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Characterization and functional analysis of chitinase family genes involved in nymph-adult transition of Sogatella furcifera

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Insect Science 2021年第4期28卷 901-916页

作者： Xibin Yang Cao Zhou Guiyun Long Hong Yang Chen Chen Daochao Jin Provincial Key Laboratory for Agricultural Pest Management of Mountainous Regions Institute of EntomologyGuizhou UniversityGuiyangChina College of Tobacco Science of Guizhou University GuiyangChina Scientific Observing and Experimental Station of Crop Pest in Guiyang Ministry of AgricultureGuiyangChina

chitinase degrades chitin in the old epidermis or peritrophic matrix of insects,which ensures normal development and *** our previous work,we comprehensively studied the function of SfCht7 in Sogatella ***,the number and function of chitinase genes in *** remain ***,we identified 12 full-length chitinase transcripts from ***,which included nine chitinase(Cht),two imaginal disc growth factor(IDGF),and one endo-β-N-acetylglucosaminidase(ENGase)*** analysis results revealed that the expression levels of eight genes(SfCht3,SfCht5,SfCht6-1,SfCht6-2,SfCht7,SfCht8,SfCht10,and SfIDGF2)with similar transcript levels peaked prior to molting of each nymph and were highly expressed in the *** on RNA interference(RNAi),description of the functions of each chitinase gene indicated that the silencing of SfCht5,SfCht10,and SfIDGF2 led to molting defects and *** inhibited the expressions of SfCht5,SfCht7,SfCht10,and SfIDGF2,which led to downregulated expressions of chitin synthase 1(SfCHS1,SfCHS1a,and SfCHS1b)and four chitin deacetylase genes(SfCDA1,SfCDA2,SfCDA3,and SfCDA4),and caused a change in the expression level of two trehalase genes(TRE1 and TRE2).Furthermore,silencing of SfCht7 induced a significant decrease in the expression levels of three wing development-related genes(SfWG,SfDpp,and SfHh).In conclusion,SfCht5,SfCht7,SfCht10,and SfIDGF2 play vital roles in nymph–adult transition and are involved in the regulation of chitin metabolism,and SfCht7 is also involved in wing development;therefore,these genes are potential targets for control of ***.

关键词： chitinase chitin deacetylase chitin synthesis RNA interference Sogatella furcifera wing development

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Biochemistry, structures and inhibition of insect chitin deacetylases

Biochemistry, structures and inhibition of insect chitin dea...

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中国化学会第一届农业化学学术讨论会

作者： Lin Liu Qing Yang State Key Laboratory of Fine Chemical Engineering School of Life Science and Biotechnology Dalian University of Technology Institute of Plant Protection Chinese Academy of Agricultural Sciences

Insect chitin deacetylases（CDAs） catalyze the removal of acetyl groups from chitin and modify this polymer during its synthesis and reorganization. CDAs are essential for insect survival and therefore represent promising targets for insecticide development. Our lab recently elucidated the biochemistry, structures and inhibition of two CDAs from Bombyx mori: Bm CDA1, which may function in cuticle modification, and Bm CDA8, which may act in modifying peritrophic membranes in the midgut. Biochemical analyses indicated that Bm CDA8 is an active enzyme that requires its substrates to occupy subsites 0, +1, and +2 for catalysis. In contrast, Bm CDA1 also required accessory proteins for catalysis. Comparing their crystal structures with those from well-studied microbial CDAs, we found that two unique loop regions contribute to the distinct architecture of their substrate-binding clefts. These comparisons revealed that both Bm CDA1 and Bm CDA8 possess a much longer and wider substratebinding cleft with a very open active site in the center than the microbial CDAs. And based on the structural uniqueness, we have been able to develop efficient inhibitors against insect CDAs. Cooperated with Prof. Kam Y. J. Zhang from RIKEN, we computationally identified 29 compounds by virtual screening. Among these, two compounds, ZINC3325306 and ZINC33253026, exhibited inhibitory activity toward Bm CDA8. Our work unveils the structural and biochemical features of insect CDAs and provides a starting point for the development of specific CDA inhibitors.

关键词： chitin deacetylase chitin chitosan protein structure inhibitor

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In vitro and in vivo application of RNA interference for targeting genes involved in peritrophic matrix synthesis in a lepidopteran system

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Insect Science 2013年第1期20卷 92-100页

作者： Umut Toprak Doug Baldwin Martin Erlandson Cedric Gillott Stephanie Harris Dwayne D. Hegedus Agriculture and Agri-Food Canada Ottawa Ontario Canada Department of Biology University of Saskatchewan Canada Department of Plant Protection Faculty of Agriculture University of Ankara Turkey Department of Food and Bioproduct Sciences University of Saskatchewan Saskatoon SK Canada

The midgut of most insects is lined with a semipermeable acellular tube, the peritrophic matrix （PM）, composed of chitin and proteins. Although various genes encoding PM proteins have been characterized, our understanding of their roles in PM structure and function is very limited. One promising approach for obtaining functional information is RNA interference, which has been used to reduce the levels of specific mRNAs using double-stranded RNAs administered to larvae by either injection or feeding. Although this method is well documented in dipterans and coleopterans, reports of its success in lepidopterans are varied. In the current study, the silencing midgut genes encoding PM proteins （insect intestinal mucin 1, insect intestinal mucin 4, PM protein 1） and the chitin biosynthetic or modifying enzymes （chitin synthase-B and chitin deacetylase 1） in a noctuid lepidopteran, Mamestra configurata, was examined in vitro and in vivo. In vitro studies in primary midgut epithelial cell preparations revealed an acute and rapid silencing （by 24 h） for the gene encoding chitin deacetylase 1 and a slower rate of silencing （by 72 h） for the gene encoding PM protein 1. Genes encoding insect intestinal mucins were slightly silenced by 72 h, whereas no silencing was detected for the gene encoding chitin synthase-B. In vivo experiments focused on chitin deacetylase 1, as the gene was silenced to the greatest extent in vitro. Continuous feeding of neonates and fourth instar larvae with double-stranded RNA resulted in silencing of chitin deacetylase 1 by 24 and 36 h, respectively. Feeding a single dose to neonates also resulted in silencing by 24 h. The current study demonstrates that genes encoding PM proteins can be silenced and outlines conditions for RNA interference by per os feeding in lepidopterans.

关键词： chitin deacetylase chitin synthase-B lepidoptera peritrophic matrix,peritrophin RNA interference

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Biocatalytic production of chitosan polymers from shrimp shells,using a recombinant enzyme produced by Pichia pastoris

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American Journal of Molecular Biology 2012年第4期2卷 341-350页

作者： Eduardo Mere Del Aguila Laidson Paes Gomes Cristina Tristao Andrade Joab Trajano Silva Vania Margaret Flosi Paschoalin Universidade Federal do Rio de Janeiro Instituto de QuímicaRio de JaneiroBrazil Universidade Federal do Rio de Janeiro Instituto de Macromoléculas Professora Eloisa ManoRio de JaneiroRJBrazil

Chitosan has a unique chemical structure with high charge density, reactive hydroxyl and amino groups, and extensive hydrogen bonding. chitin deacetylase (EC 3.***.1.41) catalyzes the hydrolysis of the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, converting it to chitosan and releasing acetate. The entire ORF of the CDA2 gene encoding one of the two isoforms of chitin deacetylase from Saccharomyces cerevisiae was cloned in Pichia pastoris. The Tg (Cda2-6xHis)p was expressed at high levels as a soluble intracellular protein after induction of the recombinant yeast culture with methanol, and purified using nickel-nitrilotriacetic acid chelate affinity chromatography, resulting in a protein preparation with a purity of >98% and an overall yield of 79%. chitin deacetylase activity was measured by a colorimetric method based on the O-phthalaldehyde reagent, which detects primary amines remaining in chitinous substrate after acetate release. The recombinant enzyme could deacetylate chitin, chitobiose, chitotriose and chitotetraose, with an optimum temperature of 50°C and pH 8.0, determined using oligochitosaccharides as the substrates. The recombinant protein was also able to deacetylate its solid natural substrate, shrimp chitin, to a limited extent, producing chitosan with a degree of acetylation (DA) of 89% as determined by Fourier transform infrared spectroscopy. The degree of deacetylation was increased by pre-hydrolysis of crystalline shrimp chitin by chitinases, which increased the deacetylation ratio triggered by chitin deacetylase, producing chito-oligosaccharides with a degree of acetylation of 33%. The results described here open the possibility to use the rCda2p, combined with chitinases, for biocatalytic conversion of chitin to chitosan with controlled degrees of deacetylation. We show herein that the crystalline chitin form can be cleanly produced in virtually quantitative yield if a combined and sequential enzyme treatment is performed.

关键词： chitin deacetylase Chitosan chitin Pichia Pastoris Deacetylation Degree

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