柞蚕蛹滞育和滞育解除过程中海藻糖酶基因表达模式及酶活力分析
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摘要
【目的】克隆柞蚕Antheraea pernyi海藻糖酶(trehalase,Treh)基因,探讨该基因在柞蚕蛹滞育和滞育解除过程中的表达模式与海藻糖酶活力变化,为阐明柞蚕蛹滞育期间糖代谢机制提供参考。【方法】利用RT-PCR技术从柞蚕蛹中克隆获得海藻糖酶基因,并对其进行生物信息学分析。采用半定量RT-PCR检测长光照(17L∶7D)处理后的滞育解除柞蚕蛹与对照滞育蛹不同组织中该基因的表达谱;采用实时定量PCR(qPCR)分析其在长光照下滞育解除过程中柞蚕蛹脂肪体中的相对表达量变化。利用3,5-二硝基水杨酸法检测脂肪体中海藻糖酶活力的变化,同时采用蒽酮比色法测定其血淋巴中海藻糖含量。【结果】克隆获得柞蚕3个海藻糖酶基因,分别命名为ApTreh1A,ApTreh1B和ApTreh2(GenBank登录号分别为:KU977455,KU977456和KU977457),开放阅读框(ORF)全长分别为1 797,1 635和1 932 bp,分别编码598,544和643个氨基酸。同源序列比对与系统进化树分析表明,ApTreh1A和ApTreh1B为可溶型海藻糖酶(Treh S),ApTreh2为膜结合型海藻糖酶(Treh M)。半定量RT-PCR检测发现,各组织中ApTreh2比ApTreh1的分布更广且表达量更高。qPCR检测发现,ApTreh1A和ApTreh1B在长光照处理后的柞蚕蛹脂肪体中,21 d时表达量都表现出快速升高[分别是对照组(12L∶12D)的2倍和4.7倍],28 d与35 d时下降,42 d时表达量再次升高;ApTreh2随着滞育的解除表达量逐渐升高,28 d时达到最高(约为对照组的2.7倍),42 d时又出现一个小高峰(约2.3倍),后期逐渐下降。长光照下脂肪体中海藻糖酶活力逐渐升高,21 d时达到最高(约18.5 U),35 d时降到最低(约11.2 U),42 d时其酶活力再次略微升高,之后呈下降趋势,与基因表达变化趋势一致。蛹血淋巴中海藻糖含量在长光照条件下呈现出升高趋势,21 d时达到最高,在整个发育时期的含量比对照组要高。【结论】本研究结果表明柞蚕蛹滞育解除过程中海藻糖酶基因表达的变化与蛹脂肪体中海藻糖酶活性、蛹血淋巴中海藻糖含量的变化趋势呈一致性,提示海藻糖酶基因的表达响应在柞蚕蛹滞育解除中发挥重要作用。
【Aim】Gene expression patterns and enzyme activities of trehalases in Antheraea pernyi pupae during diapause and diapause termination were detected so as to elucidate the relationship between carbohydrate metabolism and diapause termination during diapause of this insect. 【Methods 】 The trehalase genes were cloned from A. pernyi pupa using RT-PCR technology and subjected to bioinformatics analysis. Their expression patterns in different tissues of A. pernyi pupae during diapause termination after exposure to long photoperiod( 17 L∶ 7 D) and diapause( the control group) were analyzed by semi-quantitative RT-PCR. The relative expression levels of trehalase genes in the fat body of A. pernyi pupae during diapause termination under long photoperiod were measured using qPCR,the trehalase activity in the fat body was detected using 3,5-dinitrosalicylic acid method and the trehalose content in the haemolymph was measured using antrone chromametry method. 【Results】Three trehalase genes were cloned from A. pernyi,named ApTreh1 A,ApTreh1 B and ApTreh2 and deposited in GenBank under accession numbers KU977455,KU977456 and KU977457,respectively. Their open reading frames( ORFs) are 1 797,1 635 and 1 932 bp in length,encoding 598,544 and 643 amino acids,respectively. Homologous sequence alignment and phylogenetic analysis indicated that ApTreh1 A and ApTreh1 B are soluble trehalases( Treh S),and ApTreh2 is a membrane-bound trehalase( Treh M).Tissue-specific mRNA expression profiling using semi-quantitative RT-PCR showed that ApTreh2 had a wider distribution and higher expression level than ApTreh1. The qPCR results indicated that the expression levels of ApTreh1 A and ApTreh1 B in fat body at 21 d after exposure to long photoperiod were up-regulated and significantly higher than that of the control group( 12 L ∶ 12 D)( 2-and 4. 7-fold,respectively),while down-regulated at 28 and 35 d,and then up-regulated again at 42 d. The expression levels of ApTreh2 were up-regulated gradually,and reached the maximum( 2. 7-fold as high as that of the control group) at 28 d. At 42 d after exposure to long photoperiod,there was another expression peak( 2. 3-fold as high as that of the control group),and then down-regulated gradually. Trehalase activities in fat body increased gradually,reached the peak( about 18. 5 U) at 21 d after exposure to long photoperiod,while declined to the lowest level( 11. 2 U) at 35 d,and increased slightly at 42 d,showing the same variation trend as the gene expression. The trehalose content in the haemolymph increased under long photoperiod and reached the maximum at 21 d,and was higher than that of the control during the whole developmental stage. 【Conclusion】The results suggest that the expression of trehalase genes shows the same variation trend as the trehalase activities in the fat body and the trehalose content in the haemolymph in A. pernyi,suggesting that the expression response of trehalase genes plays a significant role in pupal diapause and diapause termination of A. pernyi.
【Aim】Gene expression patterns and enzyme activities of trehalases in Antheraea pernyi pupae during diapause and diapause termination were detected so as to elucidate the relationship between carbohydrate metabolism and diapause termination during diapause of this insect. 【Methods 】 The trehalase genes were cloned from A. pernyi pupa using RT-PCR technology and subjected to bioinformatics analysis. Their expression patterns in different tissues of A. pernyi pupae during diapause termination after exposure to long photoperiod( 17 L∶ 7 D) and diapause( the control group) were analyzed by semi-quantitative RT-PCR. The relative expression levels of trehalase genes in the fat body of A. pernyi pupae during diapause termination under long photoperiod were measured using qPCR,the trehalase activity in the fat body was detected using 3,5-dinitrosalicylic acid method and the trehalose content in the haemolymph was measured using antrone chromametry method. 【Results】Three trehalase genes were cloned from A. pernyi,named ApTreh1 A,ApTreh1 B and ApTreh2 and deposited in GenBank under accession numbers KU977455,KU977456 and KU977457,respectively. Their open reading frames( ORFs) are 1 797,1 635 and 1 932 bp in length,encoding 598,544 and 643 amino acids,respectively. Homologous sequence alignment and phylogenetic analysis indicated that ApTreh1 A and ApTreh1 B are soluble trehalases( Treh S),and ApTreh2 is a membrane-bound trehalase( Treh M).Tissue-specific mRNA expression profiling using semi-quantitative RT-PCR showed that ApTreh2 had a wider distribution and higher expression level than ApTreh1. The qPCR results indicated that the expression levels of ApTreh1 A and ApTreh1 B in fat body at 21 d after exposure to long photoperiod were up-regulated and significantly higher than that of the control group( 12 L ∶ 12 D)( 2-and 4. 7-fold,respectively),while down-regulated at 28 and 35 d,and then up-regulated again at 42 d. The expression levels of ApTreh2 were up-regulated gradually,and reached the maximum( 2. 7-fold as high as that of the control group) at 28 d. At 42 d after exposure to long photoperiod,there was another expression peak( 2. 3-fold as high as that of the control group),and then down-regulated gradually. Trehalase activities in fat body increased gradually,reached the peak( about 18. 5 U) at 21 d after exposure to long photoperiod,while declined to the lowest level( 11. 2 U) at 35 d,and increased slightly at 42 d,showing the same variation trend as the gene expression. The trehalose content in the haemolymph increased under long photoperiod and reached the maximum at 21 d,and was higher than that of the control during the whole developmental stage. 【Conclusion】The results suggest that the expression of trehalase genes shows the same variation trend as the trehalase activities in the fat body and the trehalose content in the haemolymph in A. pernyi,suggesting that the expression response of trehalase genes plays a significant role in pupal diapause and diapause termination of A. pernyi.
引文
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Ru YT,Wang Y,Wang DY,Zhou JL,Zeng J,Zhu XW,Jiang YR,Qin L,2017.Tissue-specific expression and enzyme activity variations of Antheraea pernyi hexokinase genes during diapause termination and pupa development.Acta Sericol.Sin.,43(5):773-781.[汝玉涛,王勇,王德意,周敬林,曾军,朱绪伟,姜义仁,秦利,2017.柞蚕己糖激酶基因的组织表达特征及在解除滞育和蛹发育期的表达与酶活性变化.蚕业科学,43(5):773-781]
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Silva MC,Ribeiro AF,Terra WR,Ferreira C,2009.Sequencing of Spodoptera frugiperda midgut trehalases and demonstration of secretion of soluble trehalase by midgut columnar cells.Insect Mol.Biol.,18(6):769-784.
Su ZH,Sato Y,Yamashita O,1993.Purification,c DNA cloning and Northern blot analysis of trehalase of pupal midgut of the silkworm,Bombyx mori.BBA-Gene Struct.Expr.,1173(2):217-224.
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Becker A,Schl9der P,Steele JE,Wegener G,1996.The regulation of trehalose metabolism in insects.Experientia,52(5):433-439.
Bounias M,Bahjou A,Gordoux L,Moreau R,1993.Molecular activation of a trehalase purified from the fat body of a coleopteran insect(Tenebrio molitor),by an endogenous insulin-like peptide.Biochem.Mol.Biol.Int.,31(2):249-266.
Bradford M,1976.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal.Biochem.,72(1-2):248-254.
Chen J,Tang B,Chen H,Yao Q,Huang XF,Chen J,Zhang DW,Zhang WQ,2010.Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference.PLo S ONE,5(4):e10133.
Cheng WN,Li XL,Li YP,Li JJ,Wu JX,2009.Activities of four sugar metabolic enzymes in Sitodipotsis mosellana(Gehin)(Diptera:Cecidomyiidae)larvae at different diapause stages.Acta Entomol.Sin.,52(2):133-139.[成卫宁,李修炼,李怡萍,李建军,仵均祥,2009.麦红吸浆虫不同滞育期四种糖代谢酶活力分析.昆虫学报,52(2):133-139]
Deng HS,1980.The effect of sensitization and diapause on pupa silkworms.Acta Sericol.Sin.,6(2):125-128.[邓华山,1980.关于柞蚕蛹期感光解除滞育的效应.蚕业科学,6(2):125-128]
Ding HM,Ma G,Wu SA,Zhao F,Ma CS,2011.A literature review on changes of small molecules of diapause insects during overwintering period.Chin.J.Appl.Entomol.,48(4):1060-1070.[丁惠梅,马罡,武三安,赵飞,马春森,2011.滞育昆虫小分子含量变化研究进展.应用昆虫学报,48(4):1060-1070]
Huang L,Sun LZ,Wang Y,Ru YT,Irfan M,Jiang YR,Shi SL,Yang RS,Li XS,Qin L,2016.Changes in the expression of trehalose-6-phosphate synthase gene in Antheraea pernyi(Lepidoptera:Saturniidae)during pupal diapause termination.Acta Entomol.Sin.,59(9):938-947.[黄伶,孙良振,王勇,汝玉涛,Irfan M,姜义仁,石生林,杨瑞生,李喜升,秦利,2016.柞蚕蛹解除滞育过程中海藻糖合成酶基因的表达变化.昆虫学报,59(9):938-947]
Li HJ,2016.Gene Cloning and Expression Analysis of PTTH in Antheraea pernyi.MSc Thesis,Shenyang Agricultural University,Shenyang.[李慧君,2016.柞蚕PTTH基因的克隆及表达分析.沈阳:沈阳农业大学硕士学位论文]
Li XS,Wang GB,Sun Y,Liu W,He YZ,Wang FC,Jiang YR,Qin L,2016.Transcriptome analysis of the midgut of the Chinese oak silkworm Antheraea pernyi infected with Antheraea pernyi nucleopolyhedrovirus.PLo S ONE,11(11):e0165959.
Liu XJ,Zhang HH,Li DQ,Cui M,Ma EB,Zhang JZ,2012.Sequence characterization and mRNA expression profiling of a soluble trehalase gene in Locusta migratoria(Orthoptera:Acrididae).Acta Entomol.Sin.,55(11):1264-1271.[刘晓健,张欢欢,李大琪,崔淼,马恩波,张建珍,2012.飞蝗可溶型海藻糖酶基因的序列分析及mRNA表达特性.昆虫学报,55(11):1264-1271]
Lu MX,Yang CS,1992.Dynamic temperature and photoperiodic effects of carbohydrate content in the fat body and haemolymph of the Antheraea pernyi(Lepidoptera:Saturniidae)pupae.Acta Entomol.Sin.,35(1):1-7.[陆明贤,杨长松,1992.柞蚕蛹脂肪体和血淋巴中糖类含量动态的温度和光周期效应.昆虫学报,35(1):1-7]
Mitsumasu K,Azuma M,Niimi T,Yamashita O,Yaginuma T,2005.Membrane-penetrating trehalase from silkworm Bombyx mori.Molecular cloning and localization in larval midgut.Insect Mol.Biol.,14(5):501-508.
Qin L,Li SY,2017.Chinese Tussah.China Agriculture Press,Beijing.130-132.[秦利,李树英,2017.中国柞蚕学.北京:中国农业出版社.130-132]
Ru YT,Wang Y,Wang DY,Zhou JL,Zeng J,Zhu XW,Jiang YR,Qin L,2017.Tissue-specific expression and enzyme activity variations of Antheraea pernyi hexokinase genes during diapause termination and pupa development.Acta Sericol.Sin.,43(5):773-781.[汝玉涛,王勇,王德意,周敬林,曾军,朱绪伟,姜义仁,秦利,2017.柞蚕己糖激酶基因的组织表达特征及在解除滞育和蛹发育期的表达与酶活性变化.蚕业科学,43(5):773-781]
Sauman I,Reppert SM,1996.Molecular characterization of prothoracicotropic hormone(PTTH)from the giant silkmoth Antheraea pernyi:developmental appearance of PTTH-expressing cells and relationship to circadian clock cells in central brain.Dev.Biol.,178(2):418-429.
Shukla E,Thorat LJ,Nath BB,Gaikwad SM,2015.Insect trehalase:physiological significance and potential applications.Glycobiology,25(4):357-367.
Silva MC,Ribeiro AF,Terra WR,Ferreira C,2009.Sequencing of Spodoptera frugiperda midgut trehalases and demonstration of secretion of soluble trehalase by midgut columnar cells.Insect Mol.Biol.,18(6):769-784.
Su ZH,Sato Y,Yamashita O,1993.Purification,c DNA cloning and Northern blot analysis of trehalase of pupal midgut of the silkworm,Bombyx mori.BBA-Gene Struct.Expr.,1173(2):217-224.
Takiguchi MU,Niimi T,Su ZH,Yaginuma T,1992.Trehalase from male accessory gland of an insect,Tenebrio molitor.c DNAsequencing and developmental profile of the gene expression.Biochem.J.,288(1):19-22.
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