Molecular cloning and characterization of genes encoding chitinase and N-acetyl-Î²-glucosaminidase in Dolerocypris sinensis

Gong Cheng; Shaonan Li

doi:10.14715/cmb/2019.65.6.12

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Vol. 65 No. 6: Issue 6

Issue Published : August 5, 2019

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Molecular cloning and characterization of genes encoding chitinase and N-acetyl-Î²-glucosaminidase in Dolerocypris sinensis

https://doi.org/10.14715/cmb/2019.65.6.12

Gong Cheng

Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, People's Republic of China

Shaonan Li

Institute of Pesticide and Environmental Toxicology, Zhejiang University, Hangzhou 310029, People's Republic of China

Corresponding Author(s) : Shaonan Li

tiiy93@163.com

Cellular and Molecular Biology, Vol. 65 No. 6: Issue 6
Article Published : July 31, 2019

Abstract

Chitinases and N-acetyl-Î²-glucosaminidase (NAG) are important in molting and growth of crustaceans. In ostracods, the genes encoding these enzymes have not been characterized. The aim of the present study was to clone the genes encoding chitinase (DsChi) and NAG (DsNAG) from the ostracod, Dolerocypris sinensis, elucidate the phylogenetic relationships between the cloned genes and known chitinolytic enzymes, and determine the expression patterns of these genes at different stages of growth in the presence of an environmental pollutant. The genes were amplified from the genomic DNA of the organism using polymerase chain reaction (PCR). The products from PCR were cloned and characterized with bioinformatics tools, and their expression patterns at different growth stages were determined using real-time quantitative PCR (qRT-PCR). Nine and five introns were identified in DsChi and DsNAG genes, respectively. When compared with protein sequences available in GenBank, chitinase from D. sinensis was most closely related to that of Macrobrachium nipponense (61 % homology). The NAG of D. sinensis was most closely related to that of Limulus polyphemus (55.6 % homology). Based on phylogenetic analysis of known chitinases from crustaceans and insects, the D. sinensis chitinase tightly clustered in the same branch with chitinases from species within the Malacostraca class. In contrast, NAG of D. sinensis was clustered with NAG from F. candida.The level of expression of DsChi mRNA was significantly higher than that of DsNAG throughout the period of growth (p < 0.05). Treatment of D. sinensis cells with fenoxycarb significantly downregulated the expressions of DsChi and DsNAG throughout the period of growth (p < 0.05). These results show that the protein products of DsChi and DsNAG possess remarkable biochemical properties characteristic of a chitinase and NAG, respectively.

Keywords

Dolerocypris sinensis Chitinase Chitin Acetyl-Î²-glucosaminidase Molting.

Cheng, G., & Li, S. (2019). Molecular cloning and characterization of genes encoding chitinase and N-acetyl-Î²-glucosaminidase in Dolerocypris sinensis. Cellular and Molecular Biology, 65(6), 73–80. https://doi.org/10.14715/cmb/2019.65.6.12

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References

Rinaudo, M. Chitin and chitosan: properties and applications. Prog Polym Sci 2006; 31: 603-632.
Castro R, Guerrero-Legarreta I, Bórquez R. Chitin extraction from Allopetrolisthes punctatus crab using lactic fermentation. Biotechnol Rep 2018; 20: 00287.
Liu T, Zhou Y, Chen L, Chen W, Liu L, Shen X, et al. Structural insights into cellulolytic and chitinolytic enzymes revealing crucial residues of insect Î²-N-acetyl-D-hexosaminidase. PloS one 2012; 7: 52225.
Jollès P, Muzzarelli RAA. Chitin and chitinases. Birkhauser 1999; 340.
Tanaka T, Fukui T, Atomi H, Imanaka T. Characterization of an exo-beta-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J Bacteriol 2003; 185: 5175.
Watanabe T, Kono M, Aida K, Nagasawa H. Isolation of cDNA encoding a putative chitinase precursor in the kuruma prawn Penaeus japonicus. Mol Mar Biol Biotechnol 1996; 5: 299-303.
Huang QS, Yan JH, Tang JY, Tao YM, Xie XL, Wang Y, et al. Cloning and tissue expressions of seven chitinase family genes in Litopenaeus vannamei. Fish Shellfish Immunol 2010; 29: 75-81.
Zhang J, Sun Y, Li F, Huang B, Xiang J. Molecular characterization and expression analysis of chitinase (Fcchi-3) from Chinese shrimp, Fenneropenaeus chinensis. Mol Biol Rep 2010; 37: 1913-1921.
Zhang F, Lu J, Liu P, Gao B, Li J. Cloning and Expression Analysis of the cDNA of PtCht3 in Portunus trituberculatus. Prog Fish Sci 2017; 8: 167-176.
Li X, Xu Z, Zhou G, Lin H, Zhou J, Zeng Q, et al. Molecular characterization and expression analysis of five chitinases associated with molting in the Chinese mitten crab, Eriocheir sinensis. Comp Biochem Physiol Biochem Mol Biol 2015; 187: 110-120.
Stout JD. "The Terrestrial Plankton". Tuatara 1963; 11: 57-65.
Oosterhuis SS, Baars MA, Breteler W. Release of the enzyme NAGase by the copepod Temora longicornis: characteristics and potential tool for estimating crustacean biomass production in the sea. Mar Ecol Prog Ser 2000; 196: 195-206.
Hanson M, Laurent L. NAGase activity as an indicator of aquatic ecosystem health. Aquat Ecosyst Health Manag 2005; 8: 441-450.
Martens K, Schön I, Meisch C, Horne DJ. Global diversity of ostracods (Ostracoda, Crustacea) in freshwater. Hydrobiologia 2008; 595: 185-193.
Lynn KR. Chitinases and NAGases from the american lobster ( Homarus americanus ). Comp Biochem Physiol B 1990; 96: 761-766.
Salma U, Uddowla MH, Kim M, Kim JM, Kim BK, Baek H, et al. Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp (Pandalopsis japonica): cloning and effects of eyestalk ablation on gene expression. Comp Biochem Physiol B Biochem Mol Biol 2012; 161: 197-207.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2âˆ’ Î”Î”CT method. Methods. 2001; 25: 402-408.
Ernst J, Bar-Joseph Z. STEM: a tool for the analysis of short time series gene expression data. BMC Bioinformatics 2006; 7: 191.
Zhu Q, Deng Y, Vanka P, Brown SJ, Muthukrishnan S, Kramer KJ. Computational identification of novel chitinase-like proteins in the Drosophila melanogaster genome. Bioinformatics 2004; 20: 161-169.
Zhu Q, Arakane Y, Banerjee D, Beeman RW, Kramer KJ, Muthukrishnan S. Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects. Insect Biochem Mol Biol 2008; 38: 452-466.
Kramer K. Site-directed mutagenesis and functional analysis of an active site tryptophan of insect chitinase. Insect Biochem Mol Biol 2002; 32: 1477-1488.
Zen KC, Choi HK, Krishnamachary N, Muthukrishnan S, Kramer KJ. Cloning, expression, and hormonal regulation of an insect beta-N-acetylglucosaminidase gene. Insect Biochem Mol Biol 1996; 26: 435-444.
Li L, Chen Y, Bao J, Li S. Cloning and analysis of the molecularly characterized chitinase genes of Daphnia carinata and Simocephalus vetulus. Genes Genomics 2017; 39: 1-12.
Zhang X, Noda S, Himeno R, Liu H. Cardiovascular disease-induced thermal responses during passive heat stress: An integrated computational study. Int J Numer Method Biomed Eng 2016; 23: 02768.
Fukamizo T, Kramer KJ. Mechanism of chitin oligosaccharide hydrolysis by the binary enzyme chitinase system in insect moulting fluid. Insect Biochem 1985; 15: 1-7.
Michiyoshi T, Makoto K, Manabu K. A new chitinase-related gene, BmChiR1, is induced in the Bombyx mori anterior silk gland at molt and metamorphosis by ecdysteroid. Insect Biochem Mol Biol 2002; 32: 147-151.
Enmin Z. Effects of hypoxia and sedimentary naphthalene on the activity of N-acetyl-beta-glucosaminidase in the epidermis of the brown shrimp, Penaeus aztecus. Bull Environ Contam Toxicol 2009; 82: 579-582.
Chang ES, Mykles DL. Regulation of crustacean molting: A review and our perspectives. Gen Comp Endocrinol 2011; 172: 323-330.
Claire D, Marília MI, Thierry C, Michel L, Evelyne F, Christophe L, et al. NAGase activity as an indicator of altered survival, growth and reproduction in Daphnia pulex and Daphnia magna (Crustacea: Cladocera) exposed to spinosad and diflubenzuron. Ecotoxicol Environ Saf 2011; 74: 800-810.

References

Rinaudo, M. Chitin and chitosan: properties and applications. Prog Polym Sci 2006; 31: 603-632.

Castro R, Guerrero-Legarreta I, Bórquez R. Chitin extraction from Allopetrolisthes punctatus crab using lactic fermentation. Biotechnol Rep 2018; 20: 00287.

Liu T, Zhou Y, Chen L, Chen W, Liu L, Shen X, et al. Structural insights into cellulolytic and chitinolytic enzymes revealing crucial residues of insect Î²-N-acetyl-D-hexosaminidase. PloS one 2012; 7: 52225.

Jollès P, Muzzarelli RAA. Chitin and chitinases. Birkhauser 1999; 340.

Tanaka T, Fukui T, Atomi H, Imanaka T. Characterization of an exo-beta-D-glucosaminidase involved in a novel chitinolytic pathway from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. J Bacteriol 2003; 185: 5175.

Watanabe T, Kono M, Aida K, Nagasawa H. Isolation of cDNA encoding a putative chitinase precursor in the kuruma prawn Penaeus japonicus. Mol Mar Biol Biotechnol 1996; 5: 299-303.

Huang QS, Yan JH, Tang JY, Tao YM, Xie XL, Wang Y, et al. Cloning and tissue expressions of seven chitinase family genes in Litopenaeus vannamei. Fish Shellfish Immunol 2010; 29: 75-81.

Zhang J, Sun Y, Li F, Huang B, Xiang J. Molecular characterization and expression analysis of chitinase (Fcchi-3) from Chinese shrimp, Fenneropenaeus chinensis. Mol Biol Rep 2010; 37: 1913-1921.

Zhang F, Lu J, Liu P, Gao B, Li J. Cloning and Expression Analysis of the cDNA of PtCht3 in Portunus trituberculatus. Prog Fish Sci 2017; 8: 167-176.

Li X, Xu Z, Zhou G, Lin H, Zhou J, Zeng Q, et al. Molecular characterization and expression analysis of five chitinases associated with molting in the Chinese mitten crab, Eriocheir sinensis. Comp Biochem Physiol Biochem Mol Biol 2015; 187: 110-120.

Stout JD. "The Terrestrial Plankton". Tuatara 1963; 11: 57-65.

Oosterhuis SS, Baars MA, Breteler W. Release of the enzyme NAGase by the copepod Temora longicornis: characteristics and potential tool for estimating crustacean biomass production in the sea. Mar Ecol Prog Ser 2000; 196: 195-206.

Hanson M, Laurent L. NAGase activity as an indicator of aquatic ecosystem health. Aquat Ecosyst Health Manag 2005; 8: 441-450.

Martens K, Schön I, Meisch C, Horne DJ. Global diversity of ostracods (Ostracoda, Crustacea) in freshwater. Hydrobiologia 2008; 595: 185-193.

Lynn KR. Chitinases and NAGases from the american lobster ( Homarus americanus ). Comp Biochem Physiol B 1990; 96: 761-766.

Salma U, Uddowla MH, Kim M, Kim JM, Kim BK, Baek H, et al. Five hepatopancreatic and one epidermal chitinases from a pandalid shrimp (Pandalopsis japonica): cloning and effects of eyestalk ablation on gene expression. Comp Biochem Physiol B Biochem Mol Biol 2012; 161: 197-207.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2âˆ’ Î”Î”CT method. Methods. 2001; 25: 402-408.

Ernst J, Bar-Joseph Z. STEM: a tool for the analysis of short time series gene expression data. BMC Bioinformatics 2006; 7: 191.

Zhu Q, Deng Y, Vanka P, Brown SJ, Muthukrishnan S, Kramer KJ. Computational identification of novel chitinase-like proteins in the Drosophila melanogaster genome. Bioinformatics 2004; 20: 161-169.

Zhu Q, Arakane Y, Banerjee D, Beeman RW, Kramer KJ, Muthukrishnan S. Domain organization and phylogenetic analysis of the chitinase-like family of proteins in three species of insects. Insect Biochem Mol Biol 2008; 38: 452-466.

Kramer K. Site-directed mutagenesis and functional analysis of an active site tryptophan of insect chitinase. Insect Biochem Mol Biol 2002; 32: 1477-1488.

Zen KC, Choi HK, Krishnamachary N, Muthukrishnan S, Kramer KJ. Cloning, expression, and hormonal regulation of an insect beta-N-acetylglucosaminidase gene. Insect Biochem Mol Biol 1996; 26: 435-444.

Li L, Chen Y, Bao J, Li S. Cloning and analysis of the molecularly characterized chitinase genes of Daphnia carinata and Simocephalus vetulus. Genes Genomics 2017; 39: 1-12.

Zhang X, Noda S, Himeno R, Liu H. Cardiovascular disease-induced thermal responses during passive heat stress: An integrated computational study. Int J Numer Method Biomed Eng 2016; 23: 02768.

Fukamizo T, Kramer KJ. Mechanism of chitin oligosaccharide hydrolysis by the binary enzyme chitinase system in insect moulting fluid. Insect Biochem 1985; 15: 1-7.

Michiyoshi T, Makoto K, Manabu K. A new chitinase-related gene, BmChiR1, is induced in the Bombyx mori anterior silk gland at molt and metamorphosis by ecdysteroid. Insect Biochem Mol Biol 2002; 32: 147-151.

Enmin Z. Effects of hypoxia and sedimentary naphthalene on the activity of N-acetyl-beta-glucosaminidase in the epidermis of the brown shrimp, Penaeus aztecus. Bull Environ Contam Toxicol 2009; 82: 579-582.

Chang ES, Mykles DL. Regulation of crustacean molting: A review and our perspectives. Gen Comp Endocrinol 2011; 172: 323-330.

Claire D, Marília MI, Thierry C, Michel L, Evelyne F, Christophe L, et al. NAGase activity as an indicator of altered survival, growth and reproduction in Daphnia pulex and Daphnia magna (Crustacea: Cladocera) exposed to spinosad and diflubenzuron. Ecotoxicol Environ Saf 2011; 74: 800-810.

Author biographies is not available.