代谢组学法研究转录因子SlNAC4对番茄果实代谢产物的影响
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摘要
以SlNAC4基因沉默番茄和野生型番茄为研究对象,采用高效液相色谱-串联高分辨率质谱仪对破色期番茄果实代谢产物进行代谢组学分析,探究转录因子SlNAC4对果实成熟中代谢产物的影响。利用XCMS软件和inhouse标准品、HMDB、KEGG等数据库对化合物进行定性分析,用metaX软件进行定量及差异代谢产物的筛选。对两组番茄代谢组学分析结果显示,既能与数据库物质的一级离子m/z匹配也能与数据库物质的碎片离子(二级)m/z匹配到的差异代谢物质共有67种,包括氨基酸衍生物及二肽类(19种)、维生素类(7种)、糖类及其衍生物(4种)、核酸及其衍生物(11种)、生物碱类(4种)、香精香料(4种)及有机酸(9种)等。其中有29种化合物含量上调,38种含量显著下降,表明番茄转录因子SlNAC4影响了番茄果实成熟过程中的代谢。
In this study, SlNAC4 gene-silencing and wild-type tomato fruit were used for metabonomic investigation of tomato fruit metabolites at the breaking stage by using high performance liquid chromatography coupled to high-resolution tandem mass spectrometer(HPLC-HRMS/MS) in order to evaluate the effect of transcriptional factor SlNAC4 on tomato fruit metabolites during its ripening. The compounds were qualitatively analyzed by comparison of their mass spectral data with those in HMDB and KEGG databases as well as those of in-house reference standards using the XCMS software. The quantitative analysis and differential metabolite screening were carried out using metaX software. The results showed that a total of 67 differential metabolites that could match both the mass spectral and tandem mass spectral data(m/z) were found,including 19 amino acid derivatives and dipeptides, 7 vitamins, 4 carbohydrates and their derivatives, 11 nucleic acids and their derivatives, 4 alkaloids, 4 ?avors and 9 organic acids, 29 of which were up-regulated whereas the rest were signi?cantly down-regulated, indicating that SlNAC4 transcriptional factors affects tomato fruit metabolism during the ripening process.
In this study, SlNAC4 gene-silencing and wild-type tomato fruit were used for metabonomic investigation of tomato fruit metabolites at the breaking stage by using high performance liquid chromatography coupled to high-resolution tandem mass spectrometer(HPLC-HRMS/MS) in order to evaluate the effect of transcriptional factor SlNAC4 on tomato fruit metabolites during its ripening. The compounds were qualitatively analyzed by comparison of their mass spectral data with those in HMDB and KEGG databases as well as those of in-house reference standards using the XCMS software. The quantitative analysis and differential metabolite screening were carried out using metaX software. The results showed that a total of 67 differential metabolites that could match both the mass spectral and tandem mass spectral data(m/z) were found,including 19 amino acid derivatives and dipeptides, 7 vitamins, 4 carbohydrates and their derivatives, 11 nucleic acids and their derivatives, 4 alkaloids, 4 ?avors and 9 organic acids, 29 of which were up-regulated whereas the rest were signi?cantly down-regulated, indicating that SlNAC4 transcriptional factors affects tomato fruit metabolism during the ripening process.
引文
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[19]CAPANOGLU E,BEEKWILDER J,BOYACIOGLU D,et al.Changes in antioxidant and metabolite profiles during production of tomato paste[J].Food Chemistry,2008,56(3):964-973.DOI:10.1021/jf072990e.
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[27]FERNANDA R,ROMINA P,CLAUDIA F,et al.The increase in electrical conductivity of nutrient solution enhances compositional and sensory properties of tomato fruit cv.Patron[J].Scientia Horticulturae,2019,244:388-398.DOI:10.1016/j.scienta.2018.09.059.
[28]BAGHERIAN S A A,HAMZEHZARGHANI H,IZAPANAH K,et al.Effects of potato spindle tuber viroid infection on tomato metabolic profile[J].Journal Plant Physiology,2016,201:42-53.DOI:10.1016/j.jplph.2016.06.014.
[29]ROHRMANN J,TIHGE T,ALBA R,et al.Combined transcription factor profiling,microarray analysis and metabolite profiling reveals the transcriptional control of metabolic shifts occurring during tomato fruit development[J].The Plant Journal,2011,68(6):999-1013.DOI:10.1111/j.1365-313X.2011.04750.x.
[30]TOHGE T,FERNIE A R,Metabolomics-inspired insight into developmental,environmental and genetic aspects of tomato fruit chemical composition and quality[J].Plant Cell Physiology,2015,56(9):1681-1696.DOI:10.1093/pcp/pcv093.
[2]DUVAL M,HSIEH T F,KIM S Y,et al.Molecular characterization of At NAM:a member of the Arabidopsis NAC domain superfamily[J].Plant Molecular Biology,2002,50(2):237-248.DOI:10.1093/jxb/ers178.
[3]HAO Y J,SONG Q X,CHEN H W,et al.Plant NAC-type transcription factor proteins contain a NARD domain for repression of transcriptional activation[J].Planta,2010,232(5):1033-1043.DOI:10.1007/s00425-010-1238-2.
[4]姜秀明,牛义岭,许向阳.番茄NAC基因家族的系统进化及表达分析[J].分子植物育种,2016,14(8):1948-1964.DOI:10.13271/j.mpb.014.001948.
[5]陈秀玲,王傲雪,张珍珠,等.番茄转录因子NAC家族的鉴定及生物信息学分析[J].植物生理学报,2014,50(4):461-470.DOI:10.13592/j.cnki.ppj.2013.0477.
[6]RIAHI L,ZOGHLAMI N,DEREEPER A,et al.Single nucleotide polymorphism and haplotype diversity of the gene NAC4 in grapevine[J].Industrial Crops and Products,2013,43:718-724.DOI:10.1016/j.indcrop.2012.08.021.
[7]PANDURANGAIAH M,REDDY K E,RAO G L,et al.Cloning and expression analysis of MuNAC4 transcription factor protein from horsegram(Macrotyloma uniflorum(Lam.)Verdc.)conferred salt stress tolerance in Escherichia coli[J].Acta Physiol Plant,2012,35(1):139-146.DOI:10.1007/s11738-012-1056-1.
[8]PANDURANGAIAH M,RAO G L,SUDHAKARBABU O,et al.Overexpression of horsegram(Macrotyloma uniflorum Lam.Verdc.)NAC transcriptional factor(MuNAC4)in groundnut confers enhanced drought tolerance[J].Molecular Biotechnology,2014,56(8):758-769.DOI:10.1007/s12033-014-9754-0.
[9]蒋变玲.番茄转录因子NAC与番茄红素合成及软化关系研究[D].天津:天津大学,2017:5-10.
[10]SHAN W,KUANG J F,CHEN L,et al.Molecular characterization of banana NAC transcription factors and their interactions with ethylene signalling component EIL during fruit ripening[J].Journal of Experimental Botany,2012,63(14):1047-1051.DOI:10.1093/jxb/err313.
[11]VIDAL E A,áLVAREZL J M,GUTIéRRREZ R A,et al.Nitrate regulation of AFB3 and NAC4 gene expression in Arabidopsis roots depends on NRT1.1 nitrate transport function[J].Plant Signaling&Behavior,2014,9(6):e28501.DOI:10.4161/psb.28501.
[12]HAN X M,FENG Z Q,XING D D,et al.Two NAC transcription factors from Caragana intermedia altered salt tolerance of the transgenic Arabidopsis[J].BMC Plant Biology,2015,15:208.DOI:10.1186/s12870-015-0591-5.
[13]TIKUNOV Y,LOMMEN A,VOS C H R D,et al.A novel approach for nontargeted data analysis for metabolomics.Large-scale profiling of tomato fruit volatiles[J].Plant Physiol,2005,139(3):1125-1137.DOI:10.1104/pp.105.068130.
[14]RIEDELSHEIMER C,LISEC J,CZEDIK-EYSENBERG A,et al.Genome-wide association mapping of leaf metabolicprofiles for dissecting complex traits in maize[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(23):8872-8877.DOI:10.1073/pnas.1120813109.
[15]MEULEBROEK L V,BUSSCHE J V,STEPPE K,et al.Ultrahigh performance liquid chromatography coupled to high resolution Orbitrap mass spectrometry for metabolomic profiling of the endogenous phytohormonal status of the tomato plant[J].Journal of Chromatofraphy A,2012,1260:67-80.DOI:10.1016/j.chroma.2012.08.047.
[16]THISSENU,COULIER L,OVER KAMPKM,et al.A proper metabolomics strategy supports efficient food quality improvement:a case study on tomato sensory properties[J].Food Quality and Preference,2011,22(6):499-506.DOI:10.1016/j.foodqual.2010.12.001.
[17]TIEMAN D,BLISS P,MCLNTYRE L M,et al.The chemical interactions underlying tomato flavor preferences[J].Current Biology,2012,22(11):1035-1039.DOI:10.1016/j.cub.2012.04.016.
[18]SCHWAHN K,SOUZA L P D,FEMIE A R,et al.Metabolomicsassisted refinement of the pathways of steroidal glycoalkaloid biosynthesis in the tomato clade[J].Journal Integrative Plant Biology,2014,56(9):864-875.DOI:10.1111/jipb.12274.
[19]CAPANOGLU E,BEEKWILDER J,BOYACIOGLU D,et al.Changes in antioxidant and metabolite profiles during production of tomato paste[J].Food Chemistry,2008,56(3):964-973.DOI:10.1021/jf072990e.
[20]CALAFIORE R,RUGGIERI V,RAIOLA A,et al.Exploiting genomics resources to identify candidate genes underlying antioxidants content in tomato fruit[J].Front Plant Science,2016,7:397.DOI:10.3389/fpls.2016.00397.
[21]ENFISSI E M A,BARNECHE F,AHMED I,et al.Integrative transcript and metabolite analysis of nutritionally enhanced DE-ETIOLATED1 downregulated tomato fruit[J].Plant Cell,2010,22(4):1190-1215.DOI:10.1105/tpc.110.073866.
[22]BEMER M,KARLOVA R,BALLESTER A R,et al.The tomato FRUITFULL homologs TDR4/FUL1 and MBP7/FUL2 regulate ethylene-independent aspects of fruit ripening[J].The Plant Cell,2012,24(11):4437-4451.DOI:10.1105/tpc.112.103283.
[23]URSEM R,TIKUNOV Y,BOVY A,et al.A correlation network approach to metabolic data analysis for tomato fruits[J].Euphytica,2008,161(1/2):181-193.DOI:10.1007/s10681-008-9672-y.
[24]MEULEBROEK L V,BUSSCHE J V,CLERCQ N D,et al.A metabolomics approach to unravel the regulating role of phytohormones towards carotenoid metabolism in tomato fruit[J].Metabolomics,2015,11(3):667-683.DOI:10.1007/s11306-014-0728-9.
[25]BASTíAS A,YA?EZ M,OSORIO S,et al.The transcription factor AREB1 regulates primary metabolic pathways in tomato fruits[J].Jounal Experimental Botany,2014,65(9):2351-2363.DOI:10.1093/jxb/eru114.
[26]BASTíAS A,LóPEZ-CLIMENT M,VALCARCEL M,at al.Modulation of organic acids and sugar content in tomato fruits by an abscisic acid-regulated transcription factor[J].Physiologia Plantarum,2011,141(3):215-226.DOI:10.1111/j.1399-3054.2010.01435.x.
[27]FERNANDA R,ROMINA P,CLAUDIA F,et al.The increase in electrical conductivity of nutrient solution enhances compositional and sensory properties of tomato fruit cv.Patron[J].Scientia Horticulturae,2019,244:388-398.DOI:10.1016/j.scienta.2018.09.059.
[28]BAGHERIAN S A A,HAMZEHZARGHANI H,IZAPANAH K,et al.Effects of potato spindle tuber viroid infection on tomato metabolic profile[J].Journal Plant Physiology,2016,201:42-53.DOI:10.1016/j.jplph.2016.06.014.
[29]ROHRMANN J,TIHGE T,ALBA R,et al.Combined transcription factor profiling,microarray analysis and metabolite profiling reveals the transcriptional control of metabolic shifts occurring during tomato fruit development[J].The Plant Journal,2011,68(6):999-1013.DOI:10.1111/j.1365-313X.2011.04750.x.
[30]TOHGE T,FERNIE A R,Metabolomics-inspired insight into developmental,environmental and genetic aspects of tomato fruit chemical composition and quality[J].Plant Cell Physiology,2015,56(9):1681-1696.DOI:10.1093/pcp/pcv093.