植物几丁质酶的生物功能

几丁质酶(ＥＣ3.2.1.14)是一种能降解几丁质(Ｎ乙酰氨基葡萄糖线性聚物)的糖苷酶。已经发现多种微生物、动物、植物都可产生几丁质酶。就植物而言,几丁质酶不仅存在于被子植物的双子叶植物和单子叶植物,而且也存在于裸子植物及蕨类植物中[1]。在植株中,几丁质酶可分布于根、茎、叶、花器、果实、种子诸器官。种子、根、花器中的几丁质酶含量一般较其它器官高。在正常情况下,植物中几丁质酶活性较低,但经诱导因子诱导,活性会迅速升高。真菌、细菌、病毒的侵染,真菌和植物细胞壁的组成成分,多糖等诱导物,伤害,乙烯,有机分子如水杨酸、氨基酸类…     

杆状病毒几丁质酶基因结构与功能的研究进展*

杆状病毒几丁质酶基因是杆状病毒的非必需基因 ,是高度保守的基因。该基因在杆状病毒复制晚期表达产生几丁质酶 ,该酶N端具信号肽 ,中部是酶的活性区 ,C端是酶的内质网结合区。杆状病毒几丁质酶同时具有内切和外切几丁质酶活性 ,主要功能是水解昆虫体内的组成型几丁质。杆状病毒几丁质酶对于虫体液化是必需的 ,同时它还是原组织蛋白酶 (pro V Cath)的分子伴侣 ,并与病毒侵染机制相关联。杆状病毒的几丁质酶基因与细菌的几丁质酶基因可能源于共同的祖先。     

昆虫杆状病毒几丁质酶及其应用研究进展

杆状病毒几丁质酶基因（chitinase,,ChiA）是晚期表达的非必需基因,高度保守。表达产物几丁质酶分为3个区：N-端信号肽区,中部酶活性区和C-末端酶内质网结合区。该酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的几丁质,促进虫体液化;作为组织蛋白酶原（pro-V-Cath）的分子伴侣,参与其加工和运输过程; 影响多角体的形成,并与细胞的裂解有关;还与病毒侵染机制相关联。杆状病毒ChiA与细菌ChiA源于共同的祖先,而昆虫ChiA则可能直接来自杆状病毒。在害虫生物防治中,杆状病毒ChiA可直接作为杀虫剂,或作为苏云金杆菌和杆状病毒等微生物杀虫剂的增效剂使用;杆状病毒ChiA可转入植物,获得具有持续杀虫及抗病活性的转基因植物;将杆状病毒ChiA的内质网定位序列删除、突变,或在病毒基因组中插入外源ChiA,重组病毒的杀虫活性增强。通过基因工程手段,删除病毒基因组ChiA或V-Cath,可改善杆状病毒表达系统对分泌蛋白和膜结合蛋白的表达。     

杆状病毒几丁质酶基因结构与功能的研究进展

杆状病毒几丁质酶基因是杆状病毒的非必需基因,是高度保守的基因。该基因在杆状病毒复制晚期表达产生几丁质酶,该酶N端具信号肽,中部是酶的活性区,C端是酶的内质网结合区。杆状病毒几丁质酶同时具有内切和外切几丁质酶活性,主要功能是水解昆虫体内的组成型几丁质。杆状病毒几丁质酶对于虫体液化是必需的,同时它还是原组织蛋白酶(pro-V-Cath)的分子伴侣,并与病毒侵染机制相关联。杆状病毒的几丁质酶基因与细菌的几丁质酶基因可能源于共同的祖先。     

渐狭蜡蚧菌产生的几丁质酶对芦笋茎枯病菌的抑制作用

目的:研究渐狭蜡蚧菌产生的几丁质酶及其对芦笋茎枯病菌天门冬拟茎点霉的抑制效果。方法:通过乙酰葡萄糖胺法、对硝基苯酚法和活性电泳法测定渐狭蜡蚧菌CGMCC5328的产几丁质酶特性及其粗酶液对芦笋茎枯病菌的抑菌效果。结果:渐狭蜡蚧菌CGMCC5328几丁质酶粗提取液的几丁质降解酶系活性第4 d达到高峰期;几丁质外切酶活性第6 d达到高峰期;活性染色检测到5条几丁质酶谱带,相对分子质量分别为32.9×103、42.1×103、54.1×103、65.6×103和79.6×103。用该几丁质酶粗酶液处理芦笋茎枯病菌,与灭活酶液处理相比较,抑菌效果明显,酶液处理导致菌丝畸形。结论:渐狭蜡蚧菌CGMCC5328产生的几丁质酶对芦笋茎枯病菌有抑制作用。     

一组纤维素分解菌复合系NSC-7的酶活表达特性

为了揭示一组具有降解纤维素和林丹双重功能的细菌复合系NSC-7的降解活性, 本文对该菌系的分解能力、纤维素酶活性和半纤维素酶活性进行测定。结果表明, NSC-7在14 d内, 可降解稻秆干重的 73.6％, 其中降解纤维素82.1％, 半纤维素58.2％, 木质素5.4％。用广泛采用的酶活测定方法测定了4种纤维素酶和半纤维素酶活性, 在培养的第8天, 内切酶、总纤维素酶、外切酶和b-糖苷酶活性都达到最大值, 分别为4.48 U/mL、7.51 U/mL、15.83 U/mL和25.78 U/mL。在培养的第5天, 半纤维素酶活性达到最高值为280.9 U/mL, 其平均值比纤维素酶活性高43.71倍。     

一组纤维素分解菌复合系NSC-7的酶活表达特性

为了揭示一组具有降解纤维素和林丹双重功能的细菌复合系NSC-7的降解活性, 本文对该菌系的分解能力、纤维素酶活性和半纤维素酶活性进行测定.结果表明,NSC-7在14d内,可降解稻秆干重的73.6%,其中降解纤维素82.1%,半纤维素58.2%,木质素5.4%.用广泛采用的酶活测定方法测定了4种纤维素酶和半纤维素酶活性,在培养的第8天,内切酶、总纤维素酶、外切酶和B.糖苷酶活性都达到最大值,分别为4.48U/mL、7.51U/mL、15.83U/mL和25.78U/mL.在培养的第5天,半纤维素酶活性达到最高值为280.9U/mL,其平均值比纤维素酶活性高43.71倍.     

扁豆几丁酶的特性和诱导

采用再生几丁质亲和层析和两性电解质等电聚焦电泳,纯化了扁豆荚几了酶,其分子量３０ｋＤ,等电点为９．１,主要呈内切酶活性。扁豆荚中不同组织几丁酶比活力有很大差异。在豆荚发育过程中,其酶活性变化呈单峰曲线,而比活力则持续上升,表明扁豆几丁酶活性变化与发育相关。经ＨｇＣｌ２处理后,扁豆荚壳和种子的几丁酶活性均明显提高。在扁豆荚发育的不同阶段,几丁酶的诱导特性也有明显差异,幼嫩豆荚几丁酶诱导活性的增加更为明显。     

球孢白僵菌几丁质酶诱发突变的探讨

从球孢白僵菌Beauveria bassiana 017菌株获得了几丁质水解活性较高的突变株。诱变原为紫外线及高能电子束。诱变体为分生孢子及芽生孢子。通过测定在几丁质琼脂平板上透明圈的大小、透明圈直径与菌落直径之比值,选出酶高产菌株,并通过摇瓶培养选育出EU-120高酶活性突变株,其几丁质酶活性比原始菌株提高了近3倍。并发现以上诱变原均能获得较好的诱变效果,在选育过程中加入猩红液可提高透明圈的清晰度。并通过比较几丁质酶对胶体几丁质及“晶体”几丁质的活性,探索酶调节的可能机理。     

氨基茚磷酸(AIP)和氨基氧乙酸(AOA)对茉莉酸甲酯诱导的烟草一些酶活性的影响

用茉莉酸甲酯(MJ,1mg ml^-1)处理培养在含0．1mmol／L AIP(水杨酸合成抑制剂)和／或1mmol／L AOA(乙烯合成抑制剂)的MS培养基上的烟草愈伤组织,测定某些酶的活性。结果表明：MJ明显提高过氧化物酶(POD)、β1,3-葡聚糖苷酶和几丁质外切酶的活性,略微促进苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性,抑制几丁内切酶的活性,而AOA和AIP则明显抑制MJ对POD和β1,3-葡聚糖苷酶活性的诱导作用,但对MJ诱导的PAL和PPO的活性影响很小,AOA和AIP可能作为逆境因子促进PAL的活性。AOA能部分解除MJ对几丁内切酶的抑制作用,但对MJ诱导的几丁外切酶的影响较小,而AIP抑制几丁内切酶的活性,也抑制MJ对此酶的诱导作用。因此我们认为：MJ对POD、PPO和几丁内切酶的影响可能是通过乙烯途径,对β1,3-葡聚糖苷酶和几丁外切酶的影响可能是通过水杨酸(SA)途径,而对PAL的影响可能是通过其它途径。     

Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis) in transgenic apple plants

Genes from the biocontrol fungus Trichoderma atroviride encoding the antifungal proteins endochitinase or exochitinase (N-acetyl--D-hexosaminidase) were inserted into Marshall McIntosh apple singly and in combination. The genes were driven by a modified CaMV35S promoter. The resulting plants were screened for resistance to Venturia inaequalis, the causal agent of apple scab, and for effects of enzyme expression on growth. Disease resistance was correlated with the level of expression of either enzyme when expressed alone but exochitinase was less effective than endochitinase. The level of expression of endochitinase was negatively correlated with plant growth while exochitinase had no consistent effect on this character. Plants expressing both enzymes simultaneously were more resistant than plants expressing either single enzyme at the same level; analyses indicated that the two enzymes acted synergistically to reduce disease. Selected lines, especially one expressing low levels of endochitinase activity and moderate levels of exochitinase activity, were highly resistant in growth chamber trials and had negligible reduction in vigor relative to control plants. We believe that this is the first report of resistance in plants induced by expression of an N-acetylhexosaminidase and is the first report of in planta synergy between an exochitinase and an endochitinase.     

Tissue specificity and induction of class I, II and III chitinases in barley (Hordeum vulgare)

Barley ( Hordeum vulgare L.) chitinases (EC 3.2.1.14) were found to be distributed and induced in highly tissue specific patterns. Out of 6 chitinases investigated 3 were present in leaves and only a class II chitinase (molecular mass 24 846 ± 5 Da, pI≥9.8) was markedly induced in leaves heavily infected with powdery mildew ( Erysiphe graminis f. sp. hordei ). The class II chitinase and a novel class III chitinase (molecular mass 30 kDa, pI≥9.8) were found in intercellular washing fluid of leaves, suggesting extracellular deposition. Neither of these two proteins were induced after infiltration of sodium salicylate (2 m M , pH 6.5) or nickel chloride (2 m M ). The class III chitinase showed exochitinase activity in addition to endochitinase activity. No grain specific chitinases were found in leaves after any of the stresses applied. In contrast, 3 grain specific chitinases and one of the leaf chitinases were found in in vitro cultures.     

Endo- and Exochitinase Activity in Kiwifruit Infected with Botrytis cinerea

Endo- and exochitinase activities were determined in autoclaved leaves, live leaves, and fruit of kiwifruit (Actinidia deliciosa var. deliciosa cv. Hayward [A. Chev.]Liang and Ferguson) following infection with Botrytis cinerea (Pers.). Appreciable endochitinase activity was found in healthy and diseased leaves and fruit at harvest and after cool-storage, but was absent in plant tissue killed by autoclaving. Later harvested fruit produced more endochitinase activity in storage than did early harvested fruit. Exochitinase activity was only associated with diseased tissue, and was present in diseased autoclaved leaves. The results suggest that endochitinase activity originates from the plant whilst exochitinase is associated with the pathogen. The significance of these findings is discussed.     

Deficiency of current methods in assaying endochitinase activity

Since chitin is degraded by a combination of both endo- and exochitinases, it is likely that both enzymes will be present in a crude extract. Currently used substrates for detecting endochitinase activity suffer from the fact that they could easily be digested by the contaminating exochitinase, thus giving either a false-positive or an inaccurate reading of the endochitinase activity. Using Photorhabdus luminescens, a bacterium symbiotically associated with insect-parasitic nematode Heterorhabditis bacteriophora as an exemplary system, we have identified these two chitinases by a simple "fluorimetric zymography" procedure. The exochitinase is a metalloenzyme and its activity is inhibited by 1,10-phenanthroline. Once the exochitinase activity is detected in a crude extract, its contribution must be eliminated before accurate determination of the endochitinase activity can be carried out. Specific properties of these enzymes including the pH activity profile, the requirement of metal ions for activity, and the molecular weight of the enzymes are among the factors to be considered in developing assaying procedures for endochitinase.     

The BmChi-h gene, a bacterial-type chitinase gene of Bombyx mori, encodes a functional exochitinase that plays a role in the chitin degradation during the molting process

The silkworm, Bombyx mori, has been recently demonstrated to contain a bacterial-type chitinase gene (BmChi-h) in addition to a well-characterized endochitinase gene (BmChitinase). The deduced amino acid sequence of BmChi-h showed extensive structural similarities with chitinases from bacteria such as Serratia marcescens chiA and baculoviruses (v-CHIA). Bacterial-type chitinase genes have not been found from any eukaryotes and viruses except for lepidopteran insects and lepidopteran baculoviruses. Thus, it was suggested that BmChi-h may be derived from a bacterial or baculovirus chitinase gene via horizontal gene transfer. In this report, we investigated the biological function of BmChi-h. Our enzymological study indicated that a chitinase encoded by BmChi-h has exo-type substrate preference, which is the same as S. marcescens chiA and v-CHIA, and different from BmChitinase, which has endo-type substrate preference. An immunohistochemical study revealed that BmChi-h localizes in the chitin-containing tissues during the molting stages, indicating that it plays a role in chitin degradation during molting. These results suggest that BmChi-h (exochitinase) and BmChitinase (endochitinase) may catalyze a native chitin by a concerted mechanism. Cloning and comparison of BmChi-h orthologues revealed that bacterial-type chitinase genes are highly conserved among lepidopteran insects, suggesting that the utilization of a bacterial-type chitinase during the molting process may be a general feature of lepidopteran insects.     

Chitinase in bean leaves: induction by ethylene,purification, properties,and possible function

Ethylene induced an endochitinase in primary leaves of Phaseolus vulgaris L. The enzyme formed chitobiose and higher chitin oligosaccharides from insoluble, colloidal or regenerated chitin. Less than 5% of the total chitinolytic activity was detected in an exochitinase assay proposed by Abeles et al. (1970, Plant Physiol. 47, 129–134) for ethylene-induced chitinase. In ethylene-treated plants, chitinase activity started to increase after a lag of 6 h and was induced 30 fold within 24 h. Exogenously supplied ethylene at 1 nl ml^?1 was sufficient for half-maximal induction, and enhancement of the endogenous ethylene formation also enhanced chitinase activity. Cycloheximide prevented the induction. Among various hydrolases tested, only chitinase and, to a lesser extent, β-1,3-glucanase were induced by ethylene. Induction of chitinase by ethylene occurred in many different plant species. Ethylene-induced chitinase was purified by affinity chromatography on a column of regenerated chitin. Its apparent molecular weight obtained by sodium dodecyl sulfate-gel electrophoresis was 30,000; the molecular weight determined from filtration through Sephadex G-75 was 22,000. The purified enzyme attacked chitin in isolated cell walls of Fusarium solani. It also acted as a lysozyme when incubated with Micrococcus lysodeikticus. It is concluded that ethylene-induced chitinase functions as a defense enzyme against fungal and bacterial invaders.     

Vacuolar localization of ethylene-induced chitinase in bean leaves

The localization of ethylene-induced endochitinase was studied in bean (Phaseolus vulgaris L. cv Saxa) leaves. The specific activity of chitinase in mesophyll protoplasts isolated from the leaves was as high as in tissue homogenates, indicating that most of the enzyme was located intracellularly. Vacuoles isolated and purified from the protoplasts were found to contain most of the intracellular chitinase activity.     

南昌市不同植物类群叶片氮磷浓度及其化学计量比

对南昌大学前湖校区89种主要植物叶片的N、P浓度及其化学计量比进行了研究,结果表明:乔灌、常绿、针叶、种子、裸子和单子叶植物类群的N浓度分别低于相对应的草本、落叶、阔叶、蕨类、被子和双子叶植物类群,而C3和C4植物差异不显著;乔灌、常绿和裸子植物类群的P浓度含量分别低于相对应的草本、落叶和被子植物类群,而针叶和阔叶、蕨类和种子、单子叶和双子叶、C3和C4植物类群间差异不显著;乔木、阔叶、被子和双子叶植物类群叶片N/P分别高于相对应的灌草、针叶、裸子和单子叶植物类群,而常绿和落叶、蕨类和种子、C3和C4植物类群之间差异不显著.可见,不同类型植物对N和P的吸收利用存在差异,且对不同养分供应采取不同的适应对策.结合研究区土壤养分现状,建议优先选择常绿、针叶、裸子和单子叶植物类群作为城市园林植物.     

Effects of sugar beet chitinase IV on root-associated fungal community of transgenic silver birch in a field trial

Heterogenous chitinases have been introduced in many plant species with the aim to increase the resistance of plants to fungal diseases. We studied the effects of the heterologous expression of sugar beet chitinase IV on the intensity of ectomycorrhizal (ECM) colonization and the structure of fungal communities in the field trial of 15 transgenic and 8 wild-type silver birch (Betula pendula Roth) genotypes. Fungal sequences were separated in denaturing gradient gel electrophoresis and identified by sequencing the ITS1 region to reveal the operational taxonomic units. ECM colonization was less intense in 7 out of 15 transgenic lines than in the corresponding non-transgenic control plants, but the slight decrease in overall ECM colonization in transgenic lines could not be related to sugar beet chitinase IV expression or total endochitinase activity. One transgenic line showing fairly weak sugar beet chitinase IV expression without significantly increased total endochitinase activity differed significantly from the non-transgenic controls in the structure of fungal community. Five sequences belonging to three different fungal genera (Hebeloma, Inocybe, Laccaria) were indicative of wild-type genotypes, and one sequence (Lactarius) indicated one transgenic line. In cluster analysis, the non-transgenic control grouped together with the transgenic lines indicating that genotype was a more important factor determining the structure of fungal communities than the transgenic status of the plants. With the tested birch lines, no clear evidence for the effect of the heterologous expression of sugar beet chitinase IV on ECM colonization or the structure of fungal community was found.     

Cloning and expression of a Streptomyces plicatus chitinase (chitinase-63) in Escherichia coli

4-Methylumbelliferyl (4-MU) glycosides of N-acetylglucosamine oligosaccharides were used as substrates to detect expression of a Streptomyces chitinase in Escherichia coli. Low levels of enzyme were detected when S. plicatus DNA was cloned into a bacteriophage lambda vector (EMBL-4). Subcloning into E. coli plasmids also gave low but detectable levels of enzyme expression. High level expression was achieved by resection of the cloned S. plicatus DNA with Bal31 followed by in-frame fusion to the amino-terminal peptide sequence of beta-galactosidase found in the pUC vectors. The Streptomyces chitinase was secreted into the periplasmic space of E. coli, and its signal sequence was removed. We characterized the activity of the cloned enzyme and compared it to three other purified Streptomyces plicatus chitinases with respect to hydrolysis of the 4-MU oligosaccharides. We found that two of the enzymes form 4-methylumbelliferone much more rapidly from the 4-MU disaccharide than from the trisaccharide. These same enzymes convert the 4-MU trisaccharide primarily to diacetylchitobiose and the 4-MU monosaccharide, a nonfluorescent product. The latter compound is not hydrolyzed appreciably by any of the enzymes. On the basis of these results, we suggest a new definition of "exo" and "endo" chitinase that differs from that found in the literature. We propose that exochitinase activity be defined as processive action starting at the nonreducing ends of chitin chains with release of successive diacetylchitobiose units, and that endochitinase activity be defined as random cleavage at internal points in chitin chains.