黑腹果蝇white基因在H_2O_2影响下卷翅形成中的作用及机理
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摘要
卷翅是果蝇遗传学上最常用的标记之一,但卷翅形成的具体机制还不清楚.过去的研究发现,理化刺激影响果蝇卷翅的形成.我们最近研究发现,H_2O_2处理不仅会影响果蝇的羽化率,还会使其出现卷翅现象.本研究通过改变H_2O_2浓度、果蝇培养温度和H_2O_2处理时间,探讨影响黑腹果蝇卷翅形成的具体因素,并对其超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-PX)活力进行检测,探讨H_2O_2对果蝇抗氧化能力的影响.结果表明:果蝇的羽化率与H_2O_2浓度成反比.温度、H_2O_2浓度和H_2O_2处理时间的改变均会影响果蝇翅的卷曲程度和卷翅果蝇所占的比例.其中white基因突变果蝇对这3种条件反应最明显,mini-white(white基因回复突变)果蝇却可以拯救该表型,它的反应与野生型OR相似.H_2O_2对含Cy基因的果蝇卷翅的形成也有一定的影响,可以加大果蝇翅的卷曲程度.对SOD、CAT和GSH-PX活力检测发现,H_2O_2处理会使果蝇的抗氧化能力降低.实时荧光定量PCR检测发现,H_2O_2处理会导致果蝇基因表达量发生改变.黑腹果蝇卷翅形成是一个十分复杂的过程,H_2O_2可能作为某种信号分子或是间接影响某种因子参与黑腹果蝇的卷翅形成过程.该卷翅形成过程可能与Cy基因导致的果蝇卷翅过程是同一个信号途径,两者也可能是通过不同的模式进行调控的.
Curly wing is one of the most frequently used genetic markers in Drosophila melanogaster,but its molecular mechanism is remained unclear. Previous results have showed that physicochemical stimulation would affect the formation of the cruly wing. Our recent study found that H_2O_2 could not only affect the eclosion rate of D. melanogaster,but also induce the formation of curly wing. Here,we aimed to uncover the specific factors influencing the formation of curly wing in D.melanogaster via changing the concentration of H_2O_2 and the temperature as well as the time of H_2O_2 treatment. We measured the activities of superoxide dismutase( SOD),catalase( CAT) and glutathione peroxidase( GSH-PX),in order to examine the effects of H_2O_2 on antioxidative capacity of D. melanogaster. The results showed that the eclosion rate of D. melanogaster was inversely correlated with the concentration of H_2O_2. The change of temperature,H_2O_2 concentration and the period of H_2O_2 treatment affected the degree of the curl and the proportion of the curly wing. The white mutant flies responded most significantly to these three conditions,the mini-white( white gene reverse mutation) flies could rescue the curly phenotype,and responded similarly to the wild type OR.H_2O_2 had effects on the formation of the curly wing which contained the Cy mutation,leading to increased rate of the curly wing. D. melanogaster treated with H_2O_2 would reduce the antioxidative capacity. Results from real-time quantitative PCR showed that H_2O_2 treatment resulted in a change in gene expression. The formation of curly wing was a complicated process,and H_2O_2 might act as asignaling molecule or indirectly affect certain factors in the formation of curly wing in D. melanogaster. This process might share the same signaling pathway with the Cy mutant,or might be regulated by different modulating patterns.
Curly wing is one of the most frequently used genetic markers in Drosophila melanogaster,but its molecular mechanism is remained unclear. Previous results have showed that physicochemical stimulation would affect the formation of the cruly wing. Our recent study found that H_2O_2 could not only affect the eclosion rate of D. melanogaster,but also induce the formation of curly wing. Here,we aimed to uncover the specific factors influencing the formation of curly wing in D.melanogaster via changing the concentration of H_2O_2 and the temperature as well as the time of H_2O_2 treatment. We measured the activities of superoxide dismutase( SOD),catalase( CAT) and glutathione peroxidase( GSH-PX),in order to examine the effects of H_2O_2 on antioxidative capacity of D. melanogaster. The results showed that the eclosion rate of D. melanogaster was inversely correlated with the concentration of H_2O_2. The change of temperature,H_2O_2 concentration and the period of H_2O_2 treatment affected the degree of the curl and the proportion of the curly wing. The white mutant flies responded most significantly to these three conditions,the mini-white( white gene reverse mutation) flies could rescue the curly phenotype,and responded similarly to the wild type OR.H_2O_2 had effects on the formation of the curly wing which contained the Cy mutation,leading to increased rate of the curly wing. D. melanogaster treated with H_2O_2 would reduce the antioxidative capacity. Results from real-time quantitative PCR showed that H_2O_2 treatment resulted in a change in gene expression. The formation of curly wing was a complicated process,and H_2O_2 might act as asignaling molecule or indirectly affect certain factors in the formation of curly wing in D. melanogaster. This process might share the same signaling pathway with the Cy mutant,or might be regulated by different modulating patterns.
引文
[1]Ward L.The genetics of curly wing in Drosophila.Another case of balanced lethal factors.Genetics,1923,8:276-300
[2]Waddington CH.The genetic control of wing development in Drosophila.Genetics,1940,41:75-139
[3]Gr9nke S,Bickmeyer I,Wunderlich R,et al.Curled encodes the Drosophila homolog of the vertebrate circadian deadenvlase nocturnin.Genetics,2009,183:219-232
[4]Lee JH,Budanov AV,Park EJ,et al.Sestrin as a feedback inhibitor of tor that prevents age-related pathologies.Science,2010,327:1223-1228
[5]Li H-W(李红伟),Lian L-S(连林生),Zhao C-J(赵春江),et al.Identification of a commom deletion in different curly mutants in Drosophila melanogaster.Hereditas(遗传),2006,28(10):1254-1258(in Chinese)
[6]Hurd TR,Liang FX,Lehmann R.Correction:Curly encodes dual oxidase,which acts with heme peroxidase curly su to shape the adult Drosophila wing.PLo SGenetics,2015,11(11):e1005625
[7]Bedard K,Krause HK.The nox family of ros-generating NADPH oxidases:Physiology and pathophysiology.Physiological Reviews,2007,87:245-313
[8]Sung-Hee K,Won-Jae L.Role of duox in gut inflammation:Lessons from Drosophila model of gut-microbiota interactions.Frontiers in Cellular&Infection Microbiology,2014,3:116-128
[9]O’Hare K,Murphy C,Levis R,et al.DNA sequence of the white locus of Drosophila melanogaster.Journal of Molecular Biology,1984,180:437-455
[10]Vasiliou V,Vasiliou K,Nebert DW.Human APT-binding cassette(abc)transporter family.Human Genomics,2009,3:281-290
[11]Dermauw W,Van LT.The abc gene family in arthropods:Comparative genomics and role in insecticide transport and resistance.Insect Biochemistry&Molecular Biology,2014,45:89-110
[12]Borycz J,Borycz JA,Kubów A,et al.Drosophila abc transporter mutants white,brown and scarlet have altered contents and distribution of biogenic amines in the brain.Journal of Experimental Biology,2008,211:3454-3466
[13]Anaka M,Macdonald CD,Barkova E,et al.The white gene of Drosophila melanogaster encodes a protein with a role in courtship behavior.Journal of Neurogenetics,2008,22:243-276
[14]Xiao C,Robertson RM.Timing of locomotor recovery form anoxia modulated by the white gene in Drosophila.Genetics,2016,203:511-515
[15]Hersh BM.More than meets the eye:A primer for‘timing of locomotor recovery form anoxia modulated by the white gene in Drosophila melanogaster’.Genetics,2016,204:1369-1375
[16]Hales KG,Korey CA,Larracuente AM,et al.Genetics on the fly:A primer on the Drosophila model system.Genetics,2015,201:815-842
[17]Hazelrigg T,Levis R,Rubin GM.Transformation of white locus DNA in Drosophila:Dosage compensation,zeste interaction,and position effects.Cell,1984,36:469-481
[18]De Deken X,Wang D,Many MC,et al.Cloning of two human c DNAs encoding new members of the NADPHoxidase family.Biological Chemistry,2000,275:23227-23233
[19]Dupuy C,Ohayon R,Valent A,et al.Purification of a novel flavoprotein involved in the thyroid NADPHoxidase.Biological Chemistry,1999,274:37265-37269
[20]Sun X-D(孙向东),Kuang W(况炜),Chen Y-Y(陈莹莹),et al.Vascular effects of hydrogen peroxide and its mechanism.Modern Practical Medicine(现代实用医学),2005,17(2):124-127(in Chinese)
[21]Daugherty A,Dunn JL,Rateri DL,et al.Myeloperoxidase,a catalyst for lipoprotein oxidation,is expressed in human atherosclerotic lesions.Journal of Clinical Investigation,1994,94:437-444
[22]Hazen SL,Heinecke JW.3-chlorotyrosine,a specific marker of myeloperoxidase-catalyzed oxidation,is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima.Journal of Clinical Investigation,1997,99:2075-2081
[23]Hameed A,Farooq S,Iqbal N,et al.Influence of exogenous application of hydrogen peroxide on root and seedling growth on wheat(Triticum aestivum L.).Issue:A Journal of Opinion,2004,10:58-64
[24]Hameed A,Malik SA,Iqbal N,et al.Influence of hydrogen peroxide on initial leaf and coleoptile growth in etiolated wheat(Triticum aestivum L.)seedlings.Asian Journal of Plant Sciences,2003,2:1121-1125
[25]Uchida A,Jagendorf AT,Hibino T,et al.Effects of hydrogen peroxide and nitric oxide on both salt and heat stress tolerance in rice.Plant Science,2002,163:515-523
[26]Chen XY,Ding X,Xu S,et al.Endogenous hydrogen peroxide plays a positive role in the upregulation of heme oxygenase and acclimation to oxidative stress in wheat seedling leaves.Journal of Integrative Plant Biology,2009,51:951-960
[27]Li J-T(李金亭),Zhao P-P(赵萍萍),Qiu Z-B(邱宗波),et al.Effects of exogenous hydrogen peroxide on the physiological index of wheat seedlings under salt stress.Acta Botanica Boreali-Occidentalia Sinica(西北植物学报),2012,32(9):1796-1801(in Chinese)
[28]Li SW,Xue LG,Xu SJ,et al.Hydrogen peroxide acts as a signal molecule in the adventitious root formation of mung bean seedlings.Environmental&Experimental Botany,2009,65:63-71
[29]Sun B(孙冰),Lin X-D(林欣大),Zhang C-H(张春红).Effect of salbutamol on the pupation,emergence,life span,antioxidative capacity and DNAdamage in Drosophila melanogaster.Scientia Agricultura Sinica(中国农业科学),2014,47(19):3890-3897(in Chinese)
[30]Xu L(徐莉),Mao J(毛杰),Tang Q-Y(唐启义),et al.Acute toxicity of ginger essential oil to Drosophila melanogaster.Chinese Journal of Applied Ecology(应用生态学报),2007,18(8):1827-1831(in Chinese)
[31]Pfaffl MW.A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Research,2001,29:e45
[32]Partridge L,Barrie B,Fowler K,et al.Evolution and development of body size and cell size in Drosophila melanogaster in response to temperature.Evolution,1994,48:1269-1276
[33]Wang Y-C(王荫长).Insect Physiology and Biochemistry.Beijing:China Agriculture Press,1991(in Chinese)
[34]Thummel CS.Steroid-triggered death by autophagy.Bioessays News&Reviews in Molecular Cellular&Developmental Biology,2001,23:677-682
[35]Yin VP,Thummel CS.Mechanisms of steroid-triggered programmed cell death in Drosophila.Seminars in Cell&Developmental Biology,2005,16:237-243
[36]Okazaki T,Okudaira N,Iwabuchi K,et al.Apoptosis and adhesion of hemocytes during molting stage of silkworm,bombyx mori.Zoological Science,2006,23:299-304
[37]Campbell JL,Nash HA.Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster.Journal of Neurobiology,2001,49:339-349
[38]Evans J,Day J,Cabrero P Jat,et al.A new role for a classical gene:White transports cyclic GMP.Journal of Experimental Biology,2008,211:890-899
[39]Xiao C,Robertson RM.Locomotion induced by spatial restriction in adult Drosophila.PLo S One,2015,10(9):e0135825
[2]Waddington CH.The genetic control of wing development in Drosophila.Genetics,1940,41:75-139
[3]Gr9nke S,Bickmeyer I,Wunderlich R,et al.Curled encodes the Drosophila homolog of the vertebrate circadian deadenvlase nocturnin.Genetics,2009,183:219-232
[4]Lee JH,Budanov AV,Park EJ,et al.Sestrin as a feedback inhibitor of tor that prevents age-related pathologies.Science,2010,327:1223-1228
[5]Li H-W(李红伟),Lian L-S(连林生),Zhao C-J(赵春江),et al.Identification of a commom deletion in different curly mutants in Drosophila melanogaster.Hereditas(遗传),2006,28(10):1254-1258(in Chinese)
[6]Hurd TR,Liang FX,Lehmann R.Correction:Curly encodes dual oxidase,which acts with heme peroxidase curly su to shape the adult Drosophila wing.PLo SGenetics,2015,11(11):e1005625
[7]Bedard K,Krause HK.The nox family of ros-generating NADPH oxidases:Physiology and pathophysiology.Physiological Reviews,2007,87:245-313
[8]Sung-Hee K,Won-Jae L.Role of duox in gut inflammation:Lessons from Drosophila model of gut-microbiota interactions.Frontiers in Cellular&Infection Microbiology,2014,3:116-128
[9]O’Hare K,Murphy C,Levis R,et al.DNA sequence of the white locus of Drosophila melanogaster.Journal of Molecular Biology,1984,180:437-455
[10]Vasiliou V,Vasiliou K,Nebert DW.Human APT-binding cassette(abc)transporter family.Human Genomics,2009,3:281-290
[11]Dermauw W,Van LT.The abc gene family in arthropods:Comparative genomics and role in insecticide transport and resistance.Insect Biochemistry&Molecular Biology,2014,45:89-110
[12]Borycz J,Borycz JA,Kubów A,et al.Drosophila abc transporter mutants white,brown and scarlet have altered contents and distribution of biogenic amines in the brain.Journal of Experimental Biology,2008,211:3454-3466
[13]Anaka M,Macdonald CD,Barkova E,et al.The white gene of Drosophila melanogaster encodes a protein with a role in courtship behavior.Journal of Neurogenetics,2008,22:243-276
[14]Xiao C,Robertson RM.Timing of locomotor recovery form anoxia modulated by the white gene in Drosophila.Genetics,2016,203:511-515
[15]Hersh BM.More than meets the eye:A primer for‘timing of locomotor recovery form anoxia modulated by the white gene in Drosophila melanogaster’.Genetics,2016,204:1369-1375
[16]Hales KG,Korey CA,Larracuente AM,et al.Genetics on the fly:A primer on the Drosophila model system.Genetics,2015,201:815-842
[17]Hazelrigg T,Levis R,Rubin GM.Transformation of white locus DNA in Drosophila:Dosage compensation,zeste interaction,and position effects.Cell,1984,36:469-481
[18]De Deken X,Wang D,Many MC,et al.Cloning of two human c DNAs encoding new members of the NADPHoxidase family.Biological Chemistry,2000,275:23227-23233
[19]Dupuy C,Ohayon R,Valent A,et al.Purification of a novel flavoprotein involved in the thyroid NADPHoxidase.Biological Chemistry,1999,274:37265-37269
[20]Sun X-D(孙向东),Kuang W(况炜),Chen Y-Y(陈莹莹),et al.Vascular effects of hydrogen peroxide and its mechanism.Modern Practical Medicine(现代实用医学),2005,17(2):124-127(in Chinese)
[21]Daugherty A,Dunn JL,Rateri DL,et al.Myeloperoxidase,a catalyst for lipoprotein oxidation,is expressed in human atherosclerotic lesions.Journal of Clinical Investigation,1994,94:437-444
[22]Hazen SL,Heinecke JW.3-chlorotyrosine,a specific marker of myeloperoxidase-catalyzed oxidation,is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima.Journal of Clinical Investigation,1997,99:2075-2081
[23]Hameed A,Farooq S,Iqbal N,et al.Influence of exogenous application of hydrogen peroxide on root and seedling growth on wheat(Triticum aestivum L.).Issue:A Journal of Opinion,2004,10:58-64
[24]Hameed A,Malik SA,Iqbal N,et al.Influence of hydrogen peroxide on initial leaf and coleoptile growth in etiolated wheat(Triticum aestivum L.)seedlings.Asian Journal of Plant Sciences,2003,2:1121-1125
[25]Uchida A,Jagendorf AT,Hibino T,et al.Effects of hydrogen peroxide and nitric oxide on both salt and heat stress tolerance in rice.Plant Science,2002,163:515-523
[26]Chen XY,Ding X,Xu S,et al.Endogenous hydrogen peroxide plays a positive role in the upregulation of heme oxygenase and acclimation to oxidative stress in wheat seedling leaves.Journal of Integrative Plant Biology,2009,51:951-960
[27]Li J-T(李金亭),Zhao P-P(赵萍萍),Qiu Z-B(邱宗波),et al.Effects of exogenous hydrogen peroxide on the physiological index of wheat seedlings under salt stress.Acta Botanica Boreali-Occidentalia Sinica(西北植物学报),2012,32(9):1796-1801(in Chinese)
[28]Li SW,Xue LG,Xu SJ,et al.Hydrogen peroxide acts as a signal molecule in the adventitious root formation of mung bean seedlings.Environmental&Experimental Botany,2009,65:63-71
[29]Sun B(孙冰),Lin X-D(林欣大),Zhang C-H(张春红).Effect of salbutamol on the pupation,emergence,life span,antioxidative capacity and DNAdamage in Drosophila melanogaster.Scientia Agricultura Sinica(中国农业科学),2014,47(19):3890-3897(in Chinese)
[30]Xu L(徐莉),Mao J(毛杰),Tang Q-Y(唐启义),et al.Acute toxicity of ginger essential oil to Drosophila melanogaster.Chinese Journal of Applied Ecology(应用生态学报),2007,18(8):1827-1831(in Chinese)
[31]Pfaffl MW.A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Research,2001,29:e45
[32]Partridge L,Barrie B,Fowler K,et al.Evolution and development of body size and cell size in Drosophila melanogaster in response to temperature.Evolution,1994,48:1269-1276
[33]Wang Y-C(王荫长).Insect Physiology and Biochemistry.Beijing:China Agriculture Press,1991(in Chinese)
[34]Thummel CS.Steroid-triggered death by autophagy.Bioessays News&Reviews in Molecular Cellular&Developmental Biology,2001,23:677-682
[35]Yin VP,Thummel CS.Mechanisms of steroid-triggered programmed cell death in Drosophila.Seminars in Cell&Developmental Biology,2005,16:237-243
[36]Okazaki T,Okudaira N,Iwabuchi K,et al.Apoptosis and adhesion of hemocytes during molting stage of silkworm,bombyx mori.Zoological Science,2006,23:299-304
[37]Campbell JL,Nash HA.Volatile general anesthetics reveal a neurobiological role for the white and brown genes of Drosophila melanogaster.Journal of Neurobiology,2001,49:339-349
[38]Evans J,Day J,Cabrero P Jat,et al.A new role for a classical gene:White transports cyclic GMP.Journal of Experimental Biology,2008,211:890-899
[39]Xiao C,Robertson RM.Locomotion induced by spatial restriction in adult Drosophila.PLo S One,2015,10(9):e0135825