昆虫几丁质酶基因的分子特性概述

昆虫几丁质酶是分解昆虫体壁和中肠围食膜几丁质的重要酶类。已从烟草天蛾、家蚕等多种昆虫中分离到几丁质酶的cDNA和DNA序列。昆虫几丁质酶基因有着相似的分子特性,这些特性可为构建杀虫工程菌及转几丁质酶基因植物奠定基础。作者结合自己在该领域的工作,着重就昆虫几丁质酶基因结构特点,基因的拷贝数,基因在体内的时空表达以及异源表达及活性测定等多个方面的研究方法和研究进展进行了较为全面地介绍。

转几丁质酶和葡聚糖酶基因棉花的获得及其对黄萎病的抗性

通过根癌杆菌介导法 ,将维管束特异启动子 (竹节花黄斑病毒启动子CoYMV)启动的几丁质酶和CaMV3 5S启动的 β 1,3 葡聚糖酶嵌合双价基因 ,导入陆地棉品种冀合 3 2 1和中棉所 3 5。利用卡那霉素涂抹法对转基因株的卡那霉素抗性进行初步鉴定 ,再经PCR和Southern杂交确证 ,结果显示双价抗病基因分别以 1～ 2个拷贝整合到棉花基因组内。对转基因株系T3 幼苗进行无底塑钵菌液浇根法鉴定和大田病圃鉴定 ,结果显示 :7个转化株系均表现不同程度的抗或耐黄萎病性 ,苗期鉴定结果和大田病圃调查结果基本一致 ;其中D9910、D9915和D9919等 3个转基因纯合系的大田病情指数分别为 6 5、9 4和 9 5,均达到高抗水平。对以上 3个高抗黄萎病的纯合系进行遗传分析 ,结果显示这 3个抗病纯合系的卡那霉素抗性符合一对显性基因分离规律 ,结合分子杂交验证结果 ,可以认为这3个转基因株均含有一个拷贝的双抗病基因。

Increase of β -1, 3-Glucanase and Chitinase Activities in Cotton Callus Cells Treated by Salicylic Acid and Toxin of Verticillium dahliae

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.

Molecular Cloning and Sequence Analysis of Class Ⅱ Chitinase Gene in Leymus chinensis

[ 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.

A Novel Cotton Gene Encoding a New Class of Chitinase

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.

Effect of Chitinase-Producing Strain V-8 on Controlling Cotton Fusarium Wilt

[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.

SA Induction of aGrapevine ClassⅢChitinase Gene VCH3 Promoter in Transgenic Tobacco Vascular Tissue

The 1216bp5’upstream region of the gene encoding the class Ⅲ chitinase VCH3 was isolated from grapevine(Vitis amurensis Rubr．)(Genbank accession number AF441123)and two inverse salicylic acid(SA)responsive cis-acting motifs(TGACG)were found at-1181bp and-293 bp upstream of the transcriptional start site．respectively．To characterize the vcH3promoter，four chimeric constructs varied in the length of promoter fragments from-1187bp，-892bp，-589bp and-276bpto+7bp relative to the transcriptional start site were placed to the upstream of the β-glucuronidase(GUS)coding region and transferred to Nicotlana tobacum L．CV．NC89 by Agrobacterium tumefaciens-mediated leaf discs transformation．The functional properties of each promoter fragment were examined by fluorometric and histochemical analysis of GUS activity in the transgenic tobacco root treated withSA．The VCH3(-276)GUS construct．containing only the TATA and CAAT boxes was shown to have little inducibility upontreatment with SA．However，the similarly higher level of GUS expression was observed in the VCH3(-589) GUS or VCH3(-892) GUS transgenic plants with only one cis-acting motif,while the most abundance of GUS expression was found in the full-1ength promoter(-1187bpto+7bp)with two cis-acting motifs．The seresults indicated that the twocis-acting motifs werere quired for the maximal expression of the GUS reporter gene by SA induction．In addition，the histochemical analysis of GUS activity showed that the four VCH3 promoter fragments were more active in vascular tissue than that in outer and inner cortexes of the transgenic tobacco roots treated by SA，suggesting that the region involved in vascular tissue-specific expression of VCH3 promoter upon SA inducibility appears to belocated between positions-276 bp and+7bp relative to the transcriptional start site．In general，these results indicate a potential use for the SA induction of VCH3 promoter in genetic engineering．

Isolation and Screening of Strains for Silkworm Puparium Chitin Degradation and the Optimization of Chitinase Production by Response Surface Methodology

[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.

Chitinases in Oryza sativa ssp. japonica and Arabidopsis thaliana

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.

Biocontrol Efficiency of Bacillus subtilis SL-13 and Characterization of an Antifungal Chitinase

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.

Potential role of chitinase 3-like-1 in inflammation-associated carcinogenic changes of epithelial cells

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.

Towards understanding the ecology and mechanisms of biocontrol of Clonostachys rosea IK726

Clonostachys rosea (syn. Gliocladium roseum) IK726 was originally selected as an effective biocontrol agent (BCA) against cereal seed borne diseases caused by Fusarium culmorum and Bipolaris sorokiniana. We have studied the efficacy of the antagonist against different pathogens in several crops and found that the antagonist also is able to control Alternaria radicina and A. dauci on carrot seeds and different cold-storage fungi in acorns. IK726 is also able to reduce severity of soil borne Pythium spp. in cabbage, carrot and sugar beet. In addition, growth-promoting effects of IK726 have been demonstrated in barley and tomato. In order to develop and improve application methods and control strategies, essential basic studies of ecology and the mechanisms of control of IK726 is needed and has led us to use various molecular tools. The UP-PCR technology is used for strain recognition and we have developed GUS and GFP-transformants that resembles the wildtype strain in ecological fitness parameters. Using either the GUS-transformant or UP-PCR we have found that IK726, when applied with seeds, reproduces and survives several months in the rhizosphere of field grown barley and carrot. The GFP-transformant is used to study the behavior and in situ interactions of the antagonist with pathogens and plants. Using the GFP marker, we have observed conidial germination, colonization and conidiogenesis in natural soil, in vermiculite and on carrot and barley seed and roots and on barley leaves. Moreover in situ interactions with Alternaria on carrot material have been studied. The modes of action of C. rosea are not well understood but enzymatic activity, mycoparasitism, substrate competition, antibiosis and induced resistance are thought to play a role. Barley treated with C. rosea IK726 has an enhanced chitinolytic and glucanolytic activity compared to the activity in non-treated barley in pot experiments with field soil. Identification of chitinases from IK726 and studies of their role in interactions with pathogens have therefore been addressed in a recently initiated project. Preliminary results indicate that IK726 produces three types of chitinases, which seem to be regulated by glucose. Development of degenerated primers for cloning of an endochitinase is in progress.

G protein signalling involved in host recognition and mycoparasitism-related chitinase expression in Trichoderma atroviride

Mycoparasitic species of Trichoderma are commercially applied as biological control agents against various fungal pathogens. The mycoparasitic interaction is host specific and includes recognition, attack and subsequent penetration and killing of the host. Investigations on the underlying events revealed that Trichoderma responds to multiple signals from the host (e.g. lectins or other ligands such as low molecular weight components released from the host’s cell wall) and host attack is accompanied by morphological changes and the secretion of hydrolytic enzymes and antibiotics. Degradation of the cell wall of the host fungus is-besides glucanases and proteases-mainly achieved by chitinases. In vivo studies showed that the ech42 gene (encoding endochitinase 42) is expressed before physical contact of Trichoderma with its host, probably representing one of the earliest events in mycoparasitism, whereas Nag1 (N-acetylglucosaminidase) plays a key role in the general induction of the chitinolytic enzyme system of T. atroviride . Investigations on the responsible signal transduction pathways of T. atroviride led to the isolation of several genes encoding key components of the cAMP and MAP kinase signaling pathways, as alpha and β subunits of heterotrimeric G proteins, the regulatory subunit of cAMP-dependent protein kinase, adenylate cyclase, and three MAP kinases. Analysis of knockout mutants, generated by Agrobacterium-mediated transformation, revealed that at least two alpha-subunits of heterotrimeric G proteins are participating in mycoparasitism-related signal transduction. The Tga1 G alpha subunit was shown to be involved in mycoparasitism-related processes such as chitinase expression and overproduction of toxic secondary metabolites, whereas Tga3 was found to be completely avirulent showing defects in chitinase formation and host recognition.

Purification and characterization of extracellular chitinase from a novel strain Aspergillus fumigatus CS-01

Twelve samples derived from different locations in south central area of China are treated by enrichment and spread-plate technique for initial screening. Seven chitinase-producing strains are isolated. The chitinase present in the culture supematant of strain CS-01 possesses the maximum activity of 0.118 U/mL. Analysis of the morphological feature and the ITS rDNA sequence reveals that strain CS-01 belongs to Aspergillus fumigatus. Production of the chitinase is regulated by a inducible way and the maximum activity appears at 36 h in colloidal chitin culture. Purification of the chitinase is carried out by salting out, gel filtrate chromatography and anion exchange chromatography sequentially. Native-PAGE and SDS-PAGE indicate that the chitinase from A. fumigatus CS-01 is a monomer with the relative molecular mass estimated to be 4.50 × 10^4. Its maximum activity appears at pH 5 and 55 ℃. The chitinase is stable at pH 4.0-7.5 and below 45 ℃.

Bioinformatics Analysis of Class I Chitinase in Tobacco

This study almed to analyze the characteristics of Chi I （cIass I chitinase） in tobacco by using bioinformatics methods and tooIs so as to Iay a foundation for further study on disease resistance of Chi I in tobacco. The amino acid sequence characteristics and phyIogenetic reIationships of Chi Is of Nicotiana tabacum, Ara-bidopsis thaliana, Oryza sativa and Phaseolus vulgaris from NCBI were predicted and analyzed by using bioinformatics methods. The resuIts showed that the moIec-uIar weights of Chi Is with signal peptides ranged from 28 to 37 kDa. The se-quences of Chi Is, which al contalned both N-terminal chitin binding domaln （Chit BD1）andcatalyticdomaln（CIyco hydro-19）,werehighIyconserved.Thesequences of Nt-ChiA, Nt-ChiB and Nt-ChiC, three Chi Is from Nicotiana tabacum had high simiIarity, so their phyIogenetic reIationships were also cIose. The reIationships be-tween Chi Is from Nicotiana tabacum and one of the Chi Is from Arabidopsis thaliana and Phaseolus vulgaris were cIoser than those between Chi Is from Nico-tiana tabacum and Oryza sativa. The resuIts indicated that the properties of Chi I in tobacco were almost the same as those in other pIants. This study wiI provide a basis for further researches on disease resistance proteins in tobacco.

Cloning and expression in Saccharomyces cerevisiae of chit2 gene from Beauveria bassiana

To study recycled trashes from shrimps and crabs in the sea through chitinase secreted by microorganisms,the chitinase gene chit2 was cloned and sequenced from Beauveria bassiana by the polymerase chain reaction(PCR),and was ligated into the yeast expression vector pYES2.The expression vector plasmid was transformed into Saccharomyces cerevisiae H158.Gene expression took place upon induction with 2% galactose.The measurement of enzyme activity shows that the expression production can be expressed in active forms and secreted to the medium.The enzyme activity approaches the peak of 0.63 U/mL when the culture time is 36 h.

Clustering and Expression Analysis of Chitinases in Maize and Rice

Chitinases play an important role in regulating plant growth and development, especially defending themselves from fungal pathogens. It is important to do the biological analyses in crops. In this study, the result showed that the chitinases were distributed into the whole genome in rice, and nearly the whole genome in maize expect for Chromosome 9. The clustering results showed that one out of three chitinases from maize and rice belonged to new groups, which were separated from those in the conformed Classes I-VII. The identification of most amino acid sequences was very low among the chitinases from rice and/or maize. It was inferred that the chitinases with different functions were relatively stable during plant evolution. The relationship of chitinases expression between leaf blade and anther was positively significant in maize, but not significant in rice. Additionally, the ratio of chitinases with up- or down-regulated expression in sensitive maize under Fusarium moniliforme inoculation was different from that in sensitive rice under Magnaporthe grisea inoculation. It might be result from different tissues infected by different fungi. The number of chitinases from resistant maize was less than that from sensitive maize, which inferred that the resistant pathways on F. moniliforme should be not only chitin induced Pathogen-associated molecular PTI （patterns-triggered immunity） pathway, but also might include other PTI pathways that improve tolerance to F. moniliforme. The analysis of expression pattern of chitinases from maize and rice under fungi inoculation will be contributed into further research on the defense mechanism of fungi in crops.

Partial Characterization of Chitinolytic Extract from Endophytic Streptomyces sp. and Its Effects on the Boll Weevil

The present study achieves the biochemistry partial characterization of the chitinolytic extract produced by an endophytic Streptomyces sp. strain （A8）. This extract was also tested against Anthonomus grandis, the cotton boll weevil aiming its control. The chitinase crude extract from the A8 strain was cultured for five days in a minimum liquid media supplemented with chitin. The extract was partially characterized by standard methods. The chitinolytic extract had an optimum temperature of 66 ＂C and an optimum pH between 4 to 9 （around 80% of relative activity）. We also characterized the temperature and pH stability and measured the effects of enzyme inhibitors. The filtered chitinolytic extract was added to an artificial boll weevil diet. Boll weevil development from the egg stage to the adult stage was prolonged, and the percentage of adults that emerged was approximately 66% less than on control diet. This study showed that the.larval development of A. grandis was inhibited by the presence of characterized chitinolytic extract in artificial diet. This work provides an experimental basis for using the chitinase from an endophytic bacterium Streptomyces sp. as a biocontrol alternativeto controlling the plant pest A. grandis.

Genetic transformation ofpopulusxeuramericana cv. guariento with chitinase gene

In this paper, populus xeuramericana cv. Guariento was transformed with bean chitinase by Agrobacterium tumefaciens-mediated leaf disc method. Firstly, the leaf explants were pre-cultured at 25℃ for 2d. Secondly, they were infected in Agrobacterium tumefaciens suspension （OD600=0.5） for 20 rain, then were co-cultured for 3d in the dark. Thirdly, the explants were transferred to the selection culture medium （containing Kanamycin 40 mg.L^-1 and Cefotaxime Sodium 800 mg-L1） and incubated at 25℃ until resistance buds formed. Chitinase activity was determined for the positive plants by PCR and PCR-Southern blot hybridization analysis. And, chitinase activity of positive plants was significantly higher than that of control plant, and the highest ratio of activity of NO.4 to that of control was 3.41. It showed that bean chitinase gene had been expressed in the plant genome.

Nicotine enhances migration and invasion of human esophageal squamous carcinoma cells which is inhibited by nimesulide

AIM： To study the effect of nicotine on the migration and invasion of human esophageal squamous carcinoma cells and to investigate whether nimesulide can inhibit the effect of nicotine.METHODS： The esophageal squamous carcinoma cell line （TE-13） was treated with different concentrations of nicotine （100 μg/mL and 200 μg/mL） or 200 μg/mL nicotine plus 100 μmol/L nimesulide. Cell migration and invasion were measured using migration and invasion chamber systems. COX-2 expression was determined by Western blotting. Matrix metalloproteinase-2 （MMP-2） was analyzed by zymography and ELISA.RESULTS： Nicotine （100 μg/mL, 200 μg/mL） enhanced TE-13 cells migration and invasion, and increased the protein expression of COX-2 and the activity of MMP-2. Nicotine （200 μ/mL） stimulated TE-13 cells migration and invasion which were partly blocked by nimesulide. This was associated with decreased protein expression of COX-2 and decreased activity and protein expression of MMP-2. CONCLUSION： Nicotine enhances the migration and invasion of the esophageal squamous carcinoma cell line, and nimesulide partly blocks the effect ofnicotine-enhanced esophageal squamous carcinoma cell migration and invasion.