飞蝗内表皮结构糖蛋白基因LmAbd-2的表达与功能分析
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摘要
【目的】分析飞蝗(Locusta migratoria)内表皮结构糖蛋白(endocuticle structural glycoprotein)基因LmAbd-2的序列特征和表达特性,利用RNA干扰(RNA interference,RNAi)技术结合Hematoxylin and eosin(H&E)染色方法以及透射电镜(transmission electron microscopy,TEM)技术探究其生物学功能,探讨其对飞蝗内表皮发育的影响。【方法】基于课题组飞蝗转录组数据库获得LmAbd-2的cDNA编码序列,并克隆验证。利用ExPASy工具翻译LmAbd-2氨基酸序列,然后利用SignaIP4.1Server和SMART软件分别对其信号肽和结构域进行分析;利用NetOGlyc4.0 Server对其糖基化位点进行预测,使用GENEDOC软件进行多序列比对,并利用MEGA 6.0软件中neighbor-joining(NJ)方法与其他昆虫同源序列进行聚类分析。采用荧光定量PCR(reverse-transcription quantitative PCR,RT-qPCR)分析LmAbd-2在飞蝗5龄第2天不同组织以及4龄(N4D1—N4D6)、5龄(N5D1—N5D8)和成虫(AD1—AD4)表皮不同发育时期表达模式。采用RNAi技术结合H&E染色方法以及TEM技术观察干扰LmAbd-2后对飞蝗生长发育和表皮结构的影响。【结果】序列分析结果显示,飞蝗LmAbd-2 cDNA编码序列长为683 bp,开放阅读框为459 bp,编码152个氨基酸;功能域分析发现LmAbd-2含有1个信号肽和1个几丁质结合域(chitin binding domain 4,ChtBD4),属于CPR家族RR-1亚类;序列比对分析发现其在物种间具有较高的保守性,其中与沙漠蝗(Schistocerca gregaria)SgAbd-2序列相似度为92.5%,且在N末端含有一个保守基序PTPPPIP,其中第2位苏氨酸为糖基化位点;系统发育分析结果显示LmAbd-2与SgAbd-2聚为一支,亲缘关系最近;RT-qPCR分析结果显示LmAbd-2在体壁中高表达,且在蜕皮后(内表皮形成时期)高表达;沉默LmAbd-2后飞蝗没有肉眼可见的表型,但H&E染色结果显示与对照组相比飞蝗表皮层变薄,进一步超微结构分析发现内表皮片层结构减少。【结论】LmAbd-2是一种内表皮结构糖蛋白,属于CPR家族RR-1亚类,其编码基因主要在体壁中高表达,而且在飞蝗4龄、5龄和成虫内表皮形成时期呈周期性表达;RNAi试验表明,LmAbd-2在飞蝗蜕皮过程中参与内表皮的形成。
【Objective】The objective of this study is to analyze the sequence and expression characteristics of endocuticle structural glycoprotein gene Lm Abd-2 that identified from Locusta migratoria transcriptome, explore its function by Hematoxylin and eosin(H&E) staining and transmission electron microscopy(TEM) based on RNAi with ds Lm Abd-2, and to discuss the effect on the formation of endocuticle during L. migratoria molting.【Method】The cDNA sequence of Lm Abd-2 was obtained according to the transcriptome database of L. migratoria, and was cloned by reverse-transcription PCR(RT-PCR) and sequenced. The amino acid sequence of Lm Abd-2 was translated by Ex PASy tool, and its signal peptide and structural domain were analyzed by using Signa IP4.1 Server and SMART software, respectively. Meanwhile, the O-linked glycosylation sites were predicted by using NetOGlyc4.0 Server. The homologous sequences of Abd-2 from locust and other insect species were aligned by GENEDOC software and evolutionary tree was constructed by using the MEGA 6.0 software with the neighbor-joining(NJ) method. Expression profiles of Lm Abd-2 in different tissues at day 2 of 5 th instar nymphs and developmental days of 4 th instar nymphs(N4 D1-N4 D6), 5 th instar nymphs (N5 D1-N5 D8), and the early stage of adults (AD1-AD4) were assayed by using reverse-transcription quantitative PCR(RT-qPCR). The biological function of Lm Abd-2 was analyzed by combination of RNAi and H&E staining and TEM methods.【Result】The results of sequence analysis showed that the length of the Lm Abd-2 cDNA is 683 bp and full length of its ORF is 459 bp, encoding 152 amino acids. The functional domain analysis showed that the protein encoded by Lm Abd-2 contains one signal peptide and one chitin binding domain(ChtBD4), which belongs to RR-1 subclass of the CPR family. Sequence alignment analysis showed that it is highly conserved among species, and the similarity with Sg Abd-2 is 92.5%, and it contains conserved motif(PTPPPIP) in N-terminal and has a potential O-linked glycosylation site (T116) at which glycosylation modification may occur.Phylogenetic tree analysis showed that LmAbd-2 has a close genetic relationship with SgAbd-2 of Schistocerca gregaria. The results of RT-qPCR revealed that Lm Abd-2 was highly expressed in the integument, but lower or no expression in other tested tissues, and showed periodic expression during molting. After injection of ds Lm Abd-2 on day 3 of 4 th instar nymphs and day 4 of 5 th instar nymphs, the insects could normally molt to adults and showed no macroscopic phenotype; however, the cuticle of the adults was thinner, and there were significantly fewer endocuticular lamellae than those in the control.【Conclusion】An endocuticle structural glycoprotein gene Lm Abd-2 was obtained according to the L. migratoria transcriptome database. The protein encoded by Lm Abd-2 belongs to RR-1 subclass of CPR family. LmAbd-2 was mainly expressed in integument among all the tested tissues and showed periodic expression at the early stage of 4 th instar nymphs, 5 th instar nymphs and adults. The results of RNAi suggested that Lm Abd-2 was involved in the formation of endocuticle during L. migratoria molting.
【Objective】The objective of this study is to analyze the sequence and expression characteristics of endocuticle structural glycoprotein gene Lm Abd-2 that identified from Locusta migratoria transcriptome, explore its function by Hematoxylin and eosin(H&E) staining and transmission electron microscopy(TEM) based on RNAi with ds Lm Abd-2, and to discuss the effect on the formation of endocuticle during L. migratoria molting.【Method】The cDNA sequence of Lm Abd-2 was obtained according to the transcriptome database of L. migratoria, and was cloned by reverse-transcription PCR(RT-PCR) and sequenced. The amino acid sequence of Lm Abd-2 was translated by Ex PASy tool, and its signal peptide and structural domain were analyzed by using Signa IP4.1 Server and SMART software, respectively. Meanwhile, the O-linked glycosylation sites were predicted by using NetOGlyc4.0 Server. The homologous sequences of Abd-2 from locust and other insect species were aligned by GENEDOC software and evolutionary tree was constructed by using the MEGA 6.0 software with the neighbor-joining(NJ) method. Expression profiles of Lm Abd-2 in different tissues at day 2 of 5 th instar nymphs and developmental days of 4 th instar nymphs(N4 D1-N4 D6), 5 th instar nymphs (N5 D1-N5 D8), and the early stage of adults (AD1-AD4) were assayed by using reverse-transcription quantitative PCR(RT-qPCR). The biological function of Lm Abd-2 was analyzed by combination of RNAi and H&E staining and TEM methods.【Result】The results of sequence analysis showed that the length of the Lm Abd-2 cDNA is 683 bp and full length of its ORF is 459 bp, encoding 152 amino acids. The functional domain analysis showed that the protein encoded by Lm Abd-2 contains one signal peptide and one chitin binding domain(ChtBD4), which belongs to RR-1 subclass of the CPR family. Sequence alignment analysis showed that it is highly conserved among species, and the similarity with Sg Abd-2 is 92.5%, and it contains conserved motif(PTPPPIP) in N-terminal and has a potential O-linked glycosylation site (T116) at which glycosylation modification may occur.Phylogenetic tree analysis showed that LmAbd-2 has a close genetic relationship with SgAbd-2 of Schistocerca gregaria. The results of RT-qPCR revealed that Lm Abd-2 was highly expressed in the integument, but lower or no expression in other tested tissues, and showed periodic expression during molting. After injection of ds Lm Abd-2 on day 3 of 4 th instar nymphs and day 4 of 5 th instar nymphs, the insects could normally molt to adults and showed no macroscopic phenotype; however, the cuticle of the adults was thinner, and there were significantly fewer endocuticular lamellae than those in the control.【Conclusion】An endocuticle structural glycoprotein gene Lm Abd-2 was obtained according to the L. migratoria transcriptome database. The protein encoded by Lm Abd-2 belongs to RR-1 subclass of CPR family. LmAbd-2 was mainly expressed in integument among all the tested tissues and showed periodic expression at the early stage of 4 th instar nymphs, 5 th instar nymphs and adults. The results of RNAi suggested that Lm Abd-2 was involved in the formation of endocuticle during L. migratoria molting.
引文
[1]MOUSSIAN B,SEIFARTH C,MüLLER U,BERGER J,SCHWARZH.Cuticle differentiation during Drosophila embryogenesis.Arthropod Structure and Development,2006,35(3):137-152.
[2]VINCENT J F,WEGST U G.Design and mechanical properties of insect cuticle.Arthropod Structure and Development,2004,33(3):187-199.
[3]NOH M Y,MUTHUKRISHNAN S,KRAMER K J,ARAKANE Y.Development and ultrastructure of the rigid dorsal and flexible ventral cuticles of the elytron of the red flour beetle,Tribolium castaneum.Insect Biochemistry and Molecular Biology,2017,91:21-33.
[4]WILLIS J H.Structural cuticular proteins from arthropods:Annotation,nomenclature,and sequence characteristics in the genomics era.Insect Biochemistry and Molecular Biology,2010,40(3):189-204.
[5]KAROUZOU M V,SPYROPOULOS Y,ICONOMIDOU V A,CORNMAN R S,HAMODRAKAS S J,WILLIS J H.Drosophila cuticular proteins with the R&R Consensus:Annotation and classification with a new tool for discriminating RR-1 and RR-2sequences.Insect Biochemistry and Molecular Biology,2007,37(8):754-760.
[6]CORNMAN R S,TOGAWA T,DUNN W A,HE N,EMMONS A C,WILLIS J H.Annotation and analysis of a large cuticular protein family with the R&R Consensus in Anopheles gambiae.BMC Genomics,2008,9:22.
[7]FUTAHASHI R,OKAMOTO S,KAWASAKI H,ZHONG Y S,IWANAGA M,MITA K,FUJIWARA H.Genome-wide identification of cuticular protein genes in the silkworm,Bombyx mori.Insect Biochemistry and Molecular Biology,2008,38(12):1138-1146.
[8]IOANNIDOU Z S,THEODOROPOULOU M C,PAPANDREOU NC,WILLIS J H,HAMODRAKAS S J.CutProtFam-Pred:Detection and classification of putative structural cuticular proteins from sequence alone,based on profile Hidden Markov Models.Insect Biochemistry and Molecular Biology,2014,52:51-59.
[9]DITTMER N T,TETREAU G,CAO X,JIANG H,WANG P,KANOST M R.Annotation and expression analysis of cuticular proteins from the tobacco hornworm,Manduca sexta.Insect Biochemistry and Molecular Biology,2015,62:100-113.
[10]PAN P L,YE Y X,LOU Y H,LU J B,CHENG C,SHEN Y,MOUSSIAN B,ZHANG C X.A comprehensive omics analysis and functional survey of cuticular proteins in the brown planthopper.Proceedings of the National Academy of Sciences of the United States of America,2018,115(20):5175-5180.
[11]GUAN X,MIDDLEBROOKS B W,ALEXANDER S,WASSERMANS A.Mutation of TweedleD,a member of an unconventional cuticle protein family,alters body shape in Drosophila.Proceedings of the National Academy of Sciences of the United States of America,2006,103(45):16794-16799.
[12]ARAKANE Y,LOMAKIN J,GEHRKE S H,HIROMASA Y,TOMICH J M,MUTHUKRISHNAN S,BEEMAN R W,KRAMER KJ,KANOST M R.Formation of rigid,non-flight forewings(Elytra)of a beetle requires two major cuticular proteins.PLoS Genetics,2012,8(4):e1002682.
[13]QIAO L,XIONG G,WANG R X,HE S Z,CHEN J,TONG X L,HUH,LI C L,GAI T T,XIN Y Q,LIU X F,CHEN B,XIANG Z H,LU C,DAI F Y.Mutation of a cuticular protein,BmorCPR2,alters larval body shape and adaptability in silkworm,Bombyx mori.Genetics,2014,196(4):1103-1115.
[14]MUN S,YOUNG NOH M,DITTMER N T,MUTHUKRISHNAN S,KRAMER K J,KANOST M R,ARAKANE Y.Cuticular protein with a low complexity sequence becomes cross-linked during insect cuticle sclerotization and is required for the adult molt.Scientific Reports,2015,5:10484.
[15]NOH M Y,MUTHUKRISHNAN S,KRAMER K J,ARAKANE Y.Tribolium castaneum RR-1 cuticular protein Tc CPR4 is required for formation of pore canals in rigid cuticle.PLoS Genetics,2015,11(2):e1004963.
[16]XIONG G,TONG X L,GAI T T,LI C,QIAO L,MONTEIRO A,HUH,HAN M J,DING X,WU S Y,XIANG Z H,LU C,DAI F Y.Body shape and coloration of silkworm larvae are influenced by a novel cuticular protein.Genetics,2017,207(3):1053-1066.
[17]LU J B,LUO X M,ZHANG X Y,PAN P L,ZHANG C X.An ungrouped cuticular protein is essential for normal endocuticle formation in the brown planthopper.Insect Biochemistry and Molecular Biology,2018,100:1-9.
[18]PETKAU G,WINGEN C,JUSSEN L C,RADTKE T,BEHR M.Obstructor-A is required for epithelial extracellular matrix dynamics,exoskeleton function,and tubulogenesis.The Journal of Biological Chemistry,2012,287(25):21396-21405.
[19]王燕,李大琪,刘晓健,李涛,马恩波,范仁俊,张建珍.飞蝗表皮蛋白Obstructor家族基因的分子特性及基于RNAi的功能分析.中国农业科学,2015,48(1):73-82.WANG Y,LI D Q,LIU X J,LI T,MA E B,FAN R J,ZHANG J Z.Molecular characterization and RNAi-based functional analysis of Obstructor family genes in Locusta migratoria.Scientia Agricultura Sinica,2015,48(1):73-82.(in Chinese)
[20]TAJIRI R,OGAWA N,FUJIWARA H,KOJIMA T.Mechanical control of whole body shape by a single cuticular protein obstructor-Ein Drosophila melanogaster.PLoS Genetics,2017,13(1):e1006548.
[21]JAN S,LIU S,HAFEEZ M,ZHANG X,DAWAR F U,GUO J,GAOC,WANG M.Isolation and functional identification of three cuticle protein genes during metamorphosis of the beet armyworm,Spodoptera exigua.Scientific Reports,2017,7:16061.
[22]ANDERSEN S O.Amino acid sequence studies on endocuticular proteins from the desert locust,Schistocerca gregaria.Insect Biochemistry and Molecular Biology,1998,28(5/6):421-434.
[23]ZHAO X M,GOU X,QIN Z Y,LI D Q,WANG Y,MA E B,LI S,ZHANG J Z.Identification and expression of cuticular protein genes based on Locusta migratoria transcriptome.Scientific Reports,2017,7:45462.
[24]赵小明,贾盼,勾昕,刘卫敏,马恩波,张建珍.飞蝗内表皮蛋白基因LmAbd-5的表达与功能分析.中国农业科学,2017,50(10):1817-1826.ZHAO X M,JIA P,GOU X,LIU W M,MA E B,ZHANG J Z.Expression and functional analysis of endocuticle structural glycoprotein gene LmAbd-5 in Locusta migratoria.Scientia Agricultura Sinica,2017,50(10):1817-1826.(in Chinese)
[25]ZHAO X M,QIN Z Y,LIU W M,LIU X J,MOUSSIAN B,MA E B,LI S,ZHANG J Z.Nuclear receptor HR3 controls locust molt by regulating chitin synthesis and degradation genes of Locusta migratoria.Insect Biochemistry and Molecular Biology,2018,92:1-11.
[26]SONG T Q,YANG M L,WANG Y L,LIU Q,WANG H M,ZHANGJ,LI T.Cuticular protein Lm Twdl1 is involved in molt development of the migratory locust.Insect Science,2016,23(4):520-530.
[27]杨亚亭,赵小明,秦忠玉,刘卫敏,马恩波,张建珍.飞蝗表皮蛋白基因LmNCP1的分子特性及功能分析.中国农业科学,2018,51(7):1303-1314.YANG Y T,ZHAO X M,QIN Z Y,LIU W M,MA E B,ZHANG J Z.Molecular characteristics and function analysis of cuticle protein gene LmNCP1 in Locusta migratoria.Scientia Agricultura Sinica,2018,51(7):1303-1314.(in Chinese)
[28]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCT method.Methods,2001,25(4):402-408.
[29]CHARLES J P.The regulation of expression of insect cuticle protein genes.Insect Biochemistry and Molecular Biology,2010,40(3):205-213.
[2]VINCENT J F,WEGST U G.Design and mechanical properties of insect cuticle.Arthropod Structure and Development,2004,33(3):187-199.
[3]NOH M Y,MUTHUKRISHNAN S,KRAMER K J,ARAKANE Y.Development and ultrastructure of the rigid dorsal and flexible ventral cuticles of the elytron of the red flour beetle,Tribolium castaneum.Insect Biochemistry and Molecular Biology,2017,91:21-33.
[4]WILLIS J H.Structural cuticular proteins from arthropods:Annotation,nomenclature,and sequence characteristics in the genomics era.Insect Biochemistry and Molecular Biology,2010,40(3):189-204.
[5]KAROUZOU M V,SPYROPOULOS Y,ICONOMIDOU V A,CORNMAN R S,HAMODRAKAS S J,WILLIS J H.Drosophila cuticular proteins with the R&R Consensus:Annotation and classification with a new tool for discriminating RR-1 and RR-2sequences.Insect Biochemistry and Molecular Biology,2007,37(8):754-760.
[6]CORNMAN R S,TOGAWA T,DUNN W A,HE N,EMMONS A C,WILLIS J H.Annotation and analysis of a large cuticular protein family with the R&R Consensus in Anopheles gambiae.BMC Genomics,2008,9:22.
[7]FUTAHASHI R,OKAMOTO S,KAWASAKI H,ZHONG Y S,IWANAGA M,MITA K,FUJIWARA H.Genome-wide identification of cuticular protein genes in the silkworm,Bombyx mori.Insect Biochemistry and Molecular Biology,2008,38(12):1138-1146.
[8]IOANNIDOU Z S,THEODOROPOULOU M C,PAPANDREOU NC,WILLIS J H,HAMODRAKAS S J.CutProtFam-Pred:Detection and classification of putative structural cuticular proteins from sequence alone,based on profile Hidden Markov Models.Insect Biochemistry and Molecular Biology,2014,52:51-59.
[9]DITTMER N T,TETREAU G,CAO X,JIANG H,WANG P,KANOST M R.Annotation and expression analysis of cuticular proteins from the tobacco hornworm,Manduca sexta.Insect Biochemistry and Molecular Biology,2015,62:100-113.
[10]PAN P L,YE Y X,LOU Y H,LU J B,CHENG C,SHEN Y,MOUSSIAN B,ZHANG C X.A comprehensive omics analysis and functional survey of cuticular proteins in the brown planthopper.Proceedings of the National Academy of Sciences of the United States of America,2018,115(20):5175-5180.
[11]GUAN X,MIDDLEBROOKS B W,ALEXANDER S,WASSERMANS A.Mutation of TweedleD,a member of an unconventional cuticle protein family,alters body shape in Drosophila.Proceedings of the National Academy of Sciences of the United States of America,2006,103(45):16794-16799.
[12]ARAKANE Y,LOMAKIN J,GEHRKE S H,HIROMASA Y,TOMICH J M,MUTHUKRISHNAN S,BEEMAN R W,KRAMER KJ,KANOST M R.Formation of rigid,non-flight forewings(Elytra)of a beetle requires two major cuticular proteins.PLoS Genetics,2012,8(4):e1002682.
[13]QIAO L,XIONG G,WANG R X,HE S Z,CHEN J,TONG X L,HUH,LI C L,GAI T T,XIN Y Q,LIU X F,CHEN B,XIANG Z H,LU C,DAI F Y.Mutation of a cuticular protein,BmorCPR2,alters larval body shape and adaptability in silkworm,Bombyx mori.Genetics,2014,196(4):1103-1115.
[14]MUN S,YOUNG NOH M,DITTMER N T,MUTHUKRISHNAN S,KRAMER K J,KANOST M R,ARAKANE Y.Cuticular protein with a low complexity sequence becomes cross-linked during insect cuticle sclerotization and is required for the adult molt.Scientific Reports,2015,5:10484.
[15]NOH M Y,MUTHUKRISHNAN S,KRAMER K J,ARAKANE Y.Tribolium castaneum RR-1 cuticular protein Tc CPR4 is required for formation of pore canals in rigid cuticle.PLoS Genetics,2015,11(2):e1004963.
[16]XIONG G,TONG X L,GAI T T,LI C,QIAO L,MONTEIRO A,HUH,HAN M J,DING X,WU S Y,XIANG Z H,LU C,DAI F Y.Body shape and coloration of silkworm larvae are influenced by a novel cuticular protein.Genetics,2017,207(3):1053-1066.
[17]LU J B,LUO X M,ZHANG X Y,PAN P L,ZHANG C X.An ungrouped cuticular protein is essential for normal endocuticle formation in the brown planthopper.Insect Biochemistry and Molecular Biology,2018,100:1-9.
[18]PETKAU G,WINGEN C,JUSSEN L C,RADTKE T,BEHR M.Obstructor-A is required for epithelial extracellular matrix dynamics,exoskeleton function,and tubulogenesis.The Journal of Biological Chemistry,2012,287(25):21396-21405.
[19]王燕,李大琪,刘晓健,李涛,马恩波,范仁俊,张建珍.飞蝗表皮蛋白Obstructor家族基因的分子特性及基于RNAi的功能分析.中国农业科学,2015,48(1):73-82.WANG Y,LI D Q,LIU X J,LI T,MA E B,FAN R J,ZHANG J Z.Molecular characterization and RNAi-based functional analysis of Obstructor family genes in Locusta migratoria.Scientia Agricultura Sinica,2015,48(1):73-82.(in Chinese)
[20]TAJIRI R,OGAWA N,FUJIWARA H,KOJIMA T.Mechanical control of whole body shape by a single cuticular protein obstructor-Ein Drosophila melanogaster.PLoS Genetics,2017,13(1):e1006548.
[21]JAN S,LIU S,HAFEEZ M,ZHANG X,DAWAR F U,GUO J,GAOC,WANG M.Isolation and functional identification of three cuticle protein genes during metamorphosis of the beet armyworm,Spodoptera exigua.Scientific Reports,2017,7:16061.
[22]ANDERSEN S O.Amino acid sequence studies on endocuticular proteins from the desert locust,Schistocerca gregaria.Insect Biochemistry and Molecular Biology,1998,28(5/6):421-434.
[23]ZHAO X M,GOU X,QIN Z Y,LI D Q,WANG Y,MA E B,LI S,ZHANG J Z.Identification and expression of cuticular protein genes based on Locusta migratoria transcriptome.Scientific Reports,2017,7:45462.
[24]赵小明,贾盼,勾昕,刘卫敏,马恩波,张建珍.飞蝗内表皮蛋白基因LmAbd-5的表达与功能分析.中国农业科学,2017,50(10):1817-1826.ZHAO X M,JIA P,GOU X,LIU W M,MA E B,ZHANG J Z.Expression and functional analysis of endocuticle structural glycoprotein gene LmAbd-5 in Locusta migratoria.Scientia Agricultura Sinica,2017,50(10):1817-1826.(in Chinese)
[25]ZHAO X M,QIN Z Y,LIU W M,LIU X J,MOUSSIAN B,MA E B,LI S,ZHANG J Z.Nuclear receptor HR3 controls locust molt by regulating chitin synthesis and degradation genes of Locusta migratoria.Insect Biochemistry and Molecular Biology,2018,92:1-11.
[26]SONG T Q,YANG M L,WANG Y L,LIU Q,WANG H M,ZHANGJ,LI T.Cuticular protein Lm Twdl1 is involved in molt development of the migratory locust.Insect Science,2016,23(4):520-530.
[27]杨亚亭,赵小明,秦忠玉,刘卫敏,马恩波,张建珍.飞蝗表皮蛋白基因LmNCP1的分子特性及功能分析.中国农业科学,2018,51(7):1303-1314.YANG Y T,ZHAO X M,QIN Z Y,LIU W M,MA E B,ZHANG J Z.Molecular characteristics and function analysis of cuticle protein gene LmNCP1 in Locusta migratoria.Scientia Agricultura Sinica,2018,51(7):1303-1314.(in Chinese)
[28]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2ΔΔCT method.Methods,2001,25(4):402-408.
[29]CHARLES J P.The regulation of expression of insect cuticle protein genes.Insect Biochemistry and Molecular Biology,2010,40(3):205-213.