低温胁迫差异表达基因在佛手和枳中的半定量RT-PCR分析
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摘要
以佛手(Citrus medica L.var.sarcodactylis Swingle)和枳(Poncirus trifoliata Raf.)为试材,-4℃低温处理24 h后,采用半定量RT-PCR技术,研究佛手34个低温胁迫差异表达基因在佛手和枳中的表达情况,通过比较获得佛手低温敏感相关基因,分析两者抗寒性差异原因。结果表明,佛手和枳中变化趋势不同的基因有17个,其中14个基因在佛手中表达量有变化,枳中表达量不变,3个基因在佛手与枳中表现出完全相反的变化趋势;8个基因变化趋势相同;9个基因在枳中未检测到。推测17个变化趋势不同的基因可能与佛手低温敏感相关,它们所介导的分子途径可为揭示佛手低温敏感提供有力证据;9个枳中未检测到的基因是佛手中特异表达基因,可能是引起佛手低温敏感,造成佛手和枳抗寒性差异的关键基因。
Aimed to investigate cold sensitive genes in fingered citron(Citrus medica var. sarcodactylis Swingle) and unveil the differences of cold resistance mechanism between C. medica and Poncirus trifoliata. The expression patterns of 34 cold-stress induced genes in C. medica and P. trifoliata were analyzed by semi-quantitative RT-PCR after being exposed to -4 ℃ for 24 h. The results showed that the expression levels of 17 genes were different between C.medica and P. trifoliata,among which the transcripts levels of 14 genes changed response to cold-stress,while no changes were observed in P. trifoliate simultaneously. The transcript levels of 3 genes were found with contrary changing models in two plant species. Besides,the expression of 8 genes in C. medica shared similar pattern as in P. trifoliata,and 9 genes failed to be amplified in P. trifoliata. Thus,it was suggested that the 17 genes differentially expressed in these two plant species were related to cold sensitivity of C. medica. The pathway they were involved in might be the important molecular mechanism regarding cold sensitivity of C. medica. Nine genes with different expres-sion levels induced by cold-stress in C. medica,which could not be amplified in P. trifoliata,might be the important genes involved in the cold sensibility of C. medica and the important reason resulted in the different cold resistance between C. medica and P. trifoliata.
Aimed to investigate cold sensitive genes in fingered citron(Citrus medica var. sarcodactylis Swingle) and unveil the differences of cold resistance mechanism between C. medica and Poncirus trifoliata. The expression patterns of 34 cold-stress induced genes in C. medica and P. trifoliata were analyzed by semi-quantitative RT-PCR after being exposed to -4 ℃ for 24 h. The results showed that the expression levels of 17 genes were different between C.medica and P. trifoliata,among which the transcripts levels of 14 genes changed response to cold-stress,while no changes were observed in P. trifoliate simultaneously. The transcript levels of 3 genes were found with contrary changing models in two plant species. Besides,the expression of 8 genes in C. medica shared similar pattern as in P. trifoliata,and 9 genes failed to be amplified in P. trifoliata. Thus,it was suggested that the 17 genes differentially expressed in these two plant species were related to cold sensitivity of C. medica. The pathway they were involved in might be the important molecular mechanism regarding cold sensitivity of C. medica. Nine genes with different expres-sion levels induced by cold-stress in C. medica,which could not be amplified in P. trifoliata,might be the important genes involved in the cold sensibility of C. medica and the important reason resulted in the different cold resistance between C. medica and P. trifoliata.
引文
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[24]Zhang S,Gu YQ,Singh J,et al.New insights into Oryza genome evolution:high gene colinearity and differential retrotransposon amplification[J].Plant Molecular Biology,2007,64(5):589-600.
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[26]Borutova R,Faix S,Placha I,et al.Effects of deoxynivalenol and zearalenone on oxidative stress and blood phagocytic activity in broilers[J].Plant Physiology,2008,62(4):303-312.
[27]石晓冰,魏家绵,沈允钢.叶绿体ATP合酶CF1与CF0亚基间的相互作用[J].科学通报,2001,46(18):1550-1554.
[28]Zhao Y,Davis RE,Wei W,et al.Phytoplasma genomes:Evolution through mutually complementary mechanisms,gene loss and horizontal acquisition[M]//Gross DC,Lichens-Park A,Kole C.Genomics of plant-associated bacteria.Berlin:Springer Berlin Heidelberg,2014,17:235-271.
[29]Kasetsomboon T,Kate-Ngam S,Sriwongchai T,et al.Sequence variation of avirulence gene AVR-Pita1 in rice blast fungus,Magnaporthe oryzae[J].Mycological Progress,2013,12(4):617-628.
[30]Luo LN,Herrin DL.A novel rhodanese is required to maintain chloroplast translation in Chlamydomonas[J].Plant Molecular Biology,2012,79(4):495-508.
[31]Zhang JH,Li XS,He ZM,et al.Molecular character of a phosphatase 2C(PP2C)gene relation to stress tolerance in Arabidopsis thaliana[J].Molecular Biology Reports,2013,40(3):2633-2644.
[32]Chakraborty N,Ohta M,Zhu JK.Recognition of a PP2C interaction motif in several plant protein kinases[J].Methods in Molecular Biology,2007,365(3):287-298.
[33]Stern A,Privman E,Rasis M,et al.Evolution of the metazoan protein phosphatase 2C superfamily[J].Journal of Molecular Evolution,2007,64(1):61-70.
[34]Nogueira FT,Rosa VE,Menossi M,et al.RNA expression profiles and data mining of sugarcane response to low temperature[J].Plant Physiology,2003,132(4):1811-1824.
[35]Johnston SL,Prakash R,Chen NJ,et al.An enzyme activity capable of endotransglycosylation of heteroxylan polysaccharides is present in plant primary cell walls[J].Planta,2013,237(1):173-187.
[36]Wilsker D,Petermann E,Helleday T,et al.Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control[J].Proceeding of National Academic Science(USA),2008,105(52):20752-20757.
[37]Rikhvanov EG,Fedoseeva IV,Pyatrikas DV,et al.Role of mitochondria in the operation of calcium signaling system in heat-stressed plants[J].Russian Journal of Plant Physiology,2014,61(2):141-153.
[38]Moussatova A,Kandt C,O'Mara ML,et al.ATP-binding cassette transporters in Escherichia coli[J].Biochimica et Biophysica Acta(BBA)-Biomembranes,2008,1778(9):1757-1771.
[39]Hannivoort RA,Dunning S,Vander Borght S,et al.Multidrug resistance-associated proteins are crucial for the viability of activated rat hepatic stellate cells[J].Hepatology,2008,48(2):624-634.
[40]Lu H,Klaassen C.Gender differences in mRNA expression of ATP-binding cassette efflux and bile acid transporters in kidney,liver,and intestine of 5/6 nephrectomized rats[J].Drug Metab Dispos,2008,36(1):16-23.
[41]Holt BF,Youssef B,Dangl JL.Antagonistic control of disease resistance protein stability in the plant immune system[J].Science,2005,309(5736):929-932.
[2]Chen WR,Zheng JS,Li YQ,et al.Effects of high temperature on photosynthesis,chlorophyll fluorescence,chloroplast ultrastructure,and antioxidant activities in fingered citron[J].Russian Journal of Plant Physiology,2012,59(6):732-740.
[3]郭卫东,张真真,蒋小韦,等.低温胁迫下佛手半致死温度测定和抗寒性分析[J].园艺学报,2009,36(1):81-86.
[4]Hincha DK,Zuther E.Introduction:plant cold acclimation and freezing tolerance[J].Plant Cold Acclimation Methods in Molecular Biology,2014,1166:1-6.
[5]Weiser CJ.Cold resistance and injury in woody plants[J].Science,1970,69(3952):1269-1273.
[6]Champ KI,Vicente JF,Gloria AM.The role of CBF transcriptional activators in two Citrus species(Poncirus and Citrus)with contrasting levels of freezing tolerance[J].Physiologia Plantarum,2007,129(3):529-541.
[7]Jia Y,Rio HS,Robbins AL,et al.Cloning and sequence analysis of a low temperature induced gene from trifoliata orange with unusual pre-mRNA processing[J].Plant Cell Reports,2004,23(3):159-166.
[8]Porat R.Identification of a grapefruit c DNA belonging to a unique class of citrus dehydrins and characterization of its expression patterns under temperature stress conditions[J].Physiologia Plantarum,2002,115(4):598-603.
[9]He LG,Wang HL,Liu DC,et al.Isolation and expression of a cold-responsive gene Pt CBF in Poncirus trifoliata and isolation of citrus CBF promoters[J].Biologia Plantarum,2014,56(3):484-492.
[10]Long GY,Song JY,Deng ZN,et al.Ptcorp gene induced by cold stress was identified by proteomic analysis in leaves of Poncirus trifoliata(L.)Raf[J].Molecular Biology Reports,2012,39(5):5859-5866.
[11]Sahin-Cevik M,Moore GA.Two AP2domain containing genes isolated from the cold-hardy citrus relative Poncirus trifoliata are induced in response to cold[J].Functional Plant Biology,2006,33(9):863-875.
[12]曹诣斌,石瑞,陈文荣,等.低温胁迫下佛手和枳乙烯应答因子6(ERF6)表达变化的比较分析[J].园艺学报,2011,38(10):1873-1882.
[13]叶杰君.柑橘类植物EF6和GRAS基因的低温胁迫应答及启动子序列分析[D].金华:浙江师范大学,2013.
[14]Ribeiro RV,Espinoza-Núez E.Junior JP,et al.Citrus rootstocks for improving the horticultural performance and physiological responses under constraining environments[J].Improvement of Crops in the Era of Climatic Changes,2014,33(1):22-37.
[15]Lang P,Zhang CK,Ebel RC,et al.Identification of cold acclimated genes in leaves of Citrus unshiu by mRNA differential display[J].Gene,2005,359(45):111-118.
[16]Ron P,Konstantinos P,Dafna R,et al.Isolation of a dehydrin c DNA from orange and grapefruit citrus fruit that is specifically induced by the combination of heat followed by chilling temperatures[J].Physiologia Plantarum,2004,120(2):256-264.
[17]Sanchez-Ballesta MT.Involvement of phenylalanine ammonialyase in the response of Fortune mandarin fruits to cold temperature[J].Physiologia Plantarum,2000,108(4):382-389.
[18]刘祖祺,张石诚.植物抗性生理学[M].北京:中国农业出版社,1994:8-29.
[19]Chhandak B.PCR primer design[M].New York:Human Press,2015:31-56.
[20]Chuang LY,Cheng YH,Yang CH.Specific primer design for the polymerase chain reaction[J].Biotechnology Letters,2013,35(10):1541-1549.
[21]Lommel L,Chen L,Madura K.The 26S proteasome negatively regulates the level of over all genomic nucleotide excision repair[J].Nucleic Acids Research,2000,28(6):4839-4845.
[22]Peng ZH,Staub JM,Serino G.The cellular level of PR500,a protein complex related to the 19S regulatory particle of the proteasome,is regulated in response to stresses in plants[J].Molecular Biology of the Cell,2001,12(10):383.
[23]Su YC,Xu LP,Xue BT,et al.Molecular cloning and characterization of two pathogenesis-relatedβ-1,3-glucanase genes Sc Glu A1 and Sc Glu D1 from sugarcane infected by Sporisorium scitamineum[J].Plant Cell Reports,2013,32(10):1503-1519.
[24]Zhang S,Gu YQ,Singh J,et al.New insights into Oryza genome evolution:high gene colinearity and differential retrotransposon amplification[J].Plant Molecular Biology,2007,64(5):589-600.
[25]Gawryluk RM,Gray MW.Evidence for an early evolutionary emergence ofγ-type carbonic anhydrases as components of mitochondrial respiratory complex I[J].BMC Evolutionary Biology,2010,10(4):176.
[26]Borutova R,Faix S,Placha I,et al.Effects of deoxynivalenol and zearalenone on oxidative stress and blood phagocytic activity in broilers[J].Plant Physiology,2008,62(4):303-312.
[27]石晓冰,魏家绵,沈允钢.叶绿体ATP合酶CF1与CF0亚基间的相互作用[J].科学通报,2001,46(18):1550-1554.
[28]Zhao Y,Davis RE,Wei W,et al.Phytoplasma genomes:Evolution through mutually complementary mechanisms,gene loss and horizontal acquisition[M]//Gross DC,Lichens-Park A,Kole C.Genomics of plant-associated bacteria.Berlin:Springer Berlin Heidelberg,2014,17:235-271.
[29]Kasetsomboon T,Kate-Ngam S,Sriwongchai T,et al.Sequence variation of avirulence gene AVR-Pita1 in rice blast fungus,Magnaporthe oryzae[J].Mycological Progress,2013,12(4):617-628.
[30]Luo LN,Herrin DL.A novel rhodanese is required to maintain chloroplast translation in Chlamydomonas[J].Plant Molecular Biology,2012,79(4):495-508.
[31]Zhang JH,Li XS,He ZM,et al.Molecular character of a phosphatase 2C(PP2C)gene relation to stress tolerance in Arabidopsis thaliana[J].Molecular Biology Reports,2013,40(3):2633-2644.
[32]Chakraborty N,Ohta M,Zhu JK.Recognition of a PP2C interaction motif in several plant protein kinases[J].Methods in Molecular Biology,2007,365(3):287-298.
[33]Stern A,Privman E,Rasis M,et al.Evolution of the metazoan protein phosphatase 2C superfamily[J].Journal of Molecular Evolution,2007,64(1):61-70.
[34]Nogueira FT,Rosa VE,Menossi M,et al.RNA expression profiles and data mining of sugarcane response to low temperature[J].Plant Physiology,2003,132(4):1811-1824.
[35]Johnston SL,Prakash R,Chen NJ,et al.An enzyme activity capable of endotransglycosylation of heteroxylan polysaccharides is present in plant primary cell walls[J].Planta,2013,237(1):173-187.
[36]Wilsker D,Petermann E,Helleday T,et al.Essential function of Chk1 can be uncoupled from DNA damage checkpoint and replication control[J].Proceeding of National Academic Science(USA),2008,105(52):20752-20757.
[37]Rikhvanov EG,Fedoseeva IV,Pyatrikas DV,et al.Role of mitochondria in the operation of calcium signaling system in heat-stressed plants[J].Russian Journal of Plant Physiology,2014,61(2):141-153.
[38]Moussatova A,Kandt C,O'Mara ML,et al.ATP-binding cassette transporters in Escherichia coli[J].Biochimica et Biophysica Acta(BBA)-Biomembranes,2008,1778(9):1757-1771.
[39]Hannivoort RA,Dunning S,Vander Borght S,et al.Multidrug resistance-associated proteins are crucial for the viability of activated rat hepatic stellate cells[J].Hepatology,2008,48(2):624-634.
[40]Lu H,Klaassen C.Gender differences in mRNA expression of ATP-binding cassette efflux and bile acid transporters in kidney,liver,and intestine of 5/6 nephrectomized rats[J].Drug Metab Dispos,2008,36(1):16-23.
[41]Holt BF,Youssef B,Dangl JL.Antagonistic control of disease resistance protein stability in the plant immune system[J].Science,2005,309(5736):929-932.