棉铃虫Ⅳ型几丁质酶的基因克隆、酶学性质及表达谱
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摘要
【目的】昆虫几丁质酶(chitinase, CHT)主要参与蜕皮、围食膜的降解和机体免疫防御等重要生理生长发育过程。本研究旨在对棉铃虫Helicoverpa armigeraⅣ型(group)几丁质酶基因进行克隆和表达分析,为以该基因作为棉铃虫防控的分子靶标提供理论依据。【方法】采用RT-PCR和RACE技术从棉铃虫中肠中克隆Ⅳ型几丁质酶基因,分别运用DNAMAN和MEGA软件进行多序列比对和构建系统发育树。在大肠杆菌Escherichia coli(DE3)中诱导表达其体外重组蛋白,利用Western blot进一步验证;用Ni-NTA纯化柱纯化重组蛋白,之后研究该蛋白的酶学性质。qPCR分析该基因的在棉铃虫不同发育阶段和6龄幼虫不同组织中的表达谱。【结果】克隆获得棉铃虫几丁质酶基因HaCHT4(GenBank登录号:MH500771),其cDNA长1 624 bp,ORF长1 527 bp,编码509个氨基酸,预测的分子量为55.2 kD。蛋白质序列的N末端具有信号肽,中间序列部分含有一个催化结构域(catalytic domain, CAD), C末端含有一个几丁质结合结构域(chitin binding domain, CBD)。多序列比对显示,HaCHT4具有几丁质酶的保守区域;系统发育分析表明,HaCHT4属于Ⅳ型几丁质酶。重组蛋白His-HaCHT4在大肠杆菌中成功表达。纯化的重组蛋白对胶体几丁质底物具有降解活性,最适温度和pH分别为50℃和7,动力学参数K_m和V_(max)值分别为1.76±0.35 mg/mL和0.0220±0.0012μg/mL·s。qPCR分析表明,HaCHT4在1龄和2龄幼虫期的表达量显著高于其他幼虫龄期及预蛹期;主要在中肠和脂肪体中高度表达,体壁和头部中低表达。【结论】结果提示棉铃虫HaCHT4可能参与围食膜中几丁质降解过程。这些结果为深入研究HaCHT4的功能奠定了基础,并为害虫防治提供了有用的信息。
【Aim】 Insect chitinases are mainly involved in such important physiological processes as molting, degradation of peritrophic membranes and immune defense. This study aims to clone a group IV chitinase gene from Helicoverpa armigera and to analyze its expression profiles so as to provide the theoretical basis for the control of this insect using the gene as a molecular target. 【Methods】 A group IV chitinase gene was cloned from the midgut of H. armigera using RT-PCR and RACE techniques and subjected to multiple sequence alignment by DNAMAN, and the phylogenetic tree was constructed by MEGA. Its recombinant protein was expressed in Escherichia coli(DE3) and verified by Western-blot. The enzymatic properties of the recombinant protein were characterized after purification with Ni-NTA. The expression levels of this gene in different developmental stages and tissues of the 6 th instar larvae of H.armigera were analyzed by qPCR. 【Results】 A chitinase gene was cloned from H. armigera and designated as HaCHT4(GenBank accession no.: MH500771). Its full-length cDNA is 1 624 bp in length and contains an ORF of 1 527 bp that encodes 509 amino acids with a predicted molecular weight of 55.2 kD. Its encoded protein contains a signal peptide, one catalytic domain(CAD) and one chitin binding domain(CBD). Both multiple sequence alignment and phylogenetic analysis showed that HaCHT4 has the conserved domain of chitinase and belongs to group IV chitinases. The recombinant His-HaCHT4 was successfully expressed in E. coli. The purified recombinant protein exhibited the activity of digesting chitin, with the optimum reaction temperature of 50℃ and pH value of 7. The values of kinetic parameters K_m and V_(max) of the recombinant protein were 1.76±0.35 mg/mL and 0.0220±0.0012 μg/mL·s, respectively. qPCR results revealed that the relative expression levels of HaCHT4 in the 1 st and 2 nd larval instars of H.armigera were significantly higher than those in other larval instars and the pre-pupal stage, and HaCHT4 was predominantly expressed in the midgut and fat body of the 6 th instar larva, but lowly expressed in the integument and head. 【Conclusion】 The results suggest that HaCHT4 may participate in chitin degradation in the peritrophic matrix in H. arrmigera. These results lay a foundation for the further function study of HaCHT4 and provide useful information for pest control.
【Aim】 Insect chitinases are mainly involved in such important physiological processes as molting, degradation of peritrophic membranes and immune defense. This study aims to clone a group IV chitinase gene from Helicoverpa armigera and to analyze its expression profiles so as to provide the theoretical basis for the control of this insect using the gene as a molecular target. 【Methods】 A group IV chitinase gene was cloned from the midgut of H. armigera using RT-PCR and RACE techniques and subjected to multiple sequence alignment by DNAMAN, and the phylogenetic tree was constructed by MEGA. Its recombinant protein was expressed in Escherichia coli(DE3) and verified by Western-blot. The enzymatic properties of the recombinant protein were characterized after purification with Ni-NTA. The expression levels of this gene in different developmental stages and tissues of the 6 th instar larvae of H.armigera were analyzed by qPCR. 【Results】 A chitinase gene was cloned from H. armigera and designated as HaCHT4(GenBank accession no.: MH500771). Its full-length cDNA is 1 624 bp in length and contains an ORF of 1 527 bp that encodes 509 amino acids with a predicted molecular weight of 55.2 kD. Its encoded protein contains a signal peptide, one catalytic domain(CAD) and one chitin binding domain(CBD). Both multiple sequence alignment and phylogenetic analysis showed that HaCHT4 has the conserved domain of chitinase and belongs to group IV chitinases. The recombinant His-HaCHT4 was successfully expressed in E. coli. The purified recombinant protein exhibited the activity of digesting chitin, with the optimum reaction temperature of 50℃ and pH value of 7. The values of kinetic parameters K_m and V_(max) of the recombinant protein were 1.76±0.35 mg/mL and 0.0220±0.0012 μg/mL·s, respectively. qPCR results revealed that the relative expression levels of HaCHT4 in the 1 st and 2 nd larval instars of H.armigera were significantly higher than those in other larval instars and the pre-pupal stage, and HaCHT4 was predominantly expressed in the midgut and fat body of the 6 th instar larva, but lowly expressed in the integument and head. 【Conclusion】 The results suggest that HaCHT4 may participate in chitin degradation in the peritrophic matrix in H. arrmigera. These results lay a foundation for the further function study of HaCHT4 and provide useful information for pest control.
引文
Agrawal N, Sachdev B, Rodrigues J, Sree KS, Bhatnagar RK, 2013. Development associated profiling of chitinase and microRNA of Helicoverpa armigera identified chitinase repressive microRNA. Sci. Rep., 3: 2292.
Arakane Y, Muthukrishnan S, 2010. Insect chitinase and chitinase-like proteins. Cell. Mol. Life Sci., 67(2): 201-216.
Blattner R, Gerard PJ, Spindler-Barth M, 1997. Synthesis and biological activity of allosamidin and allosamidin analogues. Pestic. Sci., 50(4): 312-318.
Frutos R, Rang C, Royer M, 2008. Managing insect resistance to plants producing Bacillus thuringiensis toxins. Crit. Rev. Biotechnol., 19(3): 227-276.
Fukamizo F, 2000. Chitinolytic enzymes catalysis, substrate binding, and their application. Curr. Protein Pept. Sci., 1(1): 105-124.
Ganbaatar O, Cao BD, Zhang YN, Bao DR, Bao WH, Wuriyanghan H, 2017. Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors. BMC Biotechnol., 17(1): 9.
Genta FA, Blanes L, Cristofoletti PT, Lago CL, Terra WR, Ferreira C, 2006. Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut. Insect Biochem. Molec. Biol., 36(10): 789-800.
Girard C, Jouanin L, 1999. Molecular cloning of a gut-specific chitinase cDNA from the beetle Phaedon cochleariae. Insect Biochem. Molec. Biol., 29(6): 549-556.
Han JH, Lee KS, Li JH, Kim I, Je YH, Kim DH, Sohn HD, Jin BR, 2005. Cloning and expression of a fat body-specific chitinase cDNA from the spider, Araneus ventricosus. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 140(3): 427-435.
Hegedus D, Erlandson M, Gillott C, Toprak U, 2009. New insights into peritrophic matrix synthesis, architecture, and function. Annu. Rev. Entomol., 54: 285-302.
Jasrapuria S, Arakane Y, Osman G, Kramer KJ, Beeman RW, Muthukrishnan S, 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. Insect Biochem. Molec. Biol., 40(3): 214-227.
Khajuria C, Buschman LL, Chen MS, Muthukrishnan S, Zhu KY, 2010. A gut-specific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia nubilalis larvae. Insect Biochem. Molec. Biol., 40(8): 621-629.
Kramer KJ, Hopkins TL, Schaefer J, 1995. Applications of solids NMR to the analysis of insect sclerotized structures. Insect Biochem. Molec. Biol., 25(10): 1067-1080.
Lehane MJ, 1997. Peritrophic matrix structure and function. Annu. Rev. Entomol., 42: 525-550.
Li DQ, Du JZ, Zhang JQ, Hao YS, Liu XJ, Wang YX, Ma EB, Zhang JZ, Sun Y, 2011. Study on expression characteristics and functions of chitinase family genes from Locusta migratoria manilensis (Meyen). Sci. Agric. Sin., 44(3): 485-492. [李大琪, 杜建中, 张建琴, 郝耀山, 刘晓健, 王亦学, 马恩波, 张建珍, 孙毅, 2011. 东亚飞蝗几丁质酶家族基因的表达特性与功能研究. 中国农业科学, 44(3): 485-492]
Madhuprakash J, El Gueddari NE, Moerschbacher BM, Podile AR, 2015. Catalytic efficiency of chitinase-D on insoluble chitinous substrates was improved by fusing auxiliary domains. PLoS ONE, 10(1): e0116823.
Mamta, Reddy KRK, Rajam MV, 2016. Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato. Plant Mol. Biol., 90(3): 281-292.
Merzendorfer H, Zimoch L, 2003. Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. J. Exp. Biol., 206(24): 4393-4412.
Nakamura T, Mine S, Hagihara Y, Ishikawa K, Ikegami T, Uegaki K, 2008. Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus. J. Mol. Biol., 381(3): 670-680.
Ohno T, Armand S, Hata T, Nikaidou N, Henrissat B, Mitsutomi M, Watanabe T, 1996. A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT6037. J. Bacteriol., 178(17): 5065-5070.
Osman GH, Assem SK, Alreedy RM, Elghareeb DK, Basry MA, Rastogi A, Kalaji HM, 2015. Development of insect resistant maize plants expressing a chitinase gene from the cotton leaf worm, Spodoptera littoralis. Sci. Rep., 5: 18067.
Pan Y, Peng L, Wang Y, Yin LJ, Ma HX, Ma GH, Chen KP, He YQ, 2012. In silico identification of novel chitinase-like proteins in the silkworm, Bombyx mori, genome. J. Insect Sci., 12: 150.
Qu MB, Ma L, Chen P, Yang Q, 2014. Proteomic analysis of insect molting fluid with a focus on enzymes involved in chitin degradation. J. Proteome Res., 13(6): 2931-2940.
Ramalho-Ortig?o JM, Traub-Csek? YM, 2003. Molecular characterization of Llchit1, a midgut chitinase cDNA from the leishmaniasis vector Lutzomyia longipalpis. Insect Biochem. Molec. Biol., 33(3): 279-287.
Shao L, Devenport M, Jacobs-Lorena M, 2001. The peritrophic matrix of hematophagous insects. Arch. Insect Biochem. Physiol., 47(2): 119-125.
Shen ZC, Jacobs-Lorena M, 1997. Characterization of a novel gut-specific chitinase gene from the human malaria vector Anopheles gambiae. J. Biol. Chem., 272(46): 28895-28900.
Shi L, Paskewitz SM, 2004. Identification and molecular characterization of two immune-responsive chitinase-like proteins from Anopheles gambiae. Insect Mol. Biol., 13(4): 387-398.
Tetreau G, Cao XL, Chen YR, Muthukrishnan S, Jiang HB, Blissard GW, Kanost MR, Wang P, 2015. Overview of chitin metabolism enzymes in Manduca sexta: identification, domain organization, phylogenetic analysis and gene expression. Insect Biochem. Molec. Biol., 62(1): 114-126.
Xia M, 2015. Molecular Cloning, Biochemical Characterization and Inhibitor Discovery of Group Ⅳ Chitinase from the Insect Ostrinia furnacalis. MSc Thesis, Dalian University of Technology, Dalian, Liaoning. [夏梦, 2015. 亚洲玉米螟Ⅳ家族几丁质酶的克隆表达、性质表征及抑制剂研究. 辽宁大连: 大连理工大学硕士学位论文]
Xiang JB, 2004. Studies on the Structure and Components of Peritrophic Membrane and the Selective Toxicity of Celangulin Ⅴ. MSc Thesis, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, Shaanxi. [相静波, 2004. 昆虫围食膜结构、组分及与苦皮藤素Ⅴ选择毒性关系的研究. 陕西杨凌: 西北农林科技大学硕士学位论文]
Xiong GH, Xing LS, Lin Z, Saha TT, Wang C, Jiang H, Zou Z, 2015. High throughput profiling of the cotton bollworm Helicoverpa armigera immunotranscriptome during the fungal and bacterial infections. BMC Biotechnol., 16(1): 321.
Yan J, Cheng Q, Narashimhan S, Li CB, Aksoy S, 2002. Cloning and functional expression of a fat body-specific chitinase cDNA from the tsetse fly, Glossina morsitans morsitans. Insect Biochem. Molec. Biol., 32(9): 979-989.
Zhang BS, Cao YQ, Zhang W, Yin YP, Wang ZK, Peng GX, Xia YX, 2007. Cloning, sequence analysis and tissue expression of the chitinase gene from the midgut of Locusta migratoria manilensis (Meyen). Acta Entomol. Sin., 50(6): 555-559. [张八生, 曹月青, 张伟, 殷幼平, 王中康, 彭国雄, 夏玉先, 2007. 东亚飞蝗中肠几丁质酶基因的克隆、序列分析及组织定位. 昆虫学报, 50(6): 555-559]
Zhang DW, Chen J, Yao Q, Pan ZQ, Chen J, Zhang WQ, 2012. Functional analysis of two chitinase genes during the pupation and eclosion stages of the beet armyworm Spodoptera exigua by RNA interference. Arch. Insect Biochem. Physiol., 79(4-5): 220-234.
Zhang JZ, Zhang X, Arakane Y, Muthukrishnan S, Kramer KJ, Ma E, Zhu KY, 2011. Comparative genomic analysis of chitinase and chitinase-like genes in the african malaria mosquito (Anopheles gambiae). PLoS ONE, 6(5): e19899.
Zhu QS, Arakane Y, Banerjee D, Beeman RW, Kramer KJ, Muthukrishnan S, 2008a. Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects. Insect Biochem. Molec. Biol., 38(4): 452-466.
Zhu QS, Arakane Y, Beeman RW, Kramer KJ, Muthukrishnan S, 2008b. Characterization of recombinant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum. Insect Biochem. Molec. Biol., 38(4): 467-477.
Zhu QS, Arakane Y, Beeman RW, Kramer KJ, Muthukrishnan S, 2008c. Functional specialization among insect chitinase family genes revealed by RNA interference. Proc. Natl. Acad. Sci. USA, 105(18): 6650-6655.
Zhu QS, Deng YP, Vanka P, Brown SJ, Muthukrishnan S, Kramer KJ, 2004. Computational identification of novel chitinase-like proteins in the Drosophila melanogaster genome. Bioinformatics, 20(2): 161-169.
Arakane Y, Muthukrishnan S, 2010. Insect chitinase and chitinase-like proteins. Cell. Mol. Life Sci., 67(2): 201-216.
Blattner R, Gerard PJ, Spindler-Barth M, 1997. Synthesis and biological activity of allosamidin and allosamidin analogues. Pestic. Sci., 50(4): 312-318.
Frutos R, Rang C, Royer M, 2008. Managing insect resistance to plants producing Bacillus thuringiensis toxins. Crit. Rev. Biotechnol., 19(3): 227-276.
Fukamizo F, 2000. Chitinolytic enzymes catalysis, substrate binding, and their application. Curr. Protein Pept. Sci., 1(1): 105-124.
Ganbaatar O, Cao BD, Zhang YN, Bao DR, Bao WH, Wuriyanghan H, 2017. Knockdown of Mythimna separata chitinase genes via bacterial expression and oral delivery of RNAi effectors. BMC Biotechnol., 17(1): 9.
Genta FA, Blanes L, Cristofoletti PT, Lago CL, Terra WR, Ferreira C, 2006. Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut. Insect Biochem. Molec. Biol., 36(10): 789-800.
Girard C, Jouanin L, 1999. Molecular cloning of a gut-specific chitinase cDNA from the beetle Phaedon cochleariae. Insect Biochem. Molec. Biol., 29(6): 549-556.
Han JH, Lee KS, Li JH, Kim I, Je YH, Kim DH, Sohn HD, Jin BR, 2005. Cloning and expression of a fat body-specific chitinase cDNA from the spider, Araneus ventricosus. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 140(3): 427-435.
Hegedus D, Erlandson M, Gillott C, Toprak U, 2009. New insights into peritrophic matrix synthesis, architecture, and function. Annu. Rev. Entomol., 54: 285-302.
Jasrapuria S, Arakane Y, Osman G, Kramer KJ, Beeman RW, Muthukrishnan S, 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. Insect Biochem. Molec. Biol., 40(3): 214-227.
Khajuria C, Buschman LL, Chen MS, Muthukrishnan S, Zhu KY, 2010. A gut-specific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia nubilalis larvae. Insect Biochem. Molec. Biol., 40(8): 621-629.
Kramer KJ, Hopkins TL, Schaefer J, 1995. Applications of solids NMR to the analysis of insect sclerotized structures. Insect Biochem. Molec. Biol., 25(10): 1067-1080.
Lehane MJ, 1997. Peritrophic matrix structure and function. Annu. Rev. Entomol., 42: 525-550.
Li DQ, Du JZ, Zhang JQ, Hao YS, Liu XJ, Wang YX, Ma EB, Zhang JZ, Sun Y, 2011. Study on expression characteristics and functions of chitinase family genes from Locusta migratoria manilensis (Meyen). Sci. Agric. Sin., 44(3): 485-492. [李大琪, 杜建中, 张建琴, 郝耀山, 刘晓健, 王亦学, 马恩波, 张建珍, 孙毅, 2011. 东亚飞蝗几丁质酶家族基因的表达特性与功能研究. 中国农业科学, 44(3): 485-492]
Madhuprakash J, El Gueddari NE, Moerschbacher BM, Podile AR, 2015. Catalytic efficiency of chitinase-D on insoluble chitinous substrates was improved by fusing auxiliary domains. PLoS ONE, 10(1): e0116823.
Mamta, Reddy KRK, Rajam MV, 2016. Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato. Plant Mol. Biol., 90(3): 281-292.
Merzendorfer H, Zimoch L, 2003. Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. J. Exp. Biol., 206(24): 4393-4412.
Nakamura T, Mine S, Hagihara Y, Ishikawa K, Ikegami T, Uegaki K, 2008. Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus. J. Mol. Biol., 381(3): 670-680.
Ohno T, Armand S, Hata T, Nikaidou N, Henrissat B, Mitsutomi M, Watanabe T, 1996. A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT6037. J. Bacteriol., 178(17): 5065-5070.
Osman GH, Assem SK, Alreedy RM, Elghareeb DK, Basry MA, Rastogi A, Kalaji HM, 2015. Development of insect resistant maize plants expressing a chitinase gene from the cotton leaf worm, Spodoptera littoralis. Sci. Rep., 5: 18067.
Pan Y, Peng L, Wang Y, Yin LJ, Ma HX, Ma GH, Chen KP, He YQ, 2012. In silico identification of novel chitinase-like proteins in the silkworm, Bombyx mori, genome. J. Insect Sci., 12: 150.
Qu MB, Ma L, Chen P, Yang Q, 2014. Proteomic analysis of insect molting fluid with a focus on enzymes involved in chitin degradation. J. Proteome Res., 13(6): 2931-2940.
Ramalho-Ortig?o JM, Traub-Csek? YM, 2003. Molecular characterization of Llchit1, a midgut chitinase cDNA from the leishmaniasis vector Lutzomyia longipalpis. Insect Biochem. Molec. Biol., 33(3): 279-287.
Shao L, Devenport M, Jacobs-Lorena M, 2001. The peritrophic matrix of hematophagous insects. Arch. Insect Biochem. Physiol., 47(2): 119-125.
Shen ZC, Jacobs-Lorena M, 1997. Characterization of a novel gut-specific chitinase gene from the human malaria vector Anopheles gambiae. J. Biol. Chem., 272(46): 28895-28900.
Shi L, Paskewitz SM, 2004. Identification and molecular characterization of two immune-responsive chitinase-like proteins from Anopheles gambiae. Insect Mol. Biol., 13(4): 387-398.
Tetreau G, Cao XL, Chen YR, Muthukrishnan S, Jiang HB, Blissard GW, Kanost MR, Wang P, 2015. Overview of chitin metabolism enzymes in Manduca sexta: identification, domain organization, phylogenetic analysis and gene expression. Insect Biochem. Molec. Biol., 62(1): 114-126.
Xia M, 2015. Molecular Cloning, Biochemical Characterization and Inhibitor Discovery of Group Ⅳ Chitinase from the Insect Ostrinia furnacalis. MSc Thesis, Dalian University of Technology, Dalian, Liaoning. [夏梦, 2015. 亚洲玉米螟Ⅳ家族几丁质酶的克隆表达、性质表征及抑制剂研究. 辽宁大连: 大连理工大学硕士学位论文]
Xiang JB, 2004. Studies on the Structure and Components of Peritrophic Membrane and the Selective Toxicity of Celangulin Ⅴ. MSc Thesis, Northwest Sci-Tech University of Agriculture and Forestry, Yangling, Shaanxi. [相静波, 2004. 昆虫围食膜结构、组分及与苦皮藤素Ⅴ选择毒性关系的研究. 陕西杨凌: 西北农林科技大学硕士学位论文]
Xiong GH, Xing LS, Lin Z, Saha TT, Wang C, Jiang H, Zou Z, 2015. High throughput profiling of the cotton bollworm Helicoverpa armigera immunotranscriptome during the fungal and bacterial infections. BMC Biotechnol., 16(1): 321.
Yan J, Cheng Q, Narashimhan S, Li CB, Aksoy S, 2002. Cloning and functional expression of a fat body-specific chitinase cDNA from the tsetse fly, Glossina morsitans morsitans. Insect Biochem. Molec. Biol., 32(9): 979-989.
Zhang BS, Cao YQ, Zhang W, Yin YP, Wang ZK, Peng GX, Xia YX, 2007. Cloning, sequence analysis and tissue expression of the chitinase gene from the midgut of Locusta migratoria manilensis (Meyen). Acta Entomol. Sin., 50(6): 555-559. [张八生, 曹月青, 张伟, 殷幼平, 王中康, 彭国雄, 夏玉先, 2007. 东亚飞蝗中肠几丁质酶基因的克隆、序列分析及组织定位. 昆虫学报, 50(6): 555-559]
Zhang DW, Chen J, Yao Q, Pan ZQ, Chen J, Zhang WQ, 2012. Functional analysis of two chitinase genes during the pupation and eclosion stages of the beet armyworm Spodoptera exigua by RNA interference. Arch. Insect Biochem. Physiol., 79(4-5): 220-234.
Zhang JZ, Zhang X, Arakane Y, Muthukrishnan S, Kramer KJ, Ma E, Zhu KY, 2011. Comparative genomic analysis of chitinase and chitinase-like genes in the african malaria mosquito (Anopheles gambiae). PLoS ONE, 6(5): e19899.
Zhu QS, Arakane Y, Banerjee D, Beeman RW, Kramer KJ, Muthukrishnan S, 2008a. Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects. Insect Biochem. Molec. Biol., 38(4): 452-466.
Zhu QS, Arakane Y, Beeman RW, Kramer KJ, Muthukrishnan S, 2008b. Characterization of recombinant chitinase-like proteins of Drosophila melanogaster and Tribolium castaneum. Insect Biochem. Molec. Biol., 38(4): 467-477.
Zhu QS, Arakane Y, Beeman RW, Kramer KJ, Muthukrishnan S, 2008c. Functional specialization among insect chitinase family genes revealed by RNA interference. Proc. Natl. Acad. Sci. USA, 105(18): 6650-6655.
Zhu QS, Deng YP, Vanka P, Brown SJ, Muthukrishnan S, Kramer KJ, 2004. Computational identification of novel chitinase-like proteins in the Drosophila melanogaster genome. Bioinformatics, 20(2): 161-169.