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胡杨miR1444b在拟南芥中正调控植物抗旱性

李双 苏艳艳 王厚领 李惠广 刘超 夏新莉 尹伟伦

李双, 苏艳艳, 王厚领, 李惠广, 刘超, 夏新莉, 尹伟伦. 胡杨miR1444b在拟南芥中正调控植物抗旱性[J]. 北京林业大学学报, 2018, 40(4): 1-9. doi: 10.13332/j.1000-1522.20180043
引用本文: 李双, 苏艳艳, 王厚领, 李惠广, 刘超, 夏新莉, 尹伟伦. 胡杨miR1444b在拟南芥中正调控植物抗旱性[J]. 北京林业大学学报, 2018, 40(4): 1-9. doi: 10.13332/j.1000-1522.20180043
Li Shuang, Su Yanyan, Wang Houling, Li Huiguang, Liu Chao, Xia Xinli, Yin Weilun. Populus euphratica miR1444b positively regulates plants response to drought stress in Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2018, 40(4): 1-9. doi: 10.13332/j.1000-1522.20180043
Citation: Li Shuang, Su Yanyan, Wang Houling, Li Huiguang, Liu Chao, Xia Xinli, Yin Weilun. Populus euphratica miR1444b positively regulates plants response to drought stress in Arabidopsis thaliana[J]. Journal of Beijing Forestry University, 2018, 40(4): 1-9. doi: 10.13332/j.1000-1522.20180043

胡杨miR1444b在拟南芥中正调控植物抗旱性

doi: 10.13332/j.1000-1522.20180043
基金项目: 

国家自然科学基金项目 31600484

转基因生物新品种培育重大专项 2018ZX08021001

国家自然科学基金项目 31570308

国家自然科学基金项目 31770649

详细信息
    作者简介:

    李双。主要研究方向:植物抗逆分子生物学。Email:shuang_LI@bjfu.edu.cn 地址:100083北京市海淀区清华东路35号北京林业大学生物科学与技术学院

    责任作者:

    夏新莉,教授,博士生导师。主要研究方向:植物抗逆分子生物学。Email:xiaxl@bjfu.edu.cn 地址:同上

    尹伟伦,教授,博士生导师。主要研究方向:植物生理与生物技术。Email:yinwl@bjfu.edu.cn 地址:同上

  • 中图分类号: S718.43;S792.119;Q943.2

Populus euphratica miR1444b positively regulates plants response to drought stress in Arabidopsis thaliana

  • 摘要: 目的miR1444b是木本植物特异miR1444家族中的一员,能够参与植物对金属胁迫的响应,但在干旱胁迫响应中的功能尚不明确。方法本实验克隆得到了胡杨MIR1444b基因(peu-MIR1444b)及其启动子(pro-peu-MIR1444b),将peu-MIR1444b基因在模式植株拟南芥中过表达,对转基因植株进行正常浇水、甘露醇模拟干旱和自然干旱条件处理,研究peu-MIR1444b的功能。结果克隆得到的pro-peu-MIR1444bpeu-MIR1444b与Phytozome数据库收录的毛果杨基因组序列相似性分别为99.61%和98.82%。利用PlantCARE数据库分析发现,pro-peu-MIR1444b序列中包含逆境相关的TC-rich repeats元件和受干旱诱导的MYB结合位点MBS。实时荧光定量PCR分析显示miR1444b在转基因拟南芥中表达量显著高于野生型,预测的靶基因葡聚糖合成酶Ⅳ(AT3G14570.2)表达量明显下调,初步确定葡聚糖合成酶Ⅳ在拟南芥中被peu-miR1444b负调控。在250 mmol/L甘露醇模拟的干旱胁迫条件下,转基因拟南芥株系TG-2和TG-5萌发率分别显著高于野生型20.83%和26.67%,根长分别显著高于野生型56.83%和52.60%(P < 0.05)。土壤自然干旱8 d测得转基因拟南芥的光合速率、气孔导度、叶片水分利用效率、PSⅡ最大光化学效率和过氧化物酶(POD)活性均显著高于野生型(P < 0.05)。结论胡杨miR1444b可以促进根系生长增加植物的吸水能力,负调控葡聚糖合成酶Ⅳ提高植物的渗透调节能力,维持植物在水分亏缺条件下的光合效率,保证植株正常生长,在拟南芥中正调控植物抗旱性。

     

  • 图  1  胡杨MIR1444b前体与phytozome上预测的ptr-MIR1444b序列比对

    Figure  1.  Comparison of P. euphratica MIR1444b precursor with that of P. trichocarpa

    图  2  预测的miR1444b靶基因AT3G14570.2相对表达量分析

    Col-0为野生型,TG-1、TG-2和TG-5分别为转基因株系1、2和5号。每个实验设置3个重复,每个数值均为3组实验数据的平均值,误差线代表标准差。不同小写字母表示P < 0.05水平差异显著。下同。

    Figure  2.  Analysis of relative gene expression of predicted miR1444b target gene AT3G14570.2

    Col-0 is wild type, TG-1, TG-2 and TG-5 are transgenic lines 1, 2 and 5, respectively. Three replicates were set up for each independent experiment, each of which was an average of three experimental sets of data and error bars representing standard deviation. Different lowercase letters indicate significant difference at P < 0.05 level. The same below.

    图  3  拟南芥在甘露醇处理下的萌发率和根长统计分析

    A.1/2MS培养基上种子萌发率;B.1/2MS+250 mmol/L甘露醇培养基上种子萌发率;C.1/2MS培养基上生长8 d的根长;D.1/2MS培养基+250 mmol/L甘露醇培养基上生长8 d的根长。每个实验设置3个重复,每个数值均为3组实验数据的平均值,误差线代表标准差;下同。

    Figure  3.  Analysis of A. thaliana germination rate and root length under mannitol treatment

    A, seed germination rate on 1/2 MS medium; B, seed germination rate on 1/2 MS medium with 250 mmol/L mannitol; C, root length after 8 days of growth on 1/2 MS medium; D, root length after 8 days of growth on 1/2 MS with 250 mmol/L mannitol. Three replicates were set up for each independent experiment, each of which was an average of three experimental sets of data and error bars represented standard deviation; the same below.

    图  4  自然干旱和复水后拟南芥光合指标测定

    A.干旱以及复水之后拟南芥的生长状况;B~D.正常浇水、干旱8 d、复水3 d野生型和转基因拟南芥的光合速率、气孔导度、叶片水分利用效率的变化。

    Figure  4.  Measurement of A. thaliana photosynthesis indexes after natural soil drought and rewatered conditions

    A, Growth status of A. thaliana after drought and rewatered conditions; B-D, measurement of photosynthetic rate, stomatal conductance and water use efficiency of wild-type and transgenic A. thaliana under normal watering, drought for 8 days and rewatered for 3 days.

    图  5  PSⅡ最大光化学效率和POD活性测定

    A.自然干旱和复水后拟南芥PSⅡ最大光化学效率;B.自然干旱和复水后过氧化物酶活性。

    Figure  5.  PSⅡ maximum photochemical efficiency and POD activity determination

    A, PSⅡ maximum photosynthetic efficiency of A. thaliana after natural drought and rewatering; B, peroxidase activity of A. thaliana after natural drought and rewatering.

    表  1  peu-MIR1444b基因启动子顺式作用元件分析

    Table  1.   Cis-acting element analysis of peu-MIR1444b gene promoter

    顺式作用元件
    Sitename
    序列
    Sequence
    功能
    Function
    AE-box AGAAACAT Part of a module for light response
    ATCT-motif AATCTAATCT Part of a conserved DNA module involved in light responsiveness
    CAAT-box CAATT Common cis-acting element in promoter and enhancer regions
    CAT-box GCCACT Cis-acting regulatory element related to meristem expression
    GA-motif ATAGATAA Part of a light responsive element
    MBS CAACTG MYB binding site involved in drought-inducibility
    MRE AACCTAA MYB binding site involved in light responsiveness
    TATA-box TATA Core promoter element around -30 of transcription start
    TC-rich repeats ATTTTCTCCA Cis-acting element involved in defense and stress responsiveness
    TGA-element AACGAC Auxin-responsive element
    ARE TGGTTT Cis-acting regulatory element essential for anaerobic induction
    CGTCA-motif CGTCA Cis-acting regulatory element involved in MeJA-responsiveness
    GAG-motif AGAGAGTCCC Part of a light responsive element
    GATA-motif AAGATAAGATT Part of a light responsive element
    TC-rich repeats GTTTTCTTAC Cis-acting element involved in defense and stress responsiveness
    TCT-motif TCTTAC Part of a light responsive element
    TCATG-motif TGACG Cis-acting regulatory element involved in MeJA-responsiveness
    下载: 导出CSV

    表  2  拟南芥中预测的靶基因

    Table  2.   Potential target genes in A. thaliana

    靶基因名称
    Target gene
    互补序列
    Complementary sequence
    期望值
    Expectation
    翻译产物
    Translation product
    AT1G23440.2 GAACGUUGAUAGAGUGUGAU 3 类焦谷氨酰肽酶Ⅰ
    Pyroglutamyl peptidase I-like, ATGSL04
    AT3G14570.2 GAGCUUUGACAGAAUGUGGA 3 葡聚糖合成酶Ⅳ
    Glucan synthase-likeⅣ
    AT1G18830.1 GAUAGUUCACCGAAUGUGAA 3 类WD40重复超家族蛋白
    Transducin/WD40 repeat-like superfamily protein
    下载: 导出CSV
  • [1] 王让会, 王晓伟, 游先祥, 等.荒漠河岸林生态系统的结构分析[J].干旱区研究, 2002, 19(2):7-11. http://d.old.wanfangdata.com.cn/Periodical/ghqyj200202002

    Wang R H, Wang X W, You X X, et al.Analysis on the structure of the desert riparian forest ecosystems[J].Arid Zone Research, 2002, 19(2):7-11. http://d.old.wanfangdata.com.cn/Periodical/ghqyj200202002
    [2] Dong Y, Wang C, Han X, et al.A novel bHLH transcription factor PeHLH35 from Populus euphratica confers drought tolerance through regulating stomatal development, photosynthesis and growth in Arabidopsis[J].Biochemical & Biophysical Research Communications, 2014, 450(1):453-458.
    [3] He F, Wang H L, Li H G, et al.PeCHYR1, a ubiquitin E3 ligase from Populus euphratica, enhances drought tolerance via ABA-induced stomatal closure by ROS production in Populus[J/OL].Plant Biotechnology Journal, 2018[2018-03-09].https://doi.org/10.1111/pbi.12893.
    [4] 李岚, 王厚领, 赵琳, 等.异源表达Peu-miR473a增强拟南芥的抗旱性[J].北京林业大学学报, 2015, 37(5):30-39. doi: 10.13332/j.1000-1522.20140461

    Li L, Wang H L, Zhao L, et al.Heterogeneous expression of Peu-miR473a gene confers drought tolerance in Arabidopsis thaliana[J].Journal of Beijing Forestry University, 2015, 37(5):30-39. doi: 10.13332/j.1000-1522.20140461
    [5] 段中鑫.胡杨microRNA Peu-miR156j和Peu-miR69o表达模式分析及功能鉴定[D].北京: 北京林业大学, 2012.

    Duan Z X.Expression pattern and functional analysis of microRNA peu-miR156j and peu-miR169o from Populous euphratica [D].Beijing: Beijing Forestry University, 2012.
    [6] 段中鑫, 覃玉蓉, 夏新莉, 等.超量表达胡杨peu-MIR156j基因增强拟南芥耐盐性[J].北京林业大学学报, 2011, 33(6):1-7. http://j.bjfu.edu.cn/article/id/9665

    Duan Z X, Qin Y R, Xia X L, et al.Overexpression of Populous euphratica peu-miR156j gene enhancing salt tolerance in Arabidopsis thaliana[J].Journal of Beijing Forestry University, 2011, 33(6):1-7. http://j.bjfu.edu.cn/article/id/9665
    [7] Park W, Li J, Song R, et al.Carpel factory, a dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana[J].Current Biology, 2002, 12(17):1484-1495. doi: 10.1016/S0960-9822(02)01017-5
    [8] Zhang Y C, Yu Y, Wang C Y, et al.Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching[J].Nature Biotechnology, 2013, 31(9):848-854. doi: 10.1038/nbt.2646
    [9] Taylor P F, Guiling S, Caitlin E B, et al.Salt and drought stresses induce the aberrant expression of microRNA genes in tobacco[J].Molecular Biotechnology, 2011, 49(2):159-165. doi: 10.1007/s12033-011-9387-5
    [10] Li B S, Qin Y R, Duan H, et al.Genome-wide characterization of new and drought stress responsive microRNAs in Populus euphratica[J].Journal of Experimental Botany, 2011, 62 (11):3765-3779. doi: 10.1093/jxb/err051
    [11] Wang T Z, Chen L, Zhao M, et al.Identification of drought-responsive microRNAs and their targets in Medicago truncatula by genome-wide high-throughput sequencing[J].BMC Genomics, 2011, 12(1):361-372. doi: 10.1186/1471-2164-12-361
    [12] Lu X, Duan H, Lian C L, et al.The role of peu-miR164 and its target Pe-NAC genes in response to abiotic stress in Populus euphratica[J].Plant Physiology & Biochemistry, 2017, 115:418-438.
    [13] Lu S F, Chiang C.Conservation and diversity of MicroRNA-associated copper-regulatory networks in Populous trichocarpa [J].Journal of Integrative Plant Biology, 2011, 53(11):879-891. doi: 10.1111/jipb.2011.53.issue-11
    [14] 崔秀娜.毛果杨微小RNA1444a的克隆、胁迫表达分析及遗传转化研究[D].阜新: 辽宁工程技术大学, 2012. http://cdmd.cnki.com.cn/Article/CDMD-10147-1014181640.htm

    Cui X N.Cloning, stress expression analysis and genetic transformation of miR1444a in Populus trichacarpa[D].Fuxin: Liaoning University of Engineering and Technology, 2012. http://cdmd.cnki.com.cn/Article/CDMD-10147-1014181640.htm
    [15] 覃玉蓉.胡杨microRNAs的克隆及其功能研究[D].北京: 北京林业大学, 2011. http://cdmd.cnki.com.cn/Article/CDMD-10022-1011134964.htm

    Qin Y R.Cloning and functional analysis of microRNAs from Populus euphratica[D].Beijing: Beijing Forestry University, 2011. http://cdmd.cnki.com.cn/Article/CDMD-10022-1011134964.htm
    [16] Zhang X, Henriques R, Lin S S, et al.Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method[J].Nature Protocols, 2006, 1 (2):641-646. doi: 10.1038/nprot.2006.97
    [17] Shi H T, Ye T, Zhu J K, et al.Constitutive production of nitric oxide leads to enhanced drought stress resistance and extensive transcriptional reprogramming in Arabidopsis[J].Journal of Experimental Botany, 2014, 65(15):4119-4131. doi: 10.1093/jxb/eru184
    [18] Si L, Guo C, Cao Y, et al.The effect of nitrobenzene on antioxidative enzyme activity and DNA damage in tobacco seedling leaf cells[J].Environmental Toxicology & Chemistry, 2012, 31 (9):2078-2084. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=1e4cd3977ce9520946e32365fbfc39de
    [19] 高润石, 王红, 高艾.茎环法与加PolyA尾法PCR在检测MicroRNA时引物设计的策略[J].毒理学杂志, 2012, 26(5):378-381. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=wsdlxzz201205016

    Gao R S, Wang H, Gao A.Strategy of primer design for detecting microRNA by stem loop method and PolyA tail PCR[J].Toxicology Journal, 2012, 26(5):378-381. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=wsdlxzz201205016
    [20] Rao G, Sui J, Zeng Y, et al.Denovo transcriptome and small RNA analysis of two Chinese willow cultivars reveals stress response genes in Salix matsudana [J/OL].PLos One, 2014, 9(10): e109122[2017-10-06]. https://doi.org/10.1371/journal.pone.0109122.
    [21] Song Y, Ma K, Ci D, et al.Sexual dimorphism floral microRNA profiling and target gene expression in andromonoecious poplar (Populus tomentosa)[J/OL].PLos One, 2013, 8(5): e62681[2017-10-06]. http://doi.org/10.1371/journal.pone.0062681.
    [22] Chen L, Yuan R, Zhang Y, et al.Genome-wide profiling of novel and conserved Populus microRNAs involved in pathogen stress response by deep sequencing[J].Planta, 2012, 235(5):873-883. doi: 10.1007/s00425-011-1548-z
    [23] Wang M Z, Li C L, Lu S F.Origin and evolution of miR1444 genes in Salicaceae[J].Scientific Reports, 2017, 7:39740. doi: 10.1038/srep39740
    [24] 杨青杰.黄龙胆类胡萝卜素生物合成基因启动子的功能分析[D].哈尔滨: 东北师范大学, 2013. http://cdmd.cnki.com.cn/Article/CDMD-10200-1013357801.htm

    Yang Q J.Functional characterization of the Gentiana lutea carotenoid biosynthetic gene promoters[D].Harbin: Northeast Normal University, 2013. http://cdmd.cnki.com.cn/Article/CDMD-10200-1013357801.htm
    [25] Youshihiro N, Kazuo N, Zabta K S, et al.Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses[J].Plant Journal for Cell & Molecular Biology, 2003, 34(2):137-148. doi: 10.1046/j.1365-313X.2003.01708.x
    [26] Gao S, Gao J, Zhu X, et al.ABF2, ABF3, and ABF4 promote ABA-mediated chlorophyll degradation and leaf senescence by transcriptional activation of chlorophyll catabolic genes and senescence-associated genes in Arabidopsis[J].Molecular Plant, 2016, 9(9):1272-1285. doi: 10.1016/j.molp.2016.06.006
    [27] 石洪萍.三角褐指藻尿苷二磷酸葡萄糖焦磷酸化酶在碳流分配中的功能[D].青岛: 中国海洋大学, 2015. http://cdmd.cnki.com.cn/Article/CDMD-10423-1015717108.htm

    Shi H P.Carbon allocation function of UDP-glucose pyrophosphorylase in Pheodactylum tricornutum [D].Qingdao: Ocean University of China, 2015. http://cdmd.cnki.com.cn/Article/CDMD-10423-1015717108.htm
    [28] Ute H, Hans W, Helmut B.A sucrose-synthase gene of Vicia faba L. :expression pattern in developing seeds in relation to starch synthesis and metabolic regulation[J].Planta, 1993, 191(3):394-401. https://www.ncbi.nlm.nih.gov/pubmed/7764025
    [29] Zhang J X, Kirkham M B.Mechanisms of chilling-induced oxidative stress injury and tolerance in developing maize seedlings:changes in antioxidant system, oxidation of proteins and lipids, and protease activities[J].The Plant Journal, 1994, 35(5):785-791.
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  • 收稿日期:  2018-01-30
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