黄野螟糖原磷酸化酶基因的时空表达动态及其对温度胁迫的响应
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摘要
[目的]本文旨在鉴定重要林业害虫黄野螟(Heortia vitessoides Moore)的糖原磷酸化酶(glycogen phosphorylase,GP)基因,并分析其序列、时空表达特征及对温度胁迫的响应,为进一步研究黄野螟的抗寒机制奠定基础。[方法]基于黄野螟转录组文库获得GP基因的全长c DNA序列,通过同源比对和系统发育树对其进行鉴定,利用RT-qPCR分析其时空表达谱及对温度胁迫的响应。[结果]将筛选出的基因命名为Hv GP(Gen Bank登录号:MG517442),其开放阅读框(ORF)长2 526 bp,共编码841个氨基酸。蛋白序列相似性比对和系统进化关系分析表明:Hv GP与亚洲玉米螟(Ostrinia furnacalis)对应蛋白的相似性最高(95%),亲缘关系最近;与烟粉虱(Bemisia tabaci)对应蛋白相似性最低(78%),亲缘关系较远。RT-qPCR分析显示:Hv GP在5龄幼虫虫态时表达量最高,为对照的1.77倍; Hv GP在黄野螟不同组织均有表达,且在组织间存在表达差异,幼虫脂肪体的表达量是其体壁的72.85倍;在成虫阶段,翅中表达量最高,足中最低。在5、10、15、20℃低温胁迫下,Hv GP的表达量均高于对照处理(25℃),在30、35、40℃高温胁迫下,Hv GP的表达量呈逐渐下降趋势。[结论]根据黄野螟不同发育阶段与不同组织表达模式推测,Hv GP可能与黄野螟的组织分化、摄食及运动等生命活动有关。Hv GP基因在温度胁迫下的响应表明Hv GP能响应低温信号,高温与Hv GP的表达量存在负相关关系。
[Objectives]This study aims to identify the glycogen phosphorylase( GP) genes and clarify its expression pattern of different stages,different tissues and response to temperature stress,and provide basis for further study on the cold resistance mechanism of Heortia vitessoides. [Methods]The full-length c DNA sequence of GP gene from H. vitessoides was obtained by analyzing the transcriptional library and further veridied by homologous alignment and phylogenetic tree. The expression pattern of different stages,different tissues and response to temperature stress were analyzed by RT-qPCR. [Results]The selected gene was named as Hv GP( Gen Bank No. MG517442). The ORF frame was 2 526 bp and encoded 841 amino acids. Homologous alignment and phylogenetic tree analysis showed that the amino acid sequence of Hv GP had the highest homology with Ostrinia furnacalis( 95%),and H. vitessoides and O. furnacalis were in the same phylogenetic tree branch. H. vitessoides and Bemisia tabaci had the lowest homology( 78%),and were the furthest apart from phylogenetic tree. The RT-qPCR analysis showed that Hv GP expression was the highest in the 5 th instar larvae. The expression of the fat body in larva was 72.85 times higher than that in epidermis. The adult tissue analysis showed that the expression of Hv GP was the highest in the wings and the lowest in the feet. The expression of Hv GP at 5,10,15 and20 ℃ were higher than that of the control group at 25 ℃,and the expression of Hv GP decreased gradually at 30,35 and 40 ℃.[Conclusions]According to the expression patterns of Hv GP in different developmental stages and tissues,we infer that Hv GP might be related to feeding,exercise,tissue differentiation and other life activities. The expression level of Hv GP after exposure to different temperature indicated that Hv GP could respond to the low temperature signal,and there was a negative correlation between high temperature and Hv GP expression.
[Objectives]This study aims to identify the glycogen phosphorylase( GP) genes and clarify its expression pattern of different stages,different tissues and response to temperature stress,and provide basis for further study on the cold resistance mechanism of Heortia vitessoides. [Methods]The full-length c DNA sequence of GP gene from H. vitessoides was obtained by analyzing the transcriptional library and further veridied by homologous alignment and phylogenetic tree. The expression pattern of different stages,different tissues and response to temperature stress were analyzed by RT-qPCR. [Results]The selected gene was named as Hv GP( Gen Bank No. MG517442). The ORF frame was 2 526 bp and encoded 841 amino acids. Homologous alignment and phylogenetic tree analysis showed that the amino acid sequence of Hv GP had the highest homology with Ostrinia furnacalis( 95%),and H. vitessoides and O. furnacalis were in the same phylogenetic tree branch. H. vitessoides and Bemisia tabaci had the lowest homology( 78%),and were the furthest apart from phylogenetic tree. The RT-qPCR analysis showed that Hv GP expression was the highest in the 5 th instar larvae. The expression of the fat body in larva was 72.85 times higher than that in epidermis. The adult tissue analysis showed that the expression of Hv GP was the highest in the wings and the lowest in the feet. The expression of Hv GP at 5,10,15 and20 ℃ were higher than that of the control group at 25 ℃,and the expression of Hv GP decreased gradually at 30,35 and 40 ℃.[Conclusions]According to the expression patterns of Hv GP in different developmental stages and tissues,we infer that Hv GP might be related to feeding,exercise,tissue differentiation and other life activities. The expression level of Hv GP after exposure to different temperature indicated that Hv GP could respond to the low temperature signal,and there was a negative correlation between high temperature and Hv GP expression.
引文
[1]茅裕婷,张蒙,靳秀芳,等.土沉香对黄野螟的抗性研究[J].华南农业大学学报,2017,38(6):89-96.Mao Y T,Zhang M,Jin X F,et al. Study on resistance of Aquilaria sinensis against Heortia vitessoides[J]. Journal of South China Agricultural University,2017,38(6):89-96(in Chinese with English abstract).
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[3] Hoffmann A A,S?rensen J G,Loeschcke V. Adaptation of Drosophila to temperature extremes:bringing together quantitative and molecular approaches[J]. Journal of Thermal Biology,2003,28(3):175-216.
[4]陈豪,梁革梅,邹朗云,等.昆虫抗寒性的研究进展[J].植物保护,2010,36(2):18-24.Chen H,Liang G M,Zou L Y,et al. Research progresses in the cold hardiness of insects[J]. Plant Protection,2010,36(2):18-24(in Chinese with English abstract).
[5]陈永杰,孙绪艮,张卫光,等.桑螟越冬幼虫体内蛋白质、氨基酸、碳水化合物的变化与抗寒性的关系[J].蚕业科学,2005,31(2):111-116.Chen Y J,Sun X G,Zhang W G,et al. Relationship between variation of protein,amino acid,low-molecular carbohydrate in over-wintering Diaphania pyloalis walker larvae and cold-hardiness[J]. Acta Sericologica Sinica,2005,31(2):111-116(in Chinese with English abstract).
[6]韩瑞东,孙绪艮,许永玉,等.赤松毛虫越冬幼虫生化物质变化与抗寒性的关系[J].生态学报,2005,25(6):1352-1356.Han R D,Sun X G,Xu Y Y,et al. The biochemical mechanism of cold-hardiness in overwintering larva of Dendrolimus spectabilis Butler(Lepidoptera:Lasiocampidae)[J]. Acta Ecologica Sinica,2005,25(6):1352-1356(in Chinese with English abstract).
[7] Zhao N,Hou M,Wang T,et al. Cloning and expression patterns of the brine shrimp(Artemia sinica)glycogen phosphorylase(GPase)gene during development and in response to temperature stress[J]. Molecular Biology Reports,2014,41(1):9-18.
[8] Steele J E. Glycogen phosphorylase in insects[J]. Insect Biochemistry,1982,12(2):131-147.
[9] Brostrom C O,Hunkeler F L,Krebs E G. The regulation of skeletal muscle phosphorylase kinase by Ca2+[J]. Journal of Biological Chemistry,1971,246(7):1961-1967.
[10] Chen C P,Denlinger D L. Activation of phosphorylase in response to cold and heat stress in the flesh fly,Sarcophaga crassipalpis[J]. Journal of Insect Physiology,1990,36(8):549-553.
[11]李毅平,龚和,朴镐用.松针瘿蚊越冬幼虫体内酶活性的时序变化[J].昆虫学报,2000,43(3):227-232.Li Y P,Gong H,Park H Y. Profile of enzymic activity in overwintering mature larvae of the pine needle gall midge,Thecodiplosis japonensis[J].Acta Entomologica Sinica,2000,43(3):227-232(in Chinese with English abstract).
[12] Joanisse D R,Storey K B. Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly,Eurosta solidaginis[J].Journal of Comparative Physiology B,1994,164(3):247-255.
[13] Cori C F,Cori G T. Mechanism of formation of hexosemonophosphate in muscle and isolation of a new phosphate ester[J]. Experimental Biology and Medicine,1936,34(5):702-705.
[14] Yamashita O,Suzuki K,Hasegawa K. Glycogen phosphorylase activity in relation to diapause initiation in Bombyx eggs[J]. Insect Biochemistry,1975,5(6):707-718.
[15]郭建青.亚洲玉米螟糖原磷酸化酶基因的克隆及表达分析[D].北京:中国农业科学院,2013.Guo J Q. Cloning and expression analysis of glycogen phosphorylase gene in Ostrinia furnacalis(Guenée)(Lepidoptera:Crambidae)[D].Beijing:Chinese Academy of Agricultural Sciences,2013(in Chinese with English abstract).
[16] Barford D,Hu S H,Johnson L N. Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP[J]. Journal of Molecular Biology,1991,218(1):233-260.
[17] Ziegler R,Ashida M,Fallon A M,et al. Regulation of glycogen phosphorylase in fat body of Cecropia silkmoth pupae[J]. Journal of Comparative Physiology,1979,131(4):321-332.
[18] Bahjou A,Gourdoux L,Moreau R,et al. In vitro regulation of glycogen phosphorylase of the larval fat body of Tenebrio molitor[J]. Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1988,89(2):233-237.
[19]刘流,贺莉芳,刘晖,等.滞育家蝇幼虫糖的变化[J].医学动物防制,2008,24(11):803-804.Liu L,He L F,Liu H,et al. The change of carbohydrate of Musca domestica larva in the diapause[J]. Chinese Journal of Pest Control,2008,24(11):803-804(in Chinese with English abstract).
[20] Buchbinder J L,Fletterick R J. Role of the active site gate of glycogen phosphorylase in allosteric inhibition and substrate binding[J]. Journal of Biological Chemistry,1996,271(37):22305-22309.
[21] Hudson J W,Golding G B,Crerar M M. Evolution of allosteric control in glycogen phosphorylase[J]. Journal of Molecular Biology,1993,234(3):700-721.
[22] Toll-Riera M,Radó-Trilla N,Martys F,et al. Role of low-complexity sequences in the formation of novel protein coding sequences[J]. Molecular Biology and Evolution,2012,29(3):883-886.
[23] Mitsumasu K,Kanamori Y,Fujita M,et al. Enzymatic control of anhydrobiosis-related accumulation of trehalose in the sleeping chironomid,Polypedilum vanderplanki[J]. FEBS Journal,2010,277(20):4215-4228.
[24]赵娜.糖原磷酸化酶基因在中国卤虫胚胎发育和温度胁迫过程中的表达模式[D].大连:辽宁师范大学,2013.Zhao N. Expression pattern of glycogen phosphorylase gene during development and in response to temperature stress from brine shrimp,Artemia sinica[D]. Dalian:Liaoning Normal University,2013(in Chinese with English abstract).
[25]易传辉,陈晓鸣,史军义,等.柑橘凤蝶滞育期间糖类物质和脂肪含量变化[J].西南农业学报,2009,22(4):1142-1145.Yi C H,Chen X M,Shi J Y,et al. Change of carbohydrate and fat contents in pupae of Papilio xuthus L. during diapause stage[J]. Southwest China Journal of Agricultural Sciences,2009,22(4):1142-1145(in Chinese with English abstract).
[26] Arrese E L,Soulages J L. Insect fat body:energy,metabolism,and regulation[J]. Annual Review of Entomology,2010,55:207-225.
[27]景晓红,郝树广,康乐.昆虫对低温的适应———抗冻蛋白研究进展[J].昆虫学报,2002,45(5):679-683.Jing X H,Hao S G,Kang L. Cold adaptation in insects:progress in antifreeze protein research[J]. Acta Entomologica Sinica,2002,45(5):679-683(in Chinese with English abstract).
[28] Kostal V,Renault D,MehrabianovaA,et al. Insect cold tolerance and repair of chill-injury at fluctuating thermal regimes:role of ion homeostasis[J].Comparative Biochemistry and Physiology Part A:Molecular and Integrative Physiology,2007,147(1):231-238.
[2]乔海莉,陆鹏飞,陈君,等.黄野螟生物学特性及发生规律研究[J].应用昆虫学报,2013,50(5):1244-1252.Qiao H L,Lu P F,Chen J,et al. Biological characteristics and occurrence patterns of Heortia vitessoides[J]. Chinese Journal of Applied Entomology,2013,50(5):1244-1252(in Chinese with English abstract).
[3] Hoffmann A A,S?rensen J G,Loeschcke V. Adaptation of Drosophila to temperature extremes:bringing together quantitative and molecular approaches[J]. Journal of Thermal Biology,2003,28(3):175-216.
[4]陈豪,梁革梅,邹朗云,等.昆虫抗寒性的研究进展[J].植物保护,2010,36(2):18-24.Chen H,Liang G M,Zou L Y,et al. Research progresses in the cold hardiness of insects[J]. Plant Protection,2010,36(2):18-24(in Chinese with English abstract).
[5]陈永杰,孙绪艮,张卫光,等.桑螟越冬幼虫体内蛋白质、氨基酸、碳水化合物的变化与抗寒性的关系[J].蚕业科学,2005,31(2):111-116.Chen Y J,Sun X G,Zhang W G,et al. Relationship between variation of protein,amino acid,low-molecular carbohydrate in over-wintering Diaphania pyloalis walker larvae and cold-hardiness[J]. Acta Sericologica Sinica,2005,31(2):111-116(in Chinese with English abstract).
[6]韩瑞东,孙绪艮,许永玉,等.赤松毛虫越冬幼虫生化物质变化与抗寒性的关系[J].生态学报,2005,25(6):1352-1356.Han R D,Sun X G,Xu Y Y,et al. The biochemical mechanism of cold-hardiness in overwintering larva of Dendrolimus spectabilis Butler(Lepidoptera:Lasiocampidae)[J]. Acta Ecologica Sinica,2005,25(6):1352-1356(in Chinese with English abstract).
[7] Zhao N,Hou M,Wang T,et al. Cloning and expression patterns of the brine shrimp(Artemia sinica)glycogen phosphorylase(GPase)gene during development and in response to temperature stress[J]. Molecular Biology Reports,2014,41(1):9-18.
[8] Steele J E. Glycogen phosphorylase in insects[J]. Insect Biochemistry,1982,12(2):131-147.
[9] Brostrom C O,Hunkeler F L,Krebs E G. The regulation of skeletal muscle phosphorylase kinase by Ca2+[J]. Journal of Biological Chemistry,1971,246(7):1961-1967.
[10] Chen C P,Denlinger D L. Activation of phosphorylase in response to cold and heat stress in the flesh fly,Sarcophaga crassipalpis[J]. Journal of Insect Physiology,1990,36(8):549-553.
[11]李毅平,龚和,朴镐用.松针瘿蚊越冬幼虫体内酶活性的时序变化[J].昆虫学报,2000,43(3):227-232.Li Y P,Gong H,Park H Y. Profile of enzymic activity in overwintering mature larvae of the pine needle gall midge,Thecodiplosis japonensis[J].Acta Entomologica Sinica,2000,43(3):227-232(in Chinese with English abstract).
[12] Joanisse D R,Storey K B. Enzyme activity profiles in an overwintering population of freeze-tolerant larvae of the gall fly,Eurosta solidaginis[J].Journal of Comparative Physiology B,1994,164(3):247-255.
[13] Cori C F,Cori G T. Mechanism of formation of hexosemonophosphate in muscle and isolation of a new phosphate ester[J]. Experimental Biology and Medicine,1936,34(5):702-705.
[14] Yamashita O,Suzuki K,Hasegawa K. Glycogen phosphorylase activity in relation to diapause initiation in Bombyx eggs[J]. Insect Biochemistry,1975,5(6):707-718.
[15]郭建青.亚洲玉米螟糖原磷酸化酶基因的克隆及表达分析[D].北京:中国农业科学院,2013.Guo J Q. Cloning and expression analysis of glycogen phosphorylase gene in Ostrinia furnacalis(Guenée)(Lepidoptera:Crambidae)[D].Beijing:Chinese Academy of Agricultural Sciences,2013(in Chinese with English abstract).
[16] Barford D,Hu S H,Johnson L N. Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP[J]. Journal of Molecular Biology,1991,218(1):233-260.
[17] Ziegler R,Ashida M,Fallon A M,et al. Regulation of glycogen phosphorylase in fat body of Cecropia silkmoth pupae[J]. Journal of Comparative Physiology,1979,131(4):321-332.
[18] Bahjou A,Gourdoux L,Moreau R,et al. In vitro regulation of glycogen phosphorylase of the larval fat body of Tenebrio molitor[J]. Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1988,89(2):233-237.
[19]刘流,贺莉芳,刘晖,等.滞育家蝇幼虫糖的变化[J].医学动物防制,2008,24(11):803-804.Liu L,He L F,Liu H,et al. The change of carbohydrate of Musca domestica larva in the diapause[J]. Chinese Journal of Pest Control,2008,24(11):803-804(in Chinese with English abstract).
[20] Buchbinder J L,Fletterick R J. Role of the active site gate of glycogen phosphorylase in allosteric inhibition and substrate binding[J]. Journal of Biological Chemistry,1996,271(37):22305-22309.
[21] Hudson J W,Golding G B,Crerar M M. Evolution of allosteric control in glycogen phosphorylase[J]. Journal of Molecular Biology,1993,234(3):700-721.
[22] Toll-Riera M,Radó-Trilla N,Martys F,et al. Role of low-complexity sequences in the formation of novel protein coding sequences[J]. Molecular Biology and Evolution,2012,29(3):883-886.
[23] Mitsumasu K,Kanamori Y,Fujita M,et al. Enzymatic control of anhydrobiosis-related accumulation of trehalose in the sleeping chironomid,Polypedilum vanderplanki[J]. FEBS Journal,2010,277(20):4215-4228.
[24]赵娜.糖原磷酸化酶基因在中国卤虫胚胎发育和温度胁迫过程中的表达模式[D].大连:辽宁师范大学,2013.Zhao N. Expression pattern of glycogen phosphorylase gene during development and in response to temperature stress from brine shrimp,Artemia sinica[D]. Dalian:Liaoning Normal University,2013(in Chinese with English abstract).
[25]易传辉,陈晓鸣,史军义,等.柑橘凤蝶滞育期间糖类物质和脂肪含量变化[J].西南农业学报,2009,22(4):1142-1145.Yi C H,Chen X M,Shi J Y,et al. Change of carbohydrate and fat contents in pupae of Papilio xuthus L. during diapause stage[J]. Southwest China Journal of Agricultural Sciences,2009,22(4):1142-1145(in Chinese with English abstract).
[26] Arrese E L,Soulages J L. Insect fat body:energy,metabolism,and regulation[J]. Annual Review of Entomology,2010,55:207-225.
[27]景晓红,郝树广,康乐.昆虫对低温的适应———抗冻蛋白研究进展[J].昆虫学报,2002,45(5):679-683.Jing X H,Hao S G,Kang L. Cold adaptation in insects:progress in antifreeze protein research[J]. Acta Entomologica Sinica,2002,45(5):679-683(in Chinese with English abstract).
[28] Kostal V,Renault D,MehrabianovaA,et al. Insect cold tolerance and repair of chill-injury at fluctuating thermal regimes:role of ion homeostasis[J].Comparative Biochemistry and Physiology Part A:Molecular and Integrative Physiology,2007,147(1):231-238.