欧美杨PdMODD基因克隆与表达特性分析

张腾倩; 张伟溪; 丁昌俊; 张静; 胡赞民; 苏晓华

doi:10.12302/j.issn.1000-2006.202007015

欧美杨PdMODD基因克隆与表达特性分析

张腾倩, 张伟溪, 丁昌俊, 张静, 胡赞民, 苏晓华

南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (2) : 43-50.

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南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (2) : 43-50. DOI: 10.12302/j.issn.1000-2006.202007015

研究论文

欧美杨PdMODD基因克隆与表达特性分析

张腾倩 ¹ ,
张伟溪 ¹ ,
丁昌俊 ¹ ,
张静 ¹ ,
胡赞民 ² ,
苏晓华 ¹^,^*

作者信息 +

Cloning and expression characteristics of PdMODD genes in Populus × euramericana

Author information +

文章历史 +

摘要

【目的】研究杨树重要胁迫响应新基因,为揭示杨树抗逆分子机制、培育优良抗逆杨树新品种提供理论依据。【方法】基于水稻MODD氨基酸序列,通过BLAST在美洲黑杨基因组中筛选得到3条基因(Podel.08G114100、Podel.10G158900和Podel.17G098500),并以其序列为参考,以‘渤丰3号’杨cDNA为模板克隆得到3条基因(PdMODD1、PdMODD2和PdMODD3)。利用生物信息学分析PdMODD蛋白的结构特征及与其同源蛋白的亲缘关系。通过基因枪轰击法分析PdMODD基因亚细胞定位。采用实时荧光定量(qRT-PCR)技术,分析PdMODD基因组织特异性和不同胁迫条件下的的表达模式。【结果】PdMODD1~3基因的开放阅读框(ORF)全长分别为1 065、1 065和1 074 bp,编码354、354和357个氨基酸,均为不稳定的亲水性蛋白。PdMODD基因均为NINJA家族成员,含有3个保守结构域,其中一个为转录抑制基序EAR。系统进化树分析显示,PdMODD1蛋白与毛果杨PtAFP2亲缘性较高并与拟南芥AtAFP1和木薯MeAFP2位于同一个分支;PdMODD2蛋白与麻疯树JcAFP2亲缘关系最近,并与拟南芥AtAFP2聚成一个分支;PdMODD3与毛果杨PtAFP3-1亲缘关系最近,与栓皮槠QsAFP3、拟南芥AtAFP4属于同一分支。亚细胞定位结果表明PdMODD基因均定位于细胞核。组织特异性分析显示,PdMODD1~3在根、茎、叶中均能表达,且在叶中表达量最高,根中表达量最低。胁迫响应表达模式结果显示,NaCl和聚乙二醇(PEG)胁迫处理下,PdMODD1~3在根、茎、叶组织中主要为显著上调表达。【结论】PdMODD1~3均为NINJA家族成员,含有保守的转录抑制基序EAR,具有组织特异性,在叶中高表达,且能被NaCl和PEG胁迫显著诱导表达,推测PdMODD1、PdMODD2 和PdMODD3可能会在‘渤丰3号’杨对盐和干旱胁迫响应中发挥重要的调节作用。

Abstract

【Objective】To explore the function of PdMODD in the growth and development of Populus × euramericana, we analyzed their sequence characteristics, subcellular localization and expression patterns in response to salt and drought stress, which will provide a theoretical basis for revealing the molecular mechanisms of stress resistance and breeding new poplar varieties. 【Method】Based on the rice MODD amino acid sequence, Podel.08G114100, Podel.10G158900 and Podel.17G098500 were selected using BLAST in the genome of Populus deltoides WV94 v2.1. Using these gene sequences as references and Populus × euramericana ‘Bofeng 3 hao’ cDNA as template, three genes (PdMODD1,PdMODD2 and PdMODD3) were cloned. The structural characteristics of the PdMODD protein and its genetic relationship with its homologous proteins were analyzed using bioinformatics methods. The subcellular localization of PdMODD genes was analyzed using particle bombardment. The expression characteristics of PdMODD genes in different tissues and their response to different stresses were analyzed using qRT-PCR. 【Results】The open reading frames of PDMODD1-PDMODD3 genes were 1 065, 1 065 and 1 074 bp, encoding 354, 354 and 357 amino acids, respectively, all of which were unstable hydrophilic proteins located in the nucleus.PdMODD genes are all members of the NINJA family and contain three conserved domains, one of which is the transcriptional repression motif EAR. Phylogenetic analysis demonstrated that PdMODD1 protein was closely related to PtAFP2 (Populus trichocarpa) and located in the same branch as AtAFP1 (Arabidopsis thaliana) and MeAFP2 (Manihot esculenta). PdMODD2 protein had a close relationship with JcAFP2 (Jatropha curcas) and converged into a branch with AtAFP2; PdMODD3 was closely related to PtAFP3-1 and located in the same branch as QsAFP3 (Quercus suber) and AtAFP4. The subcellular localization results showed that PdMODD genes were localized in the nucleus. The results of tissue specificity analysis showed that PdMODD genes were highly expressed in the leaves and the least expressed in the roots. The expression patterns of stress response showed that under NaCl and PEG stress, PdMODD1, PdMODD2 and PdMODD3 were significantly upregulated in the root, stem and leaf tissues. 【Conclusion】PdMODD1-PdMODD3 are all members of the NINJA family, with the conserved transcriptional repression motif EAR and tissue specificity. They were highly expressed in the leaves, significantly induced by NaCl and PEG stress, and were in a branch with AtAFP1, AtAFP2 and AtAFP4, which had negative regulatory effects on ABA and salt stress. It is hypothesized that PdMODD1, PdMODD2 and PdMODD3 may play an important regulatory role in response to P. euramericana ‘Bofeng 3 hao’ salt and drought stress.

导出引用

张腾倩, 张伟溪, 丁昌俊, 等. 欧美杨PdMODD基因克隆与表达特性分析[J]. 南京林业大学学报（自然科学版）. 2021, 45(2): 43-50 https://doi.org/10.12302/j.issn.1000-2006.202007015

ZHANG Tengqian, ZHANG Weixi, DING Changjun, et al. Cloning and expression characteristics of PdMODD genes in Populus × euramericana[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2021, 45(2): 43-50 https://doi.org/10.12302/j.issn.1000-2006.202007015

中图分类号： S785.5

参考文献

列表( 原文顺序 | 文献年度倒序 | 文中引用次数倒序 ) 可视化分析

[1]	KAGALE S, ROZWADOWSKI K. EAR motif-mediated transcriptional repression in plants[J]. Epigenetics, 2011,6(2):141-146.DOI: 10.4161/epi.6.2.13627. 本文引用 [2]

[2]	张健飞, 权瑞党, 黄荣峰. EAR转录抑制子结构及功能的研究[J]. 中国农业科技导报, 2011,13(4):53-57. ZHANG J F, QUAN R D, HUANG R F. Studies on structure and function of repressors with EAR motif[J]. J Agric Sci Technol, 2011,13(4):53-57.DOI: 10.3969/j.issn.1008-0864.2011.04.08. 本文引用 [2]

[3]	TANG N, MA S, ZONG W, et al. MODD mediates deactivation and degradation of OsbZIP46 to negatively regulate ABA signaling and drought resistance in rice[J]. Plant Cell, 2016,28(9):2161-2177.DOI: 10.1105/tpc.16.00171. 本文引用 [4]

[4]	GARCIA M E, LYNCH T, PEETERS J, et al. A small plant-specific protein family of ABI five binding proteins (AFPs) regulates stress response in germinating Arabidopsis seeds and seedlings[J]. Plant Mol Biol, 2008,67(6):643-658.DOI: 10.1007/s11103-008-9344-2. 本文引用 [4]

[5]	PAUWELS L, BARBERO G F, GEERINCK J, et al. NINJA connects the co-repressor TOPLESS to jasmonate signalling[J]. Nature, 2010,464(7289):788-791.DOI: 10.1038/nature08854. 本文引用 [1]

[6]	KAZAN K, MANNERS J M. JAZ repressors and the orchestration of phytohormone crosstalk[J]. Trends Plant Sci, 2012,17(1):22-31.DOI: 10.1016/j.tplants.2011.10.006. 本文引用 [1]

[7]	DE GEYTER N, GHOLAMI A, GOORMACHTIG S, et al. Transcriptional machineries in jasmonate-elicited plant secondary metabolism[J]. Trends Plant Sci, 2012,17(6):349-359.DOI: 10.1016/j.tplants.2012.03.001. 本文引用 [1]

[8]	MA S, TANG N, LI X, et al. Reversible histone H2B monoubiquitination fine-tunes abscisic acid signaling and drought response in rice[J]. Mol Plant, 2019,12(2):263-277.DOI: 10.1016/j.molp.2018.12.005. 本文引用 [1]

[9]	XIANG Y, TANG N, DU H, et al. Characterization of OsbZIP23 as a key player of the basic leucine zipper transcription factor family for conferring abscisic acid sensitivity and salinity and drought tolerance in rice[J]. Plant Physiol, 2008,148(4):1938-1952.DOI: 10.1104/pp.108.128199. 本文引用 [1]

[10]	LU G J, GAO C X, ZHENG X N, et al. Identification of OsbZIP72 as a positive regulator of ABA response and drought tolerance in rice[J]. Planta, 2009,229(3):605-615.DOI: 10.1007/s00425-008-0857-3. 本文引用 [1]

[11]	王倩姿, 王玉, 孙志梅, 等. 腐植酸类物质的施用对盐碱地的改良效果[J]. 应用生态学报, 2019,30(4):1227-1234. WANG Q Z, WANG Y, SUN Z M, et al. Amelioration effect of humic acid on saline-alkali soil[J]. Chin J Appl Ecol, 2019,30(4):1227-1234.DOI: 10.13287/j.1001-9332.201904.001.

[12]	徐淑平, 卫志明. 基因枪的使用方法介绍[J]. 植物生理学通讯, 1998,34(1):41-43. XU S P, WEI Z M. Introduction to method of microprojectile bombardment and its application[J]. Plant Physiol Commun, 1998,34(1):41-43.DOI: 10.13592/j.cnki.ppj.1998.01.014. 本文引用 [1]

[13]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J]. Methods (San Diego Calif), 2001,25(4):402-408.DOI: 10.1006/meth.2001.1262. 本文引用 [1]

[14]

刘中原, 姜波, 吕佳欣, 等. 刚毛柽柳Th2CysPr_x基因的互作蛋白及其表达模式分析[J]. 南京林业大学学报(自然科学版), 2019,43(2):86-92.

LIU Z

, JIANG

, LYU J

, et al. Interacting proteins of Tamarix hispida Th2CysPr_x and their expression pattern analysis[J]. J Nanjing For Univ (Nat Sci Ed), 2019,43(2):86-92.DOI: 10.3969/j.issn.1000-2006.201806018.

本文引用 [1]

[15]	杜娟, 柴友荣. 植物转录抑制子的结构特征及其作用机理[J]. 植物学通报, 2008,25(3):344-353. DU J, CHAI Y R. Structural features and action mechanisms of plant transcriptional repressors[J]. Chin Bull Bot, 2008,25(3):344-353.DOI: 10.3969/j.issn.1674-3466.2008.03.012. 本文引用 [1]

[16]	KAGALE S, LINKS M G, ROZWADOWSKI K. Genome-wide analysis of ethylene-responsive element binding factor-associated amphiphilic repression motif-containing transcriptional regulators in Arabidopsis[J]. Plant Physiol, 2010,152(3):1109-1134.DOI: 10.1104/pp.109.151704. 本文引用 [1]

[17]	WEIGEL R R, PFITZNER U M, GATZ C. Interaction of NIMIN₁ with NPR1 modulates PR gene expression in Arabidopsis[J]. Plant Cell, 2005,17(4):1279-1291.DOI: 10.1105/tpc.104.027441. 本文引用 [1]

[18]	CHERN M, CANLAS P E, FITZGERALD H A, et al. Rice NRR,a negative regulator of disease resistance,interacts with Arabidopsis NPR1 and rice NH₁[J]. Plant J, 2005,43(5):623-635.DOI: 10.1111/j.1365-313x.2005.02485.x. 本文引用 [1]

[19]	OHTA M, MATSUI K, HIRATSU K, et al. Repression domains of class II ERF transcriptional repressors share an essential motif for active repression[J]. Plant Cell, 2001,13(8):1959-1968.DOI: 10.1105/tpc.010127. 本文引用 [1]

[20]	LIU W, KAREMERA N J U, WU T, et al. The ethylene response factor AtERF₄ negatively regulates the iron deficiency response in Arabidopsis thaliana[J]. PLoS One, 2017,12(10):e0186580.DOI: 10.1371/journal.pone.0186580. 本文引用 [1]

[21]	YAISH M W, EL-KEREAMY A, ZHU T, et al. The APETALA-2-like transcription factor OsAP2-39 controls key interactions between abscisic acid and gibberellin in rice[J]. PLoS Genet, 2010,6(9):e1001098.DOI: 10.1371/journal.pgen.1001098. 本文引用 [1]

[22]	WAN L, ZHANG J, ZHANG H, et al. Transcriptional activation of OsDERF₁ in OsERF₃ and OsAP2-39 negatively modulates ethylene synjournal and drought tolerance in rice[J]. PLoS One, 2011,6(9):e25216.DOI: 10.1371/journal.pone.0025216. 本文引用 [1]

[23]	ZHANG H W, ZHANG J F, QUAN R D, et al. EAR motif mutation of rice OsERF₃ alters the regulation of ethylene biosynjournal and drought tolerance[J]. Planta, 2013,237(6):1443-1451.DOI: 10.1007/s00425-013-1852-x. 本文引用 [1]

[24]	PAN I C, LI C W, SU R C, et al. Ectopic expression of an EAR motif deletion mutant of SlERF₃ enhances tolerance to salt stress and Ralstonia solanacearum in tomato[J]. Planta, 2010,232(5):1075-1086.DOI: 10.1007/s00425-010-1235-5. 本文引用 [1]

[25]	HUANG J, YANG X, WANG M M, et al. A novel rice C₂H₂-type zinc finger protein lacking DLN-box/EAR-motif plays a role in salt tolerance[J]. Biochim et Biophys Acta (BBA)-Gene Struct Expr, 2007,1769(4):220-227.DOI: 10.1016/j.bbaexp.2007.02.006. 本文引用 [1]

[26]	LOPEZ-MOLINA L, MONGRAND S, KINOSHITA N, et al. AFP is a novel negative regulator of ABA signaling that promotes ABI₅ protein degradation[J]. Genes Dev, 2003,17(3):410-418.DOI: 10.1101/gad.1055803. 本文引用 [2]

[27]	OHNISHI N, HIMI E, YAMASAKI Y, et al. Differential expression of three ABA-insensitive five binding protein (AFP)-like genes in wheat[J]. Genes Genet Syst, 2008,83(2):167-177.DOI: 10.1266/ggs.83.167. 本文引用 [1]

[28]	WU J, SENG S, CARIANOPOL C, et al. Cloning and characterization of a novel Gladiolus hybridus AFP family gene (GhAFP-like) related to corm dormancy[J]. Biochem Biophys Res Commun, 2016,471(1):198-204.DOI: 10.1016/j.bbrc.2016.01.146. 本文引用 [1]

[29]	HUANG M D, WU W L. Overexpression of TMAC2,a novel negative regulator of abscisic acid and salinity responses,has pleiotropic effects in Arabidopsis thaliana[J]. Plant Mol Biol, 2007,63(4):557-569.DOI: 10.1007/s11103-006-9109-8. 本文引用 [1]

[30]	CAO M J, ZHANG Y L, LIU X, et al. Combining chemical and genetic approaches to increase drought resistance in plants[J]. Nat Commun, 2017,8(1):1183.DOI: 10.1038/s41467-017-01239-3. 本文引用 [1]