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| Phylogenetic Classification of Chitinases in Cotton Bollworm (Helicoverpa armigera), Expression and Analysis of Enzymatic Characterization on Group Ⅶ Chitinase |
| ABUDUNASIER Maimaitiailii, LI Meng-Ge, BAO Jing, SUI Hui, LIU Xiao-Ning* |
| Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830046, China |
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Abstract 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 group Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅶ and Ⅷ chitinase, respectively, and 1 chitinase belonged to the new group. Group Ⅶ chitinase (HaCHTⅦ) was rarely reported, It was cloned in this study. Fusion protein His-HaCHTⅦ 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-HaCHTⅦ 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 of michaelis constant (Km) and maximum speed (Vmax) were 0.486 2 mmol/L and 2.715 1 µg/(mL·min), respectively. Therefore, HaCHTⅦ 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.
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Received: 27 July 2018
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Corresponding Authors:
* liuxn0103@sina.com
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[1] 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[D]. 硕士学位论文, 大连理工大学, 导师: 刘田, pp. 31-32.
(Xia M, 2015. Molecular cloning, biochemical characterization and inhibitor discovery of group Ⅳ chitinase from the insects ostrinia furnacalis[D]. Thesis for M.S., Dalian University of Technology, Suppervisor: Liu T, pp. 31-32.)
[2] 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.)
[3] 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 MDCII from Musca domestica[J]. Biotechnology Bulletin, 33(2): 102-108).
[4] 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 II chitinases during development of Lymantria dispar[D]. Thesis for M.S., Taiyuan University of Technology, Suppervisor: Fan X J, pp. 14-16.)
[5] 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.)
[6] 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.
[7] Arakane Y, Muthukrishnan S.2010. Insect chitinase and chitinase-like proteins[J]. Cellular and Molecular Life Sciences, 67(2): 201-216.
[8] Arakane Y, Zhu Q, Matsumiya M, et al.2003. Properties of catalytic, linker and chitin-binding domains of insect chitinase[J]. Insect Biochemistry and Molecular Biology, 33(6): 631-648.
[9] 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.
[10] 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.
[11] 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.
[12] 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.
[13] Madhuprakash J, E l Gueddari N E, Moerschbacher B M, et al.2015. Catalytic efficiency of chitinase-D on insoluble chitinous substrates was improved by fusing auxiliary domains[J]. Plos One, 10(1): e0116823-e0116823.
[14] 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.
[15] 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.
[16] Scott K D, Wilkinson K S, Lawrence N, et al.2005. Gene-flow between populations of cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) is highly variable between years[J]. Bulletin of Entomological Research, 95(4): 381-392.
[17] Tabashnik B E, Rensburg J B J V, Carrière Y.2009. Field-evolved insect resistance to Bt crops: Definition, theory, and data[J]. Journal of Economic Entomology, 102(6): 2011-2025.
[18] 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 RNA interference[J]. Archives of Insect Biochemistry and Physiology, 79(4-5): 220-234.
[19] 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.
[20] 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.
[21] 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.
[22] 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. |
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