集胞藻PCC6803中基因slr1761突变体的构建

PCR扩增了集胞藻PCC6803的slr1761基因,进一步以PGEM-T为载体将其克隆到大肠杆菌中,构建了P1761质粒。通过DNA体外重组,以卡那霉素抗性基因插入目的基因片段,构建了既含目的基因上游及下游序列、又携带选择性标记卡那霉素抗性的PK1761质粒。该质粒转化野生型集胞藻PCC6803细胞,利用同源重组原理获得了能在含卡那霉素的培养基上正常生长的基因敲除突变株。对该突变株基因组DNA进行PCR扩增,验证了其基因结构的正确性。     

噬藻体感染相关基因的研究进展

噬藻体是感染蓝细菌(蓝藻)的病毒,能调控蓝细菌种群的丰度和多样性,在许多水生生态系统的食物网动态变化和生物地球化学循环中起关键作用。噬藻体与宿主细胞发生各种相互作用,包括吸附、入侵和复制,参与感染过程,从而完成噬藻体的生命周期。本文在综述噬藻体生命周期与基因组结构相互关联的基础上,重点介绍噬藻体与宿主蓝细菌相互作用的蛋白,如噬藻体吸附蛋白、内肽酶、穿孔素、DNA聚合酶、藻胆体降解蛋白A(NblA)、毒力因子、抗CRISPR蛋白(Acr)和小分子热休克蛋白等,分析它们的分子特性,阐述它们在噬藻体感染蓝细菌以及噬藻体-蓝细菌相互作用的分子机制。为了更好地认识驱动不同噬藻体与宿主及水生环境相互作用的策略、感染效率及生态学影响,本文不仅对这些与噬藻体感染相关的重要基因研究动态进行综述与讨论,还在了解噬藻体丰富的多样性和复杂性的基础上,提出应用新技术对噬藻体感染相关基因的功能进行广泛研究,以期扩展全球水生病毒数据库,进一步认识噬藻体与宿主的相互作用机理。     

蓝细菌ORF469的分子克隆和缺失突变工程株的构建

PCR扩增了蓝细菌Synechocystis sp．PCC 6803的ORF469(编码469个氨基酸的开放阅读框),进一步以pUC118为载体将其克隆到E．Coli中,构建了pOQ2质粒。通过DNA体外重组,以红霉素抗性基因取代部分克隆化ORF469片段,又构建丁缺失ORF469片段(保留部分上游和下游序列)的pOQ22质粒。用pOQ22质粒转化Synechocystis sp．PCC 6803野生株细胞,获ORF489缺失突变工程株,它在红霉素抗性培养基上生长正常。对缺失突变工程株DNA的PCR和Southern blot分析证明,Synechocystis sp．PCC 6803的ORF469已被删除。色素测定结果揭示Synechocystis sp．PCC 6803中ORF469表达产物控制细胞内不依赖光的叶绿素生物合成。     

猪繁殖与呼吸综合征病毒核衣壳蛋白基因的克隆与原核表达研究

采用RT-PCR方法自猪繁殖与呼吸综合征病毒基因组分离出核衣壳蛋白基因(orf 7),克隆到pMD18-T载体构建成重组质粒pMD18N并进行测序比较,结果表明,所克隆的核衣壳蛋白基因序列与PRRSV美洲型ATCC VR-2332株的同源性为100%,表明orf 7 是PRRSV基因组内很保守的序列;将orf 7亚克隆到原核表达载体pGEX-KG,构建成重组质粒pGEX-KGN,用pGEX-KGN转化表达菌株BL21,经SDS-PAGE和 Western-blot分析表明克隆在谷胱苷肽转移酶(Glutathione S-transferase(GST)下游的核衣壳蛋白基因与GST获得了高效融合表达,表达的融合蛋白GST-N分子量约为41kDa,并且有免疫学反应活性;这为猪繁殖与呼吸综合征的血清学诊断方法的建立打下了基础.     

集胞藻ORFs侧翼序列的PCR扩增和定向基因插入失活策略

针对集胞藻PCC6803的1927个待定编码基因进行了两侧序列的PCR扩增。4个亚株基因组在sll0267-sll0269区域的PCR扩增产物与Kazusa DNA数据存在差异,以叶绿素合成基因chlH和chlL为例,显示三片段连接PCR产物可有效用于集胞藻6803基因组定向插入失活。     

蓝细菌6803agp基因的分子克隆和缺失突变株的构建

PCR扩增了蓝细菌集胞藻6803(Synechocystis sp.PCC6803)的agp基因（编码ADP－葡萄糖焦磷酸羧化酶）,进一步以pUC118为载体将其克隆到大肠杆菌中,构建了pUCA质粒。通过DNA体外重组,以红霉素抗性基因部分取代agp基因片段,构建了既含agp基因上游及下游序列、又携带选择性标记－红霉素抗性的pUCAE质粒。该质粒转化野生型集胞藻6803细胞,获得了能在含红霉素的培养基上正常生长的agp基因缺失突变株。对该突变株基因组DNA进行PCR扩增,验邝了其基因结构的正确性。突变株细胞生长速度较野生型细胞快,胞内的叶绿素含量比野生型细胞高,表明该突变株具有较高的光合效率。在突变株中未检测到糖原的存在,进一步从生理水平上验证了突变株构建的正确性。     

大庆湿地可培养噬藻体的分离及其相关基因的系统进化分析

【目的】揭示大庆湿地可培养蓝藻噬菌体遗传基因多样性,分析其系统进化地位,为噬藻体生态学研究提供数据支持。【方法】以鱼腥藻(Anabaena PCC7120)为宿主,采用液体富集和双层平板法分离大庆湿地水体中可培养的噬藻体,提取噬藻体混合液的DNA,PCR扩增噬藻体编码衣壳组装蛋白的g20基因和编码T7型短尾病毒的核糖体聚合酶的pol基因,克隆测序,构建系统进化树,明确可培养噬藻体相关基因的系统进化地位。【结果】克隆测序获得1条g20基因序列,4条pol基因序列。系统进化分析表明,获得g20序列隶属于可培养噬藻体类群(Clusterδ)中。而3条pol基因与我国吉林碱性稻田水体噬藻体类群(PG-Pol-I和PG-Pol-II)更相近,另一条pol序列形成独立的进化分枝。【结论】这是首次调查大庆湿地水体侵染鱼腥藻的可培养噬藻体的g20和pol基因,初步确认以鱼腥藻(Anabaena PCC7120)为宿主的可培养噬藻体g20基因归属于Clusterδ中,而大庆湿地可培养噬藻体的pol基因与我国大安稻田水体pol基因相近。     

携带EGFP基因的戊型肝炎病毒重组质粒的构建及其感染性的验证

为了构建携带增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)基因的戊型肝炎病毒(Hepatitis E virus,HEV)重组质粒,转染人肺癌细胞A549细胞验证其感染性,PCR法分两段扩增HEV全基因组序列和EGFP基因序列,将EGFP报告基因插入到HEV ORF2基因下游,并克隆到体外转录表达载体pGEM-7Zf(+)上。然后利用脂质体转染法将重组质粒转入A549细胞,24h后在荧光显微镜下观察EGFP的表达;转染72h后,利用免疫荧光法检测HEV ORF2蛋白的表达。转染7d后将发生病变的A549细胞收集作为接种物,接种A549细胞验证携带EGFP的HEV-EGFP重组病毒的感染性。结果显示经酶切和测序鉴定携带EGFP的HEV重组表达质粒pGEM-HEV-EGFP构建成功;EGFP基因与HEV在A549细胞中可融合表达;携带EGFP的HEV重组表达质粒转染A549细胞7d后出现病变,并且连续传代3代仍具有感染性。本研究成功构建了携带EGFP的HEV全基因组重组质粒pGEM-HEV-EGFP,并成功感染A549细胞,为进一步研究HEV的复制机制及致病机理奠定基础。     

集胞藻PCC6803高效基因表达平台的构建与评价

针对蓝细菌代谢工程改造的需求,成功构建了可以在模式蓝细菌菌株集胞藻PCC6803中高效表达外源基因的3个基因组整合表达平台,以及1个可以在多株蓝细菌中表达的广宿主穿梭表达平台。该表达平台通过选用集胞藻PCC6803中1,5-二磷酸核酮糖缩化酶/氧化酶的启动子驱动外源基因的表达,应用“SD-AUG”翻译融合的策略提高外源蛋白翻译效率,以及加入终止子序列Trbc以提高转录终止效率,实现了对外源基因的高效表达。利用lacZ作为报告基因,检测了所构建表达平台pFQ20在集胞藻中的基因表达效率,结果显示β-半乳糖苷酶的活性为109 Miller。同时,基于pFQ20表达平台在集胞藻PCC6803中表达了来自大肠杆菌的硫酯酶基因tesA’,蛋白印迹实验结果显示了硫酯酶的成功表达。该表达平台为在蓝细菌中开展遗传研究及基因工程改造提供了有用的遗传工具,其构建策略为在蓝细菌中构建高效稳定的外源基因表达元件提供了借鉴。     

A novel cyanophage with a cyanobacterial nonbleaching protein A gene in the genome

A cyanophage, PaV-LD, has been isolated from harmful filamentous cyanobacterium Planktothrix agardhii in Lake Donghu, a shallow freshwater lake in China. Here, we present the cyanophage's genomic organization and major structural proteins. The genome is a 95,299-bp-long, linear double-stranded DNA and contains 142 potential genes. BLAST searches revealed 29 proteins of known function in cyanophages, cyanobacteria, or bacteria. Thirteen major structural proteins ranging in size from 27 kDa to 172 kDa were identified by SDS-PAGE and mass-spectrometric analysis. The genome lacks major genes that are necessary to the tail structure, and the tailless PaV-LD has been confirmed by an electron microscopy comparison with other tail cyanophages and phages. Phylogenetic analysis of the major capsid proteins also reveals an independent branch of PaV-LD that is quite different from other known tail cyanophages and phages. Moreover, the unique genome carries a nonbleaching protein A (NblA) gene (open reading frame [ORF] 022L), which is present in all phycobilisome-containing organisms and mediates phycobilisome degradation. Western blot detection confirmed that 022L was expressed after PaV-LD infection in the host filamentous cyanobacterium. In addition, its appearance was companied by a significant decline of phycocyanobilin content and a color change of the cyanobacterial cells from blue-green to yellow-green. The biological function of PaV-LD nblA was further confirmed by expression in a model cyanobacterium via an integration platform, by spectroscopic analysis and electron microscopy observation. The data indicate that PaV-LD is an exceptional cyanophage of filamentous cyanobacteria, and this novel cyanophage will also provide us with a new vision of the cyanophage-host interactions.     

集胞藻PCC6803铜离子诱导表达平台的构建

在集胞藻PCC6803中,基因敲除是研究基因功能的最直接有效的方法,但是对于某些生存必需的基因则无法通过这种方法获得突变株。为研究集胞藻PCC6803中此类基因的功能,在其基因组中构建了一个petE基因启动子（PpetE）控制的铜离子诱导表达的平台。将集胞藻PpetE装配在lacZ报告基因的上游,通过同源双交换整合到这种蓝藻的基因组中。通过调节培养基中铜离子的浓度发现,lacZ的表达能够人为控制。特别是当铜离子浓度在6－400nmoL／L范围时,LacZ活力随铜离子浓度增加呈S型增长关系。利用这个铜离子诱导表达平台,可以控制某些必需基因的表达：提供铜离子维持细胞生存;而撤去铜离子时则关闭基因的表达,可以观察其对生命活动的影响。     

Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803

Polyhydroxyalkanoate (PHA) synthase activity in Synechocystis sp. PCC6803 was increased two-fold by introducing the PHA biosynthetic genes of Ralstonia eutropha. The resulting recombinant Synechocystis sp. PCC6803 strain was subjected to conditions that favor PHA accumulation and the effects of various carbon sources were studied. In addition, the fine structure of both wild-type and recombinant Synechocystis sp. PCC6803 was examined using freeze-fracture electron microscopy technique. The PHA granules in the recombinant Synechocystis sp. PCC6803 were localised near the thylakoid membranes. Maximum amount of PHA accumulation was obtained in the presence of acetate, where the number of granules in the recombinant cells ranged from 4 to 6 and their sizes were in the range of 70-240 nm. In comparison to wild-type Synechocystis sp. PCC6803, recombinant cells with increased PHA synthase activity showed only a marginal increase in PHA content suggesting that PHA synthase is not the rate limiting enzyme of PHA biosynthesis in Synechocystis sp. PCC6803.     

The rnb gene of Synechocystis PCC6803 encodes a RNA hydrolase displaying RNase II and not RNase R enzymatic properties

Cyanobacteria are photosynthetic prokaryotic organisms that share characteristics with bacteria and chloroplasts regarding mRNA degradation. Synechocystis sp. PCC6803 is a model organism for cyanobacteria, but not much is known about the mechanism of RNA degradation. Only one member of the RNase II-family is present in the genome of Synechocystis sp PCC6803. This protein was shown to be essential for its viability, which indicates that it may have a crucial role in the metabolism of Synechocystis RNA. The aim of this work was to characterize the activity of the RNase II/R homologue present in Synechocystis sp. PCC6803. The results showed that as expected, it displayed hydrolytic activity and released nucleoside monophosphates. When compared to two E. coli counterparts, the activity assays showed that the Synechocystis protein displays RNase II, and not RNase R characteristics. This is the first reported case where when only one member of the RNase II/R family exists it displays RNase II and not RNase R characteristics.     

Cloning, expression, purification, and preliminary characterization of a putative hemoglobin from the cyanobacterium Synechocystis sp. PCC 6803

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a gene (slr2097, glbN) encoding a 123 amino-acid product with sequence similarity to globins. Related proteins from cyanobacteria, ciliates, and green algae bind oxygen and have a pronounced tendency to coordinate the heme iron with two protein ligands. To study the structural and functional properties of Synechocystis sp. PCC 6803 hemoglobin, slr2097 was cloned and overexpressed in Escherichia coli. Purification of the hemoglobin was performed after addition of hemin to the clarified cell lysate. Recombinant, heme-reconstituted ferric Synechocystis sp. PCC 6803 hemoglobin was found to be a stable helical protein, soluble to concentrations higher than 500 microM. At neutral pH, it yielded an electronic absorption spectrum typical of a low-spin ferric species, with maxima at 410 and 546 nm. The proton NMR spectrum revealed sharp lines spread over a chemical shift window narrower than 40 ppm, in support of low-spin hexacoordination of the heme iron. Nuclear Overhauser effects demonstrated that the heme is inserted in the protein matrix to produce one major equilibrium form. Addition of dithionite resulted in an absorption spectrum with maxima at 426, 528, and 560 nm. This reduced form appeared capable of carbon monoxide binding. Optical data also suggested that cyanide ions could bind to the heme in the ferric state. The spectral properties of the putative Synechocystis sp. PCC 6803 hemoglobin confirmed that it can be used for further studies of an ancient hemoprotein structure.     

Purification and characterization of class-I and class-II fructose-1,6-bisphosphate aldolases from the cyanobacterium Synechocystis sp. PCC 6803

The whole genome sequence database for Synechocystis sp. PCC 6803 has revealed the presence of genes encoding class-I (CI) and class-II (CII) fructose-1,6-bisphosphate aldolases (FBAs) in this organism. Two types of FBA from Synechocystis sp. PCC 6803 were separated by chromatography on phenyl-Sepharose. The activity of the enzyme in the major peak was inhibited by the presence of 25 mM EDTA; however, the activity in the minor peak was not. Therefore, the FBA in the former fractions was designated as CII-FBA, and in the latter designated as CI-FBA. CI-FBA was functionally redundant in Synechocystis sp. PCC 6803, while no disruptant for the gene encoding CII-FBA was obtained under photoautotrophic conditions. The kinetic parameters of CI- and CII-FBAs purified from Synechocystis sp. PCC 6803 in the cleavage reaction of FBP were generally similar, except in their reactivity for SBP. The SBP/FBP activity ratio of the CII-FBA was two times higher than that of the CI-FBA.     

集胞藻PCC6803膜脂循环关键基因酶学性质和生理功能

集胞藻PCC6803野生型和其脂酰ACP合酶敲除突变株的自由脂肪酸含量和组成表明膜脂的重构和降解是细胞内自由脂肪酸的来源之一。在这一过程中脂肪酶起到关键性作用。通过基因组数据库检索,发现集胞藻PCC6803基因组中只有一个脂肪酶编码基因sll1969,但是还没有其功能相关的生化证据。为了确定该基因的功能及其在脂肪酸代谢途径中的作用,加深对集胞藻PCC6803脂肪酸代谢途径的了解,文中将sll1969基因在大肠杆菌中过表达和体外纯化,得到重组蛋白Sll1969,并对其酶学性质进行初步分析。在30℃条件下,测得Sll1969以对硝基苯丁酸酯作为底物时的Km和kcat值分别为(1.16±0.01)mmol/L和(332.8±10.0)/min;该脂肪酶的最适反应温度为55℃。通过比较分析sll1969突变株中脂肪酸含量和组成变化,发现sll1969的表达量与细胞自由脂肪酸的产量呈正相关,但Sll1969不是细胞中唯一的脂肪酶。     

新型聚球藻噬藻体Yong-L2-223的分离及基因组分析

【目的】噬藻体(cyanophages)是特异性侵染蓝藻(cyanobacteria)的病毒，广泛分布于各类水体中，在调节蓝藻种群动态和密度、推动生物地球水生生态系统循环中起着重要作用。本研究的目的在于分离、鉴定噬藻体。【方法】本研究以海洋聚球藻(Synechococcus sp.) PCC 7002为指示宿主，从淡水水样中分离培养一株新型噬藻体Yong-L2-223，对其进行了宿主范围实验、全基因组测序、基因功能注释和系统进化分析。【结果】针对31株供试蓝藻的宿主范围实验，结果除指示藻PCC 7002 [属于聚球藻目(Synechococcales)]外，Yong-L2-223能够感染2株淡水蓝藻，分别是来源于滇池的绿色微囊藻(Microcystis viridis) FACHB-1342 [属于色球藻目(Chroococcales)]和水华束丝藻(Aphanizomenon flos-aquae)FACHB-1209[属于念珠藻目(Nostocales)]。既可在高盐条件下感染海洋蓝藻，又可在低盐条件下感染淡水蓝藻，Yong-L2-223具有广盐性。透射电镜观察表明，Yong-L2...