几丁质酶及其研究进展

本文从几丁质酶的分布、发育调节、可诱导性、分子生物学及抗病基因工程等方面近年来的进展进行了综合论述,并对其进一步的应用提出展望。     

链霉菌SO1菌株几丁质酶的纯化及性质

由链霉菌（Streptomycessp.）SO1菌株产生的几丁质酶（Citinase）经硫酸铵盐析、OEAE纤维素柱层析、SephadexG－100柱层桥分离纯化后,得到SDS－PAGE均一样品。用SDS－PAG测得纯化后的几丁质酶分子量为41Ku,用PAGEIEF测得等电点PI为5.4。酶反应的最适pH值为6．0,最适温度为50℃,在pH5．0～9．0、温度30～50℃时酶活性比较稳定。在相当于0.1mol／LNaCl的离子强度下酶活性最高。金属离子中的Mg²⁺、Ca     

水稻几丁质酶基因导入芥菜型油菜的研究

以芥菜型油菜 (BrassicajunceaL .)下胚轴为转化材料 ,通过根癌农杆菌 (Agrobacteriumtumefaciens)介导将水稻的几丁质酶基因 (Ricechitinasegene)导入“泸洲四陵”油菜品种中 ,获得抗潮霉素的再生转基因植株 ,并讨论了影响油菜再生及转化效率的几个因素。对部分经潮霉素筛选得到的再生植株进行了多次重复PCR检测 ,发现其中 40 %以上的潮霉素抗性植株均表现出较强的阳性反应 ,初步证明几丁质酶基因已整合到油菜细胞核基因组中。     

几丁质酶的研究进展

几丁质是自然界中含量仅次于纤维素的第二大类多糖,已从各种细菌、真菌、植物、哺乳动物和昆虫中分离并鉴定出来。随着近年来对几丁质及其衍生物的了解,发现此类多糖的物理化学特性在现实中有非常广泛的应用。几丁质酶是一类分解几丁质的酶,广泛分布于细菌、真菌、植物、哺乳动物、昆虫等物种中,并发挥着重要作用。近年来国内外工作者对这些不同来源的几丁质酶做了深入研究,为其在医学和农业上更广泛的应用提供理论基础。     

海洋微生物几丁质酶分离纯化及其抗真菌活性

以实验室筛选的海洋产几丁质酶短芽胞杆菌属（Bacillus brevis sp．）菌株Bspl,经往复式摇床振荡培养96h后,发酵液先后采取了75％的硫酸铵盐析、透析、几丁质亲和层析、SDS—PAGE等方法对几丁质粗酶液进行分离纯化和鉴定。几丁质亲和层析一步纯化后,经过SDS—PAGE电泳测定该酶的分子量为23ku,其比活力为86．65．纯化倍数为1．707、产率为32．1％。纯化的几丁质酶能抑制病原真菌的生长,对病原真菌的拮抗作用具有广谱性。同时研究了几丁质酶的稳定性,以胶态几丁质为底物,分离的几丁质酶在pH7．5,55．0℃左右具有最大酶活性;Zn^2＋、Cu^2＋和Hg^2＋能强烈抑制几丁质酶活性;Ni^＋和EDTA抑制20％-40％;然而5mmol／LCo^2＋可以使几丁质酶活性提高1．4倍;Mg^2＋、Ca^2＋等也能使酶活性增加。     

植物中几丁质酶的作用

几丁质酶 (EC3.2 .1.14)是降解几丁质的糖苷酶。很多植物包括草本植物和木本植物都能产生几丁质酶 [1] 。由于几丁质酶在植物抗真菌病害中起着重要的作用 ,因而成为近年抗真菌病害研究的热点之一 [2 ] 。随着对几丁质酶研究的深入 ,发现该酶不仅与抗真菌病害有关 ,而且在植物发育、抗胁迫及共生固氮等方面都发挥着作用。1　参与植物的发育调控植物几丁质酶基因的表达具有组织特异性 ,参与了植物的发育调控 ,尤其在早期胚胎发育过程中。胡萝卜中 ,几丁质酶 EP3 参与控制早期胚胎发育 [3 ] 。在云杉体胚发育中 ,几丁质酶也起到了调控作用。…     

微生物几丁质酶研究进展

微生物几丁质酶不仅在生物降解几丁质方面起着重要作用,而且可通过水解病原真菌的细胞壁而有效地抑制其生长。到目前为止,人们已经分离和克隆出了大量的微生物几丁质酶及其基因。尽管这些几丁质酶各不相同,但它们却具有类同的蛋白质结构域:信号肽、催化结构域和几丁质结合结构域等。本文着重介绍几丁质酶的结构和分子特征、表达和调控机理,并且分析了该酶的应用前景。     

水稻几丁质酶基因在大肠杆菌中的表达、纯化与活性分析

将克隆并确定序列的水稻(Oryza sativa L．Cpslo17)几丁质酶基因Oschi的cDNA序列(此序列已在GenBank中注册, 登录号为EU045451)插入原核表达载体pGEX-4T-1中。经酶切、序列鉴定分析后, 用该重组质粒转化大肠杆菌BL21(DE3); 经IPTG诱导获得表达, 并对Oschi蛋白表达条件进行了优化; 在大肠杆菌中表达的几丁质酶经纯化后在一定的浓度、一定的反应时间内能高效地分解几丁质。     

细菌几丁质酶研究进展

真菌病害是影响作物产量的重要原因 ,而几丁质酶能有效抑制其生长、水解其细胞壁。对研究较多的细菌几丁质酶及几丁质酶基因的分子生物学研究进展进行了综述 ,并对细菌几丁质酶基因利用存在的问题进行了探讨。     

Tobacco-expressed Brassica juncea chitinase BjCHI1 shows antifungal activity in vitro

We have previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains, and have shown that its mRNA is induced by wounding and methyl jasmonate treatment (K.-J. Zhao and M.-L. Chye, Plant Mol. Biol. 40 (1999) 1009–1018). By the presence of two chitin-binding domains, BjCHI1 resembles the precursor of UDA (Urtica dioica agglutinin) but, unlike UDA, BjCHI1 retains its chitinase catalytic domain after post-translational processing. Here, we indicate the role of BjCHI1 in plant defense by demonstrating its mRNA induction upon Aspergillus niger infection or caterpillar Pieris rapae (L.) feeding. To further investigate the biological properties of BjCHI1, we transformed tobacco with a construct expressing the BjCHI1 cDNA from the CaMV 35S promoter. Subsequently, we purified BjCHI1 from the resultant transgenic R₀ plants using a regenerated chitin column followed by fast protein liquid chromatography (FPLC). Also, the significance of the second chitin-binding domain in BjCHI1 was investigated by raising transgenic tobacco plants expressing BjCHI2, a deletion derivative of BjCHI1 lacking one chitin-binding domain. Colorimetric chitinase assays at 25 °C, pH 5, showed no significant differences between the activities of BjCHI1 and BjCHI2, suggesting that chitinase activity, due to the catalytic domain, is not enhanced by the presence of a second chitin-binding domain. Both BjCHI1 and BjCHI2 show in vitro anti-fungal activity toward Trichoderma viride, causing reductions in hyphal diameter, hyphal branching and conidia size.     

Brassica juncea chitinase BjCHI1 inhibits growth of fungal phytopathogens and agglutinates Gram-negative bacteria

Brassica juncea BjCHI1 is a plant chitinase with two chitin-binding domains. Its expression, induced in response to wounding, methyl jasmonate treatment, Aspergillus niger infection, and caterpillar Pieris rapae feeding, suggests that it plays a role in defence. In this study, to investigate the potential of using BjCHI1 in agriculture, Pichia-expressed BjCHI1 and its deletion derivatives that lack one or both chitin-binding domains were tested against phytopathogenic fungi and bacteria. Transplastomic tobacco expressing BjCHI1 was also generated and its extracts assessed. In radial growth-inhibition assays, BjCHI1 and its derivative with one chitin-binding domain showed anti-fungal activities against phytopathogens, Colletotrichum truncatum, C. acutatum, Botrytis cinerea, and Ascochyta rabiei. BjCHI1 also inhibited spore germination of C. truncatum. Furthermore, BjCHI1, but not its derivatives lacking one or both domains, inhibited the growth of Gram-negative bacteria (Escherichia coli, Ralstonia solanacearum, Pseudomonas aeruginosa) more effectively than Gram-positive bacteria (Micrococcus luteus and Bacillus megaterium), indicating that the duplicated chitin-binding domain, uncommon in chitinases, is essential for bacterial agglutination. Galactose, glucose, and lactose relieved agglutination, suggesting that BjCHI1 interacts with the carbohydrate components of the Gram-negative bacterial cell wall. Retention of chitinase and bacterial agglutination activities in transplastomic tobacco extracts implicates that BjCHI1 is potentially useful against both fungal and bacterial phytopathogens in agriculture.     

水稻几丁质酶基因在大肠杆菌中的表达、纯化与活性分析

将克隆并确定序列的水稻(Oryza sativa L．Cpslo17)几丁质酶基因Oschi的cDNA序列(此序列已在GenBank中注册, 登录号为EU045451)插入原核表达载体pGEX-4T-1中。经酶切、序列鉴定分析后, 用该重组质粒转化大肠杆菌BL21(DE3); 经IPTG诱导获得表达, 并对Oschi蛋白表达条件进行了优化; 在大肠杆菌中表达的几丁质酶经纯化后在一定的浓度、一定的反应时间内能高效地分解几丁质。     

Agglutination Activity of Fasciola gigantica DM9-1, a Mannose-Binding Lectin

The DM9 domain is a protein unit of 60–75 amino acids that has been first detected in the fruit fly Drosophila as a repeated motif of unknown function. Recent research on proteins carrying DM9 domains in the mosquito Anopheles gambiae and the oyster Crassostrea gigas indicated an association with the uptake of microbial organisms. Likewise, in the trematode Fasciola gigantica DM9-1 showed intracellular relocalization following microbial, heat and drug stress. In the present research, we show that FgDM9-1 is a lectin with a novel mannose-binding site that has been recently described for the protein CGL1 of Crassostrea gigas. This property allowed FgDM9-1 to agglutinate gram-positive and -negative bacteria with appropriate cell surface glycosylation patterns. Furthermore, FgDM9-1 caused hemagglutination across all ABO blood group phenotypes. It is speculated that the parenchymal located FgDM9-1 has a role in cellular processes that involve the transport of mannose-carrying molecules in the parenchymal cells of the parasite.     

金属离子对蜡蚧菌几丁质酶活力的影响

以蜡蚧菌(Ll)发酵液为材料,经分离纯化获得Ll几丁质酶(EC3.2.1.14)制剂.研究了金属离子对Ll几丁质酶活力的影响.结果表明,K+、Mg2+、Zn2+、Ca2+和Fe3+对几丁质酶活性有明显的促进作用,而Na+和Cu2+完全抑制几丁质酶的活性;Mn2+在低浓度时对酶有激活作用,随着浓度的升高表现出抑制作用;Fe2+和Ba2+的浓度低于0.5 mmol/L时对酶起抑制作用,而高于该浓度时则对酶有激活作用.     

薇甘菊几丁质酶基因的原核表达及活性分析

将薇甘菊Mmchi1基因cDNA序列的编码区插入到原核表达载体pET-32a(+)中,构建融合表达质粒,并在大肠杆菌中进行了融合蛋白6×His-Mmchi1的诱导表达、Western blot和酶活性分析.结果表明,0.1 mmol/L IPTG、25℃诱导4 h,在大肠杆菌Rosetta-gami(DE3)中能获得可溶性的6×His-Mmchi1;Western blot证实表达的6×His-Mmchi1能与抗6×His的单抗发生特异性反应,分子量约为55 kD,与预测的融合蛋白分子量相符;纯化后的6×His-Mmchi1最佳酶活性pH和温度分别为5.5～6.5和35～40℃.为进一步研究融合蛋白6×His-Mmchi1的功能奠定了基础.     

棘孢木霉（Trichoderma asperellum）几丁质酶基因的克隆与生物信息学分析

几丁质酶作为木霉菌防治植物病虫害的主要因子,在生物防治和环境保护等领域发挥着重要的作用.为了研究棘孢木霉（Trichoderma asperellum）的生防机制并获得与其相关的功能基因,本研究通过RT-PCR、3′-RACE及5′-TAIL- PCR技术克隆了T. asperellum 1个几丁质酶基因Tachi1,对该基因进行了生物信息学分析,并利用毕赤酵母表达系统进行表达验证. Tachi1的DNA序列长1 635 bp,含有3个内含子,包含1 275 bp的开放阅读框,编码424个氨基酸;Tachi1属于糖基水解酶18家族内切几丁质酶,包含SIGGW底物结合位点和FDGIDXDWE活性中心位点,信号肽长度为22个氨基酸,成熟肽分子量为44 kD,二级结构以α-螺旋 、β-折叠和无规则卷曲为结构元件,三级结构为(α/β)8的圆桶形结构. 转Tachi1基因酵母工程菌可高效分泌表达几丁质酶Tachi1,甲醇诱导培养8 d几丁质酶酶活可达9.25 U/mL.     

Functional analyses of the chitin-binding domains and the catalytic domain of <Emphasis Type="Italic">Brassica juncea</Emphasis> chitinase BjCHI1

We previously isolated a Brassica juncea cDNA encoding BjCHI1, a novel chitinase with two chitin-binding domains. Synthesis of its mRNA is induced by wounding, methyl jasmonate treatment, Aspergillus niger infection and caterpillar Pieris rapae feeding, suggesting that the protein has a role in defense. In that it possesses two chitin-binding domains, BjCHI1 resembles the precursor of Urtica dioica agglutinin but unlike that protein, BjCHI1 retains its chitinase catalytic domain after post-translational processing. To explore the properties of multi-domain BjCHI1, we have expressed recombinant BjCHI1 and two derivatives, which lack one (BjCHI2) or both (BjCHI3) chitin-binding domains, as secreted proteins in Pichia pastoris. Recombinant BjCHI1 and BjCHI2, showed apparent molecular masses on SDS-PAGE larger than calculated, and could be deglycosylated using -mannosidase. Recombinant BjCHI3, without the proline/threonine-rich linker region containing predicted O-glycosylation sites, did not appear to be processed by -mannosidase. BjCHI1s ability to agglutinate rabbit erythrocytes is unique among known chitinases. Both chitin-binding domains are essential for agglutination; this property is absent in recombinant BjCHI2 and BjCHI3. To identify potential catalytic residues, we generated site-directed mutations in recombinant BjCHI3. Mutation E212A showed the largest effect, exhibiting 0 of wild-type specific activity. H211N and R361A resulted in considerable (>91) activity loss, implying these charged residues are also important in catalysis. E234A showed 36 retention of activity and substitution Y269D, 50. The least affected mutants were E349A and D360A, with 73 and 68 retention, respectively. Like Y269, E349 and D360 are possibly involved in substrate binding rather than catalysis.