Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides,which are advantageous for plant immunity,animal nutrition and health.However,thermophilic endo-chitinases are scar...Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides,which are advantageous for plant immunity,animal nutrition and health.However,thermophilic endo-chitinases are scarce and the transformation from exo-to endo-activity of chitinases is still a challenging problem.In this study,to enhance the endo-activity of the thermophilic chitinase Chi304,we proposed two approaches for rational design based on comprehensive structural and evolutionary analyses.Four effective single-point mutants were identified among 28 designed mutations.The ratio of(GlcNAc)3 to(GlcNAc)2 quantity(DP3/2)in the hydrolysates of the four single-point mutants undertaking colloidal chitin degradation were 1.89,1.65,1.24,and 1.38 times that of Chi304,respectively.When combining to double-point mutants,the DP3/2 proportions produced by F79A/W140R,F79A/M264L,F79A/W272R,and M264L/W272R were 2.06,1.67,1.82,and 1.86 times that of Chi304 and all four double-point mutants exhibited enhanced endo-activity.When applied to produce chitooligosaccharides(DP≥3),F79A/W140R accumulated the most(GlcNAc)4,while M264L/W272R was the best to produce(GlcNAc)3,which was 2.28 times that of Chi304.The two mutants had exposed shallower substrate-binding pockets and stronger binding abilities to shape the substrate.Overall,this research offers a practical approach to altering the cutting pattern of a chitinase to generate functional chitooligosaccharides.展开更多
Tebufenozide,an efficient insect growth regulator(IGR)against lepidopteran pests,presents a novel mode of action with minimal non-target impact.By competing with ecdysteroids for ecdysone receptor(EcR)binding,it regul...Tebufenozide,an efficient insect growth regulator(IGR)against lepidopteran pests,presents a novel mode of action with minimal non-target impact.By competing with ecdysteroids for ecdysone receptor(EcR)binding,it regulates insect growth precisely.This study explores tebufenozide's potential as a multitarget IGR,targeting both EcR and Ostrinia furnacalis chitinase I(OfChtI).The inhibitory activity against OfChtI is comparable to that of substrates(GlcNAc)5,with an IC50 of 45.77μM.Our computational findings indicate that tebufenozide binds at the subsite1 to t1 of OfChtI through various interactions.Notably,tebufenozide establishes a pi-pi interaction with the flipped sidechain of Trp107,enabling tebufenozide to deeply penetrate into the S1 pocket,thereby obstructing substrate binding to OfChtI.These insights highlight the potency of multitarget strategies,laying the groundwork for innovative IGR designs that offer comprehensive pest management solutions.展开更多
The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of ...The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.展开更多
[Objective] The aim of this study was to isolate chitinase gene from Trichoderma atroviride strain SS003. [Method] With the aeciospore wall of armandii pine blister rust as inducer, chitinase gene was induced to expre...[Objective] The aim of this study was to isolate chitinase gene from Trichoderma atroviride strain SS003. [Method] With the aeciospore wall of armandii pine blister rust as inducer, chitinase gene was induced to express in Trichoderma atroviride cells. The cDNA fragment of chitinase gene was cloned by RT-PCR approach. [Result] The activity of chitinase induced reached 40.17 μg/10 min; and the specific fragment amplified was 834 bp in length and proved to be the fragment of chitinase gene by sequencing and sequence analysis. [Conclusion] The result showed the feasibility of isolating the full length of chitinase gene and its transformation, and further producing chitinase.展开更多
[ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the ...[ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the biological function and application of chitinasa gene. [ Method] cDNA library of Leymus chinensis leaves were constructed, and its DNA sequence was determined or analyzed, while the homology of chitinasa gene and amino acid sequence was compared with that in GenBank. [ Result] One full length cDNA fragment with length of 996 bp was cloned from cDNA library of Leymus chinensis leaves. The length of ORF was 768 bp encoding 225 amino acids （GenBank accession number： EU344908）. The encoding products lacked CBD and C-terminal extension region from the view of structure, but had structural characters of Class Ⅱ chitinase gene, which indicated that amino acid sequence had high homology compared with Class Ⅱ chitinase gene of rye and wheat. The constructed recombinant vector pQE-LcChi2 could express a protein of 27 kD through induction, which was consistent with the deduced encoding product of pQE-LcChi2 gene. [ Conclusion] LcChl2 gene is an expression gene, which can express in E. coll.展开更多
DA novel chitinase gene (GhChia7) was isolated from salicylic acid (SA)-treated cotton cotyledons and characterized by DNA sequence analysis of its cDNA and genomic DNA clone. The deduced amino acid sequence, designat...DA novel chitinase gene (GhChia7) was isolated from salicylic acid (SA)-treated cotton cotyledons and characterized by DNA sequence analysis of its cDNA and genomic DNA clone. The deduced amino acid sequence, designated as class VII chitinase, shares about 30% identity to class I or II chitinases, and does not correspond to any of the previously characterized classes I-VI chitinases. Northern blotting analysis showed that the transcripts of GhChia7 were abundant both in cotton fibers and in the roots of the seedlings. The accumulation of GhChia7 mRNA in SA-treated cotyledons reached maximum at 7.5 mmol/ L concentration after 18 h. Results indicate that GhChia7 might play an important role in cotton's active defense response.展开更多
[Objective] This study aimed to screen endophytic bacteria which is antag- onistic to cotton Fusarium wilt. [Method] Fresh cotton plants collected from cotton- growing areas in Jingzhou City, Hubei Province were used ...[Objective] This study aimed to screen endophytic bacteria which is antag- onistic to cotton Fusarium wilt. [Method] Fresh cotton plants collected from cotton- growing areas in Jingzhou City, Hubei Province were used as experimental materials to isolate endophytic bacteria. Through chitinase test and co-culturing both micro-or- ganisms side by side on the same PDA culture plate, antagonistic strains to cotton Fusarium wilt were screened. [Result] A total of 83 bacterial isolates were obtained from cotton plants grown in the fields, six of which were chitinase-productive bacte- ria. Through chitinase test and co-culturing both micro-organisms side by side on the same PDA culture plate, strain V-8 which had the strongest antagonistic effect on Fusarium oxysporum f. sp. vasinfectum was screened. Strain V-8 had a wider anti- fungal spectrum with certain inhibitory effect on all the six important pathogenic fungi including Fusarium oxysporum f. sp niveum; it colonized stably in the rhizospheric soil of cotton, with a colonization density of up to 6.2x10s cfu/g fifty days after inoc- ulation; the relative effect on controlling cotton Fusarium wilt in pot test was 73.2%. The Findings of this study suggested that strain V-8 had great potential for biological control of cotton Fusarium wilt and could be taken as a substantial material for the cloning of chitinase genes. [Conclusion] The results from this study provides bases for the control of cotton fusarium wilt, as well as the exploitation of endophytic bac- teria resources in cotton and the development of novel biological pesticides.展开更多
[Objective] The aim of this study was to optimize the conditions of chitinase-produce strains.[Method] A kind of screened chitinase-produce strain G-254 was habituated cultured,and then the single factor experiment wa...[Objective] The aim of this study was to optimize the conditions of chitinase-produce strains.[Method] A kind of screened chitinase-produce strain G-254 was habituated cultured,and then the single factor experiment was carried out to explore the effects of different carbon source,nitrogen source and inorganic salt on the activity of produced chitinase;the response surface test was used to determine the optimal conditions for chitinase production.[Result] The optimal conditions for chitinase production were：8% of glucose,5% of beef extract and 0.07% of MgSO4,and the activity of chitinase reached the maximal value（6.86 U）under these conditions.[Conclusion] The study improved the activity of chitinase produced by strain G-254 and provided good foundation for industrial production.展开更多
Mammalian chitinases and the related chilectins （ChiLs） belong to the GH18 family, which hydrolyse the glycosidic bond of chitin by a substrate-assisted mechanism. Chitin the fundamental component in the coating of ...Mammalian chitinases and the related chilectins （ChiLs） belong to the GH18 family, which hydrolyse the glycosidic bond of chitin by a substrate-assisted mechanism. Chitin the fundamental component in the coating of numerous living species is the most abundant natural biopolymer. Mounting evidence suggest that the function of the majority of the mammalian chitinases is not exclusive to catalyze the hydrolysis of chitin producing pathogens, but include crucial role specifc in the immunologic activities. The chitinases and chitinase-like proteins are expressed in response to different proinflammatory cues in various tissues by activated macrophages, neutrophils and in different monocyte-derived cell lines. The mechanism and molecular interaction of chitinases in relation to immune regulation embrace bacterial infection, infammation, dismetabolic and degenerative disease. The aim of this review is to update the reader with regard to the role of chitinases proposed in the recent innate and adaptive immunity literature. The deep scrutiny of this family of enzymes could be a useful base for further studies addressed to the development of potential procedure directing these molecules as diagnostic and prognostic markers for numerous immune and infammatory diseases.展开更多
Chitinases （EC3.2.1.14）, found in a wide range of organisms, catalyze the hydrolysis of chitin and play a major role in defense mechanisms against fungal pathogens. The alignment and typical domains were analyzed us...Chitinases （EC3.2.1.14）, found in a wide range of organisms, catalyze the hydrolysis of chitin and play a major role in defense mechanisms against fungal pathogens. The alignment and typical domains were analyzed using basic local alignment search tool （BLAST） and simple modular architecture research tool （SMART）, respectively. On the basis of the annotations of flee （Oryza sativa L.） and Arabidopsis genomic sequences and using the bio-software SignalP3.0, TMHMM2.0, TargetPl.1, and big-Pi Predictor, 25 out of 37 and 16 out of 24 open reading frames （ORFs） with chitinase activity from rice and Arabidopsis, respectively, were predicted to have signal pepfides （SPs）, which have an average of 24.8 amino acids at the N-terminal region. Some of the chitinases were secreted extracellularly, whereas some were located in the vacuole. The phylogenic relationship was analyzed with 61 ORFs and 25 known ehitinases and they were classified into 6 clusters using Clustal X and MEGA3.1. This classification is not completely consistent when compared with the traditional system that classifies the chitinases into 7 classes. The frequency of distribution of amino acid residues was distinct in different clusters. The contents of alanine, glycine, serine, and leucine were very high in each cluster, whereas the contents of methionine, histidine, tryptophan, and cysteine were lower than 20%. Each cluster had distinct amino acid characteristics. Alanine, valine, leucine, cysteine, serine, and lysine were rich in Clusters Ⅰ to Ⅵ, respectively.展开更多
The seed germination and tomato seedling tests showed that Bacillus subtilis SL-13 could promote the sprouting and seedling growth of tomato.The fresh and dry weight of tomato seedlings increased 42.86%and 18.75%,resp...The seed germination and tomato seedling tests showed that Bacillus subtilis SL-13 could promote the sprouting and seedling growth of tomato.The fresh and dry weight of tomato seedlings increased 42.86%and 18.75%,respectively.The control efficacies of the SL-13 to tomato Rhizoctonia rot were 20.65%and 35.23%in the greenhouse and field,respectively.The growth of the plant-pathogenic fungus Rhizoctonia solani was considerably inhibited in the presence of the strain SL-13 culture supernatant.The main antifungal protein was detected to be chitinase through vitro assay.The chitinase was purified with DEAE-Sepharose fast flow ion exchange column chromatography and Sephadex G-75 gel filtration for further characterization.The optimal pH and temperature for the chitinase activity were 7.0 and 50°C,respectively.It was demonstrated that the enzyme was stable at pH 5-9 and 40-60°C.70%of the enzyme activity was retained when incubated at 121°C and 0.11 MPa for 20 min,and the enzyme was not sensitive to protease K and ultraviolet radiation.Thus it is suitable for effective biological control in relatively unstable environment.展开更多
An extracellular chitinase produced by Paenibacillus pasadenensis CS0611was purified by ammoniumsulfate precipitation,HiTrap DEAE FF and HiLoad26/600Superdex200pg column chromatography.The apparent molecular mass dete...An extracellular chitinase produced by Paenibacillus pasadenensis CS0611was purified by ammoniumsulfate precipitation,HiTrap DEAE FF and HiLoad26/600Superdex200pg column chromatography.The apparent molecular mass determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis was69kDa.The optimum pH and optimum temperature of the chitinase were5.0and50°C,respectively.The enzyme showed high stability at alkaline pH values and temperaturesbelow40°C.Additionally,the metal ions Mn2+,Mg2+,and Co2+inhibited activity of the chitinase.Thechitinase was active on colloidal chitin with an apparent Km of4.41mg/mL and Vmax of1.08mg/min.Substrate spectrum analysis indicated that the chitinase reacted preferentially with the glucosidicbond between GlcNAc‐GlcNAc.The enzymatic hydrolysate was analyzed by high‐performance liquidchromatography and thin layer chromatography,and clearly showed that a subunit of(GlcNAc)2was the main hydrolysis product.展开更多
The family of mammalian chitinases includes members both with and without glycohydrolase enzymatic activity against chitin, a polymer of N-acetylglucosamine. Chitin is the structural component of fungi, crustaceans, i...The family of mammalian chitinases includes members both with and without glycohydrolase enzymatic activity against chitin, a polymer of N-acetylglucosamine. Chitin is the structural component of fungi, crustaceans, insects and parasitic nematodes, but is completely absent in mammals. Exposure to antigens containing chitin- or chitin-like structures sometimes induces strong T helper type-I responses in mammals, which may be associated with the induction of mammalian chitinases. Chitinase 3-like-1 （CHI3L1）, a member of the mammalian chitinase family, is induced specifically during the course of inflammation in such disorders as inflammatory bowel disease, hepatitis and asthma. In addition, CHI3L1 is expressed and secreted by several types of solid tumors including glioblastoma, colon cancer, breast cancer and malignant melanoma. Although the exact function of CHI3L1 in inflammation and cancer is still largely unknown, CHI3L1 plays a pivotal role in exacerbating the inflammatory processes and in promoting angiogenesis and remodeling of the extracellular matrix. CHI3L1 may be highly involved in the chronic engagement of inflammation which potentiates development of epithelial tumorigenesis presumably by activating the mitogen-activated protein kinase and the protein kinase B signaling pathways. Anti-CHI3L1 antibodies or pan-chitinase inhibitors may have the potential to suppress CHI3Ll-mediated chronic inflammation and the subsequent carcinogenic change in epithelial cells.展开更多
AIM: To assess blood chitinase 3-like 1(CHi3L1) levels for 2 mo after minimally invasive colorectal resection(MICR) for colorectal cancer(CRC). METHODS: CRC patients in an Institutional Review Board approved data/plas...AIM: To assess blood chitinase 3-like 1(CHi3L1) levels for 2 mo after minimally invasive colorectal resection(MICR) for colorectal cancer(CRC). METHODS: CRC patients in an Institutional Review Board approved data/plasma bank who underwent elective MICR for whom preoperative(PreO p), early postoperative(PostO p), and 1 or more late PostO p samples [postoperative day(POD) 7-27] available were included. Plasma CHi3L1 levels(ng/m L) were determined in duplicate by enzyme linked immunosorbent assay. RESULTS: PreOp and PostOp plasma sample were available for 80 MICR cancer patients for the study. The median PreOp CHi3L1 level was 56.8 CI: 41.9-78.6 ng/mL(n = 80). Significantly elevated(P < 0.001) median plasma levels(ng/mL) over PreOp levels were detected on POD1(667.7 CI: 495.7, 771.7; n = 79), POD 3(132.6 CI: 95.5, 173.7; n = 76), POD7-13(96.4 CI: 67.7, 136.9; n = 62), POD14-20(101.4 CI: 80.7, 287.4; n = 22), and POD 21-27(98.1 CI: 66.8, 137.4; n = 20, P = 0.001). No significant difference in plasma levels were noted on POD27-41. CONCLUSION: Plasma CHi3L1 levels were significantly elevated for one month after MICR. Persistently elevated plasma CHi3L1 may support the growth of residual tumor and metastasis.展开更多
A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular...A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer （pH 2.5）. The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2＋, Ba^2＋, Na^＋, and K^＋ enhanced the enzyme activity, whereas Fe^2＋, Ag^＋, Hg^2＋, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.展开更多
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 tra...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.展开更多
基金supported by the National Key R&D Program of China[Grant No.2021YFC2103002]the National Natural Science Foundation of China[Grant No.32202720]+1 种基金The Agricultural Science and Technology Innovation Program(CAAS-ZDRW202304)the China Agriculture Research System of MOF and MARA(CARS-41).
文摘Thermophilic endo-chitinases are essential for production of highly polymerized chitooligosaccharides,which are advantageous for plant immunity,animal nutrition and health.However,thermophilic endo-chitinases are scarce and the transformation from exo-to endo-activity of chitinases is still a challenging problem.In this study,to enhance the endo-activity of the thermophilic chitinase Chi304,we proposed two approaches for rational design based on comprehensive structural and evolutionary analyses.Four effective single-point mutants were identified among 28 designed mutations.The ratio of(GlcNAc)3 to(GlcNAc)2 quantity(DP3/2)in the hydrolysates of the four single-point mutants undertaking colloidal chitin degradation were 1.89,1.65,1.24,and 1.38 times that of Chi304,respectively.When combining to double-point mutants,the DP3/2 proportions produced by F79A/W140R,F79A/M264L,F79A/W272R,and M264L/W272R were 2.06,1.67,1.82,and 1.86 times that of Chi304 and all four double-point mutants exhibited enhanced endo-activity.When applied to produce chitooligosaccharides(DP≥3),F79A/W140R accumulated the most(GlcNAc)4,while M264L/W272R was the best to produce(GlcNAc)3,which was 2.28 times that of Chi304.The two mutants had exposed shallower substrate-binding pockets and stronger binding abilities to shape the substrate.Overall,this research offers a practical approach to altering the cutting pattern of a chitinase to generate functional chitooligosaccharides.
基金financially supported by the National Natural Science Foundation of China(No.22177132 and 21977114).
文摘Tebufenozide,an efficient insect growth regulator(IGR)against lepidopteran pests,presents a novel mode of action with minimal non-target impact.By competing with ecdysteroids for ecdysone receptor(EcR)binding,it regulates insect growth precisely.This study explores tebufenozide's potential as a multitarget IGR,targeting both EcR and Ostrinia furnacalis chitinase I(OfChtI).The inhibitory activity against OfChtI is comparable to that of substrates(GlcNAc)5,with an IC50 of 45.77μM.Our computational findings indicate that tebufenozide binds at the subsite1 to t1 of OfChtI through various interactions.Notably,tebufenozide establishes a pi-pi interaction with the flipped sidechain of Trp107,enabling tebufenozide to deeply penetrate into the S1 pocket,thereby obstructing substrate binding to OfChtI.These insights highlight the potency of multitarget strategies,laying the groundwork for innovative IGR designs that offer comprehensive pest management solutions.
文摘The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.
基金Supported by Science Foundation from Southwest Forestry College(200524M)Natural Science Foundation of Yunan Province(2002C0047M)Key Scientific and Technological Project of Yunan Province(2003NG12)~~
文摘[Objective] The aim of this study was to isolate chitinase gene from Trichoderma atroviride strain SS003. [Method] With the aeciospore wall of armandii pine blister rust as inducer, chitinase gene was induced to express in Trichoderma atroviride cells. The cDNA fragment of chitinase gene was cloned by RT-PCR approach. [Result] The activity of chitinase induced reached 40.17 μg/10 min; and the specific fragment amplified was 834 bp in length and proved to be the fragment of chitinase gene by sequencing and sequence analysis. [Conclusion] The result showed the feasibility of isolating the full length of chitinase gene and its transformation, and further producing chitinase.
基金Supported by Science and Technology Research Project of Education Department of Liaoning Province(2008120)IntroducedTalent Start-up Fund Project of Dalian Nationalities University(20056209)~~
文摘[ Objective] The aim of this study was to clone Class Ⅱ chitinase gene in Leymus chinensis grown in saline land in Heilongjiang Province and analyze its sequence, which provided a foundation for further study on the biological function and application of chitinasa gene. [ Method] cDNA library of Leymus chinensis leaves were constructed, and its DNA sequence was determined or analyzed, while the homology of chitinasa gene and amino acid sequence was compared with that in GenBank. [ Result] One full length cDNA fragment with length of 996 bp was cloned from cDNA library of Leymus chinensis leaves. The length of ORF was 768 bp encoding 225 amino acids （GenBank accession number： EU344908）. The encoding products lacked CBD and C-terminal extension region from the view of structure, but had structural characters of Class Ⅱ chitinase gene, which indicated that amino acid sequence had high homology compared with Class Ⅱ chitinase gene of rye and wheat. The constructed recombinant vector pQE-LcChi2 could express a protein of 27 kD through induction, which was consistent with the deduced encoding product of pQE-LcChi2 gene. [ Conclusion] LcChl2 gene is an expression gene, which can express in E. coll.
文摘DA novel chitinase gene (GhChia7) was isolated from salicylic acid (SA)-treated cotton cotyledons and characterized by DNA sequence analysis of its cDNA and genomic DNA clone. The deduced amino acid sequence, designated as class VII chitinase, shares about 30% identity to class I or II chitinases, and does not correspond to any of the previously characterized classes I-VI chitinases. Northern blotting analysis showed that the transcripts of GhChia7 were abundant both in cotton fibers and in the roots of the seedlings. The accumulation of GhChia7 mRNA in SA-treated cotyledons reached maximum at 7.5 mmol/ L concentration after 18 h. Results indicate that GhChia7 might play an important role in cotton's active defense response.
基金Supported by the Research Fund for Mid-career and Young Scientists of Education Department of Hubei Province(Q2011130)~~
文摘[Objective] This study aimed to screen endophytic bacteria which is antag- onistic to cotton Fusarium wilt. [Method] Fresh cotton plants collected from cotton- growing areas in Jingzhou City, Hubei Province were used as experimental materials to isolate endophytic bacteria. Through chitinase test and co-culturing both micro-or- ganisms side by side on the same PDA culture plate, antagonistic strains to cotton Fusarium wilt were screened. [Result] A total of 83 bacterial isolates were obtained from cotton plants grown in the fields, six of which were chitinase-productive bacte- ria. Through chitinase test and co-culturing both micro-organisms side by side on the same PDA culture plate, strain V-8 which had the strongest antagonistic effect on Fusarium oxysporum f. sp. vasinfectum was screened. Strain V-8 had a wider anti- fungal spectrum with certain inhibitory effect on all the six important pathogenic fungi including Fusarium oxysporum f. sp niveum; it colonized stably in the rhizospheric soil of cotton, with a colonization density of up to 6.2x10s cfu/g fifty days after inoc- ulation; the relative effect on controlling cotton Fusarium wilt in pot test was 73.2%. The Findings of this study suggested that strain V-8 had great potential for biological control of cotton Fusarium wilt and could be taken as a substantial material for the cloning of chitinase genes. [Conclusion] The results from this study provides bases for the control of cotton fusarium wilt, as well as the exploitation of endophytic bac- teria resources in cotton and the development of novel biological pesticides.
基金Supported by Science and Technology Support Project of Guangdong Academy of Agricultural Science and Technology(07-Supporting-04)Agricultural Research Projects in Guangdong Province (2007A0201000043)Earmarked Fund for Modern Agro-Industry Technology Research System of China~~
文摘[Objective] The aim of this study was to optimize the conditions of chitinase-produce strains.[Method] A kind of screened chitinase-produce strain G-254 was habituated cultured,and then the single factor experiment was carried out to explore the effects of different carbon source,nitrogen source and inorganic salt on the activity of produced chitinase;the response surface test was used to determine the optimal conditions for chitinase production.[Result] The optimal conditions for chitinase production were：8% of glucose,5% of beef extract and 0.07% of MgSO4,and the activity of chitinase reached the maximal value（6.86 U）under these conditions.[Conclusion] The study improved the activity of chitinase produced by strain G-254 and provided good foundation for industrial production.
文摘Mammalian chitinases and the related chilectins （ChiLs） belong to the GH18 family, which hydrolyse the glycosidic bond of chitin by a substrate-assisted mechanism. Chitin the fundamental component in the coating of numerous living species is the most abundant natural biopolymer. Mounting evidence suggest that the function of the majority of the mammalian chitinases is not exclusive to catalyze the hydrolysis of chitin producing pathogens, but include crucial role specifc in the immunologic activities. The chitinases and chitinase-like proteins are expressed in response to different proinflammatory cues in various tissues by activated macrophages, neutrophils and in different monocyte-derived cell lines. The mechanism and molecular interaction of chitinases in relation to immune regulation embrace bacterial infection, infammation, dismetabolic and degenerative disease. The aim of this review is to update the reader with regard to the role of chitinases proposed in the recent innate and adaptive immunity literature. The deep scrutiny of this family of enzymes could be a useful base for further studies addressed to the development of potential procedure directing these molecules as diagnostic and prognostic markers for numerous immune and infammatory diseases.
基金This work was supported by the 863 Program (No. 2002AA245041), the National Natural Science Foundation of China (No. 30260006), and the R&D Foundation of Yunnan Province (No. 2003GP06).
文摘Chitinases （EC3.2.1.14）, found in a wide range of organisms, catalyze the hydrolysis of chitin and play a major role in defense mechanisms against fungal pathogens. The alignment and typical domains were analyzed using basic local alignment search tool （BLAST） and simple modular architecture research tool （SMART）, respectively. On the basis of the annotations of flee （Oryza sativa L.） and Arabidopsis genomic sequences and using the bio-software SignalP3.0, TMHMM2.0, TargetPl.1, and big-Pi Predictor, 25 out of 37 and 16 out of 24 open reading frames （ORFs） with chitinase activity from rice and Arabidopsis, respectively, were predicted to have signal pepfides （SPs）, which have an average of 24.8 amino acids at the N-terminal region. Some of the chitinases were secreted extracellularly, whereas some were located in the vacuole. The phylogenic relationship was analyzed with 61 ORFs and 25 known ehitinases and they were classified into 6 clusters using Clustal X and MEGA3.1. This classification is not completely consistent when compared with the traditional system that classifies the chitinases into 7 classes. The frequency of distribution of amino acid residues was distinct in different clusters. The contents of alanine, glycine, serine, and leucine were very high in each cluster, whereas the contents of methionine, histidine, tryptophan, and cysteine were lower than 20%. Each cluster had distinct amino acid characteristics. Alanine, valine, leucine, cysteine, serine, and lysine were rich in Clusters Ⅰ to Ⅵ, respectively.
基金Supported by the National Natural Science Foundation of China（20776017） the Xinjiang Uygur Autonomous Region High-tech Research and Development Project（20081108）+1 种基金 the Fok Ying Tung Education Foundation（101071） the Xinjiang Bingtuan Key Science and Technology Industry Project（2008GG24）
文摘The seed germination and tomato seedling tests showed that Bacillus subtilis SL-13 could promote the sprouting and seedling growth of tomato.The fresh and dry weight of tomato seedlings increased 42.86%and 18.75%,respectively.The control efficacies of the SL-13 to tomato Rhizoctonia rot were 20.65%and 35.23%in the greenhouse and field,respectively.The growth of the plant-pathogenic fungus Rhizoctonia solani was considerably inhibited in the presence of the strain SL-13 culture supernatant.The main antifungal protein was detected to be chitinase through vitro assay.The chitinase was purified with DEAE-Sepharose fast flow ion exchange column chromatography and Sephadex G-75 gel filtration for further characterization.The optimal pH and temperature for the chitinase activity were 7.0 and 50°C,respectively.It was demonstrated that the enzyme was stable at pH 5-9 and 40-60°C.70%of the enzyme activity was retained when incubated at 121°C and 0.11 MPa for 20 min,and the enzyme was not sensitive to protease K and ultraviolet radiation.Thus it is suitable for effective biological control in relatively unstable environment.
基金supported by the National Natural Science Foundation of China (21336002,21376096,21676104)~~
文摘An extracellular chitinase produced by Paenibacillus pasadenensis CS0611was purified by ammoniumsulfate precipitation,HiTrap DEAE FF and HiLoad26/600Superdex200pg column chromatography.The apparent molecular mass determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis was69kDa.The optimum pH and optimum temperature of the chitinase were5.0and50°C,respectively.The enzyme showed high stability at alkaline pH values and temperaturesbelow40°C.Additionally,the metal ions Mn2+,Mg2+,and Co2+inhibited activity of the chitinase.Thechitinase was active on colloidal chitin with an apparent Km of4.41mg/mL and Vmax of1.08mg/min.Substrate spectrum analysis indicated that the chitinase reacted preferentially with the glucosidicbond between GlcNAc‐GlcNAc.The enzymatic hydrolysate was analyzed by high‐performance liquidchromatography and thin layer chromatography,and clearly showed that a subunit of(GlcNAc)2was the main hydrolysis product.
基金Supported by NIH (DK64289,DK74454,DK43351 and DK80070)grants from the Eli and Edythe L.Broad Medical FoundationAmerican Gastroenterological Association Foundation for Digestive Health and Nutrition to Mizoguchi E
文摘The family of mammalian chitinases includes members both with and without glycohydrolase enzymatic activity against chitin, a polymer of N-acetylglucosamine. Chitin is the structural component of fungi, crustaceans, insects and parasitic nematodes, but is completely absent in mammals. Exposure to antigens containing chitin- or chitin-like structures sometimes induces strong T helper type-I responses in mammals, which may be associated with the induction of mammalian chitinases. Chitinase 3-like-1 （CHI3L1）, a member of the mammalian chitinase family, is induced specifically during the course of inflammation in such disorders as inflammatory bowel disease, hepatitis and asthma. In addition, CHI3L1 is expressed and secreted by several types of solid tumors including glioblastoma, colon cancer, breast cancer and malignant melanoma. Although the exact function of CHI3L1 in inflammation and cancer is still largely unknown, CHI3L1 plays a pivotal role in exacerbating the inflammatory processes and in promoting angiogenesis and remodeling of the extracellular matrix. CHI3L1 may be highly involved in the chronic engagement of inflammation which potentiates development of epithelial tumorigenesis presumably by activating the mitogen-activated protein kinase and the protein kinase B signaling pathways. Anti-CHI3L1 antibodies or pan-chitinase inhibitors may have the potential to suppress CHI3Ll-mediated chronic inflammation and the subsequent carcinogenic change in epithelial cells.
基金Supported by Mr.Wade Thompson and family donation funds to the Divisions of Colon and Rectal surgery,Department of Surgery,Mount Sinai West Hospital,New York,NY 10019
文摘AIM: To assess blood chitinase 3-like 1(CHi3L1) levels for 2 mo after minimally invasive colorectal resection(MICR) for colorectal cancer(CRC). METHODS: CRC patients in an Institutional Review Board approved data/plasma bank who underwent elective MICR for whom preoperative(PreO p), early postoperative(PostO p), and 1 or more late PostO p samples [postoperative day(POD) 7-27] available were included. Plasma CHi3L1 levels(ng/m L) were determined in duplicate by enzyme linked immunosorbent assay. RESULTS: PreOp and PostOp plasma sample were available for 80 MICR cancer patients for the study. The median PreOp CHi3L1 level was 56.8 CI: 41.9-78.6 ng/mL(n = 80). Significantly elevated(P < 0.001) median plasma levels(ng/mL) over PreOp levels were detected on POD1(667.7 CI: 495.7, 771.7; n = 79), POD 3(132.6 CI: 95.5, 173.7; n = 76), POD7-13(96.4 CI: 67.7, 136.9; n = 62), POD14-20(101.4 CI: 80.7, 287.4; n = 22), and POD 21-27(98.1 CI: 66.8, 137.4; n = 20, P = 0.001). No significant difference in plasma levels were noted on POD27-41. CONCLUSION: Plasma CHi3L1 levels were significantly elevated for one month after MICR. Persistently elevated plasma CHi3L1 may support the growth of residual tumor and metastasis.
基金the Science Technology Plan Foundation of Hebei Province, China (07225533)the Doctor Foundation from Agricultural University of Hebei (050031)
文摘A novel thermostable extracellular chitinase was purified from the culture filtrate of Thermomyces lanuginosus SY2 by using diethylaminoethyl Sepharose chromatography and Phenyl-Sepharose chromatography. The molecular size of the purified chitinase was estimated to be 48 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chitinase exhibited optimum catalytic activity at pH 4.5 and 55℃. The enzyme was stable at 50℃, and its half-life time at 65℃ was 25 rain. The thermostable chitinase was obtained with 60% of the full activity, when it was incubated in the buffer （pH 2.5）. The enzyme showed the unique properties for thermostability and pH stability since it was one of the most thermostable chitinases so far isolated in fungi. Ca^2＋, Ba^2＋, Na^＋, and K^＋ enhanced the enzyme activity, whereas Fe^2＋, Ag^＋, Hg^2＋, and ethylene diamine tetraacetic acid caused obvious inhibition. The N-terminal amino acids were AQGYLSVQYFVNWAI. Degenerate primers based on the N-terminal sequences of purified chitinase and a cDNA fragment encoding the chitinase gene were obtained through reverse transcriptase-polymerase chain reaction amplication. The RACE was used to generate full-length cDNA clones. The cDNA of chit contained an open reading frame of 1 326 bp encoding 442 amino acids. The gene chit has been registered in GenBank with accession number DQ092332. The alignment results of putative amino acid sequence showed the lower similarity to other chitinases in family-18 except for the catalytic domain containing two conserved motifs related with catalytic activity of chitinase.
基金supported by the National Natural Science Foundation of China(32070168,31700141,31872027)Provincial Natural Science Foundation of Hunan(2019JJ50234)+2 种基金Changsha Science and Technology Project(kq1901033)Double first-class construction project of Hunan Agricultural UniversitySakura Science Plan of Japan Science Technology Agency(JST)。
文摘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.
