绿色荧光蛋白gfp基因在钝顶螺旋藻(Spirulina platensis)A9藻株中的表达

将钝顶螺旋藻(Spirulina platensis)A9藻株在24℃培养,经2 mmol/L的EDTA预处理24 h;采用功率300 W的超声波处理70 s获得单细胞样品,以本实验室构建的携带gfp基因的质粒p215t转化A9藻株单细胞藻液,利用Amp作为选择标记,使单细胞在平板上再生长出单藻落,获得17株具有Amp抗性的转化藻株,转化率3.73‰。在390 nm紫光激发下,生长30天的转化藻丝体发出稳定绿色荧光;培养45天后具有绿色荧光的藻丝出现断裂、具有荧光藻丝长度缩短的现象。实验结果初步表明:报告基因gfp在螺旋藻中得到稳定有效的表达,可以采用单细胞再生形成单藻落技术进行螺旋藻的基因克隆。     

钝顶螺旋藻luxAB载体的构建

实验拟构建钝顶螺旋藻luxAB载体,为螺旋藻遗传转化操作系统的建立提供技术参考和支持。使用EcoRI和SmaI双酶切质粒pUCΩGUS,胶回收获得含有Ubil启动子基因及amp基因的载体大片段;根据质粒pRL1063a中luxAB基因的序列设计引物,以质粒pRL1063a为模板（SalI酶切）,PCR扩增luxAB基因片段;在T4 DNA连接酶的作用下将载体大片段和luxAB基因片段进行体外连接重组并转化感受态细胞,构建成新型质粒载体pUCΩluxAB。     

钝顶螺旋藻细胞的超微结构

钝顶螺旋藻是一种丝状多细胞蓝藻。经透射电源观察证实,细胞的核区无核膜,核仁,细胞质内无线粒体、叶绿体、高尔基体等细胞器分化。     

钝顶螺旋藻FtsZ在大肠杆菌中的表达和定位

FtsZ是与真核微管蛋白类似的原核骨架蛋白,能在细胞分裂位点聚合组装成环状结构而调控细胞分裂过程。为了研究钝顶螺旋藻（Spirulina platensis）FtsZ蛋白的功能,构建了钝顶螺旋藻FtsZ与绿色荧光蛋白GFP融合表达的质粒,并在大肠杆菌中进行了表达和定位研究,结果发现,表达融合蛋白GFP-FtsZ的大肠杆菌细胞由短杆状变为长丝状,且菌丝体长度与融合蛋白的表达量呈正比。在荧光显微镜下观察到融合蛋白GFP-FtsZ在长丝状体细菌中呈有规律的点状分布,这说明FtsZ蛋白功能高度保守,钝顶螺旋藻FtsZ蛋白能识别大肠杆菌分裂位点并装配成环状结构调控大肠杆菌细胞分裂,FtsZ蛋白的过量表达能抑制大肠杆菌正常的细胞分裂而导致长丝状体细胞的形成。     

利用云南永胜县程海湖碱水驯化培养钝顶螺旋藻的研究

本实验利用云南永胜县程海湖的碱水配制培养基,在当地的气候条件下驯化培养钝顶螺旋藻(Spirulina platensis),筛选得CH-1,CH-2和CH-3三种培养基配方。室外连续培养共240天,其中采用CH-2培养基室外大量培养90天,培养面积75平方米,连续测定20天的干藻粉产量为12.5克/米~2·天,纯度为95%,干藻粉的蛋白质含量55—63%。除色氨酸未测定外,含有另外的17种氨基酸。从程海碱水驯化培养的钝顶螺旋藻群体中,筛选获得一个螺旋藻新品系——Spirulinaplatensis str. CH-1。与原品系比较,其特点是藻丝和螺距都较长,螺旋更宽,具有上浮性较好的优势,适合在用程海碱水配制的培养基中生长。实验结果表明,利用程海丰富的湖水资源和适宜的气候条件,发展螺旋藻生产是可行的。     

钝顶螺旋藻FtsZ在大肠杆菌中的表达和定位北大核心CSCD

FtsZ是与真核微管蛋白类似的原核骨架蛋白,能在细胞分裂位点聚合组装成环状结构而调控细胞分裂过程。为了研究钝顶螺旋藻(Spirulina platensis)FtsZ蛋白的功能,构建了钝顶螺旋藻FtsZ与绿色荧光蛋白GFP融合表达的质粒,并在大肠杆菌中进行了表达和定位研究,结果发现,表达融合蛋白GFP-FtsZ的大肠杆菌细胞由短杆状变为长丝状,且菌丝体长度与融合蛋白的表达量呈正比。在荧光显微镜下观察到融合蛋白GFP-FtsZ在长丝状体细菌中呈有规律的点状分布,这说明FtsZ蛋白功能高度保守,钝顶螺旋藻FtsZ蛋白能识别大肠杆菌分裂位点并装配成环状结构调控大肠杆菌细胞分裂,FtsZ蛋白的过量表达能抑制大肠杆菌正常的细胞分裂而导致长丝状体细胞的形成。     

钝顶螺旋藻表面微观形貌的原子力显微镜研究

该文应用原子力显微镜(AFM)纳米级的分辨率对钝顶螺旋藻(Spirulina platensis)表面微观形貌进行了研究,获得了扫描范围为5.000×5.000μm、1.000×1.000μm和400.0×400.0nm三组清晰、稳定的图像,并对其进行了线性分析。结果表明:螺旋藻表面由紧密且无序堆积的突起结构组成,其高度小于20nm;突起结构高度从3nm~15nm不等,平均高度约为8~9nm。此法用于生物体表面,操作简单、快速、灵敏度好且样品无损伤,结果令人满意。     

钝顶螺旋藻富硒培养条件的优化

硒是人和动物必需的微量元素 ,补硒可以防治多种疾病。有机硒具有低毒、高生物利用度的优点 ,目前主要寄希望于生物转化的途径来获得有机硒[1 ] 。植物对硒的生物有机化作用已有综述[2 ] ,并开发有富硒酵母[3 ] 、富硒菇类[4] 、富硒大蒜、富硒黄芪、富硒西洋参、富硒麦芽、富硒茶以及富硒鸡蛋、富硒牛奶等[5] 。螺旋藻是一种很有开发利用前景的藻类 ,但其含硒量极微 ,实验报道富硒螺旋藻对60 Ｃｏ γ射线胸部照射大鼠诱发肺炎和早期肺、肝纤维增生有防治作用[6] 。在培养液中添加亚硒酸钠可以实现藻类对硒的富集和转化 ,而且螺旋藻对无机硒…     

钝顶螺旋藻(Spirulina platensis)光系统Ⅰ的分离表征及稳定性

采用蔗糖密度梯度离心法对钝顶螺旋藻光合膜蛋白(PSI)进行分离纯化,并对其光谱学性质、热稳定性及光合放氧活性进行分析表征。结果发现,采用蔗糖密度精度离心法,可以成功分离出4条色素蛋白质复合体条带,其中最下层条带为完整的PSI三聚体,其每毫克叶绿素a光合放氧活性达到420μmol/h。当温度达到50℃左右时,分离得到的PSI在溶液开始变性失活。     

钝顶螺旋藻富集转化硒及硒在藻体中的分布

钝顶螺旋藻在４℃、避光、隔离空气条件下,对硒的吸附量死体显著高于活体。藻体对硒的最大富集量为６２７．４μg.g^-1(DW),培养基中硒浓度在３００μg.ml^-1以下时有机硒转化率大于８０％。藻体中的硒主要分布在水溶性组化率大于８０％。藻体中的硒主要分布在水溶性组分中,占总硒的６５％以上,蛋白质硒占有机硒的７１．９％,多糖、核酸也有一定量硒分布。     

钝顶螺旋藻突变株FBL细胞超微结构

利用透射电镜技术观察钝顶螺旋藻出发株和突变株FBL的细胞超微结构。观察结果表明L出发株和突变株均为多细胞丝状体,细胞间横隔膜清晰,细胞壁均由四层结构组成,细胞质膜内陷形成类囊体,类囊体由双层膜堆积而成,膜上附着藻胆体,类囊体与细胞壁呈垂直方向排列,细胞质内包含有充气液泡等细胞器。与出发株相比,突变株细胞壁表面较光滑,四层结构电子密度较深;类囊体膜增多、变发达;羧化体数量增多;横隔膜收缢明显。     

富硒螺旋藻中硒别藻蓝蛋白的纯化及其特性

从富硒螺旋藻(Se richSpirulina platensis,Se-SP)中分离纯化高纯度的含硒别藻蓝蛋白(Se-containingallophycocyanin,Se-APC)并观察其生化特性。羟基磷灰石和DEAE-52柱层析方法结合制备电泳技术纯化Se-APC;光谱扫描、Native-PAGE、SDS-PAGE和IEF方法鉴定Se-APC生化特性;2,3-DAN荧光光度法检测蛋白质中Se含量。结果发现3种高纯度Se-APC的光谱特征分别与APCI、APCII、APCIII吻合;电泳鉴定它们可能都是(αβ)3,α、β亚基分别为18.3和15.7 kDa,其pI值分别为:4.76、4.85和5.02;3种Se-APC中Se含量分别为316、273和408μg/g,Se-APC经0.5mol/L NaSCN解聚和β-巯基乙醇变性处理后,蛋白质中Se含量依次减低并趋于稳定。结果提示Se-SP中APC可结合Se,APC中Se含量与其分子聚态有关,亚基中含Se量稳定,可能是以共价键方式结合,Se-APC生物活性及硒在蛋白质中的结合位点值得深入研究。     

Mutants of Spirulina platensis resistant to valine inhibition

Abstract Growth of the cyanobacterium Spirulina platensis is inhibited by low concentrations of valine. Spontaneous valine-resistant mutants were isolated from S. platensis and a preliminary characterization of three of them indicates that one (strains DR2) is defective in valine uptake and two (strains DR5 and DR9) carry alterations in a valine-mediated mechanism of synthesis of acetohydroxy acid synthase, the first common enzyme of the pathway.     

A new model of phycobilisome in Spirulina platensis

Phycobilisomes (PBS) were isolated from blue-green alga Spirulina platensis. Scanning tunneling microscope was used to investigate the three-dimensional structure of PBS deposited on freshly cleaved highly oriented py-rolytic graphite (HOPG) in ambient condition at room temperature. The results showed that the rods of PBS radiated from the core to different directions in the space other than arrayed in one plane, which was different from the typical hemi-discoidal model structure. The diameter of PBS was up to 70 nm, and the rod was approximately 50 nm in length. Similar results were observed in Langmuir-Blodgett (LB) film of PBS. The dissociated PBS could reaggregate into rod-like structures and easily form two-dimensional membrane while being absorbed on HOPG, however, no intact PBS was observed. The filling-space model structure of PBS in Spirulina platensis with STM from three-dimensional real space at nanometer scale was found, which showed that this new structural model of PBS surely exists in blue     

Production and regeneration of spheroplasts from the cyanobacterium Spirulina platensis

Abstract The development of a micro-method for the production and regeneration of spheroplasts starting from S. platensis trichomes is presented. The influence of the growth stage along with different treatments and conditions on the efficiency of spheroplast formation and regeneration are analyzed.     

Isolation and Photosynthesis of Spheroplasts in Spirulina platensis

Spirulina platensis is a nonheterocystic filamentous blue-green alga (cyanobacterium). Large quantity of highly qualified spheroplasts were obtained by improved isolation method. The spheroplast has a wrinkled and porous surface. Their diameter ranged from 3.8 btm to 4. 6 μm. The activity of photosynthetic oxygen evolution in the spheroplasts was about 40 % of the intact cell. The absorption spectra of the filaments and spheroplasts at room temperature revealed that they had the same pigments, Chla, PC, PEC and carotenoid. In spheroplasts the relative content of PC and carotenoid decreased, and that of PEC increased. It implicated that the light absorption of Spirulina platensis could be influenced by the cell wall. Some differences existed between the original cells and spheroplasts in the low temperature fluorescence emission spectra. F757 of spheroplasts excited by 436 nm was reduced obviously and that excited by 580 nm was disapeared. F728/F685 and F640/F685 enhanced, and F693/F685 was reduced. F728/F640 was lower than that of the original cells. These results indicated that removing the cell wall may inhibit the PS Ⅱ activity and influence the F695 from core antenna pigment system.     

Separation and nanoencapsulation of antitumor polypeptide from Spirulina platensis

Spirulina platensis is a multicellular edible blue‐green alga with abundant proteins (～60%). No report is available on the antitumor polypeptides from the whole proteins of S. platensis. In this study, for the first time, an antitumor polypeptide Y2 from trypsin digest of S. platensis proteins was obtained by using freeze‐thawing plus ultrasonication extraction, hydrolysis with four enzymes (trypsin, alcalase, papain, and pepsin), and gel filtration chromatography. The results showed that the degree of hydrolysis can be ordered as: trypsin (38.5%) > alcalase (31.2%) > papain (27.8%) > pepsin (7.1%). For MCF‐7 and HepG2 cells, at 250 µg/mL, the maximum inhibitory rate of Y2 was 97%, while standard drug 5‐FU was 55 and 97%, respectively. Furthermore, the nanoencapsulation of Y2 with chitosan (CS) was also investigated. After nanoencapsulation, the maximum encapsulation efficiency and polypeptides contents are 49 and 15%, respectively; and the antitumor activity is basically not lost. These data demonstrated the potential of nanopolypeptides (Y2‐CS) in food and pharmaceutical applications. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1230–1238, 2013