松墨天牛Mfe2基因的克隆、原核表达和表达谱分析
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摘要
【目的】Mfe2为过氧化物酶体多功能酶类型2(peroxisomal multifunctional enzyme type 2)基因,在脂肪酸的β氧化中起到了极其重要的作用。本研究旨在获得和鉴定松墨天牛Monochamus alternatus的Mfe2基因,原核表达其编码蛋白,并分析其在该虫不同虫态和成虫不同组织中的表达模式。【方法】采用在载体与目的片段之间设计具有重叠区引物的快速克隆方法克隆松墨天牛Mfe2基因,并对克隆所得序列进行生物信息学分析;IPTG诱导原核表达,并以Western blot结果验证Mfe2基因表达的融合蛋白的成功表达;使用RT-q PCR技术分析这一基因在天牛不同虫态和成虫不同组织中的表达谱。【结果】克隆到松墨天牛Mfe2基因,并命名为Ma Mfe2(Gen Bank登录号:MF944109),其ORF序列全长为2 148 bp,编码716个氨基酸,预测分子量大小为77.56 k D。生物信息分析显示,Ma Mfe2具有AICARFT_IMPCHas保守结构域序列,与光肩星天牛Anoplophora glabripennis过氧化物酶体多功能酶2氨基酸序列一致性最高(为91%)。Western blot检测显示大肠杆菌Escherichia coli BL21(DE3)中Ma Mfe2表达的蛋白与抗体有特异性结合。发育表达谱结果表明,Ma Mfe2在松墨天牛蛹期表达量最高,在卵和幼虫期表达量较低;组织表达谱结果表明,在成虫不同组织中,Ma Mfe2在脂肪体中表达量最高,在前胸中表达量最低。【结论】松墨天牛Mfe2基因Ma Mfe2编码过氧化物酶体多功能酶,在不同虫态和成虫组织中存在差异表达。本研究为进一步探究该基因在松墨天牛中的功能奠定了基础。
【Aim】Mfe2,peroxisomal multifunctional enzyme type 2 gene,plays a very important role in the β-oxidation of fatty acids. This study aims to clone and identify the Mfe2 gene from the Japanese sawyer beetle,Monochamus alternatus,to express its encoded protein in prokaryotic expression system,and to explore its expression patterns in different developmental stages and adult tissues of the beetle.【Methods】The recombinant plasmid was constructed to clone the Mfe2 gene of M. alternatus using thefast cloning method through designing primers with overlapping region between the vector and target fragment,and the cloned sequence was analyzed with various bioinformatics tools. The expressed Mfe2 protein in prokaryotic expression system was inducted with IPTG,and the fusion protein was verified by Western blotting. The expression profiles of this gene in different developmental stages and adult tissues of M. alternatus were investigated using the RT-q PCR technology. 【Results】Mfe2 was cloned from M.alternatus and named Ma Mfe2( Gen Bank accession no. : MF944109). Its open reading frame is 2 148 bp,encoding a protein of 716 amino acids, with an estimated molecular mass of 77. 56 k D.Bioinformatics analysis showed that Ma Mfe2 has an AICARFT_IMPCHas domain,with 91% amino acid sequence identity with peroxisomal multifunctional enzyme type 2 of Anoplophora glabripennis. The expressed protein in BL21( DE3) strains of Escherichia coli bound specifically with its antibody in Western blot analysis. Developmental expression profiles showed that the expression level of Ma Mfe2 was the highest in the pupal stage,and lower in egg and larval stages,and tissue expression profiles revealed that the expression level of Ma Mfe2 was the highest in the fat body and the lowest in the protothorax of adults. 【Conclusion】Ma Mfe2 encodes a peroxisomal multifunctional enzyme in M. alternatus,and its expression level is various in different developmental stages and adult tissues. This study provides the basis for further research on the function of this gene in M. alternatus.
【Aim】Mfe2,peroxisomal multifunctional enzyme type 2 gene,plays a very important role in the β-oxidation of fatty acids. This study aims to clone and identify the Mfe2 gene from the Japanese sawyer beetle,Monochamus alternatus,to express its encoded protein in prokaryotic expression system,and to explore its expression patterns in different developmental stages and adult tissues of the beetle.【Methods】The recombinant plasmid was constructed to clone the Mfe2 gene of M. alternatus using thefast cloning method through designing primers with overlapping region between the vector and target fragment,and the cloned sequence was analyzed with various bioinformatics tools. The expressed Mfe2 protein in prokaryotic expression system was inducted with IPTG,and the fusion protein was verified by Western blotting. The expression profiles of this gene in different developmental stages and adult tissues of M. alternatus were investigated using the RT-q PCR technology. 【Results】Mfe2 was cloned from M.alternatus and named Ma Mfe2( Gen Bank accession no. : MF944109). Its open reading frame is 2 148 bp,encoding a protein of 716 amino acids, with an estimated molecular mass of 77. 56 k D.Bioinformatics analysis showed that Ma Mfe2 has an AICARFT_IMPCHas domain,with 91% amino acid sequence identity with peroxisomal multifunctional enzyme type 2 of Anoplophora glabripennis. The expressed protein in BL21( DE3) strains of Escherichia coli bound specifically with its antibody in Western blot analysis. Developmental expression profiles showed that the expression level of Ma Mfe2 was the highest in the pupal stage,and lower in egg and larval stages,and tissue expression profiles revealed that the expression level of Ma Mfe2 was the highest in the fat body and the lowest in the protothorax of adults. 【Conclusion】Ma Mfe2 encodes a peroxisomal multifunctional enzyme in M. alternatus,and its expression level is various in different developmental stages and adult tissues. This study provides the basis for further research on the function of this gene in M. alternatus.
引文
Arnold K,Bordoli L,Kopp J,Schwede T,2006.The SWISS-MODELworkspace:a web-based environment for protein structure homology modelling.Bioinformatics,22(2):195-201.
Battistuzzi FU,Schneider KA,Spencer MK,Fisher D,Chaudhry S,Escalante AA,2016.Profiles of low complexity regions in apicomplexa.BMC Evol.Biol.,16:47.
Benkert P,Biasini M,Schwede T,2011.Toward the estimation of the absolute quality of individual protein structure models.Bioinformatics,27(3):343-350.
Ferdinandusse S,Ylianttila MS,Gloerich J,Koski MK,Oostheim W,Waterham HR,Hiltunen JK,Wanders RJA,2006.Mutational spectrum of D-bifunctional protein deficiency and structure-based genotype-phenotype analysis.Am.J.Hum.Genet.,78(1):112-124.
Haataja TJ,Koski MK,Hiltunen JK,Glumoff T,2011.Peroxisomal multifunctional enzyme type 2 from the fruitfly:dehydrogenase and hydratase act as separate entities,as revealed by structure and kinetics.Biochem.J.,435(3):771-781.
Hashimoto T,1999.Peroxisomalβ-oxidation enzymes.Neurochem.Res.,24(4):551-563.
Huyghe S,Mannaerts GP,Baes M,Veldhoven PP,2006a.Peroxisomal multifunctional protein-2:the enzyme,the patients and the knockout mouse model.Biochim.Biophys.Acta,1761(9):973-994.
Huyghe S,Schmalbruch H,Gendt KD,Verhoeven G,Guillou F,Veldhoven PP,Baes M,2006b.Peroxisomal multifunctional protein2 is essential for lipid homeostasis in Sertoli cells and male fertility in mice.Endocrinology,147(5):2228-2236.
Konkol’ováJ,Petroviˇc R,Chandoga J,RepiskM,ZelinkováH,Kr2iakováJ,KolníkováM,KantarskáD,utovskS,B9hmer D,2015.Peroxisomal D-bifunctional protein deficiency:first case reports from Slovakia.Gene,568(1):61-68.
Leenders F,Prescher G,Dolez V,Begue A,Launoit Y,Adamski J,1996a.Assignment of human 17β-hydroxysteroid dehydrogenase IVto chromosome 5q2 by fluorescence in situ hybridization.Genomics,37(3):403-404.
Leenders F,Tesdropf JG,Markus M,Engel T,Seedorf U,Adamski J,1996b.Procine 80-k Da protein reveals intrinsic 17β-hydroxysteroid dehydrogenase,fatty acyl-Co A-hydratase,and sterol transfer activities.J.Biol.Chem.,271(10):5438-5442.
Lenz C,Haerty W,Golding GB,2014.Increased substitution rates surrounding low-complexity regions within primate proteins.Genome Biol.Evol.,6(3):655-665.
Lin T,Cai ZL,Wu HJ,2015.Transcriptome analysis of the Japanese pine sawyer beetle,Monochamus alternatus(Coleoptera:Cerambycidae)by high-throughput Illumina sequencing.J.AsiaPacific Entomol.,18(3):439-445.
Maehara N,Aikawa T,Kanzaki N,2015.Relationship between pine wilt disease development in asymptomatic carrier trees of Bursaphelenchus xylophilus(Nematoda:Apelenchoididae)and their use by Monochamus alternatus(Coleoptera:Cerambycidae).Appl.Entomol.Zool.,50(1):33-39.
Novikov D,Noubhani MD,Vermeersch JR,Fournier B,Mannaerts GP,Veldhoven PP,1997.The human peroxisomal multifunctional protein involved in bile acid synthesis:activity measurement,deficiency in Zellweger syndrome and chromosome mapping.Biochim.Biophys.Acta,1360(3):229-240.
Novikov DK,Vanhove GF,Carchon H,Asselberghs S,Eyssen HJ,Veldhoven PP,Mannaerts GP,1994.Peroxisomalβ-oxidation.Purification of four novel 3-hydroxyacyl-Co A dehydrogenases from rat liver peroxisomes.J.Biol.Chem.,269(43):27125-27135.
Panda O,Akagi AE,Artyukhin AB,Judkins JC,Le HH,Mahanti P,Cohen SM,Sternberg PW,Schroeder FC,2017.Biosynthesis of modular ascarosides in C.elegans.Angew.Chem.Int.Ed.,56(17):4729-4733.
Pierce SB,Walsh T,Chisholm KM,Lee MK,Thornton AM,Fiumara A,Opitz JM,Levy-Lahad E,Klevit RE,King MC,2010.Mutations in the DBP-deficiency protein HSD17B4 cause ovarian dysgenesis,hearing loss,and ataxia of Perrault Syndrome.Am.J.Hum.Genet.,87(2):282-288.
Qin YM,Haapalainen AM,Kilpelauomo SH,Marttila MS,Koski MK,Glumoff T,Novikov DK,Hiltunen JK,2000.Human peroxisomal multifunctional enzyme type 2.J.Biol.Chem.,275(7):4965-4972.
Qin YM,Poutanen MH,Helander HM,Kvist AP,Siivari KM,Schmitz W,Conzelmann E,Hellman U,Hiltunen JK,1997.Peroxisomal multifunctional enzyme ofβ-oxidation metabolizing D-3-hydroxyacylCo A esters in rat liver:molecular cloning,expression and characterization.Biochem.J.,321(1):21-28.
Teale SA,Wickham JD,Zhang FP,Su J,Chen Y,Xiao W,Hanks LM,Millar JG,2011.A male-produced aggregation pheromone of Monochamus alternatus(Coleoptera:Cerambycidae),a major vector of pine wood nematode.J.Econ.Entomol.,104(5):1592-1598.
Togashi K,2016.Effects of crowding on larval diapause and adult body size in Monochamus alternatus alternatus(Coleoptera:Cerambycidae).Can.Entomol.,149(2):159-173.
Velasco AM,Becerra A,Morales RH,Delaye L,Jimenez-Corona ME,Ponce-de-Leon S,Lazcano A,2013.Low complexity regions(LCRs)contribute to the hypervariability of the HIV-1 gp120protein.J.Theor.Biol.,338(37):80-86.
Verheijden S,Bottelbergs A,Krysko O,Krysko DV,Beckers L,Munter SD,Veldhoven PP,Wyns S,Kulik W,Nave KA,Ramer MS,Carmeliet P,Kassmann CM,Baes M,2013.Peroxisomal multifunctional protein-2 deficiency causes neuroinflammation and degeneration of Purkinje cells independent of very long chain fatty acid accumulation.Neurobiol.Dis.,58(10):258-269.
Wu SQ,Zhu XL,Liu ZX,Shao ES,Rebeca CL,Guo YJ,Xiong YT,Mou YN,Xu RX,Hu X,Liang GH,Zou SQ,Guan X,Zhang FP,2016.Identification of genes relevant to pesticides and biology from global transcriptome data of Monochamus alternatus Hope(Coleoptera:Cerambycidae)larvae.PLo S ONE,11(1):e0147855.
Zhang X,Feng L,Chinta S,Singh P,Wang Y,Nunnery JK,Butcher RA,2015.Acyl-Co A oxidase complexes control the chemical message produced by Caenorhabditis elegans.Proc.Natl.Acad.Sci.USA,112(13):3955-3960.
Zhao LL,Zhang XX,Wei YN,Zhou J,Zhang W,Qin PJ,Chinta S,Kong XB,Liu YP,Yu HY,Hu SN,Zou Z,Butcher RA,Sun JH,2016.Ascarosides coordinate the dispersal of a plant-parasitic nematode with the metamorphosis of its vector beetle.Nat.Commun.,7:12341.
Battistuzzi FU,Schneider KA,Spencer MK,Fisher D,Chaudhry S,Escalante AA,2016.Profiles of low complexity regions in apicomplexa.BMC Evol.Biol.,16:47.
Benkert P,Biasini M,Schwede T,2011.Toward the estimation of the absolute quality of individual protein structure models.Bioinformatics,27(3):343-350.
Ferdinandusse S,Ylianttila MS,Gloerich J,Koski MK,Oostheim W,Waterham HR,Hiltunen JK,Wanders RJA,2006.Mutational spectrum of D-bifunctional protein deficiency and structure-based genotype-phenotype analysis.Am.J.Hum.Genet.,78(1):112-124.
Haataja TJ,Koski MK,Hiltunen JK,Glumoff T,2011.Peroxisomal multifunctional enzyme type 2 from the fruitfly:dehydrogenase and hydratase act as separate entities,as revealed by structure and kinetics.Biochem.J.,435(3):771-781.
Hashimoto T,1999.Peroxisomalβ-oxidation enzymes.Neurochem.Res.,24(4):551-563.
Huyghe S,Mannaerts GP,Baes M,Veldhoven PP,2006a.Peroxisomal multifunctional protein-2:the enzyme,the patients and the knockout mouse model.Biochim.Biophys.Acta,1761(9):973-994.
Huyghe S,Schmalbruch H,Gendt KD,Verhoeven G,Guillou F,Veldhoven PP,Baes M,2006b.Peroxisomal multifunctional protein2 is essential for lipid homeostasis in Sertoli cells and male fertility in mice.Endocrinology,147(5):2228-2236.
Konkol’ováJ,Petroviˇc R,Chandoga J,RepiskM,ZelinkováH,Kr2iakováJ,KolníkováM,KantarskáD,utovskS,B9hmer D,2015.Peroxisomal D-bifunctional protein deficiency:first case reports from Slovakia.Gene,568(1):61-68.
Leenders F,Prescher G,Dolez V,Begue A,Launoit Y,Adamski J,1996a.Assignment of human 17β-hydroxysteroid dehydrogenase IVto chromosome 5q2 by fluorescence in situ hybridization.Genomics,37(3):403-404.
Leenders F,Tesdropf JG,Markus M,Engel T,Seedorf U,Adamski J,1996b.Procine 80-k Da protein reveals intrinsic 17β-hydroxysteroid dehydrogenase,fatty acyl-Co A-hydratase,and sterol transfer activities.J.Biol.Chem.,271(10):5438-5442.
Lenz C,Haerty W,Golding GB,2014.Increased substitution rates surrounding low-complexity regions within primate proteins.Genome Biol.Evol.,6(3):655-665.
Lin T,Cai ZL,Wu HJ,2015.Transcriptome analysis of the Japanese pine sawyer beetle,Monochamus alternatus(Coleoptera:Cerambycidae)by high-throughput Illumina sequencing.J.AsiaPacific Entomol.,18(3):439-445.
Maehara N,Aikawa T,Kanzaki N,2015.Relationship between pine wilt disease development in asymptomatic carrier trees of Bursaphelenchus xylophilus(Nematoda:Apelenchoididae)and their use by Monochamus alternatus(Coleoptera:Cerambycidae).Appl.Entomol.Zool.,50(1):33-39.
Novikov D,Noubhani MD,Vermeersch JR,Fournier B,Mannaerts GP,Veldhoven PP,1997.The human peroxisomal multifunctional protein involved in bile acid synthesis:activity measurement,deficiency in Zellweger syndrome and chromosome mapping.Biochim.Biophys.Acta,1360(3):229-240.
Novikov DK,Vanhove GF,Carchon H,Asselberghs S,Eyssen HJ,Veldhoven PP,Mannaerts GP,1994.Peroxisomalβ-oxidation.Purification of four novel 3-hydroxyacyl-Co A dehydrogenases from rat liver peroxisomes.J.Biol.Chem.,269(43):27125-27135.
Panda O,Akagi AE,Artyukhin AB,Judkins JC,Le HH,Mahanti P,Cohen SM,Sternberg PW,Schroeder FC,2017.Biosynthesis of modular ascarosides in C.elegans.Angew.Chem.Int.Ed.,56(17):4729-4733.
Pierce SB,Walsh T,Chisholm KM,Lee MK,Thornton AM,Fiumara A,Opitz JM,Levy-Lahad E,Klevit RE,King MC,2010.Mutations in the DBP-deficiency protein HSD17B4 cause ovarian dysgenesis,hearing loss,and ataxia of Perrault Syndrome.Am.J.Hum.Genet.,87(2):282-288.
Qin YM,Haapalainen AM,Kilpelauomo SH,Marttila MS,Koski MK,Glumoff T,Novikov DK,Hiltunen JK,2000.Human peroxisomal multifunctional enzyme type 2.J.Biol.Chem.,275(7):4965-4972.
Qin YM,Poutanen MH,Helander HM,Kvist AP,Siivari KM,Schmitz W,Conzelmann E,Hellman U,Hiltunen JK,1997.Peroxisomal multifunctional enzyme ofβ-oxidation metabolizing D-3-hydroxyacylCo A esters in rat liver:molecular cloning,expression and characterization.Biochem.J.,321(1):21-28.
Teale SA,Wickham JD,Zhang FP,Su J,Chen Y,Xiao W,Hanks LM,Millar JG,2011.A male-produced aggregation pheromone of Monochamus alternatus(Coleoptera:Cerambycidae),a major vector of pine wood nematode.J.Econ.Entomol.,104(5):1592-1598.
Togashi K,2016.Effects of crowding on larval diapause and adult body size in Monochamus alternatus alternatus(Coleoptera:Cerambycidae).Can.Entomol.,149(2):159-173.
Velasco AM,Becerra A,Morales RH,Delaye L,Jimenez-Corona ME,Ponce-de-Leon S,Lazcano A,2013.Low complexity regions(LCRs)contribute to the hypervariability of the HIV-1 gp120protein.J.Theor.Biol.,338(37):80-86.
Verheijden S,Bottelbergs A,Krysko O,Krysko DV,Beckers L,Munter SD,Veldhoven PP,Wyns S,Kulik W,Nave KA,Ramer MS,Carmeliet P,Kassmann CM,Baes M,2013.Peroxisomal multifunctional protein-2 deficiency causes neuroinflammation and degeneration of Purkinje cells independent of very long chain fatty acid accumulation.Neurobiol.Dis.,58(10):258-269.
Wu SQ,Zhu XL,Liu ZX,Shao ES,Rebeca CL,Guo YJ,Xiong YT,Mou YN,Xu RX,Hu X,Liang GH,Zou SQ,Guan X,Zhang FP,2016.Identification of genes relevant to pesticides and biology from global transcriptome data of Monochamus alternatus Hope(Coleoptera:Cerambycidae)larvae.PLo S ONE,11(1):e0147855.
Zhang X,Feng L,Chinta S,Singh P,Wang Y,Nunnery JK,Butcher RA,2015.Acyl-Co A oxidase complexes control the chemical message produced by Caenorhabditis elegans.Proc.Natl.Acad.Sci.USA,112(13):3955-3960.
Zhao LL,Zhang XX,Wei YN,Zhou J,Zhang W,Qin PJ,Chinta S,Kong XB,Liu YP,Yu HY,Hu SN,Zou Z,Butcher RA,Sun JH,2016.Ascarosides coordinate the dispersal of a plant-parasitic nematode with the metamorphosis of its vector beetle.Nat.Commun.,7:12341.