棉铃虫几丁质酶的系统分类及Ⅶ型几丁质酶表达与酶学性质分析
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摘要
昆虫几丁质酶(chitinase, CHT)主要参与蜕皮、围食膜的降解、细胞增殖和机体免疫防御等重要生理生长发育过程。本研究从NCBI下载棉铃虫(Helicoverpa armigera)及其他昆虫的几丁质酶氨基酸序列,分析结构域和构建系统进化树,结果表明,棉铃虫的6个几丁质酶分别属于Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅶ、Ⅷ型几丁质酶,1个几丁质酶属新的类型(new group)。到目前为止,Ⅶ型几丁质酶HaCHTⅦ研究较少,本研究克隆了HaCHTⅦ基因,在大肠杆菌(Escherichia coli) transetta(DE3)中表达和纯化了融合蛋白His-HaCHTⅦ,并测定了其对胶体几丁质和N-乙酰壳二糖底物的降解活性,结果表明,His-HaCHTⅦ对胶体几丁质没有降解活性,而对N-乙酰壳二糖具有降解活性,最适p H值和温度分别为7和25℃,动力学参数米氏常数(Km)和最大速率(Vmax)分别为0.486 2 mmol/L和2.715 1μg/(mL·min),说明HaCHTⅦ在棉铃虫体内可能将寡聚糖降解为N-乙酰基葡糖糖,为合成新的几丁质提供起始底物而参与昆虫的生长发育。本研究结果将有助于更好地理解棉铃虫Ⅶ型几丁质酶的功能,也为害虫防治提供有益的信息。
Insect chitinases are mainly involved in important physiological growth processes such as molting,degradation of peritrophic membranes, cell proliferation and immune defense. In this study, seven chitinases from Helicoverpa armigera and other insect chitinases sequences were downloaded from NCBI. Their domains and constructed phylogenetic tree were analyzed. The results showed that 6 of chitinases belonged to groupⅠ, Ⅱ, Ⅲ, Ⅳ, Ⅶ and Ⅷ chitinase, respectively, and 1 chitinase belonged to the new group. Group Ⅶchitinase(HaCHT Ⅶ) was rarely reported, It was cloned in this study. Fusion protein His-HaCHT Ⅶ was expressed in Escherichia coli transetta(DE3) and purified. The degradation activity of fusion protein against colloidal chitin and N-acetylated chitobiose was determined. Results showed that His-HaCHT Ⅶ had no degradative activity on colloidal chitin, and had the degradative activity on N-acetylchitobiose. The optimum reaction pH value and temperature condition was at 7 and 25℃, respectively. The kinetic parameter ofmichaelis constant(Km) and maximum speed(Vmax) were 0.486 2 mmol/L and 2.715 1 μg/(mL · min),respectively. Therefore, HaCHT Ⅶ was probably to degrade oligosaccharides to N-acetylglucosamine in H.armigera and then provided substrate to synthesize the new chitin component in the development and growth.Present findings may lead us to a better understand of the function of the group Ⅶ chitinase from H. armigera and may provide useful information for pest control.
Insect chitinases are mainly involved in important physiological growth processes such as molting,degradation of peritrophic membranes, cell proliferation and immune defense. In this study, seven chitinases from Helicoverpa armigera and other insect chitinases sequences were downloaded from NCBI. Their domains and constructed phylogenetic tree were analyzed. The results showed that 6 of chitinases belonged to groupⅠ, Ⅱ, Ⅲ, Ⅳ, Ⅶ and Ⅷ chitinase, respectively, and 1 chitinase belonged to the new group. Group Ⅶchitinase(HaCHT Ⅶ) was rarely reported, It was cloned in this study. Fusion protein His-HaCHT Ⅶ was expressed in Escherichia coli transetta(DE3) and purified. The degradation activity of fusion protein against colloidal chitin and N-acetylated chitobiose was determined. Results showed that His-HaCHT Ⅶ had no degradative activity on colloidal chitin, and had the degradative activity on N-acetylchitobiose. The optimum reaction pH value and temperature condition was at 7 and 25℃, respectively. The kinetic parameter ofmichaelis constant(Km) and maximum speed(Vmax) were 0.486 2 mmol/L and 2.715 1 μg/(mL · min),respectively. Therefore, HaCHT Ⅶ was probably to degrade oligosaccharides to N-acetylglucosamine in H.armigera and then provided substrate to synthesize the new chitin component in the development and growth.Present findings may lead us to a better understand of the function of the group Ⅶ chitinase from H. armigera and may provide useful information for pest control.
引文
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仙笑笑.2013.金纹细蛾I型几丁质酶的表达、纯化及酶学活性测定[D].硕士学位论文,太原理工大学.导师:赵秋勇,pp.2-6.(Expression,purification and characterization of a chitinase from Lithocolletis ringoniella[D].Thesis for M.S.,Taiyuan University of Technology,Suppervisor:Zhao Q Y,pp.2-6.)
杨尉锦,国果,吴沁怡,等.2017.家蝇几丁质酶基因MDCⅡ重组表达质粒的构建及表达模式研究.生物技术通报,33(2):102-108.(Yang Y J,Guo G,Wu Q Y,et al.2017.Construction of the recombinant expression plasmid and expression pattern of chitinase gene MDCIIfrom Musca domestica[J].Biotechnology Bulletin,33(2):102-108).
张常.2014.Ⅰ型和Ⅱ型几丁质酶在舞毒蛾发育过程中的功能研究[D].硕士学位论文,太原理工大学.导师:范晓军,pp.14-16.(Functional study on group I and group IIchitinases during development of Lymantria dispar[D].Thesis for M.S.,Taiyuan University of Technology,Suppervisor:Fan X J,pp.14-16.)
张严峻,谭军,林玉清.2000.低温和几丁质酶处理对棉铃虫围食膜的影响[J].中国生物防治学报,16(4):152-155.(Zhang Y J,Tan J,Lin Y Q.2000.Effects of low temperature and chitinase treatment on the peritrophic membrane of Helicoverpa armigera[J].Chinese Journal of Biological Control,16(4):152-155.)
Aktar M W,Sengupta D,Chowdhury A.2009.Impact of pesticides use in agriculture:Their benefits and hazards[J]Interdisciplinary Toxicology,2(1):1-12.
Arakane Y,Muthukrishnan S.2010.Insect chitinase and chitinase-like proteins[J].Cellular and Molecular Life Sciences,67(2):201-216.
Arakane Y,Zhu Q,Matsumiya M,et al.2003.Properties of catalytic,linker and chitin-binding domains of insec chitinase[J].Insect Biochemistry and Molecular Biology,33(6):631-648.
Genta F A,Blanes L,Cristofoletti P T,et al.2009.Purification,characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut[J].Insect Biochemistry and Molecular Biology,39(12):861-874.
Jasrapuria S,Arakane Y,Osman G,et al.2010.Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny,expression and function[J].Insect Biochemistry and Molecular Biology,40(3):214-227.
Kawamura K,Shibata T,Saget O,et al.1999.A new family of growth factors produced by the fat body and active on drosophila imaginal disc cells[J].Development,126(2)211-219.
Khajuria C,Buschman L L,Chen M S,et al.2010.A gut-specific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia nubilalis larvae[J].Insect Biochemistry and Molecular Biology,40(8):621-629.
Madhuprakash J,E l Gueddari N E,Moerschbacher B M,e al.2015.Catalytic efficiency of chitinase-D on insoluble chitinous substrates was improved by fusing auxiliary domains[J].Plos One,10(1):e0116823-e0116823.
Merzendorfer H,Zimoch L.2003.Chitin metabolism in insects:structure,function and regulation of chitin synthases and chitinases[J].The Journal of experimental biology,206(24):4393-4412.
Nakamura T,Mine S,Hagihara Y,et al.2008.Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus[J].Journal of Molecular Biology,381(3):670-680.
Scott K D,Wilkinson K S,Lawrence N,et al.2005.Geneflow between populations of cotton bollworm Helicoverpa armigera(Lepidoptera:Noctuidae)is highly variable between years[J].Bulletin of Entomological Research,95(4):381-392.
Tabashnik B E,Rensburg J B J V,Carrière Y.2009.Fieldevolved insect resistance to Bt crops:Definition,theory,and data[J].Journal of Economic Entomology,102(6):2011-2025.
Zhang D,Chen J,Yao Q,et al.2012.Functional analysis of two chitinase genes during the pupation and eclosion stages of the beet armyworm Spodoptera exigua by RNAinterference[J].Archives of Insect Biochemistry and Physiology,79(4-5):220-234.
Zhang J,Zhang X,Arakane Y,et al.2011.Comparative genomic analysis of chitinase and chitinase-like genes in the african malaria mosquito(Anopheles gambiae)[J].Plos One,6(5):e19899-e19913.
Zhu Q,Arakane Y,Banerjee D,et al.2008a.Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects[J].Insect Biochemistry and Molecular Biology,38(4):452-466.
Zhu Q,Arakane Y,Beeman R W,et al.2008b.Characterization of recombinant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum[J].Insect Biochemistry and Molecular Biology,38(4):467-477.
Zhu Q,Arakane Y,Beeman R W,et al.2008c.Functional specialization among insect chitinase family genes revealed by RNA interference[J].Proceedings of the National Academy of Sciences of the USA,105(18):6650-6655.
仙笑笑.2013.金纹细蛾I型几丁质酶的表达、纯化及酶学活性测定[D].硕士学位论文,太原理工大学.导师:赵秋勇,pp.2-6.(Expression,purification and characterization of a chitinase from Lithocolletis ringoniella[D].Thesis for M.S.,Taiyuan University of Technology,Suppervisor:Zhao Q Y,pp.2-6.)
杨尉锦,国果,吴沁怡,等.2017.家蝇几丁质酶基因MDCⅡ重组表达质粒的构建及表达模式研究.生物技术通报,33(2):102-108.(Yang Y J,Guo G,Wu Q Y,et al.2017.Construction of the recombinant expression plasmid and expression pattern of chitinase gene MDCIIfrom Musca domestica[J].Biotechnology Bulletin,33(2):102-108).
张常.2014.Ⅰ型和Ⅱ型几丁质酶在舞毒蛾发育过程中的功能研究[D].硕士学位论文,太原理工大学.导师:范晓军,pp.14-16.(Functional study on group I and group IIchitinases during development of Lymantria dispar[D].Thesis for M.S.,Taiyuan University of Technology,Suppervisor:Fan X J,pp.14-16.)
张严峻,谭军,林玉清.2000.低温和几丁质酶处理对棉铃虫围食膜的影响[J].中国生物防治学报,16(4):152-155.(Zhang Y J,Tan J,Lin Y Q.2000.Effects of low temperature and chitinase treatment on the peritrophic membrane of Helicoverpa armigera[J].Chinese Journal of Biological Control,16(4):152-155.)
Aktar M W,Sengupta D,Chowdhury A.2009.Impact of pesticides use in agriculture:Their benefits and hazards[J]Interdisciplinary Toxicology,2(1):1-12.
Arakane Y,Muthukrishnan S.2010.Insect chitinase and chitinase-like proteins[J].Cellular and Molecular Life Sciences,67(2):201-216.
Arakane Y,Zhu Q,Matsumiya M,et al.2003.Properties of catalytic,linker and chitin-binding domains of insec chitinase[J].Insect Biochemistry and Molecular Biology,33(6):631-648.
Genta F A,Blanes L,Cristofoletti P T,et al.2009.Purification,characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut[J].Insect Biochemistry and Molecular Biology,39(12):861-874.
Jasrapuria S,Arakane Y,Osman G,et al.2010.Genes encoding proteins with peritrophin A-type chitin-binding domains in Tribolium castaneum are grouped into three distinct families based on phylogeny,expression and function[J].Insect Biochemistry and Molecular Biology,40(3):214-227.
Kawamura K,Shibata T,Saget O,et al.1999.A new family of growth factors produced by the fat body and active on drosophila imaginal disc cells[J].Development,126(2)211-219.
Khajuria C,Buschman L L,Chen M S,et al.2010.A gut-specific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia nubilalis larvae[J].Insect Biochemistry and Molecular Biology,40(8):621-629.
Madhuprakash J,E l Gueddari N E,Moerschbacher B M,e al.2015.Catalytic efficiency of chitinase-D on insoluble chitinous substrates was improved by fusing auxiliary domains[J].Plos One,10(1):e0116823-e0116823.
Merzendorfer H,Zimoch L.2003.Chitin metabolism in insects:structure,function and regulation of chitin synthases and chitinases[J].The Journal of experimental biology,206(24):4393-4412.
Nakamura T,Mine S,Hagihara Y,et al.2008.Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus[J].Journal of Molecular Biology,381(3):670-680.
Scott K D,Wilkinson K S,Lawrence N,et al.2005.Geneflow between populations of cotton bollworm Helicoverpa armigera(Lepidoptera:Noctuidae)is highly variable between years[J].Bulletin of Entomological Research,95(4):381-392.
Tabashnik B E,Rensburg J B J V,Carrière Y.2009.Fieldevolved insect resistance to Bt crops:Definition,theory,and data[J].Journal of Economic Entomology,102(6):2011-2025.
Zhang D,Chen J,Yao Q,et al.2012.Functional analysis of two chitinase genes during the pupation and eclosion stages of the beet armyworm Spodoptera exigua by RNAinterference[J].Archives of Insect Biochemistry and Physiology,79(4-5):220-234.
Zhang J,Zhang X,Arakane Y,et al.2011.Comparative genomic analysis of chitinase and chitinase-like genes in the african malaria mosquito(Anopheles gambiae)[J].Plos One,6(5):e19899-e19913.
Zhu Q,Arakane Y,Banerjee D,et al.2008a.Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects[J].Insect Biochemistry and Molecular Biology,38(4):452-466.
Zhu Q,Arakane Y,Beeman R W,et al.2008b.Characterization of recombinant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum[J].Insect Biochemistry and Molecular Biology,38(4):467-477.
Zhu Q,Arakane Y,Beeman R W,et al.2008c.Functional specialization among insect chitinase family genes revealed by RNA interference[J].Proceedings of the National Academy of Sciences of the USA,105(18):6650-6655.