飞蝗几丁质脱乙酰基酶基因的分子特性和生物学功能
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摘要
【目的】几丁质脱乙酰基酶(chitin deacetylase,CDA)是昆虫几丁质代谢系统的重要酶系,研究飞蝗(Locusta migratoria)几丁质脱乙酰基酶基因的分子特性和生物学功能,为新型农药靶标筛选提供科学依据。【方法】基于飞蝗转录组数据库,获得几丁质脱乙酰基酶基因的c DNA序列,将其与飞蝗基因组进行比对,绘制基因结构图。将其与赤拟谷盗CDAs序列进行比对,并采用Blast P和SMART软件进行功能域预测。将已鉴定的飞蝗CDAs分别与赤拟谷盗、果蝇、冈比亚按蚊、家蚕、中华稻蝗和云杉卷叶蛾的同源序列进行聚类分析,采用MEGA 5.02软件中的neighbor-joining(NJ)法构建系统发育树。采用reverse transcription quantitative PCR(RT-q PCR)方法检测分析CDA在飞蝗5龄若虫不同组织部位和不同发育日龄表皮中的表达特性,进一步采用RNA干扰(RNAi)技术研究其对飞蝗蜕皮发育的影响。采用化学检测法测定其几丁质含量。利用透射电镜(TEM)观察其对表皮几丁质排列的影响。【结果】在飞蝗转录组数据库中搜索获得3条几丁质脱乙酰基酶基因全长c DNA序列,生物信息学分析发现其均具有信号肽,开放阅读框包含几丁质结合区(Ch BD)和几丁质脱乙酰基催化域(CDA)2个功能域。与赤拟谷盗CDAs序列比对结果表明飞蝗2条CDAs部分序列存在差异,且与赤拟谷盗Tc CDA5的2个剪切子差异序列位置一致,显示2条序列为2个剪切子。聚类分析结果表明,3条序列分别与6种昆虫CDA4和CDA5以较高的置信度聚为一支。分别将其命名为Lm CDA4、Lm CDA5a和Lm CDA5b。不同组织部位表达结果表明Lm CDA4和Lm CDA5在飞蝗前肠、后肠和表皮中高表达,Lm CDA5在脂肪体中也有较高的表达;CDAs在5龄不同天数表皮表达结果显示Lm CDA4和Lm CDA5在5龄第1和第2天的表皮中表达量较高,之后逐步下降。注射ds Lm CDA4或ds Lm CDA524 h后,与对照组相比,基因表达量显著降低,沉默效率达到98.1%和95.6%,但均无可见表型,都可正常蜕皮至成虫。几丁质含量和透射电镜分析显示,上述基因RNAi后不影响表皮几丁质含量及排列。【结论】飞蝗几丁质脱乙酰基酶4和5基因主要在飞蝗表皮和前/后肠表达,生物学功能研究表明这2个基因不参与飞蝗蜕皮发育过程,靶基因沉默对飞蝗生长发育及表皮结构无影响。
【Objective】 Chitin deacetylase(CDA) is a key enzyme involved in chitin metabolism. The objective of this paper is to study the molecular characteristics and biological function of chitin deacetylase genes in Locusta migratoria, and to provide a theoretical basis for screening novel targets for pest control.【Method】Based on the transcriptome of L. migratoria, three c DNA sequences putatively encoded Lm CDAs were obtained, and the gene structure was analyzed by comparing with the locust genome database. Alignment was performed with the CDAs from Tribolium castaneum and the functional domains were predicted by Blast P and SMART software. The homologous sequences from T. castaneum, Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Oxya chinensis and Choristoneura fumiferana were selected to perform clustering analysis with Lm CDA4, Lm CDA5 a and Lm CDA5 b, and phylogenetic tree was constructed by using MEGA 5.02 software with the neighbor-joining(NJ) method. The RT-q PCR was applied to detect the relative expression of Lm CDA4 and Lm CDA5 in different tissues and developmental stages of the 5th instar nymphs. The RNA interference(RNAi) was performed to study the biological function of Lm CDA4 and Lm CDA5 during the molting process of L. migratoria. The chitin content was detected by chemical method. The transmission electron microscope(TEM) was applied to study whether the Lm CDA4 and Lm CDA5 have effects on the cuticle structure.【Result】The three full length c DNA sequences putatively encoded chitin deacetylase were identified from locust transcriptome database. The bioinformatics analysis showed that they possess the signal peptide, the open reading frame contains two functional domains: chitin binding domain(Ch BD) and chitin deacetylase catalytic domain(CDA). Alignment with the CDAs from T. castaneum showed that partial sequences of two CDAs from L. migratoria were different, and the splicing sites are similar to two alternatively spliced variants of Tc CDA5 from T. castaneum, which indicated two CDAs were alternatively spliced variants. The phylogenetic analysis showed that three Lm CDAs were closely grouped with the CDA4 and CDA5 from other six insect species with high bootstrap value, and named as Lm CDA4, Lm CDA5 a and Lm CDA5 b, respectively. Tissues expression pattern showed that both Lm CDA4 and Lm CDA5 were mainly expressed in the integument, foregut and hindgut. Besides, Lm CDA5 also had a high expression in the fat body. Expression of CDAs in integuments of different days of 5th instar nymph showed that both of Lm CDA4 and Lm CDA5 were highly expressed in the 1st and 2nd days of 5th instar nymph, then decreased gradually. After injection of ds Lm CDA4 and ds Lm CDA5, the expression of target gene was significantly reduced at 24 h compared with ds GFP injected controls, and the silence efficiency of gene was 98.1% and 95.6%, respectively. Phenotypic analysis showed no visible phenotypes were observed and both treatment and control insects could molt to adult successfully. Chitin content detection and TEM observation suggested that silencing the expression of both Lm CDA4 and Lm CDA5 did not affect the chitin content and chitin organization.【Conclusion】Lm CDA4 and Lm CDA5 are highly expressed in the integument, foregut and hindgut. Biologic functional analysis suggested both of them are not essential in the molting process of L. migratoria, silencing of their expression showed no effect on locust development and cuticle structure.
【Objective】 Chitin deacetylase(CDA) is a key enzyme involved in chitin metabolism. The objective of this paper is to study the molecular characteristics and biological function of chitin deacetylase genes in Locusta migratoria, and to provide a theoretical basis for screening novel targets for pest control.【Method】Based on the transcriptome of L. migratoria, three c DNA sequences putatively encoded Lm CDAs were obtained, and the gene structure was analyzed by comparing with the locust genome database. Alignment was performed with the CDAs from Tribolium castaneum and the functional domains were predicted by Blast P and SMART software. The homologous sequences from T. castaneum, Drosophila melanogaster, Anopheles gambiae, Bombyx mori, Oxya chinensis and Choristoneura fumiferana were selected to perform clustering analysis with Lm CDA4, Lm CDA5 a and Lm CDA5 b, and phylogenetic tree was constructed by using MEGA 5.02 software with the neighbor-joining(NJ) method. The RT-q PCR was applied to detect the relative expression of Lm CDA4 and Lm CDA5 in different tissues and developmental stages of the 5th instar nymphs. The RNA interference(RNAi) was performed to study the biological function of Lm CDA4 and Lm CDA5 during the molting process of L. migratoria. The chitin content was detected by chemical method. The transmission electron microscope(TEM) was applied to study whether the Lm CDA4 and Lm CDA5 have effects on the cuticle structure.【Result】The three full length c DNA sequences putatively encoded chitin deacetylase were identified from locust transcriptome database. The bioinformatics analysis showed that they possess the signal peptide, the open reading frame contains two functional domains: chitin binding domain(Ch BD) and chitin deacetylase catalytic domain(CDA). Alignment with the CDAs from T. castaneum showed that partial sequences of two CDAs from L. migratoria were different, and the splicing sites are similar to two alternatively spliced variants of Tc CDA5 from T. castaneum, which indicated two CDAs were alternatively spliced variants. The phylogenetic analysis showed that three Lm CDAs were closely grouped with the CDA4 and CDA5 from other six insect species with high bootstrap value, and named as Lm CDA4, Lm CDA5 a and Lm CDA5 b, respectively. Tissues expression pattern showed that both Lm CDA4 and Lm CDA5 were mainly expressed in the integument, foregut and hindgut. Besides, Lm CDA5 also had a high expression in the fat body. Expression of CDAs in integuments of different days of 5th instar nymph showed that both of Lm CDA4 and Lm CDA5 were highly expressed in the 1st and 2nd days of 5th instar nymph, then decreased gradually. After injection of ds Lm CDA4 and ds Lm CDA5, the expression of target gene was significantly reduced at 24 h compared with ds GFP injected controls, and the silence efficiency of gene was 98.1% and 95.6%, respectively. Phenotypic analysis showed no visible phenotypes were observed and both treatment and control insects could molt to adult successfully. Chitin content detection and TEM observation suggested that silencing the expression of both Lm CDA4 and Lm CDA5 did not affect the chitin content and chitin organization.【Conclusion】Lm CDA4 and Lm CDA5 are highly expressed in the integument, foregut and hindgut. Biologic functional analysis suggested both of them are not essential in the molting process of L. migratoria, silencing of their expression showed no effect on locust development and cuticle structure.
引文
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[18]闫晓平,赵丹,郭巍,王伟,张雅昆,郜玉杰,赵坤莉.美国白蛾几丁质脱乙酰酶的克隆、表达及酶学性质.中国农业科学,2017,50(5):849-858.YAN X P,ZHAO D,GUO W,WANG W,ZHANG Y K,GAO Y J,ZHAO K L.Cloning,expression and enzymatic characterization of chitin deacetylases from Hyphantria cunea.Scientia Agricultura Sinica,2017,50(5):849-858.(in Chinese)
[19]于荣荣,丁国伟,郭亚平,马恩波,张建珍.中华稻蝗几丁质脱乙酰基酶2基因的分子特性和生物学功能.中国农业科学,2014,47(7):1321-1329.YU R R,DING G W,GUO Y P,MA E B,ZHANG J Z.Molecular characterization and functional analysis of chitin deacetylase 2 gene in Oxya chinensis.Scientia Agricultura Sinica,2014,47(7):1321-1329.(in Chinese)
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[21]WANG S,JAYARAM A S,HEMPHALA J,SENTI K A,TSAROUHAS V,JIN H,SAMAKOVLIS C.Septate-junctiondependent luminal deposition of chitin deacetylases restricts tube elongation in the Drosophila trachea.Current Biology,2006,16(2):180-185.
[22]LUSCHNIG S,B?TZ T,ARMBRUSTER K,KRASNOW M A.Serpentine and vermiform encode matrix proteins with chitin binding and deacetylation domains that limit tracheal tube length in Drosophila.Current Biology,2006,16(2):186-194.
[23]YU H Z,LIU M H,WANG X Y,YANG X,WANG W L,GENG L,YU D,LIU X L,LIU G Y,XU J P.Identification and expression profiles of chitin deacetylase genes in the rice leaf folder,Cnaphalocrocis medinalis.Journal of Asia-Pacific Entomology,2016,19(3):691-696.
[24]KARIU T,SMITH A,YANG X,PAL U.A chitin deacetylase-like protein is a predominant constituent of tick peritrophic membrane that influences the persistence of Lyme disease pathogens within the vector.PLo S ONE,2013,8(10):e78376.
[25]LIU X J,LI F,LI D Q,MA E B,ZHANG W,ZHU K Y,ZHANG J Z.Molecular and functional analysis of UDP-N-acetylglucosamine pyrophosphorylases from the migratory locust,Locusta migratoria.PLo S ONE,2013,8(8):e71970.
[26]ZHANG J,ZHU K Y.Characterization of a chitin synthase c DNA and its increased m RNA level associated with decreased chitin synthesis in Anopheles quadrimaculatus exposed to diflubenzuron.Insect Biochemistry and Molecular Biology,2006,36(9):712-725.
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[2]MA E B,HE Y P,ZHU K Y.Comparative studies of acetylcholinesterases purified from two field populations of the oriental migratory locust(Locusta migratoria manilensis):implications of insecticide resistance.Pesticide Biochemistry and Physiology,2004,78(1):67-77.
[3]MOUSSIAN B,SCHWARZ H,BARTOSZEWSKI S,NüSSLEINVOLHARD C.Involvement of chitin in exoskeleton morphogenesis in Dorsophila melanogaster.Journal of Morphology,2005,264(1):117-130.
[4]TSIGOS L,MARTINOU A,KAFETZOPOULOS D,BOURIOTIS V.Chitin deacetylases:new,versatile tools in biotechnology.Trends in Biotechnology,2000,18(7):305-312.
[5]ZHU K Y,MERZENDORFER H,ZHANG W Q,ZHANG J Z,MUTHUKRISHNAN S.Biosynthesis,turnover and function of chitin in insects.Annual Review of Entomology,2016,61(1):177-196.
[6]ARAKANE Y,DIXIT R,BEGUM K,PARK Y,SPECHT C A,MERZENDORFER H,KRAMER K J,MUTHUKRISHNAN S,BEEMAN R W.Analysis of functions of the chitindeacetylase gene family in Tribolium castaneum.Insect Biochemistry and Molecular Biology,2009,39(5/6):355-365.
[7]DIXIT R,ARAKANE Y,SPECHT C A,RICHARD C,KRAMER K J,BEEMAN R W,MUTHUKRISHNAN S.Domain organization and phylogenetic analysis of proteins fromthe chitin deacetylase gene family of Tribolium castaneum and three other species of insects.Insect Biochemistry and Molecular Biology,2008,38(4):440-451.
[8]GUO W,LI G X,PANG Y,WANG P.A novel chitin-binding protein identified from the peritrophic membrance of the cabbage looper,Trichoplusia ni.Insect Biochemistry and Molecular Biology,2005,35(11):1224-1234.
[9]TOPRAK U,BALDWIN D,ERLANDSON M,GILLOTT C,HOU X,COUTU C,HEGEDUS D D.A chitin deacetylase and putative insect intestinal lipases are components of the Mamestra configurata(Lepidoptera:Noctuidae)peritrophic matrix.Insect Molecular Biology,2008,17(5):573-585.
[10]CAMPBELL P M,CAO A T,HINES E R,EAST P D,GORDON K H.Proteomic analysis of the peritrophic matrix from the gut of the caterpillar,Helicoverpa armigera.Insect Biochemistry and Molecular Biology,2008,38(10):950-958.
[11]ZHONG X W,WANG X H,TAN X,XIA Q Y,XIANG Z H,ZHAO P.Identification and molecular characterization of a chitin deacetylase from Bombyx mori peritrophic membrane.International Journal of Molecular Sciences,2014,15(2):1946-1961.
[12]SANDOVAL-MOJICA A F,SCHARF M E.Gut genes associated with the peritrophic matrix in Reticulitermes flavipes(Blattodea:Rhinotermitidae):Identification and characterization.Archives of Insect Biochemistry and Physiology,2016,92(2):127-142.
[13]GANGISHETTI U,VEERKAMP J,BEZDAN D,SCHWARZ H,LOHMANN I,MOUSSIAN B.The transcription factor Grainy head and the steroid hormone ecdysone cooperate during differentiation of the skin of Drosophila melanogaster.Insect Molecular Biology,2012,21(3):283-295.
[14]QUAN X G,LADD T,JUN D,WEN F Y,DOURCET D,CUSSON M,KRELL P J.Characterization of a spruce budworm chitin deacetylase gene:stage-and tissue-specific expression,and inhibition using RNA interference.Insect Biochemistry and Molecular Biology,2013,43(8):683-691.
[15]XI Y,PAN P L,YE Y X,YU B,ZHANG C X.Chitin deacetylase family genes in the brown planthopper,Nilaparvata lugens(Hemiptera:Delphacidae).Insect Molecular Biology,2014,23(6):695-705.
[16]YU R R,LIU W M,LI D Q,ZHAO X M,DING G W,ZHANG M,MA E B,ZHU K Y,LI S,MOUSSIAN B,ZHANG J Z.Helicoidal organization of chitin in the cuticle of the migratory locust requires the function of the chitin deacetylase2 enzyme(Lm CDA2).Journal of Biological Chemistry,2016,291(47):24352-24363.
[17]赵盼,张学尧,刘晓健,赵小明,于荣荣,董玮,马恩波,张建珍,张敏.飞蝗几丁质脱乙酰基酶的真核表达、亲和纯化及酶活性.中国农业科学,2017,50(6):1057-1066.ZHAO P,ZHANG X Y,LIU X J,ZHAO X M,YU R R,DONG W,MA E B,ZHANG J Z,ZHANG M.Eukaryotic expression,affinity purification and enzyme activity of chitin deacetylase in Locusta migratoria.Scientia Agricultura Sinica,2017,50(6):1057-1066.(in Chinese)
[18]闫晓平,赵丹,郭巍,王伟,张雅昆,郜玉杰,赵坤莉.美国白蛾几丁质脱乙酰酶的克隆、表达及酶学性质.中国农业科学,2017,50(5):849-858.YAN X P,ZHAO D,GUO W,WANG W,ZHANG Y K,GAO Y J,ZHAO K L.Cloning,expression and enzymatic characterization of chitin deacetylases from Hyphantria cunea.Scientia Agricultura Sinica,2017,50(5):849-858.(in Chinese)
[19]于荣荣,丁国伟,郭亚平,马恩波,张建珍.中华稻蝗几丁质脱乙酰基酶2基因的分子特性和生物学功能.中国农业科学,2014,47(7):1321-1329.YU R R,DING G W,GUO Y P,MA E B,ZHANG J Z.Molecular characterization and functional analysis of chitin deacetylase 2 gene in Oxya chinensis.Scientia Agricultura Sinica,2014,47(7):1321-1329.(in Chinese)
[20]丁国伟,于荣荣,杨美玲,马恩波,杨静,张建珍.中华稻蝗几丁质脱乙酰基酶1基因的分子特性及功能.昆虫学报,2014,57(11):1265-1271.DING G W,YU R R,YANG M L,MA E B,YANG J,ZHANG J Z.Molecular characterization and functional analysis of chitin deacetylase 1 gene in Oxya chinensis(Orthoptera:Acrididae).Acta Entomologica Sinica,2014,57(11):1265-1271.(in Chinese)
[21]WANG S,JAYARAM A S,HEMPHALA J,SENTI K A,TSAROUHAS V,JIN H,SAMAKOVLIS C.Septate-junctiondependent luminal deposition of chitin deacetylases restricts tube elongation in the Drosophila trachea.Current Biology,2006,16(2):180-185.
[22]LUSCHNIG S,B?TZ T,ARMBRUSTER K,KRASNOW M A.Serpentine and vermiform encode matrix proteins with chitin binding and deacetylation domains that limit tracheal tube length in Drosophila.Current Biology,2006,16(2):186-194.
[23]YU H Z,LIU M H,WANG X Y,YANG X,WANG W L,GENG L,YU D,LIU X L,LIU G Y,XU J P.Identification and expression profiles of chitin deacetylase genes in the rice leaf folder,Cnaphalocrocis medinalis.Journal of Asia-Pacific Entomology,2016,19(3):691-696.
[24]KARIU T,SMITH A,YANG X,PAL U.A chitin deacetylase-like protein is a predominant constituent of tick peritrophic membrane that influences the persistence of Lyme disease pathogens within the vector.PLo S ONE,2013,8(10):e78376.
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