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Barley chitinase genes expression revamp resistance against whitefly (Bemisia Tabaci) in transgenic cotton (Gossypium hirsutum L.)

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Journal of Cotton Research 2024年第1期7卷 90-100页

作者： BASHIR Samina YAQOOB Amina BASHIR Rohina BUKHARI Shanila SHAHID Naila AZAM Saira BAKHSH Allah HUSNAIN Tayyab SHAHID Ahmad Ali RAO Abdul Qayyum Centre of Excellence in Molecular Biology University of the PunjabLahore 53700Pakistan Department of Botany Kinnaird College for Women UniversityLahore 54000Pakistan Department of Botany Government College UniversityFaisalabad 38000Pakistan Institute of Botany University of the PunjabLahore 54590Pakistan.

Background chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton(Gossypium hirsutum)through Agrobacterium-mediated transformation.Fifty-five putative transgenic cotton plants were obtained,out of which fifteen plants successfully survived and were shifted to the field.Using gene-specific primers,amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation.These five plants were further evalu-ated for their mRNA expression levels.The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field,were selected to raise their subsequent progenies.Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10(2)for the ch1 gene and 3.7-fold in P2(1)for the chi2 gene.Fluorescent in situ hybridization(FISH)confirmed a single copy number of ch1 and chi2(hemizygous)on chromosome no.6.Furthermore,the efficacy of transgenes on whitefly was evaluated through an insect bioassay,where after 96 h of infestation,mortality rates of whitefly were calculated to be 78%–80%in transgenic cotton plants.The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant.Conclusion Based on these findings,it can be concluded that the chemically synthesized barley chitinase genes(ch1 and chi2)have the potential to be effective against insects with chitin exoskeletons,including whiteflies.The transgenic cotton plants expressing these genes showed increased resistance to whiteflies,resulting in reduced egg numbers and higher mortality rates.

关键词： chitinase Cotton White fly Transgene Bioassay

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 metamorphosis.In our previous work,we comprehensively studied the function of SfCht7 in Sogatella furcifera.However,the number and function of chitinase genes in S.furcifera remain unknown.Here,we identified 12 full-length chitinase transcripts from S.furcifera,which included nine chitinase(Cht),two imaginal disc growth factor(IDGF),and one endo-β-N-acetylglucosaminidase(ENGase)genes.Expression 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 integument.Based 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 lethality.RNAi 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 S.furcifera.

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

Identification of a chitinase from the hepatopancreas of Chinese black sleeper(Bostrychus sinensis)

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Acta Oceanologica Sinica 2021年第6期40卷 50-60页

作者： Yulei Chen Zhipeng Tao Minghui Zhang Lechang Sun Guangming Liu Minjie Cao College of Food and Biological Engineering Jimei UniversityXiamen 361021China Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources Xiamen 361102China

Chinese black sleeper(Bostrychus sinensis)is a fish that lives both in seawater and freshwater,feeds on crustaceans,aquatic insects and occasionally shellfish.The existence of digestive enzyme in viscera to act on chitinous exoskeleton of the prey is of interest.In this study,a chitinase was purified to homogeneity using ammonium sulfate precipitation,DEAE-Sephacel ion exchange,Sephacryl S-200 HR and Superdex 200 gel filtration columns.The purified protein presents a molecular mass of 58 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and results in a single band on native PAGE.According to peptide mass fingerprinting,two peptides containing a total of 20 amino acid residues,were 95%identical to a chitinase from yellow perch(Perca flavescens)and 100%identical to the chitinase from greater amberjack(Seriola dumerili).The purified chitinase showed optimum activity at pH 6.0,and was stable at acidic conditions and temperature below 55℃.The enzymatic activity was quite stable in the presence of NaCl,even at 1 mol/L.The chitinase was capable of degrading chitosan into low molecular mass chitooligosaccharides(COS)with sizes in a range of 200-700 Da,and the circular dichroism profile of the COS greatly differed from native chitosan.Full-length cDNA encoding the present chitinase was cloned and the transcript levels of chitinase in various tissues were determined by quantitative real-time PCR.The results showed that the transcript level of chitinase was highest in esophagus and hepatopancreas.

关键词： Bostrychus sinensis chitinase purification characterization molecular cloning chitosan

Thiazolylhydrazone dervatives as inhibitors for insect N-acetyl-β-D-hexosaminidase and chitinase

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Chinese Chemical Letters 2020年第5期31卷 1271-1275页

作者： Huibin Yang Huitang Qi Zesheng Hao Xusheng Shao Tian Liu Qing Yang Xuhong Qian Shanghai Key Laboratory of Chemical Biology Institute of Pesticides and PharmaceuticalsSchool of PharmacyEast China University of Science and TechnologyShanghai 200237China State Key Laboratory of the Discovery and Development of Novel Pesticide Shenyang Sinochem Agrochemicals Research and Development Co.Ltd.Shenyang 110021China School of Bioengineering Dalian University of TechnologyDalian 116024China

Insect chitinase and N-acetyl-β-D-hexosaminidases(Hex)are potential targets for developing new pesticides.Here,a series of thiazolylhydrazonesⅠ(with substituted group R1 at N3)andⅡ(with substituted group R1 at N2)were designed,synthesised and evaluated as competitive inhibitors of OfHexl and OfChi-h,from the agricultural pest Ostrinia furnacalis,DerivativesⅠ-3 d andⅡ-3 d,with phenoxyethyl group at R1,demonstrated the best inhibitory activities against OfHexl and OfChi-h.Molecular docking analysis indicated that the branched conformation compound II-3 d(Ki=1.5μmol/L)formed more hydrogen bonds with OfHexl than the stretched conformation compound I-3 d(Ki=5.9μmol/L).The differences in compounds’binding conformations with OfChi-h explained differences in inhibitory activity of compounds I-3 d(Ki=1.9μmol/L)and II-3 d(Ki=4.1μmol/L).This work suggests a novel scaffold for developing specific Hex and Chi-h inhibitors.

关键词： N-acetyl-β-D-hexosaminidase chitinase Inhibitor Chitin Structure-activity relationship Thiazolylhydrazone derivatives Molecular docking

Cloning, expression and biocharacterization of OfCht5, the chitinase from the insect Ostrinia furnacalis

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Insect Science 2013年第2期20卷 147-157页

作者： Qingyue Wu Tian Liu Qing Yang Department of Bioscience and Biotechnology Dalian University of Technology Dafian China

chitinase catalyzes β,4-glycosidic linkages in chitin and has attracted re- search interest due to it being a potential pesticide target and an enzymatic tool for preparation of N-acetyl-β D-glucosamine. An individual insect contains multiple genes encoding chitinases, which vary in domain architectures, expression patterns, physiological roles and biochemical properties. Herein, Of ChtS, the glycoside hydrolase family 18 chiti- nase from the widespread lepidopteran pest Ostrinia furnacalis, was cloned, expressed in the yeast Pichia pastoris and biochemically characterized in an attempt to facilitate both pest control and biomaterial preparation. Complementary DNA sequence analysis indicated that Of CHT5 consisted of an open reading frame of 1 665-bp nucleotides. Phy- logenic analysis suggested Of Cht5 belongs to the Group I insect chitinases. Expression of Of Cht5 in Pichia pastoris resulted in highest specific activity after 120 h of induction with methanol. Through two steps of purification, consisting of ammonium sulfate pre- cipitation and metal chelating chromatography, about 7 mg of the recombinant Of Cht5 was purified to homogeneity from 1 L culture supernatant. Of Cht5 effectively converted colloidal chitin into chitobiose, but had relatively low activity toward a-chitin. When chi- tooligosaccharides [（GlcNAc）n, n = 3-6] were used as substrates, Of Cht5 was observed to possess the highest catalytic efficiency parameter toward （GlcNAc）4 and predominan- tely hydrolyzed the second glycosidic bond from the non-reducing end. Together with fl-N-acetyl-D-hexosaminidase OfHexl, Of Cht5 achieved its highest efficiency in chitin degradation that yielded N-acetyl-β D-glucosamine, a valuable pharmacological reagent and food supplement, within a molar concentration ratio of Of Cht5 versus Of Hexl in the range of 9 ： 1-15 ： 1. This work provides an alternative to existing preparation of chitinase for pesticides and other applications.

关键词： chitin chitinase glycoside hydrolase N-acetyl-β-D-glucosamine Ostriniafurnacalis pest control

Identification of the key chitinase genes in Tetranychus cinnabarinus (Boisduval) based on the expression and sequence characteristic analysis

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Journal of Integrative Agriculture 2017年第4期16卷 892-899页

作者： XU Hao-ran HE Lin XIAO Wei SHEN Guang-mao Key Laboratory of Entomology and Pest Control Engineering College of Plant Protection Southwest University Chongqing 400715 P.R.China

Chitin is an important content in the exoskeletons of arthropods, and its hydrolyzation is catalyzed by chitinases during the process of molting, thus, the chitinases are considered as ideal target to interfere the growth of arthropods. This study intends to clarify the characteristic of the chitinases during the development of Tetranychus cinnabarinus, and screen out important genes as potential control targets. The results showed that the total enzyme concentration of chitinases was significantly higher in larva, the first and second nymph than that in egg and adult. Base on the transcriptome data, six unigenes encoding chitinases were identified and their expression patterns in different developmental stages were detected. The expressions of TcCHIT1 and TcCHITIO showed high abundance during the molting process and their expression change during the developmental stages was consistent with the enzyme concentration. The full-length of these two genes were further cloned, and the structural characteristics of their proteins were analyzed by constructing the three-dimensional structure model. The results provide basic information to understand the characteristic of chitinases in T. cinnabarinus and might be considered as target for control.

关键词： Tetranychus cinnabarinus chitinase gene expression protein structure

chitinase转基因拟南芥对棉铃虫核型多角体病毒（HearNPV）增效作用研究

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Chitinase转基因拟南芥对棉铃虫核型多角体病毒（HearNPV）增效作...

作者：王毛西北农林科技大学

学位级别：硕士

棉铃虫核型多角体病毒（Hear NPV）含有的几丁质酶在昆虫液化和病毒与寄主传播水平上有着重要作用。此外,原核表达的几丁质酶还可以增强Hear NPV的感染效率和杀虫毒力。而几丁质是昆虫消化道一个重要的结构,是昆虫体内中肠中天然屏障围... 详细信息

棉铃虫核型多角体病毒（Hear NPV）含有的几丁质酶在昆虫液化和病毒与寄主传播水平上有着重要作用。此外,原核表达的几丁质酶还可以增强Hear NPV的感染效率和杀虫毒力。而几丁质是昆虫消化道一个重要的结构,是昆虫体内中肠中天然屏障围食膜的重要组成成分,即在昆虫自身保护其免受肠内容物的伤害中扮演重要角色。在这项研究中,以拟南芥（Arabidopsis thaliana）为植物载体,利用根癌农杆菌（Agrobacterium tumefaciens）介导的花序侵染法对拟南芥转化了chitinase外源基因,并进行了其对棉铃虫核型多角体病毒（Helicoverpa armigera nucleopolyhedrovirus,Hear NPV）增效作用研究,取得如下结果:1、利用花序侵染法转化拟南芥,获得了6株表现型为阳性的chitinase转基因拟南芥株系,通过PCR检测初步确定为阳性的转化拟南芥株系。2、通过RT-PCR检测,表明目的基因chitinase在6个转基因株系A-F中都有表达,并且株系A的相对表达量最高,株系D的相对表达量最低。3、chitinase转基因拟南芥对Hear NPV的增效作用试验表明,饲喂株系A的棉铃虫感染Hear NPV后的LT50和LT90比取食野生型拟南芥对照组分别缩短了36.17%和40.33%。饲喂株系D的棉铃虫感染Hear NPV后的LT50和LT90比对照组分别缩短了18.08%和25.14%。4、感染相同剂量的棉铃虫在取食转基因植物后病毒在细胞中DNA拷贝数显著高于取食野生型植物的棉铃虫,其中取食转基因株系A的棉铃虫感染Hear NPV后其细胞体内病毒复制能力最强也最快,其次是取食株系D的棉铃虫。5、取食转基因拟南芥的棉铃虫病毒产量明显高于取食野生型拟南芥,且取食转基因拟南芥株系A的棉铃虫病毒产量明显高于取食转基因株系D的棉铃虫。6、感染HearNPV的棉铃虫无论取食野生型拟南芥、株系A或株系D,其日均虫重均无显著性差异;单取食转基因和野生型拟南芥未感染病毒的棉铃虫日累积增重没有显著差异,说明转基因植物单独饲喂对棉铃虫不会有很大影响。结果表明,chitinase转基因拟南芥在饲喂同时感染Hear NPV的棉铃虫时有利用病毒粒子更快更多的进入昆虫中肠细胞,且有助于病毒Hear NPV在棉铃虫体内的增值,以及提高病毒对棉铃虫的杀虫速度和杀虫效率。

关键词：拟南芥 chitinase 转基因植物 HearNPV 棉铃虫增效

Effect of Cultural Conditions on chitinase Production from Biocontrol Bacterium Against Aflatoxin

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Journal of Harbin Institute of Technology(New Series) 2015年第3期22卷 82-86页

作者： Kai Wang Peisheng Yan Lixin Cao School of Municipal and Environmental Engineering Harbin Institute of Technology School of Marine Science and Technology Harbin Institute of Technology

chitinase is one of the most important mycolytic enzymes with industrial significance. Statistical methods are employed to optimize cultural conditions with the increased production of chitinase for the selected Serratia marcescens JPP1,which are obtained from peanut hulls in Jiangsu Province, China and exhibit antagonistic activity against aflatoxins. Using single-factor experiments the effects of cultural conditions( broth content,inoculum size and rotation speed) on chitinase production from S. marcescens JPP1 are evaluated. Central composite design of Response Surface Methodology is used to optimize the levels of factors for the best yield of enzymes production. The optimized cultural conditions for obtaining the highest level of chitinase production are23. 2 mL broth content,116 r / min rotation speed and 4. 3% inoculum size. A quadratic regression model of chitinase production is built( R2= 0. 970 9) and the verification experiments confirm its validity. The maximum chitinase production obtained after the optimization is 29. 58 U / mL for a 1. 4-fold increase.

关键词： chitinase aflatoxin optimization Central Composite Design

3H-31, A Non-structural Protein of Heliothis virescens ascovirus 3h,Inhibits the Host Larval Cathepsin and chitinase Activities

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Virologica Sinica 2021年第5期36卷 1036-1051页

作者： Huan Yu Yi-Yi Ou-Yang Chang-Jin Yang Ni Li Madoka Nakai Guo-Hua Huang Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Insect Pests Hunan Agricultural UniversityChangsha 410128China College of Plant Protection Hunan Agricultural UniversityChangsha 410128China Tokyo University of Agriculture and Technology SaiwaiFuchuTokyo 183-8509Japan

3h-31 of Heliothis virescens ascovirus 3h(Hv AV-3h)is a highly conserved gene of ascoviruses.As an early gene of Hv AV-3h,3h-31 codes for a non-structural protein(3H-31)of Hv AV-3h.In the study,3h-31 was initially transcribed and expressed at 3 h post-infection(hpi)in the infected Spodoptera exigua fat body cells(Se FB).3h-31 was further inserted into the bacmid of Autographa californica nucleopolyhedrovirus(Ac MNPV)to generate an infectious baculovirus(Ac MNPV-31).In vivo experiments showed that budded virus production and viral DNA replication decreased with the expression of 3H-31,and lucent tubular structures were found around the virogenic stroma in the Ac MNPV-31-infected Se FB cells.In vivo,both LD50and LD90values of Ac MNPV-31 were significantly higher than those of the wild-type Ac MNPV(Ac MNPV-wt)in third instar S.exigua larvae.An interesting finding was that the liquefaction of the larvae killed by the infection of Ac MNPV-31 was delayed.chitinase and cathepsin activities of Ac MNPV-31-infected larvae were significantly lower than those of Ac MNPV-wt-infected larvae.The possible regulatory function of the chitinase and cathepsin for 3H-31 was further confirmed by RNAi,which showed that larval cathepsin activity was significantly upregulated,but chitinase activity was not significantly changed due to the RNAi of 3h-31.Based on the obtained results,we assumed that the function of 3H-31 was associated with the inhibition of host larval chitinase and cathepsin activities,so as to restrain the hosts in their larval stages.

关键词： Cathepsin chitinase Heliothis virescens ascovirus 3h(HvAV-3h) Recombinant virus 3h-31