蓝藻高CO2需求突变株的研究进展

对蓝藻CO2浓缩机制(CO2 concentrating mechanism,CCM)的认识,最终要揭示其作用的生理生化和分子生物学基础,蓝藻高CO2需求突变株的研究为这个目标的实现提供了一条最为有效的途径,1986年,Marcus等第一次把筛选光合作用突变株的方法加以修改,利用化学诱变筛选到第一个单细胞蓝藻Synechococcus PCC7942的高CO2需求突变株,为这一领域的研究在方法学上开辟了一个新的途径。       

聚球藻Synechococcus sp.PCC7942高CO2需求突变株的筛选及其超？…

以单细胞蓝藻球藻Ｓｙｎｅｃｈｏｃｏｃｃｕｓ ｓｐ．ＰＣＣ７９４２为材料,利用甲基磺酸乙酯（ＥＭＳ）进行化学诱变获得了一个高ＣＯ２需求突变株。它能在４％ＣＯ２下生长而不能在空气中生长。对突变株的初检表明：其回复突变率约为１０＾－７。该突变株从高ＣＯ２条件下转到空气中后,细胞在２～３ｄ内逐渐趋于死亡;其光合作用对外源无机碳的依赖性高于野生型细胞,碳酸酐酶活性也低于野生型细胞。在超微结构水平,突变株细胞     

聚球藻Synechococcus sp.PCC7942高CO_2需求突变株的筛选及其超微结构观察(英文)

以单细胞蓝藻聚球藻Synechococcussp.PCC7942为材料,利用甲基磺酸乙酯(EMS)进行化学诱变获得了一个高CO2 需求突变株。它能在 4%CO2 下生长而不能在空气中生长。对突变株的初检表明:其回复突变率约为 10 -7。该突变株从高CO2 条件下转到空气中后,细胞在 2～ 3d内逐渐趋于死亡;其光合作用对外源无机碳的依赖性高于野生型细胞,碳酸酐酶活性也低于野生型细胞。在超微结构水平,突变株细胞内出现了不同类型的异常羧体:有的为棒状;有的为不规则状;有的为 空羧体",而且,类囊体周围糖原颗粒增多。进一步说明该突变株在CO2 吸收和利用功能上有缺陷。此外,对低碳条件对羧体的诱导及羧体的生物发生也作了一些探讨     

蓝藻浓缩二氧化碳的机制

综述了蓝藻的CO2逍缩机制研究进展,并简要介绍了一些重要的研究手段。     

湖泊微拟球藻高含油突变株的构建及筛选

研究旨在建立湖泊微拟球藻(Nannochloropsis)的遗传转化体系, 然后根据外源基因随机插入的特性, 挑取抗性转化子构建突变体库, 并从突变体库中筛选高含油突变株。以湖泊微拟球藻自身β-tublin基因启动子和三角褐指藻fcpA终止子驱动和终止来源于细菌的sh ble抗性选择基因, 构建了一个转化载体pPha-T1-TUB。将线性化后的质粒以电穿孔的方法转化湖泊微拟球藻, 通过1 μg/mL zeocin的抗性平板筛选, 并经液体培养基连续传代后, 得到了可以稳定遗传的转化子。我们从这些转化子中筛选得到了数株油含量高于野生型, 且生长也优于野生型的突变株。其中, 2个突变株K26和G5的总脂中多不饱和脂肪酸含量更低, 其脂肪酸组成更符合作为生物柴油原料的标准。研究通过随机插入构建突变体库的方法为快速获取优良目的性状的高产油突变株提供了一个有效手段。     

球孢白僵菌高壳聚酶突变株的筛选

以球孢白僵菌（Beauveria bassiana)1316为出发菌株,通过紫外线和高能电子束分别诱变分生孢子,采用平板透明圆法初筛和摇瓶培养复筛的方法,经3轮诱变,筛选到5株高产突变株。其中一株最高产的定名为Beauveria bassiana 1316-V1,其壳聚糖酶活力是原始菌株的16倍;而且经传代培养,其高产特性能够稳定遗传。     

盐胁迫对于聚球藻PCC 7942大片段DNA删除突变株的生理效应

在对聚球藻PCC 7942开展基因组简化的过程中, 发现一些非必需大片段的删除可导致耐盐胁迫能力降低。突变株?Synpcc7942_0233-0253和?Synpcc7942_2169-2187, 其基因组中分别删除了18和14 kb的区域, 在BG11培养液中的生长与野生型没有显著差异, 但在含有0.4 mol/L NaCl的培养液中的生长相对于野生型被严重抑制。进一步分析发现, 在盐胁迫下2个突变株的光合放氧速率比野生型显著下降, 光系统Ⅰ和Ⅱ电子传递速率均低于野生型, 呼吸耗氧速率与野生型相比却维持在较高水平。这些结果说明, 蓝藻基因组中有些非必需基因实际上对于适应胁迫条件是需要的。     

伪狂犬病病毒鄂A株TK－／gG－／LacZ＋突变株的构建

为了以国内地方分离株鄂A株为亲本构建伪狂犬病病毒双基因缺失株,采用外切酶Ⅲ和绿豆核酸酶酶切,构建了缺失主要毒力基因TK基因部分编码区的重组质粒pSTK1-4,进一步改造成为转移质粒pUCPB4。用HindⅢ将质粒pUCPB4线性化,然后与用EcoRI消化的伪狂犬病病毒鄂A株TK^-/LacZ^ 突变株基因组DNA共转染PK－15细胞,等完全病变后,收毒作空斑试验,PCR筛选TK缺失的重组病毒。重组病毒空斑纯化3次,随机挑取空斑进行PCR扩增,证实所获得的病毒为均一的无TK^-/LacZ^ 突变株污染的TK缺失的重组病毒,分别以TK缺失株病毒与鄂A野毒株为模板对TK基因进行PCR扩增,扩增产物经酶切分析和测序后发现：TK缺失重组病毒的TK基因缺失了205个碱基,XhoⅠ和SalⅠ位点消失,SmaⅠ酶切片段发生变化,两种病毒在PK－15细胞上形成空斑的大小和增殖 滴度无明显的差别。进一步提取TK缺失突变株基因组DNA,与含gG-LacZ的转移质粒pUSKZ通过磷酸钙法共转染PK－15细胞,待完全病变后,在X-gal存在下筛选蓝斑,将桃取的蓝斑纯化3次后,对纯化的重组病毒进行TK、LacZ扩增,结果既能扩增出较以鄂A野毒株为模板扩增的要小的TK基因片段,同时又能扩增出LacZ基因,证实所得到的重组病毒为TK^-/gG^-/LacZ^ 突变株。此双缺失突变株的构建成功,为在我国最终根除伪狂犬病提供了有用的工具。     

聚球藻7942高效泌氨突变种的获得及其泌氨,谷氨酰胺合成酶活性,光合和生

将含有Ａｎａｂａｅｎａｓｐ．ＰＣ７１２０反义ｇｌｎＡ基因片段的穿梭表达质粒ｐＤＣ－ＡＴＧＳ转化单细胞蓝藻聚球藻Ｓｙｎｅ－ｃｈｏｃｏｃｃｕｓｓｐ．ＰＣＣ７９４２,通过同源重组,外源ＤＮＡ定位整合到染色体上。经过抗菌素筛选,获得一种高效泌氨的Ｓｙｎｅｃｈｏｃｏｃｃｕｓｓｐ．７９４２突变种。     

暹罗鱼腥藻空间飞行突变株的光合特性分析

对经空间飞行搭载而获得的暹罗鱼腥藻突变株的分析发现,与对照相比,它在生长率和光合效率方面明显较高。进一步分析其光合色素的组成,叶绿素荧光及PSII/PSI比值,发现突变株的PC/Chl比值明显低于对照藻株,而叶绿素荧光高于对照,PSII/PSI比值是对照藻株的1.7倍,在其它光合色素的比例上也有差异。分析这些结果表明,突变株与对照株在光合特征上有差异可能是突变株在色素系统的改变引起光能捕捉和光能利用上更为高效的原因。     

ISOLATION OF ccmKLMN GENES FROM THE MARINE CYANOBACTERIUM, SYNECHOCOCCUS SP. PCC7002 (CYANOPHYCEAE), AND EVIDENCE THAT CcmM IS ESSENTIAL FOR CARBOXYSOME ASSEMBLY

A high CO₂ requiring mutant of the marine cyanobacterium Synechococcus PCC7002 was generated using a random gene-tagging procedure. This mutant demonstrated a reduced photosynthetic affinity for inorganic carbon (C_i) and accumulated high internal levels of C_i that could not be used for photosynthesis. Analysis of the mutant genomic DNA showed that the mutagenesis had disrupted a cluster of genes involved in the cyanobacterial CO₂ concentrating mechanism (CCM), the so-called ccm genes. These characteristics are consistent with a cyanobacterial mutant with defects in carboxysome assembly and/or functioning. Further genomic analyses indicated that the genes of the Synechococcus PCC7002 operon, ccmKLMN , are structurally similar to those of two closely related cyanobacteria, Synechococcus PCC7942 and Synechocystis PCC6803. The Synechococcus PCC7002 ccmM gene, which encodes a polypeptide with a predicted size of 70 kDa, was the direct target of the mutagenesis event. The CcmM protein has two distinct regions: an N-terminal region that shows similarity to an archaeon gamma carbonic anhydrase and a C-terminal region that contains repeated domains demonstrating sequence similarity to the small subunit of Rubisco. Physiological analysis of a ccmM -defined mutant showed that these cells were essentially identical to the original mutant; they required high CO₂ concentrations for growth, they had a low photosynthetic affinity for C_i, and they internalized C_i to high levels. Moreover, ultrastructural examination showed that both the original and the defined mutants lack carboxysomes. Thus, our results demonstrate that the ccmM gene of Synechococcus PCC7002 encodes a polypeptide that is essential for carboxysome assembly and therefore for proper functioning of the cyanobacterial CCM.     

PHYSIOLOGICAL CHARACTERIZATION OF A SYNECHOCOCCUS SP. (CYANOPHYCEAE) STRAIN PCC 7942 IRON‐DEPENDENT BIOREPORTER FOR FRESHWATER ENVIRONMENTS1

The complex chemical speciation of Fe in aquatic systems and the uncertainties associated with biological assimilation of Fe species make it difficult to assess the bioavailability of Fe to phytoplankton in relation to total dissolved Fe concentrations in natural waters. We developed a cyanobacterial Fe‐responsive bioreporter constructed in Synechococcus sp. strain PCC 7942 by fusing the Fe‐responsive isiAB promoter to Vibrio harveyi luxAB reporter genes. A comprehensive physiological characterization of the bioreporter has been made in defined Fraquil medium at free ferric ion concentrations ranging from pFe 21.6 to pFe 19.5. Whereas growth and physiological parameters are largely constrained over this range of Fe bioavailability, the bioreporter elicits a luminescent signal that varies in response to Fe deficiency. A dose‐response characterization of bioreporter luminescence made over this range of Fe^{3 +} bioavailability demonstrates a sigmoidal response with a dynamic linear range extending between pFe 21.1 and pFe 20.6. The applicability of using this Fe bioreporter to assess Fe availability in the natural environment has been tested using water samples from Lake Huron (Laurentian Great Lakes). Parallel assessment of dissolved Fe and bioreporter response from these samples reinforces the idea that measures of dissolved Fe should not be considered alone when assessing Fe availability to phytoplankton communities.     

棕色固氮菌nifS敲除菌株的构建

从Azotobacter vinelandii中通过PCR扩增了5’和3’端分别缺失264bp和261bp的nifS'片段,克隆至载体pUCl8,形成重组质粒pUCS,再通过同源重组的方法,将pUCS插入Azotobacter vinelandii的nifS中,形成够阻断突变体SUl,经Southern杂交和：PCR扩增,证明所得确为nifS阻断突变株。SUl在外加氮源的BBGN培养基中能够快速生长,但在Burk's无氮培养基中,生长却极其缓慢,表明,nifS基因的破坏,已造成SU1的固氮能力接近完全丧失。该突变体的成功构建,为进一步从中纯化固氮酶两组分,研究nifS对固氮酶结构及功能的影响及iscS与nifS之间的关系奠定了良好的基础。     

一株耐碱Mn-SOD高产细菌的分离、基因克隆及序列分析

首次从长白山温泉土中筛选得到了一株高产耐碱SOD的细菌,通过抑制剂试验确定了该菌所产SOD类型为Mn-SOD。通过形态特征、生理生化特征及16SrDNA基因序列分析,与芽孢杆菌Bacillus sp.MO6同源性高达100%,与地衣芽孢杆菌Bacillus licheniformis同源性为99%。根据地衣芽孢杆菌Mn-SOD序列,并结合GenBank中已发表的多种细菌Mn-SOD基因保守区,分别设计引物,PCR扩增获得600bp的Mn-SOD全基因序列和430bp的核心片段,克隆sodA全基因序列,构建重组质粒pMD18-SOD。     

PURIFICATION AND CHARACTERIZATION OF A HOMODIMERIC ENOLASE FROM SYNECHOCOCCUS PCC 6301 (CYANOPHYCEAE)1

Enolase from Synechococcus PCC 6301 was purified 1450‐fold to electrophoretic homogeneity and a final specific activity of 68 μmol of phosphoenolpyruvate produced·min^?1·mg protein^?1. Analytical gel filtration and nondenaturing and SDS‐gel electrophoresis demonstrated that this enolase exists as a 118‐kDa homodimer composed of 56‐kDa subunits. The purified enzyme displayed 1) a broad pH‐activity profile with maximal activity occurring at pH 8.0 and 7.5 for the forward and reverse reactions, respectively, 2) a forward‐to‐reverse maximal activity ratio of about 1.6, 3) a K_m (2‐phosphoglycerate) of 0.28 mM, and 4) an absolute requirement for a divalent metal cation cofactor that was best satisfied by Mg²⁺ (K_m=0.62 mM). Enolase activity increased by about 200% after the first purification step (60° C heat treatment), whereas addition of increasing amounts of a clarified extract led to a progressive 70% inhibition in the activity of the purified enzyme. This was reflected by a reduction in enolase's V_max from 73 to 22 U·mg^?1 and forward‐to‐reverse activity ratio from 1.6 to 1.3. This inhibition was negated when the clarified extract was either preincubated with trypsin or warmed to approximately 40° for 5 min. Results are indicative of a heat‐labile enolase inhibitor protein in Synechococcus PCC 6301. By contrast, the purified enolase lost no activity when incubated at 70° C for up to 5 min. This study represents the first purification of enolase from the Cyanophyceae. Characterization of the purified enzyme's physical and kinetic features has provided insights into the structural and functional properties of cyanobacterial enolase.     

鱼腥藻Anabaena PCC7120原生质球和液泡的同步诱导

植物和真菌的液泡,都是通过原生质体途径被分离后才得以深入研究.要分离蓝藻液泡,首先要求制备蓝藻原生质体(球),而这一技术在蓝藻方面长期不过关.最近十年来,作者在这方面进行了不断的努力,先后在蓝藻原生质球制备1、培养再生和融合2等方面获得进展,这方面的研究导致了蓝藻液泡的重新发现3.所发现的液泡由无机盐诱导产生,经电镜检查为单位膜所包围,故确定为液泡.借助较为成功的原生质球制备技术,首先分离了无机盐诱导形成的液泡,在此基础上进一步发现和分离了非诱导液泡4,在分离非诱导液泡的试验过程中发现了原生质球和液泡的同步诱导作用.       

EFFECT OF IRON NUTRITION ON THE MARINE CYANOBACTERIUM SYNECHOCOCCUS GROWN ON DIFFERENT N SOURCES AND IRRADIANCES1

The effects of Fe deficiency on the marine cyanobacterium Synechococcus sp. were examined in batch cultures grown on nitrate or ammonium as a sole nitrogen source under two different irradiances. Fe-stressed cells showed lower chlorophyll a content and cellular C and N quotas. Light limitation increased the critical iron concentration below which both suppression of growth rate and changes in cellular composition were observed. At a limiting irradiance (26 μmol.m⁻².s⁻¹), this critical value was ∼10 nM, a 10 times increase compared to high-light cultures. Moreover, at low light the cellular chlorophyll a concentration was higher than at saturating light (110 μmol.m⁻².s⁻¹), this difference being most pronounced under Fe-stressed conditions. Cells grown on ammonium showed a lower half-saturation constant for Fe (K_s) compared to cells grown on nitrate, indicating Synechococcus sp. has the ability to grow faster on ammonium than on nitrate in a low Fe environment at high light. Consequently, in high-nutrient and low-chlorophyll regions where Fe limits new production, cyanobacteria most likely grow on regenerated ammonium, which requires less energy for assimilation. The K_s for growth on Fe at low light was significantly higher than at high light compared with the cells grown on the same N source, suggesting the cells require more Fe at low light. Therefore, if cells that are already Fe-limited also become light-limited, their iron stress level will increase even more. For cyanobacteria this is the first report of a study combining the interactions of Fe limitation, light limitation, and nitrogen source (NO₃⁻ vs. NH₄⁺).