埃及伊蚊表皮结构基因AaCPR100A序列特征和表达特性分析
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摘要
Aa CPR100A是本实验室从埃及伊蚊RNAseq数据库中获得的预测与表皮结构相关的基因。本文拟通过生物信息学和分子生物学技术,探究埃及伊蚊表皮Aa CPR100A基因的序列特点,并分析其在埃及伊蚊的时空表达特征。埃及伊蚊表皮Aa CPR100A蛋白序列Aa CPR100A ORF长度为765 bp,编码254个氨基酸,蛋白分子量约为28.6 k Da,1~16位氨基酸残基为信号肽,其氨基酸序列中含有表皮蛋白CPR家族中的RR-1基序。埃及伊蚊与白纹伊蚊、致倦库蚊、冈比亚按蚊和黑腹果蝇的氨基酸同源性分别为95%、72%、61%、54%。qRT-PCR结果显示Aa CPR00A基因在表皮和卵期表达量最高,并随发育阶段呈现显著递减变化。结果表明,埃及伊蚊Aa CPR100A表达具有时空和组织特异性特征。
AaCPR100 A is a predicted cuticularization-related gene isolated from RNAseq data. The bioinformatics and molecular biology methods would be used to explore its characterization and spatiotemporal expression. Nucleotide sequence analysis of Aa CPR100 A indicated that the open reading frame consisted of 765 base pairs,encoding 254 amino acid sequences with a calculated molecular weight of 28. 6 k Da. The amino acid analysis showed it has 1-16 amino acid residues as the signal peptide and contains RR-1 motif of CPR protein family. A homological comparison revealed that Aa CPR100 A amino acid sequence in Aedes aegypti was 95%,72%,61% and 72% identical to Ae. albopictus,Culex quinquefasciatus,Anopheles gambiae and Drosophila melanogaster respectively. QRT-PCR analysis revealed that Aa CPR100 A was expressed at all development stages,but higher expression in egg stage and cuticle tissues. These findings showed that Aa CPR100 A gene might be involved in the early cuticle formation of Ae. aegypti.
AaCPR100 A is a predicted cuticularization-related gene isolated from RNAseq data. The bioinformatics and molecular biology methods would be used to explore its characterization and spatiotemporal expression. Nucleotide sequence analysis of Aa CPR100 A indicated that the open reading frame consisted of 765 base pairs,encoding 254 amino acid sequences with a calculated molecular weight of 28. 6 k Da. The amino acid analysis showed it has 1-16 amino acid residues as the signal peptide and contains RR-1 motif of CPR protein family. A homological comparison revealed that Aa CPR100 A amino acid sequence in Aedes aegypti was 95%,72%,61% and 72% identical to Ae. albopictus,Culex quinquefasciatus,Anopheles gambiae and Drosophila melanogaster respectively. QRT-PCR analysis revealed that Aa CPR100 A was expressed at all development stages,but higher expression in egg stage and cuticle tissues. These findings showed that Aa CPR100 A gene might be involved in the early cuticle formation of Ae. aegypti.
引文
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Bendtsen JD,Nielsen H,von Heijne G,Brunak S.Improved prediction of signal peptides:Signal P 3.0.J Mol Biol.,2004,340(4):783-795.
Charles JP.The regulation of expression of insect cuticle protein genes.Insect Biochem Mol Biol,2010,40(3):205-213.
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Nene V,Wortman JR,Lawson D et al.Genome sequence of Aedes aegypti,a major arbovirus vector.Science,2007,316(5832):1 718-1 723.
Reid WR,Zhang L,Liu F,Liu N.The transcriptome profile of the mosquito Culex quinquefasciatus following permethrin selection.PLo S One,2012,7(10):e47163.
Riaz MA,Chandor-Proust A,Dauphin-Villemant C,Poupardin R,Jones CM,Strode C,Regent-Kloeckner M,David JP,Reynaud S.Molecular mechanisms associated with increased tolerance to the neonicotinoid insecticide imidacloprid in the dengue vector Aedes aegypti.Aquatic Toxicology,2013,126:326-337.
Soares MP,Elias-Neto M,Simoes ZL,Bitondi MM.A cuticle protein gene in the honeybee:expression during development and in relation to the ecdysteroid titer.Insect Biochem Mol Biol.,2007,37(12):1 272-1 282.
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Thompson JD,Gibson TJ,Higgins DG.Multiple sequence alignment using Clustal W and Clustal X.Curr Protoc Bioinformatics,2002,Chapter 2:Unit 23.
Vannini L,Willis JH.Localization of RR-1 and RR-2 cuticular proteins within the cuticle of Anopheles gambiae.Arthropod Struct Dev,2017,46(1):13-29.
Willis JH.Structural cuticular proteins from arthropods:annotation,nomenclature,and sequence characteristics in the genomics era.Insect Biochem Mol Biol,2010,40(3):189-204.
Wood O,Hanrahan S,Coetzee M,Koekemoer L,Brooke B.Cuticle thickening associated with pyrethroid resistance in the major malaria vector Anopheles funestus.Parasit Vectors,2010,3:67.
Andersen SO.Amino acid sequence studies on endocuticular proteins from the desert locust,Schistocerca gregaria.Insect Biochem Mol Biol,1998,28(5-6):421-434.
Andersen SO.Studies on proteins in post-ecdysial nymphal cuticle of locust,Locusta migratoria,and cockroach,Blaberus craniifer.Insect Biochem Mol Biol,2000,30(7):569-577.
Bendtsen JD,Nielsen H,von Heijne G,Brunak S.Improved prediction of signal peptides:Signal P 3.0.J Mol Biol.,2004,340(4):783-795.
Charles JP.The regulation of expression of insect cuticle protein genes.Insect Biochem Mol Biol,2010,40(3):205-213.
de Castro E,Sigrist CJ,Gattiker A,Bulliard V,LangendijkGenevaux PS,Gasteiger E,Bairoch A,Hulo N.Scan Prosite:detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins.Nucleic Acids Res.,2006,34(Web Server issue):W362-365.
Futahashi R,Okamoto S,Kawasaki H,Zhong YS,Iwanaga M,Mita K,Fujiwara H.Genome-wide identification of cuticular protein genes in the silkworm,Bombyx mori.Insect Biochem Mol Biol.,2008,38(12):1 138-1 146.
Ioannidou ZS,Theodoropoulou MC,Papandreou NC,Willis JH,Hamodrakas SJ.Cut Prot Fam-Pred:detection and classification of putative structural cuticular proteins from sequence alone,based on profile hidden Markov models.Insect Biochem Mol Biol,2014,52:51-59.
Karouzou MV,Spyropoulos Y,Iconomidou VA,Cornman RS,Hamodrakas SJ,Willis JH.Drosophila cuticular proteins with the R&R Consensus:annotation and classification with a new tool for discriminating RR-1 and RR-2 sequences.Insect Biochem Mol Biol.,2007,37(8):754-760.
Liao C,Upadhyay A,Liang J,Han Q,Li J.3,4-Dihydroxyphenylacetaldehyde synthase and cuticle formation in insects.Dev Comp Immunol,2017,91:34-43.
Livak KJ,Schmittgen TD.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method.Methods,2001,25(4):402-408.
Luz C,Batagin I.Potential of oil-based formulations of Beauveria bassiana to control Triatoma infestans.Mycopathologia,2005,160(1):51-62.
Nene V,Wortman JR,Lawson D et al.Genome sequence of Aedes aegypti,a major arbovirus vector.Science,2007,316(5832):1 718-1 723.
Reid WR,Zhang L,Liu F,Liu N.The transcriptome profile of the mosquito Culex quinquefasciatus following permethrin selection.PLo S One,2012,7(10):e47163.
Riaz MA,Chandor-Proust A,Dauphin-Villemant C,Poupardin R,Jones CM,Strode C,Regent-Kloeckner M,David JP,Reynaud S.Molecular mechanisms associated with increased tolerance to the neonicotinoid insecticide imidacloprid in the dengue vector Aedes aegypti.Aquatic Toxicology,2013,126:326-337.
Soares MP,Elias-Neto M,Simoes ZL,Bitondi MM.A cuticle protein gene in the honeybee:expression during development and in relation to the ecdysteroid titer.Insect Biochem Mol Biol.,2007,37(12):1 272-1 282.
Tamura K,Stecher G,Peterson D,Filipski A,Kumar S.MEGA6:Molecular Evolutionary Genetics Analysis version 6.0.Mol Biol Evol,2013,30(12):2 725-2 729.
Thompson JD,Gibson TJ,Higgins DG.Multiple sequence alignment using Clustal W and Clustal X.Curr Protoc Bioinformatics,2002,Chapter 2:Unit 23.
Vannini L,Willis JH.Localization of RR-1 and RR-2 cuticular proteins within the cuticle of Anopheles gambiae.Arthropod Struct Dev,2017,46(1):13-29.
Willis JH.Structural cuticular proteins from arthropods:annotation,nomenclature,and sequence characteristics in the genomics era.Insect Biochem Mol Biol,2010,40(3):189-204.
Wood O,Hanrahan S,Coetzee M,Koekemoer L,Brooke B.Cuticle thickening associated with pyrethroid resistance in the major malaria vector Anopheles funestus.Parasit Vectors,2010,3:67.