华北大黑鳃金龟几丁质脱乙酰酶HoCDA5的分离及定位分析
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摘要
为了获得华北大黑鳃金龟(Holotrichia oblita)幼虫几丁质脱乙酰酶5(HoCDA5)及其在不同组织中的表达情况,本研究利用RACE-PCR方法扩增获得编码华北大黑鳃金龟几丁质脱乙酰酶的cDNA基因hocda5(GenBank登录号:ANI87833),该基因的开放阅读框为1140bp,编码379个氨基酸,属于第V类CDA蛋白。在大肠杆菌BL21(DE3)细胞中成功表达约42kDa的蛋白,制备了HoCDA5蛋白的多克隆抗体并对其进行效价测定。利用western blot方法分析了HoCDA5蛋白在不同组织,不同发育时期的表达情况。结果表明:该蛋白主要分布于围食膜,中肠,并且在中肠的前部含量最高,在幼虫的粪便中也检测到了较大量的HoCDA5蛋白,推测华北大黑鳃金龟幼虫的围食膜为I型,HoCDA5可能随着围食膜的新陈代谢排出体外。本研究成功克隆和表达了HoCDA5重组蛋白,制备了多克隆抗体,对该蛋白进行了组织定位分析,为进一步明确HoCDA5蛋白的功能提供了理论依据。
To study the expression and localization of HoCDA5 protein in different tissues from the larvae of Holotrichia oblita,hocda5 gene was isolated by rapid amplification of cDNA ends PCR(RACE-PCR).The ORF of hocda5 gene encoded chitin deacetylase was 1140 bp in length(GenBank:ANI87833).The deduced protein sequence showed that HoCDA5 was synthesized as a preprotein of 379 amino acid residues with the predicted molecular weight of 42 kDa,containning the only chitin deacetylase domain and belonging to the group V CDA.The hocda5 was successfully expressed in prokaryotic cells BL21(DE3).The recombinant protein of HoCDA5 was used to generate specific polyclonal antiserum.The titer of polyclonal antiserum ofHoCDA5 protein was identified.The expression of HoCDA5 protein in different tissues and development stages was detected by western blot.The results showed that HoCDA5 was detected in the midgut and PM,as well as in fecal matter,and it was more abundant in the anterior of the midgut,which indicated that HoCDA5 is involved in updating the PM.In this study,we successfully prepared the polyclonal antibodies of HoCDA5,and analyzed the localization of HoCDA5 from H.oblita,which provided theoretical basis for further investigation of the function of HoCDA5 protein.
To study the expression and localization of HoCDA5 protein in different tissues from the larvae of Holotrichia oblita,hocda5 gene was isolated by rapid amplification of cDNA ends PCR(RACE-PCR).The ORF of hocda5 gene encoded chitin deacetylase was 1140 bp in length(GenBank:ANI87833).The deduced protein sequence showed that HoCDA5 was synthesized as a preprotein of 379 amino acid residues with the predicted molecular weight of 42 kDa,containning the only chitin deacetylase domain and belonging to the group V CDA.The hocda5 was successfully expressed in prokaryotic cells BL21(DE3).The recombinant protein of HoCDA5 was used to generate specific polyclonal antiserum.The titer of polyclonal antiserum ofHoCDA5 protein was identified.The expression of HoCDA5 protein in different tissues and development stages was detected by western blot.The results showed that HoCDA5 was detected in the midgut and PM,as well as in fecal matter,and it was more abundant in the anterior of the midgut,which indicated that HoCDA5 is involved in updating the PM.In this study,we successfully prepared the polyclonal antibodies of HoCDA5,and analyzed the localization of HoCDA5 from H.oblita,which provided theoretical basis for further investigation of the function of HoCDA5 protein.
引文
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[19]李少雅,赵丹,孙晓彤,等.对美国白蛾几丁质脱乙酰酶(hccda5)基因原核表达影响因素的研究[J].河北农业大学学报,2016,38(4):81-85.
[20]姚庆学,张勇,丁岩.金龟子防治研究的回顾与展望[J].东北林业大学学报,2003,31(1):64-66.
[21]Bolognesi R,Terra W R,Ferreira C.Peritrophic membrane role in enhancing digestive efficiency.Theoretical and experimental models[J].J Insect Physiol,2008,54(10-11):1413-1422.
[22]Shi X,Chamankhah M,Visal-Shah S,et al.Modeling the structure of the type I peritrophic matrix:characterization of a Mamestra configurataintestinal mucin and a novel peritrophin containing 19chitin binding domains[J].Insect Biochem Mol Biol,2004,34(10):1101-1115.
[2]Terra W R.The origin and functions of the insect peritrophic membrane and peritrophic gel[J].Archives of Insect Biochemistry and Physiology,2001,47(2):47-61.
[3]Kramer K J,Muthukrishnan S.Insect chitinases:Molecular biology and potential use as biopesticides[J].Insect Biochemistry and Molecular Biology,1997,27(11):887-900.
[4]Cohen E.Chitin synthesis and inhibition:a revisit[J].Pest Management Science,2001,57(10):946-950.
[5]Zhao Y,Park R D,Muzzarelli R A A.Chitin Deacetylases:Properties and Applications[J].Mar Drugs,2010,8(1):24-46.
[6]Caufrier F,Martinou A,Dupont C,et al.Carbohydrate esterase family 4enzymes:substrate specificity[J].Carbohydrate Research,2003,338(7):687-692.
[7]Guo W,Li G X,Pang Y,et al.A novel chitin-binding protein identified from the peritrophic membrane of the cabbage looper,Trichoplusia ni[J].Insect Biochemistry and Molecular Biology,2005,35(11):1224-1234.
[8]Luschnig S,Batz T,Armbruster K,et al.Serpentine and vermiform encode matrix proteins with chitin binding and deacetylation domains that limit tracheal tube length in Drosophila[J].Current Biology,2006,16(2):186-194.
[9]Wang S Q,Jayaram S A,Hemphala J,et al.Septatejunction-dependent luminal deposition of chitin deacetylases restricts tube elongation in the Drosophila trachea[J].Current Biology,2006,16(2):180-185.
[10]Dixit R,Arakane Y,Specht C A,et al.Domain organization and phylogenetic analysis of proteins from the chitin deacetylase gene family of Tribolium castaneum and three other species of insects[J].Insect Biochemistry and Molecular Biology,2008,38(4):440-451.
[11]Arakane Y,Dixit R,Begum K,et al.Analysis of functions of the chitin deacetylase gene family in Tribolium castaneum[J].Insect Biochemistry and Molecular Biology,2009,39(5-6):355-365.
[12]Toprak U,Baldwin D,Erlandson M,et al.A chitin deacetylase and putative insect intestinal lipases are components of the Mamestra configurata(Lepidoptera:Noctuidae)peritrophic matrix[J].Insect Molecular Biology,2008,17(5):573-585.
[13]Jakubowska A K,Caccia S,Gordon K H,et al.Downregulation of a chitin deacetylase-like protein in response to baculovirus infection and its application for improving baculovirus infectivity[J].J Virol,2010,84(5):2547-2555.
[14]Quan G,Ladd T,Duan J,et al.Characterization of a spruce budworm chitin deacetylase gene:Stage-and tissue-specific expression,and inhibition using RNA interference[J].Insect Biochemistry and Molecular Biology,2013,43(8):683-691.
[15]Zhong X W,Wang X H,Tan X,et al.Identification and Molecular Characterization of a Chitin Deacetylase fromBombyx mori Peritrophic Membrane[J].International Journal of Molecular Sciences,2014,15(2):1946-1961.
[16]Xi Y,Pan P L,Ye Y X,et al.Chitin deacetylase family genes in the brown planthopper,Nilaparvata lugens(Hemiptera:Delphacidae)[J].Insect Molecular Biology,2014,23(6):695-705.
[17]李景,杨宝东,郭巍.甜菜夜蛾几丁质脱乙酰酶基因(secda5)的原核表达及多克隆抗体制备[J].北京农学院学报,2016,31(1):38-41.
[18]赵丹.暗黑鳃金龟幼虫中肠蛋白基因的分离及表达分析[D].保定:河北农业大学,2014.
[19]李少雅,赵丹,孙晓彤,等.对美国白蛾几丁质脱乙酰酶(hccda5)基因原核表达影响因素的研究[J].河北农业大学学报,2016,38(4):81-85.
[20]姚庆学,张勇,丁岩.金龟子防治研究的回顾与展望[J].东北林业大学学报,2003,31(1):64-66.
[21]Bolognesi R,Terra W R,Ferreira C.Peritrophic membrane role in enhancing digestive efficiency.Theoretical and experimental models[J].J Insect Physiol,2008,54(10-11):1413-1422.
[22]Shi X,Chamankhah M,Visal-Shah S,et al.Modeling the structure of the type I peritrophic matrix:characterization of a Mamestra configurataintestinal mucin and a novel peritrophin containing 19chitin binding domains[J].Insect Biochem Mol Biol,2004,34(10):1101-1115.