巨大芽孢杆菌分子伴侣GroES和GroEL新基因的克隆及同源性分析

巨大芽孢杆菌作为革兰氏阳性细菌的一种,是良好的重组蛋白的表达宿主.本研究利用PCR技术从巨大芽孢杆菌基因组克隆出一条1.9Kb的基因片段.核酸序列分析结果表明,该片段全长1984bp,包含2个ORF,分别与芽孢杆菌来源的GroES和GroEL基因有高度的相似性.氨基酸序列比对发现,GroES蛋白与枯草芽孢杆菌来源的GroES蛋白氨基酸序列同源性为91%,GroEL蛋白氨基酸序列同源性为90%.     

不同寄主植物的烟粉虱(Bemisia tabaci)内共生菌传毒相关GroEL蛋白的一致性

通过对7种寄主植物上B型烟粉虱北京种群的内共生菌传毒相关groEL基因进行PCR扩增和测序,结合已有的相关序列,构建了groEL基因及其编码的GroEL蛋白的分子系统树。结果表明：烟粉虱内共生菌产生的groEL基因是一个非常保守的基因,北京不同寄主植物的烟粉虱内共生菌与IsraelB型烟粉虱内共生菌的groEL基因亲源关系非常近,位于同一进化分支,其编码的GroEL蛋白的分子系统树也基本上是一致的。不同物种的groEL基因及其编码的GroEL蛋白分别位于不同的分支,说明groEL基因及其编码的GroEL蛋白的分子系统树可以用于分析物种间的进化关系。氨基酸序列比较表明：烟粉虱内共生菌GroEL具有原核GroEL的保守氨基酸、ATP酶活性位点、多肽结合位点和GroES连接位点,为典型的hsp60。不同来源烟粉虱内共生菌GroEL有少数几个保守氨基酸发生了置换,可能不是GroEL功能的重要位点。说明在容易变异的细菌基因组中,groEL基因为了维持其正常重要的生理功能,会通过保持功能位点的稳定性来应对不同生态因素的影响。     

巨大芽孢杆菌α-淀粉酶基因核苷酸序列分析

巨大芽孢杆菌(Bacillus megaterium)AS1.127的淀粉酶基因的全碱基序列已被测定。结构基因由1982bp的单一开读框架组成。由DNA序列推测出的前体酶蛋白由659个氨基酸组成,N-端33个氨基酸为信号肽。成熟酶分子由626个氨基酸组成,分子量为68.676kD。该淀粉酶属糖化型α-淀粉酶。并与枯草杆菌(B.subtilis)168产生的糖化型α-淀粉酶之间有83.3%的同源性。分析发现两种菌产生的酶分子的N-端3/4的同源性为90.4%,而C-端1/4的同源性只有70%。序列排比结果说明在淀粉酶基因的趋异进化过程中,基因突变和遗传重组都曾起过作用。     

枯草芽孢杆菌群β-甘露聚糖酶基因克隆及同源性分析

本文对33株枯草芽孢杆菌群菌株进行β-甘露聚糖酶活性筛选,其中的32株具有β-甘露聚糖酶活性,只有1株无β-甘露聚糖酶活性.通过基因克隆测序的方法获得33株枯草芽孢杆菌群菌株β-甘露聚糖酶基因编码区全序列,对酶基因进行同源性分析并构建系统发育树;在β-甘露聚糖酶基因系统发育树中,33株枯草芽孢杆菌群菌株聚为3个分支,分别是枯草芽孢杆菌分支、地衣芽孢杆菌分支和解淀粉芽孢杆菌分支;枯草芽孢杆菌、地衣芽孢杆菌和解淀粉芽孢杆菌β-甘露聚糖酶基因种内同源性大于91%,而种间同源性为60%69%.     

解淀粉芽孢杆菌YN-1抑菌蛋白TasA基因的克隆及原核表达

本文以解淀粉芽孢杆菌YN-1菌株为研究对象,利用PCR方法从基因组DNA中扩增出编码TasA抑菌基因的全长DNA,并构建pGEX-4T2/TasA原核表达载体,获得TasA-GST融合表达的抑菌蛋白。测序结果表明,解淀粉芽孢杆菌YN-1TasA基因核苷酸序列全长为786bp(GenBank登录号:EU131674),编码261个氨基酸残基;同源性分析表明,它与解淀粉芽孢杆菌B.amyloliquefaciens FZB42(YP_001421886)序列的同源性最高,达98%,预测蛋白分子量为31kD。SDS-PAGE分析表明,TasA基因能在大肠杆菌BL21中表达,Western印迹分析pGEX-4T2/TasA原核表达载体,检测到约57kD的TasA-GST融合外源蛋白,与预测的融合蛋白分子量大小相符。表达产物细胞裂解液上清经Ni柱层析,表明浓度为500mmol/L咪唑洗脱缓冲液时获得较高浓度和纯度的纯化蛋白。进一步抑菌活性分析表明表达产物对黄瓜灰霉病病原菌检测平板上显示出较好的抑菌活性。本研究结果将为今后深入研究TasA抑菌蛋白基因以及抗病转基因工程提供了参考。     

喜盐芽孢杆菌D8基因文库的构建及甘氨酸甜菜碱转运蛋白betH基因的筛选

喜盐芽孢杆菌(Halobacillus)D8是一株产生芽孢的革兰氏阳性中度嗜盐菌,能够耐受2 5 %NaCl。以其总DNASau3AI部分酶切的片段为供体、pUC18为载体,构建了该菌株的基因文库,共获得约90 0 0个重组质粒。通过菌落原位杂交、菌落PCR检测及DNA序列测定,从该文库中筛选到含有完整的甘氨酸甜菜碱次级转运系统基因的重组质粒,将此基因命名为betH基因。序列分析发现,betH基因的大小为15 15bp ,编码由5 0 5个氨基酸组成的BetH蛋白,分子量为5 6 1kD。经蛋白疏水性分析,推测为含有12个跨膜区的跨膜蛋白,与Oceanobacillusiheyensis甘氨酸甜菜碱转运蛋白、枯草芽孢杆菌(Bacillussubtilis)OpuD、楚氏喜盐芽孢杆菌(Halobacillustrueperi)BetH、单核细胞增生利斯特氏菌(Listeriamonocytogenes)BetL、嗜盐海球菌(Marinococcushalophilus)BetM和耐盐芽孢杆菌(Bacillushalodurans)甘氨酸甜菜碱转运蛋白的氨基酸同源性分别为6 4 %、5 1%、4 9%、4 8%、4 3%和4 4 %。     

解淀粉芽孢杆菌TF28抗菌蛋白TasA基因序列分析及蛋白质结构预测

本研究以解淀粉芽孢杆菌TF28为材料,采用PCR方法从基因组DNA中扩增出抗菌蛋白Tas A基因,利用生物信息学方法对Tas A基因序列及其编码的蛋白质结构进行分析和预测。结果表明Tas A基因全长786 bp,含有一个完整的开放阅读框和一个终止密码子,编码261个氨基酸,经Blast比对,该基因与其它解淀粉芽孢杆菌Tas A基因同源性为95%~99%,与B.amyloliquefaciens FZB42(CP 000560.1)Tas A基因序列同源性最高为99%,与B.amyloliquefaciens DSM7(FN597644.1)的同源性最低为95%。预测该基因编码蛋白质分子量为28 k D,等电点为6.35,是含有信号肽和跨膜结构的亲水蛋白,蛋白结构中含有糖基化和磷酸化位点,二级结构中以琢螺旋、茁折叠和无规则卷曲为主。     

巨大芽孢杆菌b-淀粉酶基因的克隆、表达和酶学性质分析

从巨大芽孢杆菌(Bacillus megaterium)的全基因组DNA文库中筛选出一个b-淀粉酶基因amyG, 分析测定了其核苷酸序列并进行了诱导表达; 其中amyG编码的蛋白有545个氨基酸、分子量为60.194 kD, 与已报道的巨大芽孢杆菌DSM319的b-淀粉酶序列有着94.5%的同源性。经氨基酸序列比较分析发现, AmyG从N末端到C末端依次由信号肽域、糖基水解酶催化功能域和淀粉结合域3个功能域组成。其中催化功能域里含有第14家族糖基水解酶常见的几个高度保守的酶催化活性区。经多步纯化, 重组酶的比活共提高了7.4倍, 获得凝胶电泳均一的蛋白样品; 经SDS-PAGE电泳测定, 酶AmyG的分子量为57 kD。该酶的最适反应温度为60oC, 最适反应pH为7.0; 在温度不超过60oC时, 酶活较稳定; AmyG能迅速降解淀粉生成麦芽糖, 属于外切b-糖苷酶。     

二化螟β1微管蛋白基因cDNA序列的克隆与序列分析

微管是真核生物体内分布最为广泛的一类蛋白,是由a-和β-两种不同的微管蛋白组成的异源二聚体.微管参与许多细胞功能,如细胞形态发生、细胞生长和分裂等.以二化螟3龄幼虫为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),扩增得到该虫的β微管蛋白基因的cDNA序列一条.cDNA序列含1 862个碱基,开放读码框1 344个碱基,编码氨基酸447个,分子量约为50.2kD,等电点4.82.氨基酸序列中1～4个氨基酸MREI为β微管蛋白转录后调控信号,是微管蛋白特异片段;氨基酸序列的140～146GGGTGSG位存在一个微管蛋白标志信号片段.序列比对表明,克隆的β微管蛋白基因与其他昆虫的β微管蛋白基因在核苷酸和氨基酸水平上都是高度同源的,与家蚕Bombyx moriβ1微管蛋白的氨基酸序列同源性达到99.1%,与烟草天蛾β1微管蛋白的氨基酸序列同源性达到98.7%,与果蝇Drosophila melanogasterβ1微管蛋白的氨基酸序列同源性达到96.9%.该基因cDNA序列已经登录Genbank并获得登录号为EU429675.     

炭疽杆菌芽孢外壁胶原样蛋白(BclA)的多态性分析

炭疽杆菌芽孢外壁胶原样蛋白（BclA）是芽孢外壁发状菌丝的主要结构成分,也是芽孢的主要免疫原。从国内分离的3株炭疽杆菌中克隆出BclA基因并进行了序列分析,结果发现有2株（A16R和40048）的BclA与国外报道菌株长度不同,分别含有388个和322个氨基酸,72个和50个GXX三氨基酸重复序列,5个和3个含21个氨基酸的（GPT）5 GDTGTT重复序列（BclA重复）。另一株40022的BclA与国外报道的53169株完全一敛,含有370个氨基酸,66个GXX重复,5个BclA重复。对我国炭疽杆菌BclA蛋白多态性的分析为进行炭疽杆菌的基因分型以及研究炭疽芽孢的免疫原性和致病机理打下基础。     

Molecular cloning of groESL locus, and purification and characterization of chaperonins, GroEL and GroES, from Bacillus brevis

The groESL locus of a protein-hypersecreting bacterium, Bacillus brevis, was cloned by PCR using primers designed based on the DNA sequence of a B. subtilis homolog. GroEL protein was purified to apparent homogeneity and its ATPase activity was characterized: it hydrolyzed ATP, CTP, and TTP in this order of reaction rate, and its specific activity for ATP was 0.1 micromole/min/mg protein. Purified GroEL forms a tetradecamer. GroEL was estimated to contain 22% alpha-helix, 24% beta-sheet, and 19% turn structures, by CD measurement. GroES protein was also highly purified to examine its chaperonin activity. GroEL protected from thermal inactivation of and showed refolding-promoting activity for malate dehydrogenase, strictly depending on the presence of ATP and GroES.     

Molecular cloning and characterization of an alkalophilic Bacillus sp. C125 gene homologous to Bacillus subtilis sec Y.

A 1.8 kb HindIII DNA fragment containing the secY gene of alkalophilic Bacillus sp. C125 has been cloned into plasmid pUC119 using the B. subtilis secY gene as a probe. The complete nucleotide sequence of the cloned DNA indicated that it contained one complete ORF and parts of two other ORFs. The similarity of these ORFs to the sequences of the B. subtilis proteins indicated that they were the genes for ribosomal protein L15-SecY-adenylate kinase, in that order. The gene product of the alkalophilic Bacillus sp. C125 secY homologue was composed of 431 amino acids and its M(r) value has been calculated to be 47,100. The distribution of hydrophobic amino acids in the gene product suggested that the protein was a membrane integrated protein with ten transmembrane segments. The total amino acid sequence of alkalophilic Bacillus sp. C125 secY homologue showed 69.7% homology with that of B. subtilis secY. Regions of remarkably high homology (78% identity) were present in transmembrane regions, and cytoplasmic domains (73% identity) with less homologous regions present in extracellular domains (43% identity).     

Cloning, sequencing, and functional expression in Escherichia coli of chaperonin (groESL) genes from Vibrio cholerae.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat-shock genes, the groESL heat-shock operon from a Vibrio cholerae TSI-4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1,632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature-sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.     

Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

The genes of Lactococcus lactis subsp. lactis involved in histidine biosynthesis were cloned and characterized by complementation of Escherichia coli and Bacillus subtilis mutants and DNA sequencing. Complementation of E. coli hisA, hisB, hisC, hisD, hisF, hisG, and hisIE genes and the B. subtilis hisH gene (the E. coli hisC equivalent) allowed localization of the corresponding lactococcal genes. Nucleotide sequence analysis of the 11.5-kb lactococcal region revealed 14 open reading frames (ORFs), 12 of which might form an operon. The putative operon includes eight ORFs which encode proteins homologous to enzymes involved in histidine biosynthesis. The operon also contains (i) an ORF encoding a protein homologous to the histidyl-tRNA synthetases but lacking a motif implicated in synthetase activity, which suggests that it has a role different from tRNA aminoacylation, and (ii) an ORF encoding a protein that is homologous to the 3'-aminoglycoside phosphotransferases but does not confer antibiotic resistance. The remaining ORFs specify products which have no homology with proteins in the EMBL and GenBank data bases.     

Characterization of a region of the Enterococcus faecalis plasmid pAM beta 1 which enhances the segregational stability of pAM beta 1-derived cloning vectors in Bacillus subtilis.

The nucleotide sequence of a 2.13-kb EcoRI-HindIII, pAM beta 1-derived fragment, isolated from the gram-positive cloning vector pHV1431, has been determined and shown to encode two ORFs. ORF H encodes for a protein of 23,930 Da which exhibits substantial homology to bacterial site-specific recombinases, particularly the resolvases of the gram-positive transposons Tn917 (30.3% identity) and Tn552 (31.6% identity) and the clostridial plasmid pIP404 (27.1% identity). The second ORF (I) is incomplete and encodes a polypeptide which has significant homology with Escherichia coli topoisomerase I (26.0% identity). Insertion of either the entire 2.13-kb EcoRI-HindIII fragment or a 0.73-kb EcoRI-DraI subfragment encoding only the resolvase into the pAM beta 1-based cloning vector pMTL500E causes a significant enhancement of segregational stability (from 6.5 X 10(-2) to 3.0-4.0 X 10(-3) plasmid loss per cell per generation). Improved segregational stability is mirrored by a reduction in plasmid polymerization. The introduction of a stop codon into the resolvase coding region negates its ability to promote segregational stability. It is proposed that the identified determinant stabilizes pAM beta 1-based vectors in Bacillus subtilis by maintaining the plasmid population in the monomeric state, thereby reducing the chances of producing plasmid-free segregants.