豇豆几丁质酶N端序列测定及与其它植物的比较

通过自动 Edman降解程序 ,测定了经诱导、纯化的豇豆几丁质酶 N端 1 0个氨基酸的序列 ,并将该序列与其它植物几丁质酶 N端相应部分的氨基酸序列进行了比较分析。结果表明 ,该豇豆 ( Vigna sesquipedalis)几丁质酶 N端 1 0个氨基酸的序列为 EQCGSQAGGA,与 类几丁质酶同一部分同感序列同源性高达 1 0 0 % ;而与 、 及 类几丁质酶的相应序列均无同源性。结合考虑此酶的等电点 ( 8.3)及分子量 ( 33k D) ,可推测该豇豆几丁质酶属于 类几丁质酶。其 N端序列的高度保守性提示 ,该段序列可作为 类几丁质酶的一段主要特征序列 ,并可据其合成核酸探针 ,以分离、克隆其它 类几丁质酶编码基因。     

烟夜蛾几丁质酶基因的克隆与表达

运用RT-PCR技术扩增编码烟夜蛾Helicoverpaassulta(Guen啨e)幼虫几丁质酶基因的cDNA片段,将其克隆至pMD18-T载体,获得该基因的成熟蛋白阅读框序列。将该基因重组到表达型质粒pGEX-4T-2中,并转化入原核细胞中表达,序列测定结果表明,烟夜蛾幼虫几丁质酶基因的成熟蛋白阅读框全长1338bp,编码445个氨基酸残基,预测分子量和等电点分别为50.1kDa和9.26;推导的氨基酸序列与其近缘种棉铃虫几丁质酶氨基酸序列的一致性达99%,与其他6种昆虫几丁质酶的氨基酸序列也高度一致(65%~76%),并具有几丁质酶的典型特征。将该基因克隆到原核表达载体pGEX-4T-2上并转化BL21,SDS-PAGE和Western印迹分析表明,经IPTG诱导,76kDa附近没有特异蛋白条带出现,表明烟夜蛾几丁质酶基因不能在原核表达载体pGEX-4T-2中表达。     

一株拮抗香蕉枯萎病的内生细菌的分离及其几丁质酶基因信号肽的分泌活性分析

目的：旨在分离并选择一株香蕉内生细菌作为内生基因工程生防菌,并克隆其几丁质酶基因的信号肽序列。方法：从香蕉植株杆下部分离并选择了一株拮抗香蕉枯萎病且具有分泌几丁质酶能力的内生细菌,对该菌株进行了形态观察、生理生化测定和16S rDNA序列分析,克隆了其几丁质酶基因的编码序列并预测了其信号肽,构建了含有信号肽和不含信号肽的几丁质酶的表达菌株BL-chi1和BL-chi2。结果：结合形态观察、生理生化特征和16S rDNA序列比对分析确定该菌株为Klebsiella属,将该菌株命名为KKWB 5;BL-chi1和BL-chi2经IPTG诱导后,均表达了与预期蛋白大小一致的蛋白,同时BL-chi1诱导后的培养基上清中出现一条约45kDa的条带,而BL-chi2和空载体的BL-pET22b诱导后的培养基上清中均无此条带;几丁质水解试验发现,BL-chi1诱导后的培养基上清中的蛋白经浓缩和纯化后都能在几丁质平板上形成透明水解圈。结论：该几丁质酶的信号肽能被BL21（DE3）所识别,将几丁质酶分泌到培养基中,并且分泌的几丁质酶具有水解几丁质的生物学活性。内生菌KKWB-5的分离及其几丁质酶分泌信号肽序列的克隆为进一步构建内生工程菌来防治香蕉枯萎病打下了基础。     

环状芽孢杆菌几丁质酶基因序列分析、表达和生物活性测定

真菌病害一直是影响作物的主要病害之一 ,每年造成巨大经济损失。几丁质酶可水解许多病原真菌细胞壁所含有的主要成分—几丁质 ,是研究得最多的抗真菌蛋白质。许多几丁质酶基因已从微生物中克隆到 ,芽孢杆菌是一类重要的几丁质酶产生菌。环状芽孢杆菌可产生并分泌多种多糖降解酶类 ,包括几丁质酶、β 1 ,3 葡聚糖酶、β 1 ,6葡聚糖酶和半纤维素酶[1] 。Ｗａｔａｎａｂｅ克隆了环状芽孢杆菌ＷＬ 1 2菌株的几丁质酶基因ｃｈｉＡ和ｃｈｉＤ ,对该几丁质酶基因的结构和功能进行了深入研究[2～ 4 ] 。我国的陈三凤克隆了黄杆菌的几丁质酶基因 ,…     

小菜蛾几丁质酶基因的克隆及表达分析

昆虫几丁质酶参与昆虫蜕皮、消化、防御及免疫等多种生理过程,在昆虫的变态和发育中发挥着重要作用。本研究采用反转录聚合酶链式反应(RT-PCR)和快速扩增cDNA末端(RACE)技术克隆获得小菜蛾Plutella xylostella几丁质酶基因,命名为PxyChi。该基因开放阅读框长1677 bp,编码558个氨基酸,推测的蛋白分子量为62.03 kDa。氨基酸序列分析表明,小菜蛾几丁质酶第1-19位氨基酸为蛋白的信号肽,且该序列具有典型昆虫几丁质酶的4个保守氨基酸序列。蛋白结构域分析表明,PxyChi属于几丁质酶Group II家族。进化树分析表明:PxyChi与同属鳞翅目二化螟Chilo suppressalis的几丁质酶亲缘关系最近,为75.5%。不同发育时期的荧光定量PCR分析表明,PxyChi在小菜蛾各个发育时期的表达量不同,其中PxyChi在2、3、4龄幼虫、蛹和成虫中的表达量分别是1龄幼虫的28.22、0.76、0.50、84.83和286倍,PxyChi在成虫的表达量最高。暗示PxyChi蛋白可能参与小菜蛾蛹期旧表皮降解和成虫翅的发育等生理过程。本研究可为探索几丁质酶在小菜蛾发育中的功能提供理论依据。     

苏云金杆菌几丁质酶新基因的筛选和全长基因的扩增

以煮沸冻融法制备PCR扩增模板,利用苏云金芽孢杆菌（Bacillus thuringiensis,Bt）几丁质酶基因特异引物进行15个Bt血清变种的扩增分析,获得9个几丁质酶全长基因扩增产物。经克隆和序列测定,从Bt serovar.entomocidus HD109、Bt serovar canadensis HD224、Bt serovar、alesti HD16和Bt serovar.toumanoffi HD201等4个菌株中分离了几丁质酶新基因。     

原核表达的天花粉蛋白和另外两种蛋白具有体外抗真菌活性

将天花粉蛋白、烟草几丁质酶和烟草β１,３葡聚糖酶的结构基因分别克隆到原核表达系统中进行表达,对三种基因的原核表达产物的粗提取物分别进行体外抗菌活性检测,发现三种蛋白质均有抗真菌活性。三种蛋白中任意两种蛋白的组合,其抗真菌活性显著高于单一组分的抗真菌活性。三种蛋白共同作用时,获得了更好的抗真菌效果     

粉棒束孢几丁质酶基因cDNA全序列克隆及结构特征分析

通过设计基因保守区的特异性简并引物,运用SMARTRACERT-PCR技术,首次从粉棒束孢中克隆出完整的几丁质酶基因。该基因cDNA全长1549bp,5'端非翻译区89bp,3'端非翻译区有188bp,开放阅读框(ORF)1272bp,编码423个氨基酸。信号肽长度为22个氨基酸。信号肽很可能需要两次剪切。成熟的蛋白理论分子量为43.9kDa,理论等电点为5.67。氨基酸序列具有几丁质酶18族的两个高度保守的活性区域,一个是酶作用活性位点,另一个是几丁质结合区域。该蛋白可归于几丁质酶18族V类。成熟蛋白的氨基酸序列与裂虫壳AAV98691、白色扁丝霉CAA45468、菌生轮枝孢AAP45631、莱氏野村菌AAP04616和球孢白僵菌AAN41261的同源性分别为91%,89%,80%,76%和75%。     

地衣芽孢杆菌MY75菌株几丁质酶基因的异源表达及特性

【目的】地衣芽孢杆菌MY75菌株的几丁质酶基因的异源表达,并对表达蛋白的特性进行研究。【方法】制备MY75菌株培养上清粗蛋白,利用酶谱分析确定具有几丁质酶活的蛋白分子量。将该蛋白进行飞行时间质谱分析,确定其部分氨基酸序列,设计PCR引物对MY75菌株的几丁质酶基因进行克隆及异源表达。对表达蛋白的最适反应温度及pH,温度耐受性及金属离子对酶活力的影响等特性进行了研究,并测定了表达蛋白对真菌孢子萌发的抑制活性和对甜菜夜蛾幼虫的杀虫增效作用。【结果】酶谱分析证明MY75菌株培养上清液中仅含有一种55kDa的几丁质酶。将该编码基因chiMY克隆及序列分析后发现,基因长度为1797bp,编码599个氨基酸。在大肠杆菌中异源表达的几丁质酶ChiMY蛋白的分子量为67kDa。质谱分析证明,55kDa蛋白与67kDa蛋白序列相同。ChiMY最适pH和最适温度分别为7.0和50°C,为中性几丁质酶。Li+,Na+,和Mg2+离子对表达蛋白的酶活力具有促进作用,Mn2+,Cr3+,Zn2+和Ag+离子则能显著抑制酶活力,Cu2+和Fe3+离子完全抑制酶活性。生物测定的结果显示,异源表达的MY75几丁质酶能够抑制小麦赤霉及黑曲霉的孢子萌发,并且对苏云金芽孢杆菌的杀虫活力具有增效作用。【结论】地衣芽孢杆菌MY75菌株中仅有一种55kDa几丁质酶,其编码基因能够在大肠杆菌中大量表达,表达蛋白分子量与野生型蛋白之间有显著差异,由此证明MY75菌株中存在着几丁质酶的剪切加工过程。明确了地衣芽孢杆菌几丁质酶ChiMY具有抑制真菌活性及杀虫增效作用。上述全部研究结论在国内首次报道。     

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。     

哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

Cloning and Nucleotide Sequence of the Mycodextranase Gene from Streptomyces sp. J-13-3

Mycodextranase (EC 3.2.1.61) is an α-glucanase that cleaves α-1,4-bonds of alternating α-1,3- and α-1,4-linked D-glucan (nigeran). The gene encoding mycodextranase from Streptomyces sp. J-13-3 was cloned by hybridization with a degenerate oligonucleotide probe from the amino-terminal amino acid sequence of the enzyme and its nucleotide structure was analyzed. The open reading frame consisted of 1,803 base pairs encoding a signal peptide of 60 amino acids and a mature protein of 540 amino acids with a calculated molecular weight of 56,078. The deduced amino acid sequence showed weak similality to a chitinase homolog from Streptomyces lividans and a chitinase from Xanthomonas sp.     

A gene encoding a hevein-like protein from elderberry fruits is homologous to PR-4 and class V chitinase genes

We isolated SN-HLPf (Sambucus nigra hevein-like fruit protein), a hevein-like chitin-binding protein, from mature elderberry fruits. Cloning of the corresponding gene demonstrated that SN-HLPf is synthesized as a chimeric precursor consisting of an N-terminal chitin-binding domain corresponding to the mature elderberry protein and an unrelated C-terminal domain. Sequence comparisons indicated that the N-terminal domain of this precursor has high sequence similarity with the N-terminal domain of class I PR-4 (pathogenesis-related) proteins, whereas the C terminus is most closely related to that of class V chitinases. On the basis of these sequence homologies the gene encoding SN-HLPf can be considered a hybrid between a PR-4 and a class V chitinase gene.     

Cloning and expression of chitinase A gene from Streptomyces cyaneus SP-27: the enzyme participates in protoplast formation of Schizophyllum commune

Chitinase A of Streptomyces cyaneus SP-27 or chitinase I of Bacillus circulans KA-304 showed the protoplast-forming activity when combined with alpha-1,3-glucanase of B. circulans KA-304. The gene of chitinase A was cloned. It consisted of 903 nucleotides encoding 301 amino acid residues, including a putative signal peptide (35 amino acid residues). The deduced N-terminal moiety of chitinase A showed sequence homology with the chitin-binding domain of chitinase F from Streptomyces coelicolor and chitinase 30 from Streptomyces olivaceoviridisis. The C-terminal moiety also showed high sequence similarity to the catalytic domain of several Streptomyces family 19 chitinases as well as that of chitinase I of B. circulans KA-304. Recombinant chitinase A was expressed in Escherichia coli Rosetta-gami B (DE 3). The properties of the recombinant enzyme were almost the same as those of chitinase A purified from a culture filtrate of S. cyaneus SP-27. The recombinant enzyme was superior to B. circulans KA-304 chitinase I not only in respect to protoplast forming activity in a mixture containing alpha-1,3-glucanase, but also to antifungal activity and powder chitin-hydrolyzing activity.     

Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.     

Cloning and nucleotide sequence of the mycodextranase gene from Streptomyces sp. J-13-3

Mycodextranase (EC 3.2.1.61) is an alpha-glucanase that cleaves alpha-1,4-bonds of alternating alpha-1,3- and alpha-1,4-linked D-glucan (nigeran). The gene encoding mycodextranase from Streptomyces sp. J-13-3 was cloned by hybridization with a degenerate oligonucleotide probe from the amino-terminal amino acid sequence of the enzyme and its nucleotide structure was analyzed. The open reading frame consisted of 1,803 base pairs encoding a signal peptide of 60 amino acids and a mature protein of 540 amino acids with a calculated molecular weight of 56,078. The deduced amino acid sequence showed weak similality to a chitinase homolog from Streptomyces lividans and a chitinase from Xanthomonas sp.     

The photoconvertible water-soluble chlorophyll-binding protein of Chenopodium album is a member of DUF538, a superfamily that distributes in Embryophyta

Various plants possess hydrophilic chlorophyll (Chl) proteins known as water-soluble Chl-binding proteins (WSCPs). WSCPs exist in two forms: Class I and Class II, of which Class I alone exhibits unique photoconvertibility. Although numerous genes encoding Class II WSCPs have been identified and the molecular properties of their recombinant proteins have been well characterized, no Class I WSCP gene has been identified to date. In this study, we cloned the cDNA and a gene encoding the Class I WSCP of Chenopodium album (CaWSCP). Sequence analyses revealed that CaWSCP comprises a single exon corresponding to 585 bp of an open reading frame encoding 195 amino acid residues. The CaWSCP protein sequence possesses a signature of DUF538, a protein superfamily of unknown function found almost exclusively in Embryophyta. The recombinant CaWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (CaWSCP-His) that removes Chls from the thylakoid. Under visible light illumination, the reconstituted CaWSCP-His was successfully photoconverted into a different pigment with an absorption spectrum identical to that of native CaWSCP. Interestingly, while CaWSCP-His could bind both Chl a and Chl b, photoconversion occurred only in CaWSCP-His reconstituted with Chl a.     

Cloning and expression of a gene encoding a root specific nitrate reductase in bean (Phaseolus vulgaris)

A structural gene encoding nitrate reductase (NR) in the legume Phaseolus vulgaris cv. Saxonia has been cloned and sequenced. The NR gene encodes a protein of 881 amino acids with a MW of 99.2 kDa. The coding sequence is interrupted by three introns, which are located in the molybdopterin cofactor binding domain. In the flanking regions the signals of a functional eukaryotic gene are present. The gene is the smallest NR gene so far identified in higher plants. Comparison to other NRs shows homology ranging from 65 to 74% at the amino acid level. The similarity is highest for the three functional domains, and lowest in the N-terminal end of the protein. mRNA studies demonstrate that the gene is nitrate inducible and expressed exclusively in the roots of bean. Southern blot analysis indicates the presence of a second NR gene in bean. This gene may encode a NR enzyme expressed in leaves.     

Diversity of laccase-like multicopper oxidase genes in Morchellaceae: identification of genes potentially involved in extracellular activities related to plant litter decay

Despite the important role played by soil-inhabiting ascomycetes in plant litter decay processes, studies on the diversity and function of their laccase-like multicopper oxidase (LMCO) genes are scarce. In the present work, the LMCO gene diversity in 15 strains representing nine Morchellaceae and one Discinaceae species was evaluated by PCR. One to six different genes were found within the species, representing 26 different sequence types. Cluster analysis revealed LMCO genes belonging to four main gene families encoding different protein classes (Class I-IV). To identify the genes related to extracellular activities and potentially involved in litter decay processes, liquid cultures were induced by different aromatic compounds. Morchella conica and Verpa conica showed the strongest LMCO activity enhancement in the presence of the naturally occurring phenolic compound guaiacol, and their expressed LMCO genes were identified by sequencing. Only genes belonging to the gene families encoding the Class II and III proteins were expressed. Both genes (Class II and III) of the mycorrhizal-like strain M. conica were exclusively expressed in the presence of guaiacol. In contrast to the saprotrophic strain V. conica, the gene encoding the Class III protein was constitutively expressed as it was also found in control cultures without guaiacol.