基于转录组研究MPEF对毕赤酵母的致死机理
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摘要
探究微芯片脉冲电场(microchip pulse electric field,MPEF)技术对毕赤酵母的致死效果和致死机理。在400 V电压和80个脉冲条件下,MPEF技术可以很好地钝化毕赤酵母。借助新一代高通量测序技术对MPEF处理前后毕赤酵母基因的表达水平进行分析,将差异表达基因在基因本体(gene ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)数据库中进行比对。测序结果表明,MPEF组和对照组差异变化显著的基因共794个,其中上调基因361个,下调基因433个;在MPEF作用下,半胱天冬蛋白酶活性、丙酮酸转运等显著上调,而核苷酸代谢、核糖体生物合成、蛋白质转录翻译、RNA聚合酶活性、超氧化物歧化酶活性、跨膜运输调节、维生素代谢等显著下调。采用实时荧光定量聚合酶链式反应对6个差异基因进行验证,得到与转录组测序结果一致的基因表达量变化趋势。毕赤酵母体内细胞结构的破坏、基因损伤、蛋白质合成和功能受限以及酶活性的改变可能是MPEF处理导致其死亡的主要原因。
In this work, the effect of microchip pulse electric field(MPEF) treatment on inactivating Pichia and the underlying mechanism were investigated. A good inactivation of Pichia was achieved after 80 pulses at 400 V. The differentially expressed genes of Pichia before and after MPEF treatment were analyzed using new generation of highthroughput sequencing, and they were aligned to the gene ontology(GO) and the Kyoto Encyclopedia of Genes and Genomes(KEGG) databases. Results showed that a total of significantly differentially expressed 794 genes between the MPEF and control groups were found, 361 genes of which were up-regulated while 433 were down-regulated. After MPEF treatment,caspase activity and pyruvate transport raised significantly whereas nucleotide metabolism, ribosomal biosynthesis, protein transcription and translation, RNA polymerase activity, superoxide dismutase activity, transmembrane transport regulation,vitamin metabolism were significantly down-regulated. Furthermore, 6 differentially expressed genes were confirmed by real-time fluorescence quantitative polymerase chain reaction(q-PCR), achieving the same gene expression patterns as the transcriptome sequencing results. Destruction of cell structure, gene damage, restriction of protein synthesis and function,and changes in enzyme activities might be the main causes of death for Pichia after MPEF treatment.
In this work, the effect of microchip pulse electric field(MPEF) treatment on inactivating Pichia and the underlying mechanism were investigated. A good inactivation of Pichia was achieved after 80 pulses at 400 V. The differentially expressed genes of Pichia before and after MPEF treatment were analyzed using new generation of highthroughput sequencing, and they were aligned to the gene ontology(GO) and the Kyoto Encyclopedia of Genes and Genomes(KEGG) databases. Results showed that a total of significantly differentially expressed 794 genes between the MPEF and control groups were found, 361 genes of which were up-regulated while 433 were down-regulated. After MPEF treatment,caspase activity and pyruvate transport raised significantly whereas nucleotide metabolism, ribosomal biosynthesis, protein transcription and translation, RNA polymerase activity, superoxide dismutase activity, transmembrane transport regulation,vitamin metabolism were significantly down-regulated. Furthermore, 6 differentially expressed genes were confirmed by real-time fluorescence quantitative polymerase chain reaction(q-PCR), achieving the same gene expression patterns as the transcriptome sequencing results. Destruction of cell structure, gene damage, restriction of protein synthesis and function,and changes in enzyme activities might be the main causes of death for Pichia after MPEF treatment.
引文
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[35]陈罡.谷氨酸纳对哺乳仔猪蛋白质和脂肪代谢影响的研究[D].长沙:湖南农业大学,2013:34-35.
[36]DéMOULIN D,CARLIER M F,BIBETTE J,et al.Power transduction of actin filaments ratcheting in vitro against a load[J].Proceedings of the National Academy of Sciences,2014,111(50):17845-17850.DOI:10.1073/pnas.1414184111.
[37]DE POUPLANA L R,SANTOS M A S,ZHU J H,et al.Protein mistranslation:friend or foe?[J].Trends in Biochemical Sciences,2014,39(8):355-362.DOI:10.1016/j.tibs.2014.06.002.
[38]ZHANG W,CHEN C,SHI H,et al.Curcumin is a biologically active copper chelator with antitumor activity[J].Phytomedicine,2016,23(1):1-8.DOI:10.1016/j.phymed.2015.11.005.
[39]KHAN H Y,ZUBAIR H,FAISAL M,et al.Plant polyphenol induced cell death in human cancer cells involves mobilization of intracellular copper ions and reactive oxygen species generation:a mechanism for cancer chemopreventive action[J].Molecular Nutrition&Food Research,2014,58(3):437-446.DOI:10.1002/mnfr.201300417.
[40]CHUBUKOV V,GEROSA L,KOCHANOWSKI K,et al.Coordination of microbial metabolism[J].Nature Reviews Microbiology,2014,12(5):327-340.DOI:10.1038/nrmicro3238.
[41]GREEN D R,GALLUZZI L,KROEMER G.Metabolic control of cell death[J].Science,2014,345:1250256.DOI:10.1126/science.1250256.
[2]MCAULET C M,SINGH T K,HARO-MAZA J F,et al.Microbiological and physicochemical stability of raw,pasteurised or pulsed electric field-treated milk[J].Innovative Food Science&Emerging Technologies,2016,38:365-373.DOI:10.1016/j.ifset.2016.09.030.
[3]AADIL R M,ZENG X A,ALI A,et al.Influence of different pulsed electric field strengths on the quality of the grapefruit juice[J].International Journal of Food Science&Technology,2015,50(10):2290-2296.DOI:10.1111/ijfs.12891.
[4]吴碧秀,吴剑峰,李灼坤.高压脉冲电场技术在果蔬汁中杀菌的研究进展[J].现代食品,2016,7(13):15-16.DOI:10.16736/j.cnki.cn41-1434/ts.2016.13.007.
[5]KAMAT V,PANDEY S,PAKNIKAR K,et al.A facile one-step method for cell lysis and DNA extraction of waterborne pathogens using a microchip[J].Biosensors and Bioelectronics,2018,99:62-69.DOI:10.1016/j.bios.2017.07.040.
[6]YAN S,CHEN P,ZENG X,et al.Integrated multifunctional electrochemistry microchip for highly efficient capture,release,lysis,and analysis of circulating tumor cells[J].Analytical Chemistry,2017,89(22):12039-12044.DOI:10.1021/acs.analchem.7b02469.
[7]SANCHEZ-MORENO C,DE ANCOS B,PLAZA L,et al.Nutritional approaches and health-related properties of plant foods processed by high pressure and pulsed electric fields[J].Critical Reviews in Food Science and Nutrition,2009,49(6):552-576.DOI:10.1080/10408390802145526.
[8]LIU L,ZHAO L,YANG J,et al.Low-voltage pulsed electric field sterilization on a microfluidic chip[J].Electroanalysis,2013,25(5):1301-1309.DOI:10.1002/elan.201200648.
[9]ZHU N,WANG Y,ZHU Y,et al.Design of a treatment chamber for low-voltage pulsed electric field sterilization[J].Innovative Food Science&Emerging Technologies,2017,42:180-189.DOI:10.1016/j.ifset.2017.07.016.
[10]CHUECA B,PAGáN R,GARCíA-GONZALO D.Transcriptomic analysis of Escherichia coli MG1655 cells exposed to pulsed electric fields[J].Innovative Food Science&Emerging Technologies,2015,29:78-86.DOI:10.1016/j.ifset.2014.09.003.
[11]ROZALI S N M,MILANI E A,DEED R C,et al.Bacteria,mould and yeast spore inactivation studies by scanning electron microscope observations[J].International Journal of Food Microbiology,2017,263:17-25.DOI:10.1016/j.ijfoodmicro.2017.10.008.
[12]WANG L H,WANG M S,ZENG X A,et al.Temperaturemediated variations in cellular membrane fatty acid composition of Staphylococcus aureus in resistance to pulsed electric fields[J].Biochimica et Biophysica Acta(BBA)-Biomembranes,2016,1858(8):1791-1800.DOI:10.1016/j.bbamem.2016.05.003.
[13]YUN O,LIU Z W,ZENG X A,et al.Salmonella typhimurium resistance on pulsed electric fields associated with membrane fluidity and gene regulation[J].Innovative Food Science&Emerging Technologies,2016,36:252-259.DOI:10.1016/j.ifset.2016.06.013.
[14]MORTAZAVI A,WILLIAMS B A,MCCUE K,et al.Mapping and quantifying mammalian transcriptomes by RNA-Seq[J].Nature Methods,2008,5(7):621-628.DOI:10.1038/nmeth.1226.
[15]张石磊,翟向和,王春光,等.小檗碱对禽源大肠杆菌抑菌作用及耐药消除作用的转录组学分析[J].中国畜牧兽医,2017,44(5):1518-1525.DOI:10.16431/j.cnki.1671-7236.2017.05.036.
[16]朱健铭,翁幸鐾,姜如金,等.黄芩水煎剂对尿道致病性大肠埃希菌的转录组影响分析[J].中草药,2017,48(9):1791-1801.DOI:10.7501/j.issn.0253-2670.2017.09.015.
[17]赵心清,张明明,徐桂红,等.酿酒酵母乙酸耐性分子机制的功能基因组进展[J].生物工程学报,2014,30(3):368-380.DOI:10.13345/j.cjb.130469.
[18]GARCíA D,GóMEZ N,CONDóN S,et al.Pulsed electric fields cause sublethal injury in Escherichia coli[J].Letters in Applied Microbiology,2003,36(3):140-144.DOI:10.1046/j.1472-765X.2003.01282.x.
[19]WAITE-CUSIC J G,DIONO B H S,YOUSEF A E.Screening for Listeria monocytogenes surrogate strains applicable to food processing by ultrahigh pressure and pulsed electric field[J].Journal of Food Protection,2011,74(10):1655-1661.DOI:10.4315/0362-028X.JFP-11-099.
[20]KANEHISA M,GOTO S,SATO Y,et al.Data,information,knowledge and principle:back to metabolism in KEGG[J].Nucleic Acids Research,2013,42(D1):D199-D205.DOI:10.1093/nar/gkt1076.
[21]PERNI S,CHALISE P R,SHAMA G,et al.Bacterial cells exposed to nanosecond pulsed electric fields show lethal and sublethal effects[J].International Journal of Food Microbiology,2007,120(3):311-314.DOI:10.1016/j.ijfoodmicro.2007.10.002.
[22]WANG M S,ZENG X A,SUN D W,et al.Quantitative analysis of sublethally injured Saccharomyces cerevisiae cells induced by pulsed electric fields[J].LWT-Food Science and Technology,2015,60(2):672-677.DOI:10.1016/j.lwt.2014.09.028.
[23]ZHAO W,YANG R,SHEN X,et al.Lethal and sublethal injury and kinetics of Escherichia coli,Listeria monocytogenes and Staphylococcus aureus in milk by pulsed electric fields[J].Food Control,2013,32(1):6-12.DOI:10.1016/j.foodcont.2012.11.029.
[24]RAIVIO T L,LEBLANC S K D,PRICE N L.The Escherichia coli Cpx envelope stress response regulates genes of diverse function that impact antibiotic resistance and membrane integrity[J].Journal of Bacteriology,2013,195(12):2755-2767.DOI:10.1128/JB.00105-13.
[25]NEPPER J F,WEIBEL D B.Membrane fluidity modulates biofilm formation in E.coli via the Rcs phosphorelay[J].Biophysical Journal,2017,112(3):281.DOI:10.1016/j.bpj.2016.11.1523.
[26]LIU Z W,ZENG X A,NGADI M,et al.Effect of cell membrane fatty acid composition of Escherichia coli on the resistance to pulsed electric field(PEF)treatment[J].LWT-Food Science and Technology,2017,76:18-25.DOI:10.1016/j.lwt.2016.10.019.
[27]FERRARIO M,GUERRERO S,ALZAMORA S M.Study of pulsed light-induced damage on Saccharomyces cerevisiae in apple juice by flow cytometry and transmission electron microscopy[J].Food and Bioprocess Technology,2014,7(4):1001-1011.DOI:10.1007/s11947-013-1121-9.
[28]DAKIK P,TITORENKO V I.Communications between mitochondria,the nucleus,vacuoles,peroxisomes,the endoplasmic reticulum,the plasma membrane,lipid droplets,and the cytosol during yeast chronological aging[J].Frontiers in Genetics,2016,7:177.DOI:10.3389/fgene.2016.00177.
[29]ROOS W P,KAINA B.DNA damage-induced cell death:from specific DNA lesions to the DNA damage response and apoptosis[J].Cancer Letters,2013,332(2):237-248.DOI:10.1016/j.canlet.2012.01.007.
[30]BRAR G A,WEISSMAN J S.Ribosome profiling reveals the what,when,where and how of protein synthesis[J].Nature Reviews Molecular Cell Biology,2015,16(11):651-664.DOI:10.1038/nrm4069.
[31]SANO R,REED J C.ER stress-induced cell death mechanisms[J].Biochimica et Biophysica Acta(BBA)-Molecular Cell Research,2013,1833(12):3460-3470.DOI:10.1016/j.bbamcr.2013.06.028.
[32]MCILWAIN D R,BERGER T,MAK T W.Caspase functions in cell death and disease[J].Cold Spring Harbor Perspectives in Biology,2013,5(4):a008656.DOI:10.1101/cshperspect.a008656.
[33]CANOVA M J,MOLLE V.Bacterial serine/threonine protein kinases in host-pathogen interactions[J].Journal of Biological Chemistry,2014,289(14):9473-9479.DOI:10.1074/jbc.R113.529917.
[34]SHAHID F,FAROOQUI Z,ABIDI S,et al.Oral administration of thymoquinone mitigates the effect of cisplatin on brush border membrane enzymes,energy metabolism and antioxidant system in rat intestine[J].Biomedicine&Pharmacotherapy,2017,94:1111-1120.DOI:10.1016/j.biopha.2017.08.044.
[35]陈罡.谷氨酸纳对哺乳仔猪蛋白质和脂肪代谢影响的研究[D].长沙:湖南农业大学,2013:34-35.
[36]DéMOULIN D,CARLIER M F,BIBETTE J,et al.Power transduction of actin filaments ratcheting in vitro against a load[J].Proceedings of the National Academy of Sciences,2014,111(50):17845-17850.DOI:10.1073/pnas.1414184111.
[37]DE POUPLANA L R,SANTOS M A S,ZHU J H,et al.Protein mistranslation:friend or foe?[J].Trends in Biochemical Sciences,2014,39(8):355-362.DOI:10.1016/j.tibs.2014.06.002.
[38]ZHANG W,CHEN C,SHI H,et al.Curcumin is a biologically active copper chelator with antitumor activity[J].Phytomedicine,2016,23(1):1-8.DOI:10.1016/j.phymed.2015.11.005.
[39]KHAN H Y,ZUBAIR H,FAISAL M,et al.Plant polyphenol induced cell death in human cancer cells involves mobilization of intracellular copper ions and reactive oxygen species generation:a mechanism for cancer chemopreventive action[J].Molecular Nutrition&Food Research,2014,58(3):437-446.DOI:10.1002/mnfr.201300417.
[40]CHUBUKOV V,GEROSA L,KOCHANOWSKI K,et al.Coordination of microbial metabolism[J].Nature Reviews Microbiology,2014,12(5):327-340.DOI:10.1038/nrmicro3238.
[41]GREEN D R,GALLUZZI L,KROEMER G.Metabolic control of cell death[J].Science,2014,345:1250256.DOI:10.1126/science.1250256.