小麦TaGAPDH5基因的亚细胞定位和表达分析
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摘要
利用生物信息学方法预测中国春小麦TaGAPDH5的理化性质、亚细胞定位等分子特征,发现该蛋白的相对分子质量为31 541.87,理论等电点(pI)为6.08,为酸性亲水性蛋白,没有信号肽和跨膜结构域.采用基因枪法将TaGAPDH5-GFP融合载体瞬时转化洋葱表皮细胞,发现TaGAPDH5分布于细胞质.通过PLEXdb和Genevestigator数据库下载中国春小麦幼苗、分蘖、拔节等生育期的芯片表达数据,构建TaGAPDH5的芯片表达谱,发现TaGAPDH5在小麦整个生长周期均持续较高水平表达且具有一定组织特异性.采用实时荧光定量PCR(qRT-PCR)技术研究其不同时期的组织表达特性,发现TaGAPDH5从孕穗期到开花期,在同一时期的不同组织中和不同发育时期的同一组织中表达差异较大.通过构建诱导表达谱,发现TaGAPDH5基因能响应多种非生物胁迫.因此推测TaGAPDH5在小麦生长发育过程中发挥重要作用.
Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) plays pivotal roles in plant metabolism as a ubiquitous protein. However, the precise role of GAPDH in plant is not completely understood. The characteristics of physicochemical properties and subcellular localization of TaGAPDH5 in wheat cv. Chinese spring were analyzed using the bioinformatics software.The acid hydrophilic protein with a molecular weight of 31541.87 and an isoelectric point(pI) of 6.08 have no signal peptide,transmembrane domains. After construction of transient expression vector TaGAPDH5-GFP, particle bombardment with onion epidermal cells indicated that TaGAPDH5 was localized in the cytoplasm. Microarray data of TaGAPDH5 in wheat were downloaded from PLEXdb and Genevestigator with their accession numbers, expression analysis of TaGAPDH5 by microarray revealed that TaGAPDH5 showed remarkable tissue specificity and expressed constantly throughout the plant developmental stages. Quantitative RT-PCR(qRT-PCR) was used to detect organizational expression characteristics of different periods of TaGAPDH5 gene, the result revealed that TaGAPDH5 was differentially expressed between different tissues in the same period,or in the same tissues in different developmental periods. The present study has laid a foundation for subsequently exploring molecular mechanism of regulation of gene expression and signal transmission under drought stress. Relative expression analysis revealed that TaGAPDH5 could be significantly induced by abiotic stresses. Therefore, TaGAPDH5 may play a important role in wheat metabolism.
Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) plays pivotal roles in plant metabolism as a ubiquitous protein. However, the precise role of GAPDH in plant is not completely understood. The characteristics of physicochemical properties and subcellular localization of TaGAPDH5 in wheat cv. Chinese spring were analyzed using the bioinformatics software.The acid hydrophilic protein with a molecular weight of 31541.87 and an isoelectric point(pI) of 6.08 have no signal peptide,transmembrane domains. After construction of transient expression vector TaGAPDH5-GFP, particle bombardment with onion epidermal cells indicated that TaGAPDH5 was localized in the cytoplasm. Microarray data of TaGAPDH5 in wheat were downloaded from PLEXdb and Genevestigator with their accession numbers, expression analysis of TaGAPDH5 by microarray revealed that TaGAPDH5 showed remarkable tissue specificity and expressed constantly throughout the plant developmental stages. Quantitative RT-PCR(qRT-PCR) was used to detect organizational expression characteristics of different periods of TaGAPDH5 gene, the result revealed that TaGAPDH5 was differentially expressed between different tissues in the same period,or in the same tissues in different developmental periods. The present study has laid a foundation for subsequently exploring molecular mechanism of regulation of gene expression and signal transmission under drought stress. Relative expression analysis revealed that TaGAPDH5 could be significantly induced by abiotic stresses. Therefore, TaGAPDH5 may play a important role in wheat metabolism.
引文
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[20] NEBENFüHR A. Identifying Subcellular Protein Localization with Fluorescent Protein Fusions After Transient Expression in Onion Epidermal Cells[M]. New Jersey:Humana Press,2014.
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[24] ANOMAN A D, FLORES-TORNERO M, ROSATELLéZ S, et al. The specific role of plastidial glycolysis in photosynthetic and heterotrophic cells under scrutiny through the study of glyceraldehyde-3-phosphate dehydrogenase[J]. Plant Signaling&Behavior,2016,11(3):e1128614.
[25] ANOMAN A D, MUnOZ-BERTOMEU J, ROSATéLLEZ S, et al. Metabolism of Heterotrophic Cells in Arabidopsis[J]. Plant Physiology,2015,169(3):1619-37.
[26] PIATTONI C V, DML F, DELLAFERRERA I, et al.Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase Is Phosphorylated during Seed Development[J]. Frontiers in Plant Science,2017,8(190):522.
[27] CHAO HUANG, JINGQI YUAN. Using radial basis function on the general form of Chou's pseudo amino acid composition and PSSM to predict subcellular locations of proteins with both single and multiple sites[J].BioSystems,2013,113(1):50-57.
[28] KENICHIRO IMAI, KENTA NAKAI. Prediction of subcellular locations of proteins:Where to proceed[J].Protemics,2010,10(22):3970-3983.
[29] ZAFFAGNINI M, FERMANI S, COSTA A, et al. Plant cytoplasmic GAPDH:redox post-translational modifications and moonlighting properties[J]. Frontiers in Plant Science,2013,4(20):450.
[30] YANG S S, ZHAI Q H. Cytosolic GAPDH:a key mediator in redox signal transduction in plants[J]. Biologia Plantarum, 2017,61(3):1-10.
[2] LIU T F, FANG H, LIU J, et al. Cytosolic glyceraldehyde-3-phosphate dehydrogenases play crucial roles in controlling cold‐induced sweetening and apical dominance of potato(Solanum tuberosum L.)tubers[J]. Plant Cell&Environment,2017,40(12):3043-3054.
[3] SIROVER M A. On the functional diversity of glyceraldehyde-3-phosphate dehydrogenase:Biochemical mechanisms and regulatory control[J]. Biochimica et biophysica acta,2011,1810(8):741-751.
[4] WANG J. Selection and validation of reference genes for quantitative gene expression analyses in black locust(Robinia pseudoacacia L.)using real-time quantitative PCR[J]. Plos One, 2018,13(3):e0193076.
[5] MORIGASAKI S, SHIMDAD K, IKNER A, et al. Glycolytic enzyme GAPDH promotes peroxide stress signaling through multistep phosphorelay to a MAPK cascade[J].Molecular cell,2008, 30(1):108-113.
[6] MUNOZ-BERTOMEU J, CASCALES-MINANA B,MULET J M, et al. Plastidial glyceraldehyde-3-phosphate dehydrogenase deficiency leads to altered root development and affects the sugar and amino acid balance in Arabidopsis[J]. Plant Physiology,2009, 151(2):541-558.
[7] MUnOZ-BERTOMEU J, CASCALES-MInANA B, IRLES-SEGURA A, et al. The plastidial glyceraldehyde-3-phosphate dehydrogenase is critical for viable pollen development in Arabidopsis[J]. Plant Physiology,2010,152(4):1830-1841.
[8] LEE M N, HA S H, KIM J, et al. Glycolytic flux signals to mTOR through glyceraldehyde-3-phosphate dehydrogenase-mediated regulation of rheb.[J]. Molecular&Cellular Biology, 2009,29(14):3991-4001.
[9] GANI Z, BORADIA V M, RAGHU RAM J, et al. Purification and characterization of glyceraldehyde-3-phosphate-dehydrogenase(GAPDH)from pea seeds[J]. Protein Expression Purification, 2016,127:22-27.
[10] FANG Y, XIONG L. General mechanisms of drought response and their application in drought resistance improvement in plants[J]. Cellular&Molecular Life Sciences Cmls,2015,72(4):673.
[11] KAPPACHERY S, BANIEKAL-HIERMATH G, YU J W, et al. Effect of over-and under-expression of glyceraldehyde 3-phosphate dehydrogenase on tolerance of plants to water-deficit stress[J]. Plant Cell Tissue and Organ Culture,2015,121:97-107.
[12] VELASCO R, SALAMINI F, BARTELS D. Dehydration and ABA increase mRNA levels and enzyme activity of cytosolic GAPDH in the resurrection plant craterostigma plantagineum[J].Plant molecular biology,1994,26(1):541-546.
[13] ZHAI Z, GOMEZ-MEJIBA S E, RAMIREZ D C. Free radical mechanism of myeloperoxidase-induced GAPDH oxidation, aggregation and proteotoxic stress in activated macrophages[J]. Free Radical Biology&Medicine,2010,49:S174-S174.
[14] GUO L, MA F F, WEI F, et al. Cytosolic phosphorylating glyceraldehyde-3-phosphate dehydrogenases affect Arabidopsis cellular metabolism and promote seed oil accumulation[J]. Plant Cell,2014,26(7):3023-3035.
[15] ZHANG X H, RAO X L, SHI H T, et al. Overexpression of a cytosolic glyceraldehyde-3-phosphate dehydrogenase gene OsGAPC3, confers salt tolerance in rice[J]. Plant Cell Tissue&Organ Culture,2011,107(1):1.
[16] LAO Y M, LU Y, JIANG J G, et al. Six regulatory elements lying in the promoter region imply the functional diversity of chloroplast GAPDH in Duanliella bardawil[J].Journal of Agricultural&Food Chemistry,2012,60(36):9211-9220.
[17]张琳.小麦长武134GAPDH基因在3种非生物胁迫下的表达分析[D].杨凌:西北农林科技大学,2014.
[18]张阳璞.长武134小麦GAPDH基因过表达载体的构建及拟南芥的遗传转化[D].杨凌:西北农林科技大学,2014.
[19] LIU H, ZHOU X, DONG N, et al. Expression of a wheat MYB gene in transgenic tob acco enhances resistance to Ralstonia solanacearum and to drought and salt stresses[J]. Functional Integrative Genomics,2011,11(3):431-443.
[20] NEBENFüHR A. Identifying Subcellular Protein Localization with Fluorescent Protein Fusions After Transient Expression in Onion Epidermal Cells[M]. New Jersey:Humana Press,2014.
[21] YANG J Q, KONG F N, CHAO L I, et al. Cloning and Expression Analysis of PyAQP1 in Pyropia yezoensis[J].Periodical of Ocean University of China,2016,46(8):79-86.
[22] WANG G, WANG G, ZHANG X, et al. Isolation of high quality RNA from cereal seeds containing high levels of starch.[J]. Phytochemical Analysis,2012,23(2):159–163.
[23] SCHMITTGEN T D, LIVAK K J. Analyzing real-time PCR data by the comparative C(T)method[J]. Nature Protocols,2008,3(6):1101-1108.
[24] ANOMAN A D, FLORES-TORNERO M, ROSATELLéZ S, et al. The specific role of plastidial glycolysis in photosynthetic and heterotrophic cells under scrutiny through the study of glyceraldehyde-3-phosphate dehydrogenase[J]. Plant Signaling&Behavior,2016,11(3):e1128614.
[25] ANOMAN A D, MUnOZ-BERTOMEU J, ROSATéLLEZ S, et al. Metabolism of Heterotrophic Cells in Arabidopsis[J]. Plant Physiology,2015,169(3):1619-37.
[26] PIATTONI C V, DML F, DELLAFERRERA I, et al.Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase Is Phosphorylated during Seed Development[J]. Frontiers in Plant Science,2017,8(190):522.
[27] CHAO HUANG, JINGQI YUAN. Using radial basis function on the general form of Chou's pseudo amino acid composition and PSSM to predict subcellular locations of proteins with both single and multiple sites[J].BioSystems,2013,113(1):50-57.
[28] KENICHIRO IMAI, KENTA NAKAI. Prediction of subcellular locations of proteins:Where to proceed[J].Protemics,2010,10(22):3970-3983.
[29] ZAFFAGNINI M, FERMANI S, COSTA A, et al. Plant cytoplasmic GAPDH:redox post-translational modifications and moonlighting properties[J]. Frontiers in Plant Science,2013,4(20):450.
[30] YANG S S, ZHAI Q H. Cytosolic GAPDH:a key mediator in redox signal transduction in plants[J]. Biologia Plantarum, 2017,61(3):1-10.