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作物学报 ›› 2018, Vol. 44 ›› Issue (9): 1347-1356.doi: 10.3724/SP.J.1006.2018.01347

• 研究论文 • 上一篇    下一篇

大豆GmHDL57基因的克隆及抗盐功能鉴定

柯丹霞(),彭昆鹏,张孟珂,贾妍,王净净   

  1. 信阳师范学院生命科学学院 / 大别山农业生物资源保护与利用研究院, 河南信阳 464000
  • 收稿日期:2017-11-03 接受日期:2018-06-12 出版日期:2018-09-10 网络出版日期:2018-06-30
  • 通讯作者: 柯丹霞
  • 基金资助:
    本研究由国家自然科学基金项目(31400213);河南省科技攻关计划项目(182102110448);信阳师范学院青年骨干教师资助计划项目(2015);信阳师范学院“南湖学者奖励计划”青年项目和信阳师范学院研究生科研创新基金资助

Cloning and Salt Resistance Function Identification of GmHDL57 Gene from Glycine max

Dan-Xia KE(),Kun-Peng PENG,Meng-Ke ZHANG,Yan JIA,Jing-Jing WANG   

  1. College of Life Sciences / Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains, Xinyang Normal University, Xinyang 464000, Henan, China
  • Received:2017-11-03 Accepted:2018-06-12 Published:2018-09-10 Published online:2018-06-30
  • Contact: Dan-Xia KE
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31400213);Science and Technology Research Projects of Henan Province(182102110448);Funding Scheme for Young Core Teachers of Xinyang Normal University(2015);Nanhu Scholars Program for Young Scholars of Xinyang Normal University, and the Scientific Research Foundation of Graduate School of Xinyang Normal University

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摘要：

HD-Zip I类转录因子在植物抵御非生物胁迫过程中发挥重要功能, 本研究克隆得到1个大豆HD-Zip I类基因GmHDL57 (Glycine max homeodomain-leucine zipper protein 57)。序列分析表明, GmHDL57基因包含1个1038 bp的开放读码框, 编码345个氨基酸, 具有HD-Zip类家族蛋白典型的保守结构域。基因时空表达分析表明, 大豆GmHDL57基因在大豆植株的各个不同时期及不同器官中均有表达, 在花中表达量最高。采用实时荧光定量PCR技术分析了4种非生物胁迫(脱落酸、NaCl、PEG、冷)对幼苗期大豆根中GmHDL57基因表达的影响。结果表明, 该基因表达量受高盐胁迫诱导显著升高, 在脱落酸及干旱胁迫下上升幅度较小, 但在冷胁迫下呈下降趋势。盐胁迫前后GmHDL57基因在根中的表达量明显高于茎和叶, 在盐胁迫48 h时达到峰值, 72 h和96 h时表达量缓慢下降。此外, 构建GmHDL57基因的植物超表达载体, 利用根癌农杆菌转化法获得转基因百脉根, 200 mmol L -1 NaCl处理条件下, 转基因百脉根的株高、根长、叶绿素含量、根系活力以及阳离子K +、Ca 2+含量显著高于野生型, 而丙二醛含量、相对质膜透性以及Na +的含量明显低于野生型。以上研究结果表明, GmHDL57基因参与了大豆对非生物胁迫的应答过程, 过量表达GmHDL57基因能够显著提高百脉根的抗盐能力。

关键词: 大豆, GmHDL57基因, 非生物胁迫, 抗盐性

Abstract:

The HD-Zip I class transcription factor plays an important role in plant resistance to abiotic stresses. An HD-Zip I class gene GmHDL57 (Glycine max homeodomain-leucine zipper protein 57) was cloned from soybean in this study. Sequence analysis showed that GmHDL57 gene contained a 1038 bp ORF, encoding 345 amino acids, and featured with HD-Zip family proteins’ typical conserved domain. GmHDL57 was expressed in different organs of soybean plants and the highest expression occurred in flowers. The effects of abiotic stresses (abscisic acid, NaCl, PEG, and cold) on GmHDL57 gene expression in soybean seedling stage were analyzed by real-time quantitative PCR. The expression level of GmHDL57 gene was obviously increased under high salinity stress and less affected by ABA and drought stress but decreased by cold stress. The expression level of GmHDL57 gene in roots was significantly higher than that in stems and leaves before and after salt stress, and reached the peak at 48 h, then decreased slowly at 72 h and 96 h after salt stress. The overexpression vector of GmHDL57 was constructed and transformed into Agrobacterium tumefaciens strain EHA105 to obtain the stable transgenic Lotus japonicus plants. After being treated with 200 mmol L -1 NaCl for 14 d, the shoot height, root length, chlorophyll content, root activity as well as K + and Ca 2+ content increased significantly in transgenic plants compared with the wild type. The malondialdehyde content, relative membrane permeability and Na +content were obviously reduced in transgenic plants compared with the wild type. It is hypothesized that the GmHDL57 gene participates in the abiotic stress response of soybean, and over-expression of GmHDL57 gene could enhance resistance to saline in Lotus japonicus.

Key words: Glycine max, GmHDL57 gene, abiotic stress, salt resistance

图1

GmHDL57蛋白质的三级空间结构预测"

图2

大豆GmHDL57与其他植物同源蛋白的保守序列比对分析 Glycine max: 栽培大豆; Glycine soja: 野生大豆; Cajanus cajan: 木豆; Vigna angularis: 赤豆; Phaseolus vulgaris: 菜豆; Vigna radiata: 绿豆; Cicer arietinum: 鹰嘴豆; Lupinus angustifolius: 狭叶羽扇豆; Medicago truncatula: 蒺藜苜蓿; Arachis duranensis: 蔓花生; Arachis ipaensis: 落花生。黑线部分为同源异型框结构域序列, 虚线部分为同源异型框结合类亮氨酸拉链结构域序列。"

图3

大豆GmHDL57与同系物的系统进化分析 Glycine max: 栽培大豆; Glycine soja: 野生大豆; Cajanus cajan: 木豆; Vigna angularis: 赤豆; Phaseolus vulgaris: 菜豆; Vigna radiata: 绿豆; Cicer arietinum: 鹰嘴豆; Lupinus angustifolius: 狭叶羽扇豆; Medicago truncatula: 蒺藜苜蓿; Arachis duranensis: 蔓花生; Arachis ipaensis: 落花生。标尺代表遗传相似性, 表明不同物种间同系物进化关系的远近。"

图4

大豆GmHDL57基因在不同器官中的表达分析横坐标依次为: 幼叶、花、1 cm豆荚、开花后10 d、14 d荚壳、开花后10 d、14 d、21 d、25d、28 d、35 d、42 d种子、根和根瘤; DAF代表开花后的天数。"

图5

大豆GmHDL57基因在非生物胁迫下的表达分析"

图6

盐胁迫下GmHDL57基因在大豆不同组织中的表达特征分析"

图7

转基因百脉根阳性植株检测及抗盐表型鉴定 A: PCR检测植株中GUS基因的表达; B: RT-PCR检测植株中GmHDL57基因的表达; C: 不同盐浓度处理14 d后百脉根的生长状态。Lj 9-5, Lj 1-1: 转基因株系。M: DL2000 DNA marker。"

图8

盐胁迫对转基因百脉根株高(A)和根长(B)的影响 Lj 9-5, Lj 1-1: 转基因株系。*代表差异显著(P<0.05), **代表差异极显著(P<0.01)。"

图9

盐胁迫下转基因百脉根的生理指标 A: 丙二醛含量; B: 相对质膜透性; C: 叶绿素含量; D: 根系活力。Lj 9-5, Lj 1-1: 转基因株系。*代表差异显著(P<0.05), **代表差异极显著(P<0.01)。"

图10

盐胁迫下转基因百脉根的阳离子含量 A: 叶片中Na+含量; B: 根中Na+含量; C: 叶片中K+含量; D: 根中K+含量; E: 叶片中Ca2+含量; F: 根中Ca2+含量。Lj 9-5, Lj 1-1: 转基因株系。*代表差异显著(P<0.05), **代表差异极显著(P<0.01)。"

[1] Ariel F D, Manavella P A, Dezar C A, Chan R L . The true story of the HD-Zip family. Trends Plant Sci, 2007,12:419-426
[2] Mukherjee K, Brocchieri L, Burglin T R . A comprehensive classification and evolutionary analysis of plant homeobox genes. Mol Biol Evol, 2009,26:2775-2794
doi: 10.1093/molbev/msp201 pmid: 2775110
[3] Harris J C, Hrmova M, Lopato S, Langridge P . Modulation of plant growth by HD-Zip class I and II transcription factors in response to environmental stimuli. New Phytol, 2011,190:823-837
[4] Henriksson E ,Olsson A S B, Johannesson H, Johansson H, Hanson J, Engström P, Söderman E. , Homeodomain leucine zipper class I genes in Arabidopsis expression patterns and phylogenetic relationships. Plant Physiol, 2005,139:509-518
[5] Olsson A S B, Engstrom P, Soderman E . The homeobox genes ATHB12 and ATHB7 encode potential regulators of growth in response to water deficit in Arabidopsis. Plant Mol Biol, 2004,55:663-677
[6] Himmelbach A, Hoffmann T, Leube M, Höhener B, Grill E . Homeodomain protein ATHB6 is a target of the protein phosphatase ABI1 and regulates hormone responses in Arabidopsis. EMBO J, 2002,21:3029-3038
[7] Ré D A, Dezar C A, Chan R L, Baldwin I T, Bonaventure G . Nicotiana attenuata NaHD20 plays a role in leaf ABA accumulation during water stress, benzylacetone emission from flowers, and the timing of bolting and flower transitions. J Exp Bot, 2011,62:155-166
[8] Ariel F, Diet A, Verdenaud M, Gruber V, Frugier F, Chan R, Crespi M . Environmental regulation of lateral root emergence in Medicago truncatula requires the HD-Zip I transcription factor HB1. Plant Cell, 2010,22:2171-2183
[9] 李明娜, 龙瑞才, 杨青川, 沈益新, 康俊梅, 张铁军 . 紫花苜蓿盐诱导HD-Zip类转录因子MsHB2的克隆及功能分析. 中国农业科学, 2014,47:622-632
Li M N, Long R C, Yang Q C, Shen Y X, Kang J M, Zhang T J . Cloning and function analysis of a salt-stress-induced HD-Zip transcription factor MsHB2 from alfalfa.. Sci Agric Sin, 2014,47:622-632 (in Chinese with English abstract)
[10] Cao L, Yu Y, Duanmu H Z, Chen C, Duan X B, Zhu P H, Chen R R, Li Q, Zhu Y M, Ding X D . A novel Glycine soja homeodomain-leucine zipper (HD-Zip) I gene, Gshdz4, positively regulates bicarbonate tolerance and responds to osmotic stress in Arabidopsis. BMC Plant Biol, 2016,16:184
[11] Chen X, Chen Z, Zhao H, Zhao Y, Cheng B, Xiang Y . Genome-wide analysis of soybean HD-zip gene family and expression profiling under salinity and drought treatments. PLoS One, 2014,9:e87156
[12] Belamkar V, Weeks N T, Bharti A K, Farmer A D, Graham M A, Cannon S B . Comprehensive characterization and RNA-Seq profiling of the HD-Zip transcription factor family in soybean ( Glycine max) during dehydration and salt stress. BMC Genomics, 2014,15:950
[13] Wang Y J, Li Y D, Luo G Z, Tian A G, Wang H W, Zhang J S, Chen S Y . Cloning and characterization of an HD-Zip I gene GmHZ1 from soybean. Planta, 2005,221:831-843
doi: 10.1007/s00425-005-1496-6
[14] 柯丹霞, 李祥永, 王磊, 程琳, 刘永辉, 李小艳, 王慧芳 . 大豆GmHAT5的克隆及其转基因百脉根的抗盐分析. 中国农业科学, 2017,50:1559-1570
Ke D X, Li X Y, Wang L, Cheng L, Liu Y H, Li X Y, Wang H F . Isolation of GmHAT5 from Glycine max and analysis of saline tolerance for transgenic Lotus japonicus.. Sci Agric Sin, 2017,50:1559-1570 (in Chinese with English abstract)
[15] 柯丹霞, 李祥永 . 结瘤信号途径中相关调控蛋白的研究进展. 信阳师范学院学报(自然科学版), 2015,28:621-626
Ke D X, Li X Y . Research progress of key regulatory proteins in nodulation pathway. J Xinyang Nor Univ (Nat Sci Edn), 2015,28:621-626 (in Chinese with English abstract)
[16] Marquez A J . Lotus japonicus Handbook. The Netherlands: Springer, 2005. pp 279-284
[17] Agalou A, Purwantomo S, Overnas E, Johannesson H, Zhu X Y ,Estiati A, de Kam R J, Engström P, Slamet-Loedin I H, Zhu Z, Wang M, Xiong L Z, Meijer A H, Ouwerkerk P B. , A genome-wide survey of HD-Zip genes in rice and analysis of drought-responsive family members. Plant Mol Biol, 2008,66:87-103
[18] Zhang S X, Haider I, Kohlen W, Jiang L, Bouwmeester H, Meijer A H, Schluepmann H, Liu C M, Ouwerkerk P B . Function of the HD-Zip I gene Oshox22 in ABA-mediated drought and salt tolerances in rice. Plant Mol Biol, 2012,80:571-585
doi: 10.1007/s11103-012-9967-1 pmid: 23109182
[19] Zhao Y, Zhou Y Q, Jiang H Y, Li X Y, Gan D F, Peng X J, Zhu S W, Cheng B J . Systematic analysis of sequences and expression patterns of drought-responsive members of the HD-Zip gene family in maize. PLoS One, 2011,6:e28488
[20] Hu R B, Chi X Y, Chai G H, Kong Y Z, He G, Wang X Y, Shi D C, Zhang D Y, Zhou G K . Genome-wide identification, evolutionary expansion, and expression profile of homeodomain- leucine zipper gene family in poplar (Populus trichocarpa). PLoS One, 2012,7:e31149
[21] Liu W, Fu R, Li Q, Li J, Wang L N, Ren Z H . Genome-wide identification and expression profile of homeodomain-leucine zipper class I gene family in Cucumis sativus. Gene, 2013,531:279-287
[22] Fu R, Liu W, Li Q, Li J, Wang L N, Ren Z H . Comprehensive analysis of the homeodomain-leucine zipper IV transcription factor family in Cucumis sativus. Genome, 2013,56:395-405
[23] Elhiti M, Stasolla C . Structure and function of homeodomain-leucine zipper (HD-Zip) proteins. Plant Signal Behav, 2009,4:86-88
doi: 10.4161/psb.4.2.7692 pmid: 2637487
[24] Sahu B B, Shaw B P . Isolation, identification and expression analysis of salt-induced genes in Suaeda maritime, a natural halophyte, using PCR-based suppression subtractive hybridization. BMC Plant Biol, 2009,9:69
[25] 王臻昱, 才华, 柏锡, 纪巍, 李勇, 魏正巍, 朱延明 . 野生大豆GsGST19基因的克隆及其转基因苜蓿的耐盐碱性分析. 作物学报, 2013,38:971-979
Wang Z Y, Cai H, Bai X, Ji W, Li Y, Wei Z W, Zhu Y M . Isolation of GsGST19 from Glycine soja and analysis of saline- alkaline tolerance for transgenic Medicago sativa.. Acta Agron Sin, 2013,38:971-979 (in Chinese with English abstract)
[26] 魏正巍, 朱延明, 化烨, 才华, 纪巍, 柏锡, 王臻昱, 文益东 . 转GsPPCK1基因苜蓿植株的获得及其耐碱性分析. 作物学报, 2013,39:68-75
Wei Z W, Zhu Y M, Hua Y, Cai H, Ji W, Bai X, Wang Z Y, Wen Y D . Transgenic alfalfa with GsPPCK1 and its alkaline tolerance analysis.. Acta Agron Sin, 2013,39:68-75 (in Chinese with English abstract)
[27] 赵阳, 朱延明, 柏锡, 纪巍, 吴婧, 唐立郦, 才华 . 转GsCBRLK/SCMRP双价基因苜蓿耐碱性及氨基酸含量分析. 作物学报, 2014,40:431-438
Zhao Y, Zhu Y M, Bai X, Ji W, Wu J, Tang L L, Cai H . Over-expressing GsCBRLK/SCMRP enhances alkaline tolerance and methionine content in transgenic Medicago sativa. . Acta Agron Sin, 2014,40:431-438 (in Chinese with English abstract)
[28] Yang T, Poovaiah B W . Hydrogen peroxide homeostasis: activation of plant catalase by calcium/calmodulin. Proc Nat Acad Sci USA, 2002,99:4097-4102
doi: 10.1073/pnas.052564899 pmid: 122654
[29] 朱娉慧, 陈冉冉, 于洋, 宋雪薇, 李慧卿, 杜建英, 李强, 丁晓东, 朱延明 . 碱胁迫相关基因GsWRKY15的克隆及其转基因苜蓿的耐碱性分析. 作物学报, 2017,43:1319-1327
Zhu P H, Chen R R, Yu Y, Song X W, Li H Q, Du J Y, Li Q, Ding X D, Zhu Y M . Cloning of gene GsWRKY15 related to alkaline stress and alkaline tolerance of transgenic plants.. Acta Agron Sin, 2017,43:1319-1327 (in Chinese with English abstract)
[30] Olhoft P M, Flagel L E, Donovan C M . Efficient soybean transformation using hygromycin B selection in the cotyledonary-node method. Planta, 2003,216:723-735
doi: 10.1007/s00425-002-0922-2 pmid: 12624759
[31] 王昌陵, 赵军, 李英慧, 范云六, 张丽娟, 刘章雄, 关荣霞, 吕淑霞, 常汝镇, 邱丽娟 . 转录因子ABP9 转化大豆(Glycine max L.)及遗传转化条件优化. 中国农业科学, 2008,41:1908-1916
Wang C L, Zhao J, Li Y H, Fan Y L, Zhang L J, Liu Z X, Guan R X, Lyu S X, Chang R Z, Qiu L J . Transforming transcription factor ABP9 into Soybean and optimization of the transformation system. Sci Agric Sin, 2008,41:1908-1916 (in Chinese with English abstract)
[32] Devi M K, Sakthivela G, Giridhar P . Protocol for augmented shoot organogenesis in selected variety of soybean. J Exp Biol, 2012,50:729-734
[33] 杨权, 王月月, 刘炎光, 蒋春志, 张孟臣, 张洪霞, 张洁, 王冬梅 . 大豆子叶节遗传转化体系优化及抗逆基因AtNHX5的转化研究. 大豆科学, 2015,34:205-211
Yang Q, Wang Y Y, Liu Y G, Jiang C Z, Zhang M C, Zhang H X, Zhang J, Wang D M . Study on optimization of soybean cotyledonary node genetic transformation system and the transformation of resistance gene AtNHX5.. Soybean Sci, 2015,34:205-211 (in Chinese with English abstract)
[34] 柯丹霞, 熊文真, 彭昆鹏, 李祥永 . 抗盐基因Gm01g04890大豆子叶节遗传转化研究. 信阳师范学院学报(自然科学版), 2017,30(1):46-51
Ke D X, Xiong W Z, Peng K P, Li X Y . Study on genetic transformation of salt resistant gene Gm01g04890 in soybean. J Xinyang Nor Univ(Nat Sci Edn), 2017,30(1):46-51 (in Chinese with English abstract)
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