来源于蓝藻、铜绿微囊藻的尿苷二磷酸葡萄糖脱氢酶基因的克隆和鉴定

以已知的尿苷二磷酸葡萄糖脱氢酶基因的保守区为基础,自行设计一对简并引物,该对引物从形成水华的蓝藻（Synechocystis PCC6803)铜绿微囊藻FACHB 905株(Microcystis aeruginosa FACHB 905)的基因组DNA中扩增到一个476bp的DNA片段。通过TAIL-PCR和连接介导的PCR两种方法分离该片段的侧翼序列,最后得到大小约2．5kb的DNA片段。序列分析揭示其中有一个编码462个氨基酸的开放阅读框,我们将此开放阅读框对应的蛋白命名为Mud。该Mud蛋白的氨基酸序列与蓝藻(73％相同,87％相似)和细菌(Bacillus subtilis)(51％相同,67％相似)的尿苷二磷酸葡萄糖脱氢酶氨基酸序列表现高度的同源性。将该mud基因克隆于p-GEX-4T-1融合表达载体并在大肠杆菌中表达GST—Mud融合蛋白,经过酶活力测定发现,GST—Mud蛋白具有一定的尿苷二磷酸葡萄糖脱氢酶活性。用抗GST-Mud蛋白的多抗对Maeruginosa FACHB 905的胞质蛋白组分进行Western印迹分析,结果显示一条分子量大小约49kD的专一条带,这个分子量与从基因推断出的蛋白分子量大小基本一致。综上所述,我们认为从微囊藻克隆到的Mud蛋白基因是尿苷二磷酸葡萄糖脱氢酶基因,该酶在其他生物如植物和细菌中参与多糖合成,是多糖合成的关键酶之一,而在藻类中对尿苷二磷酸葡萄糖脱氢酶开展研究却是首次报道。     

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。     

毛木耳漆酶基因的克隆、序列分析及其鉴定

本文利用PCR和RACE技术首次从毛木耳AP4菌株中获得编码漆酶基因的cDNA及其基因组全长序列,基因组大小为2514 bp.通过比较该漆酶基因的cDNA和基因组DNA的全长序列,发现该基因包含14个外显子和13个内含子.cDNA序列的全长为1972 bp,其包含一个完整的ORE长度为1860 bp,编码619氨基酸,推测的分子量大小为68 kD,等电点pI为5.15.在氨基酸序列的氨基末端存在一个信号肽序列,同时该基因还包括含铜氧化酶的三个功能结构域KOG1263、SufI和pfam00394.氨基酸序列与GenBank中登录的真菌漆酶蛋白序列比对表明:该氨基酸序列与其它真菌漆酶蛋白序列有较高的同源性,氨基酸序列相同性最高达41%,相似性为58%,并且含有真菌漆酶的四个保守的Cu-bind结构域.将获得的漆酶基因lacl与毕赤酵母表达载体pPIC9K连接,构建重组质粒pYH3660,将其转化到毕赤酵母中,经甲醇诱导该基因在第10天产酶高达123 IU/L,并通过Native SDS-PAGE电泳获得预期大小的漆酶蛋白条带.结构分析和功能验证均表明:本研究获得的基因lacl为漆酶基因.     

嗜铁钩端螺旋菌(Leptospirillum ferriphilum)gyrB基因的PCR扩增、克隆与序列分析

根据GenBank和ICB数据库中gyrB蛋白氨基酸序列的两个保守区域TPGMYIG和QRY(F)KGLGEM设计简并引物,以L.ferriphilum菌株YSK基因组DNA为模板,PCR扩增出获得大小约为2.2kb的DNA片段.序列分析表明,菌株YSK的扩增片断的开放阅读框(ORF)能够推导出一个编码分子量约为82....     

绿色木霉内切几丁质酶cDNA的克隆及其编码蛋白的序列分析

根据已克隆的内切几丁质酶基因序列的同源性比较,设计引物,采用PCR技术从绿色木霉基因组中分离出一个大小为1467bp的特异DNA片段,采用RT．PeR技术从绿色木霉总RNA中分离出大小约1276bp的eDNA片段。序列对比后发现该内切几丁质酶DNA含有三个内含子,大小分别为52bp,69bp,64bp。同源性分析表明其全长eDNA序列和已经报道的内切几丁质酶序列的同源性高达95％以上,预测其编码蛋白的氨基酸序列含424个氨基酸残基,分子量为46kDa,氨基酸序列分析表明该内切几丁质酶164～172位氨基酸是其活性中心,用同源建模法模拟其空间结构模型,为进一步研究其作用机制奠定了良好基础。     

甲醇利用菌SDM11中glyA基因的克隆及其在大肠杆菌中的表达

对甲醇降解菌Methylobacterium.sp SDM11中的glyA基因进行克隆及特性研究,以获得更多的丝氨酸羟甲基转移酶(serine hydroxymethyltransferase,SHMT)资源。根据GenBank中已报道的Methylobacterium extorquensAM1中的glyA基因序列(登录号:L33463)设计引物,以SDM11的基因组DNA为模板,PCR扩增glyA基因。利用pETblue-2载体将该基因在大肠杆菌BL21(DE3)中得到表达。PCR扩增到一个1.40 kb大小的DNA片段,经过blast软件比对分析,发现该片段与已报道的Methylobacterium extorquensAM1的glyA基因的序列相似性为95%,氨基酸序列的相似性为98%。该基因编码468个氨基酸,预测的分子量大小为52.2 kD,等电点为7.02,发现纯化后的目标蛋白具有SHMT酶活性,并初步测定了酶活力。     

枯草芽孢杆菌葡萄糖脱氢酶基因的克隆及其序列分析

根据Lampel报道的葡萄糖脱氢酶基因序列设计合成两条引物,以野生型枯草芽孢杆菌染色体DNA为模板,PCR扩增得到含有葡萄糖脱氢酶基因的大约780bp的DNA片段,将其克隆到pUC-T载体中。序列分析表明,克隆得到的葡萄糖脱氢酶基因含有783bp,编码261个氨基酸的蛋白质。得到的基因序列与文献报道的进行比较,其核苷酸同源率为75．5％,编码氨基酸序列的同源率为83．9％。     

三种水华蓝藻对不同磷浓度生理响应的比较研究

本实验研究了铜绿微囊藻(Microcystis aeruginosa FACHB469)、水华鱼腥藻(Anabaena flos-aquae FACHB245)和浮游颤藻(Oscillatoria planctonicaFACHB708)对磷浓度变化的生理响应。结果表明,在缺磷条件下,A. flos-aquae对低磷环境的适应能力较强,O. planctonica其次,M. aeruginosa最差;在磷充足条件,微囊藻对磷过量吸收的能力明显高于其他两种蓝藻。三种蓝藻胞外碱性磷酸酶活性(APA)与培养基中磷浓度呈负相关性,其产生碱性磷酸酶(AP)的能力由高至低为:A. flos-aquae>O. planctonica>M. aeruginosa。磷缺陷时A. flos-aquae产生的胞外APA约是M. aeruginosa的10倍,是O. planctonica的5倍。     

应用RT-qPCR技术定量检测湖泊水体中蓝藻方法的比较

利用RT-qPCR技术建立了对湖泊水体中的微囊藻和蓝藻的SYBR Green Ⅰ荧光定量PCR检测方法,在所建立的方法中,对以微囊藻藻蓝蛋白基因、蓝藻16S rRNA基因、微囊藻16S rRNA基因分别作为RT-qPCR检测的目的基因所得结果进行了比较,并对实验室培养的微囊藻和太湖的环境样品进行了检测。结果表明,用藻蓝蛋白基因作为检测目的基因,以M.aeruginosa PCC 7806基因组DNA作为标准品的测定方法与显微镜计数的结果有较好的相关性和一致性,并具有简便、快速、特异性高的特点,可以满足检测的要求。     

用简并寡核苷酸探针筛选对虾白斑综合征病毒DNA多聚酶基因片段

根据一些病毒的DNA多聚酶氨基酸序列中特有的保守序列VYGDTD设计的简并寡核苷酸,经地高辛标记后与对虾白斑综合征病毒基因库克隆杂交,筛选出一段长度为707 bp的EcoR I基因片段,该片段在一个开放阅读框内.并含DNA多聚酶B家族特有的保守序列YGDTDS.经与基因库比较,其氨基酸序列与藻类DNA病毒科(Phycodnaviridae)的几株藻类病毒的DNA多聚酶片段有部分相似,因此推测该核苷酸片段为对虾白斑综合征病毒DNA多聚酶基因的部分序列.     

UDP-glucose dehydrogenase from bovine liver: primary structure and relationship to other dehydrogenases.

The primary structure of bovine liver UDP-glucose dehydrogenase (UDPGDH), a hexameric, NAD(+)-linked enzyme, has been determined at the protein level. The 52-kDa subunits are composed of 468 amino acid residues, with a free N-terminus and a Ser/Asn microhetergeneity at one position. The sequence shares 29.6% positional identity with GDP-mannose dehydrogenase from Pseudomonas, confirming a similarity earlier noted between active site peptides. This degree of similarity is comparable to the 31.1% identity vs. the UDPGDH from type A Streptococcus. Database searching also revealed similarities to a hypothetical sequence from Salmonella typhimurium and to "UDP-N-acetyl-mannosaminuronic acid dehydrogenase" from Escherichia coli. Pairwise identities between bovine UDPGDH and each of these sequences were all in the range of approximately 26-34%. Multiple alignment of all 5 sequences indicates common ancestry for these 4-electron-transferring enzymes. There are 27 strictly conserved residues, including a cysteine residue at position 275, earlier identified by chemical modification as the expected catalytic residue of the second half-reaction (conversion of UDP-aldehydoglucose to UDP-glucuronic acid), and 2 lysine residues, at positions 219 and 338, one of which may be the expected catalytic residue for the first half-reaction (conversion of UDP-glucose to UDP-aldehydoglucose). A GXGXXG pattern characteristic of the coenzyme-binding fold is found at positions 11-16, close to the N-terminus as with "short-chain" alcohol dehydrogenases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and nucleotide sequence of the glpD gene encoding sn-glycerol-3-phosphate dehydrogenase of Pseudomonas aeruginosa.

Nitrosoguanidine-induced Pseudomonas aeruginosa mutants which were unable to utilize glycerol as a carbon source were isolated. By utilizing PAO104, a mutant defective in glycerol transport and sn-glycerol-3-phosphate dehydrogenase (glpD), the glpD gene was cloned by a phage mini-D3112-based in vivo cloning method. The cloned gene was able to complement an Escherichia coli glpD mutant. Restriction analysis and recloning of DNA fragments located the glpD gene to a 1.6-kb EcoRI-SphI DNA fragment. In E. coli, a single 56,000-Da protein was expressed from the cloned DNA fragments. An in-frame glpD'-'lacZ translational fusion was isolated and used to determine the reading frame of glpD by sequencing across the fusion junction. The nucleotide sequence of a 1,792-bp fragment containing the glpD region was determined. The glpD gene encodes a protein containing 510 amino acids and with a predicted molecular weight of 56,150. Compared with the aerobic sn-glycerol-3-phosphate dehydrogenase from E. coli, P. aeruginosa GlpD is 56% identical and 69% similar. A similar comparison with GlpD from Bacillus subtilis reveals 21% identity and 40% similarity. A flavin-binding domain near the amino terminus which shared the consensus sequence reported for other bacterial flavoproteins was identified.     

The amino acid sequence of low-potential cytochrome c550 from the cyanobacterium Microcystis aeruginosa

The low-potential cytochrome c550 has been purified from the cyanobacterium Microcystis aeruginosa and its amino acid sequence has been determined. The protein contains 135 amino acid residues with the Cys-X-X-Cys-His heme binding site at residues 37 to 41. The sequence from residue 28 to 45 shows similarity to cytochrome c553 residues 1 to 18 when the heme binding sites are aligned. Another region of similarity is in the carboxyl-terminal regions of these two proteins. The two aligning regions of cytochrome c553 correspond to helical segments in other related cytochromes. A partial sequence of cytochrome c550 from Aphanizomenon flos-aquae was obtained and showed a 48% identity to the sequence of the M. aeruginosa cytochrome. The single methionine residue in cytochrome c550 of M. aeruginosa occurs at position 119 but there is no methionine in this region in the A. flos-aquae cytochrome, indicating that methionine is not the sixth ligand to the heme iron atom. Histidine 92 is a possible sixth ligand in M. aeruginosa cytochrome c550. The far-uv circular dichroism spectrum indicates that this protein is approximately 17% alpha helix, 42% beta-pleated sheet, and 41% random coil.     

Cloning of the Microcystis aeruginosa M228 lectin (MAL) gene

We have cloned and characterized the gene encoding Microcystis aeruginosa (strain M228) lectin (MAL). The gene contains 1551 nucleotides and an open reading frame for a protein of 517 amino acids with a predicted molecular weight of 55,159 Da. The carboxy-terminal region of MAL has three tandemly repeated homologous domains composed of 61 amino acids. These regions show similarity to the corresponding regions of the alpha-amylase of Clostridium beijerinckii (23% identity). The mal gene lies adjacent to an ORF that display homology to cytochrome P-450 and polyketide synthase. Southern hybridization showed that the genomic DNA of the strain M228 contained, in addition to MAL gene (mal), at least two other mal like gene.     

NADP(+)-isocitrate dehydrogenase from the cyanobacterium Anabaena sp. strain PCC 7120: purification and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene.

NADP(+)-isocitrate dehydrogenase (NADP(+)-IDH) from the dinitrogen-fixing filamentous cyanobacterium Anabaena sp. strain PCC 7120 was purified to homogeneity. The native enzyme is composed of two identical subunits (M(r), 57,000) and cross-reacts with antibodies obtained against the previously purified NADP(+)-IDH from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. Anabaena NADP(+)-IDH resembles in its physicochemical and kinetic parameters the typical dimeric IDHs from prokaryotes. The gene encoding Anabaena NADP(+)-IDH was cloned by complementation of an Escherichia coli icd mutant with an Anabaena genomic library. The complementing DNA was located on a 6-kb fragment. It encodes an NADP(+)-IDH that has the same mobility as that of Anabaena NADP(+)-IDH on nondenaturing polyacrylamide gels. The icd gene was subcloned and sequenced. Translation of the nucleotide sequence gave a polypeptide of 473 amino acids that showed high sequence similarity to the E. coli enzyme (59% identity) and with IDH1 and IDH2, the two subunits of the heteromultimeric NAD(+)-IDH from Saccharomyces cerevisiae (30 to 35% identity); however, a low level of similarity to NADP(+)-IDHs of eukaryotic origin was found (23% identity). Furthermore, Anabaena NADP(+)-IDH contains a 44-residue amino acid sequence in its central region that is absent in the other IDHs so far sequenced. Attempts to generate icd mutants by insertional mutagenesis were unsuccessful, suggesting an essential role of IDH in Anabaena sp. strain PCC 7120.     

Biosynthesis of glycosaminoglycan precursors: evidences for different tissue specific forms of UDP-glucose dehydrogenase

UDP-glucose dehydrogenase (UDPGDH) was extracted and partially purified from different rat tissues and the kinetic parameters and some properties of the enzyme were determined and compared. The pH optimum ranged between 8.6 and 9.4 for liver and kidney UDPGDH and between 8.4 and 8.6 for skin and lung UDPGDH. Liver and kidney enzymes showed a similar affinity for both UDPG and NAD. Lung and skin enzymes also showed similar affinity for both substrates, which differed however from that of liver and kidney UDPGDH. Both liver and kidney enzymes had a higher heat stability and a different electrophoretic mobility compared to skin and lung UDPGDH. These data suggest the existence of different tissue specific forms of the enzyme.     

A mannan binding lectin is involved in cell-cell attachment in a toxic strain of Microcystis aeruginosa

Microcystin, a hepatotoxin that represents a serious health risk for humans and livestock, is produced by the bloom-forming cyanobacterium Microcystis aeruginosa in freshwater bodies worldwide. Here we describe the discovery of a lectin, microvirin (MVN), in M. aeruginosa PCC7806 that shares 33% identity with the potent anti-HIV protein cyanovirin-N from Nostoc ellipsosporum. Carbohydrate microarrays were employed to demonstrate the high specificity of the protein for high-mannose structures containing alpha(1-->2) linked mannose residues. Lectin binding analyses and phenotypic characterizations of MVN-deficient mutants suggest that MVN is involved in cell-cell recognition and cell-cell attachment of Microcystis. A binding partner of MVN was identified in the lipopolysaccharide fraction of M. aeruginosa PCC7806. MVN is differentially expressed in mutants lacking the hepatotoxin microcystin. Additionally, MVN-deficient mutants contain much lower amounts of microcystin than the wild-type cells. We discuss a possible functional correlation between microcystin and the lectin and possible implications on Microcystis morphotype formation. This study provides the first experimental evidence that microcystins may have an impact on Microcystis colony formation that is highly important for the competitive advantage of Microcystis over other phytoplankton species.     

Cloning and expression studies of the Dunaliella salina UDP-glucose dehydrogenase cDNA.

The enzyme UDP-glucose dehydrogenase (EC 1.1.1.22) converts UDP-glucose to UDP-glucuronate. Plant UDP-glucose dehydrogenase (UGDH) is an important enzyme in the formation of hemicellulose and pectin, the components of primary cell walls. A cDNA, named DsUGDH, (GeneBank accession number: AY795899) corresponding to UGDH was cloned by RT-PCR approach from Dunaliella salina. The cDNA is 1941-bp long and has an open reading frame encoded a protein of 483 amino acids with a calculated molecular weight of 53 kDa. The derived amino acids sequence shows high homology with reported plants UGDHs, and has highly conserved amino acids motifs believed to be NAD binding site and catalytic site. Although UDP-glucose dehydrogenase is a comparatively well characterized enzyme, the cloning and characterization of the green alga Dunaliella salina UDP-glucose dehydrogenase gene is very important to understand the salt tolerance mechanism of Dunaliella salina. Northern analyses indicate that NaCl can induce the expression the DsUGDH.