因型筛选及相关基因的克隆. 石河子大学硕士学位论文, 新疆石河子, 2011
Han L. Nitrogen Efficiency of Genotype Selection and Related Gene Cloning in Cotton. MS Thesis of Shihezi University, 2011, Shihezi, China (in Chinese with English abstract)
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