香蕉冷胁迫相关MicroRNA差异表达分析
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摘要
研究香蕉miRNA在冷胁迫处理下的动态表达,初步探索miRNA在香蕉冷胁迫过程中的可能作用。利用茎环引物实时荧光RT-PCR(stem-loop qRT-PCR)策略,检测冷胁迫响应miRNA在5℃冷胁迫处理不同时间点香蕉幼苗叶片中的表达模式。结果表明,在检测的15个香蕉miRNAs中,不同的miRNA在同一冷胁迫时间下的表达量存在很大差异。根据其表达模式,可将15个miRNA分成4组。组1和组2中的12个miRNA(miR159e、miR159f、miR160-3p、miR397a、miR399j、miR164e、miR444a、miR408、miR5179、miR530、miR535和miR5538),在5℃冷胁迫处理下表达量总体为上调,其在胁迫过程中的平均表达水平高于对照CK(0 h),说明冷胁迫诱导此类miRNA的表达。第3组包括miR156l、miR162a和miR166a,其在5℃冷胁迫处理各时间点的表达量均低于对照,为下调表达,说明冷胁迫抑制此类miRNA的表达。第4组仅含1个miRNA,即miR156a,除在冷胁迫处理12 h表达量明显上调外,其余时间点均下调表达。对这些miRNAs作用的靶基因进行预测和分析发现,它们主要参与植物的新陈代谢、信号传导和生长发育过程等。这为进一步验证与低温胁迫相关的目的miRNA的功能提供理论依据。
To explore the potential roles of miRNA in banana under cold stress, we analyzed the dynamic expression of cold responsive miRNAs in banana. The expression profiling of cold-responsive miRNAs in banana leaves was detected by stem-loop qRT-PCR after 0, 2, 4, 8, 12 and 24 h of cold stress at 5℃. The results showed that fifteen potential cold-responsive miRNA were expressed and exhibited different expression patterns at different treatment stages. The 15 miRNA were divided into 4 groups according to their expression pattern. Generally, the expressions of Group I(miR159 e, miR159 f, miR160-3 p, miR397 a, miR399 j, miR408,miR5179, miR530, miR535 and miR5538) and II(miR164 e and miR444 a) were up-regulated at 5℃ cold stress. During the cold stress, the average expression level of these miRNA was higher than the control(0 h).miR156 l, miR162 a and miR166 a were in Group III, and their expression was reduced during the whole period of the cold treatment. Group IV had only one miRNA(miR156 a), its expression was significantly increased only at 12 h and reduced at the rest of the time points under cold stress. The analysis of the target genes of these miRNA revealed that these miRNA participated in the signal transduction, growth and development and stress response under abiotic stress. These findings provide valuable information for further functional characterization of miRNA associated with cold stress in banana.
To explore the potential roles of miRNA in banana under cold stress, we analyzed the dynamic expression of cold responsive miRNAs in banana. The expression profiling of cold-responsive miRNAs in banana leaves was detected by stem-loop qRT-PCR after 0, 2, 4, 8, 12 and 24 h of cold stress at 5℃. The results showed that fifteen potential cold-responsive miRNA were expressed and exhibited different expression patterns at different treatment stages. The 15 miRNA were divided into 4 groups according to their expression pattern. Generally, the expressions of Group I(miR159 e, miR159 f, miR160-3 p, miR397 a, miR399 j, miR408,miR5179, miR530, miR535 and miR5538) and II(miR164 e and miR444 a) were up-regulated at 5℃ cold stress. During the cold stress, the average expression level of these miRNA was higher than the control(0 h).miR156 l, miR162 a and miR166 a were in Group III, and their expression was reduced during the whole period of the cold treatment. Group IV had only one miRNA(miR156 a), its expression was significantly increased only at 12 h and reduced at the rest of the time points under cold stress. The analysis of the target genes of these miRNA revealed that these miRNA participated in the signal transduction, growth and development and stress response under abiotic stress. These findings provide valuable information for further functional characterization of miRNA associated with cold stress in banana.
引文
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[41]袁进成,宋晋辉,马海莲,等.转玉米ZmABI3-L基因增加拟南芥的抗旱和耐盐性[J].草业学报,2016,25(2):124-131.
[42] Ma C, Burd S, Lers A. miR408 is involved in abiotic stressresponses in Arabidopsis[J]. The Plant Journal,2015,84:169-187.
[2] Subbaraya U. Potential and constraints of using wild Musa[A].//In:Subbaraya U. Farmers’knowledge of wild Musa in Indian[M]. Foodand Agriculture Organization of the United Nations,2006:33-36.
[3] Baurens F C, Bocs S, Rouard M, et al. Mechanisms of haplotypedivergence at the RGA08 nucleotide-binding leucine-rich repeatgene locus in wild banana(Musa balbisiana)[J]. BMC PlantBiology,2010,10:149-165.
[4] Ravi I, Uma S, Vaganan M M, et al. Phenotyping bananas fordrought resistance[J]. Frontiers in Physiology,2013,4:9.
[5] Bartel D P. MicroRNAs:genomics, biogenesis, mechanism, andfunction[J]. Cell,2004,116(2):281-297.
[6] Sunkar R, Li Y F, Jagadeeswaran G. Functions of microRNAs inplant stress responses[J]. Trends in Plant Science,2012,17(4):196-203.
[7] Sunkar R, Zhu J K. Novel and stress-regulated microRNAs andother small RNAs from Arabidopsis[J]. Plant Cell,2004,16(8):2001-2019.
[8] Zhou X, Wang G, Sutoh K, et al. Identification of cold-induciblemicroRNAs in plants by transcriptome analysis[J]. Biochimica etBiophysica Acta,2008,1779(11):780-788.
[9] Liu H H, Tian X, Li Y J, et al. Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana[J]. RNA,2008,14(5):836-843.
[10] Lu S, Sun Y H, Chiang V L. Stress-responsive microRNAs inPopulus[J]. Plant Journal,2008,55(1):131-151.
[11] Zhang J, Xu Y, Huan Q, et al. Deep sequencing of Brachypodiumsmall RNAs at the global genome level identifies microRNAsinvolved in cold stress responses[J]. BMC genomics,2009,10:449-465.
[12] Lv D K, Bai X, Li Y, et al. Profiling of cold-stress-responsivemiRNAs in rice by microarrays[J]. Gene,2010,459(1-2):39-47.
[13] Tang Z, Zhang L, Xu C, et al. Uncovering small RNA-mediatedresponses to cold stress in a wheat thermosensitive genic male-sterile line by deep sequencing[J]. Plant Physiology,2012,159(2):721-738.
[14] Thiebaut F, Rojas C A, Almeida K L, et al. Regulation of miR319during cold stress in sugarcane[J]. Plant Cell Environment,2012,35(3):502-512.
[15] Wang J Y, Liu J H, Jia C H, et al. Cold stress responsivemicroRNAs and their targets in Musa balbisiana[J]. Frontiers ofAgricultural Science and Engineering,2016,3(4):335-345.
[16]柴娟,冯仁军,史后蕊,等.一种快速提取香蕉叶片总核酸、总RNA和总DNA的新方法[J].热带作物学报,2014,35(1):104-109.
[17] Chen C, Ridzon D A, Broomer A J, et al. Real-time quantificationof microRNAs by stem-loop RT-PCR[J]. Nucleic Acids Research,2005,33:e179.
[18] Livak K J, Schmittgen T D. Analysis of relative gene expressiondata using real-time quantitative PCR and 2-ΔΔCTMethod[J].Methods,2001,25:402-408.
[19] Xu S X, Liu N, Mao W H, et al. Identification of chilling-responsive microRNAs and their targets in vegetable soybean(Glycine max L.)[J]. Scientific reports,2016,6:26619.
[20] Zhang X N, Li X, Liu J H. Identification of conserved and novelcold-responsive microRNAs in Trifoliate Orange(Poncirustrifoliata(L)Raf.)using high-throughput sequencing[J]. PlantMolecular Biology Reporter,2014,32:328-341.
[21] Sun X, Fan G, Su L, et al. Identification of cold-induciblemicroRNAs in grapevine[J]. Frontiers in Plant Science,2015,6:595.
[22] Giacomelli J I, Weigel D, Chan R L, et al. Role of recentlyevolved miRNA regulation of sunflower HaWRKY6 inresponse to temperature damage[J]. New Phytologist,2012,195(4):766-773.
[23] Chen Z, Gao X, Zhang J. Alteration of osa-miR156e expressionaffects rice plant architecture and strigolactones(SLs)pathway[J].Plant Cell Report,2015,34(5):767-781.
[24] Cui LG, Shan JX, Shi M, et al. The miR156-SPL9-DFR pathwaycoordinates the relationship between development and abiotic stresstolerance in plants[J]. Plant Journal,2014,80(6):1108-1117.
[25] Olsen A N, Ernst H A, Leggio L L, et al. NAC transcription factors:structurally distinct, functionally diverse[J]. Trends in Plant Science,2005,10(2):79-87.
[26] Hu H H, You J, Fang Y J, et al. Characterization of transcriptionfactor gene SNAC2 conferring cold and salt tolerance in rice[J].Plant Molecular Biology,2008,67(1):169-181.
[27] Ciechanover A. The ubiquitin-proteasome pathway:on proteindeath and cell life[J].EMBO Journal,1998,17:7151-7160.
[28] Jung Y J, Lee I H, Nou I S, et al. BrRZFP1 a Brassica rapa C3HC4-type RING zinc finger protein involved in cold, salt anddehydration stress[J]. Plant biology,2013,15:274-283.
[29] Kim J, Jung J H, Reyes J L, et al. MicroRNA directed cleavage ofATHB15 mRNA regulates vascular development in Arabidopsisinflorescence stems[J]. The Plant Journal,2005,42(1):84-94.
[30] Zeng X, Xu Y, Jiang J, et al. Identification of cold stress responsivemicroRNAs in two winter turnip rape(Brassica rapa L.)by highthroughput sequencing[J]. BMC Plant Biology,2018,18:52.
[31] Dong C H, Pei H. Over-expression of miR397 improves planttolerance to cold stress in Arabidopsis thaliana[J]. Journal of PlantBiology,2014,57:209-217.
[32] Li Y F, Zheng Y, Addo-Quaye C, et al. Transcriptome-wideidentification of microRNA targets in rice[J]. The Plant Journal,2010,62:742-759.
[33] Owttrim G. RNA helicases:diverse roles in prokaryotic response toabiotic stress[J]. RNA Biology,2013,1:96-110.
[34] Gong Z, Lee H, Xiong L, et al. RNA helicase-like protein as anearly regulator of transcription factors for plant chilling andfreezing tolerance[J]. Proceedings of the National Academy ofSciences of the United States of America,2002,99(17):11507-11512.
[35] Gong Z, Dong C H, Lee H, et al. A DEAD box RNA helicase isessential for mRNA export and important for development andstress responses in Arabidopsis[J]. Plant Cell,2005,17(1):256-267.
[36] Macovei A, Tuteja N. MicroRNAs targeting DEAD-box helicasesare involved in salinity stress response in rice(Oryza sativa L.)[J].BMC Plant Biology,2012,12:183.
[37] Tuteja N, Sahoo R, Garg B, et al. OsSUV3 dual helicase functionsin salinity stress tolerance by maintaining photosynthesis andantioxidant machinery in rice(Oryza sativa L. cv.‘IR64’)[J]. PlantJournal,2013,76:115-127.
[38] Mary E G, Tim L, Julian P. A Small plant-specific protein family ofABI five binding proteins(AFPs)regulates stress response ingerminating Arabidopsis seeds and seedings[J]. Plant MolecularBiology,2008,67:643-658.
[39] Kobayashi F, Maeta E, Terashima A, et al. Positive role of a wheatHvABI5 ortholog in abiotic stress response of seedlings[J].Physiologia Plantarum,2008,134:74-86.
[40] Yang X, Yang Y N, Xue L J, et al. Rice ABI5-Like1 regulatesabscisic acid and auxin responses by affecting the expression ofABRE-containing genes[J]. Plant Physiology,2011,156(3):1397-1409.
[41]袁进成,宋晋辉,马海莲,等.转玉米ZmABI3-L基因增加拟南芥的抗旱和耐盐性[J].草业学报,2016,25(2):124-131.
[42] Ma C, Burd S, Lers A. miR408 is involved in abiotic stressresponses in Arabidopsis[J]. The Plant Journal,2015,84:169-187.