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。     

多变鱼腥藻半稳定状态连续培养的若干特征

分别使用恒化装置和全玻璃发酵罐连续培养多变鱼腥藻。在稳定条件下加以周期性的光照(L∶D=12∶12),经5—6d得到半稳定状态连续培养物。在光照阶段,培养物的生物量增长速度约等于稀释速度D的两倍,叶绿素的增长速度略小于D的两倍,糖原的累积速度几乎高出D~5的一个数量级。细胞内糖原和蛋白质含量变化的时间过程反映出培养物在光-暗条件下的碳氮物质合成与消耗相互联系的代谢特点。半稳定状态连续培养受培养物内在的生理活性支配,生长参数的变化符合方程X=X_(oe)~((μ-D)~t),但不同于恒浊培养和恒化培养。外界因素的强弱变化只改变生长参量变化的强度。本法适用于研究微藻的生态生理。     

多变鱼腥藻在不同氮源培养条件下光能转换效率的研究

依公式PE=KY,其中K等于生物量的热值(每克干重千卡),Y等于产率即每吸收千卡光能所产生的生物量的干重,测试了在无氮和有结合氮培养下的多变鱼腥藻(Anabaena variabilis)的光能转化效率。结果指出在无氮培养下的最高PEN2为8．1％,在结合氮(NH4Cl)培养下PENH =4+为5.8％。对这种差异性作了简要讨论。     

多变鱼腥藻在不同氮源培养条件下光能转换效率的研究

依公式PE=KY,其中K等于生物量的热值(每克干重千卡),Y等于产率即每吸收千卡光能所产生的生物量的干重,测试了在无氮和有结合氮培养下的多变鱼腥藻(Anabaena variabilis)的光能转化效率。结果指出在无氮培养下的最高PE_(N2)为8.1%,在结合氮(NH_4Cl)培养下PE_(NH_4~ )为5.8%。对这种差异性作了简要讨论。     

hGM-CSF基因穿梭表达载体的构建及其在鱼腥藻7120中的克隆

人粒-巨噬细胞集落刺激因子(hGM-CSF)作为一种造血生长因子,能够刺激T细胞和巨噬细胞增殖、成熟和分化,具有极其重要的免疫调解功能.本研究运用PCR方法,从质粒pAG-MT-8中克隆该基因,并在其5′端添加有利于在蓝藻细胞中高效表达的SD序列,然后插入到表达载体(pRL-439)强启动子PpsbA的下游,进一步与穿梭表达载体pDC-08相连构建成穿梭表达载体pDC-GM.利用三亲接合转移方法将该穿梭表达载体(pDC-GM)转入丝状鱼腥藻7120,通过相应抗生素筛选后得到能稳定遗传的转基因藻.以该转基因藻的基因组DNA为模板进行PCR检测,结果表明hGM-CSF基因已转入鱼腥藻7120.这是首次尝试把蓝藻作为制备重组hGM-CSF的新宿主,具有潜在的经济价值和社会效益.     

多变鱼腥藻藻胆体的分离和荧光鉴定其完整性与解离程度

完整藻胆体的室温荧光峰位于678nm附近,而不完整藻胆体其峰位于673nm以下。在液氮温度下,完整藻胆体的F686与F666相对荧光强度比值超过10,F686与F655之比值超过20。不完整藻胆体的F686与F666和F686与F655之比值远低于完整藻胆体。可用室温荧光峰的波长位置和液氮温度下F686与F655和F666的相对荧光强度比值来判断藻胆体的完整性和解离程度。而液氮温度下F686与F655,F666之比值是更灵敏的指标。     

多变鱼腥藻可逆氢酶的诱导及其特性

在NH4^ 或在无氮培养条件下,对多变鱼腥藻的营养细胞照光并通过N3气24小时,均可诱导出可逆氢酶,照光和厌氧是在多变鱼腥藻营养胞中诱导出可逆氢酶的必要条件。－－在NH4^ 或无氮两种培养条件下所诱导出的可逆氢酶的性质基本相同。（1）当提供还原甲基紫精做它的电子供体时,它们能够放氢;当提供苄基紫精做它的电子受体时,它们都可以吸氢。（2）它们都是热稳定的,并对O2都是不敏感的。（3）它们的放氢活性的最适pH相同,为pH7－7．5。（4）在NH4^ 和无氮培养条件下,所诱导的可逆氢酶的Km值（对于放氢）分别为300umol/l和295umol/l,大致相同,如此高的Km值表示它们在细胞中的代谢功能可能是放氢。（5）CO抑制它们的放氢活性,而C2H2对它们的放氢活性没有影响,在NH4^ 培养条件下,从从变鱼腥藻营养细胞所诱导出的可逆氢酶的放氢活性可达1530nmol H2/mg. 干重．小时。它比在无氮培养条件下所诱导的可逆氢酶的放氢活性高3－5倍,以上实验结果表明,多变鱼腥藻在无氮培养条件下,异形胞的出现影响营养细胞中可逆氢酶的合成和活性的调节。     

重组人粒细胞集落刺激因子(rhG-CSF)基因在鱼腥藻中的克隆

为了将rhG-CSF基因在鱼腥藻PCC 7120中克隆,用于制备口服制剂,利用DNA重组技术,在不改变阅读杠的前提下,将hG-CSF基因进行突变,并插入到pUC-19载体上,构建中间载体pUC=G-CSF;将pUC-G-CSF插入到pRL-489的启动子PpsbA的下游,构建穿梭表达载体pRL-G-CSF;通过三亲接合转移方法,将pRL-G-CSF转入丝状体蓝藻鱼腥藻PCC 7120内。本试验得到了有抗生素性的鱼腥藻,并用PCR技术检测到rhG-CSF基因在转基因鱼腥藻中存在。     

多变鱼腥藻可逆氢酶的诱导及其特性

在NH4^+或在无氮培养条件下,对多变鱼腥藻的营养细胞照光并通过N3气24小时,均可诱导出可逆氢酶,照光和厌氧是在多变鱼腥藻营养胞中诱导出可逆氢酶的必要条件。－－在NH4^+或无氮两种培养条件下所诱导出的可逆氢酶的性质基本相同。（1）当提供还原甲基紫精做它的电子供体时,它们能够放氢;当提供苄基紫精做它的电子受体时,它们都可以吸氢。（2）它们都是热稳定的,并对O2都是不敏感的。（3）它们的放氢活性的最适pH相同,为pH7－7．5。（4）在NH4^+和无氮培养条件下,所诱导的可逆氢酶的Km值（对于放氢）分别为300umol/l和295umol/l,大致相同,如此高的Km值表示它们在细胞中的代谢功能可能是放氢。（5）CO抑制它们的放氢活性,而C2H2对它们的放氢活性没有影响,在NH4^+培养条件下,从从变鱼腥藻营养细胞所诱导出的可逆氢酶的放氢活性可达1530nmol H2/mg. 干重．小时。它比在无氮培养条件下所诱导的可逆氢酶的放氢活性高3－5倍,以上实验结果表明,多变鱼腥藻在无氮培养条件下,异形胞的出现影响营养细胞中可逆氢酶的合成和活性的调节。     

PpsbA驱动的类金属硫蛋白基因smtA在鱼腥藻的表达和镉离子抗性的提高

金属硫蛋白 (Metallothionein,简称MT)是一类富含半胱氨酸、具有很强金属结合能力的小分子量蛋白。       

A mutant of the cyanobacterium Anabaena variabilis ATCC 29413 lacking cyanophycin synthetase: growth properties and ultrastructural aspects

The gene cphA encoding cyanophycin synthetase was interrupted in Anabaena variabilis ATCC 29413 by insertional mutagenesis. The mutant lacked cyanophycin granules and the polar nodules of heterocysts. The mutant grew as fast as the wild-type irrespective of the nitrogen source at low light intensity whereas growth on N(2) was somewhat reduced in high light. It is concluded that cyanophycin metabolism and polar nodules are not essential for aerobic N(2) fixation.     

PHYSIOLOGICAL RESPONSES OF ANABAENA VARIABILIS (CYANOPHYCEAE) TO INSTANTANEOUS EXPOSURE TO VARIOUS COMBINATIONS OF LIGHT INTENSITY AND TEMPERATURE1

Light intensity and temperature interactions have a complex effect on the physiological process rates of the filamentous bluegreen alga Anabaena variabilis Kütz. The optimum temperature for photosynthesis increased with increasing light intensity from 10°C at 42 μE·m^?2·s^?1 to 35°C at 562 μE·m^?2·s^?1. The light saturation parameter, I_K, increased with increasing temperatures. The maximum photosynthetic rate (2.0 g C·g dry wt.^?1·d^?1) occurred at 35°C and 564 μE·m^?2·s^?1. At 15°C, the maximum rate was 1.25 g C·g dry wt.^?1·d^?1 at 332 μE·m^?2·s^?1. The dark respiration rate increased exponentially with temperature. Under favorable conditions of light intensity and temperature the percent of extracellular release of dissolved organic carbon was less than 5% of the total C fixed. This release increased to nearly 40% under combinations of low light intensity and high temperature. A mathematical model was developed to simulate the interaction of light intensity and temperature on photosynthetic rate. The interactive effects were represented by making the light-saturation parameters a function of temperature.     

Identification and initial utilization of a portion of the smaller plasmid of Anabaena variabilis ATCC 29413 capable of replication in Anabaena sp. strain M-131

Summary Anabaena variabilis ATCC 29413 contains two cryptic plasmids. Clones of the smaller (41 kb) plasmid, designated pRDS1, in cosmid vectors were used to construct a physical map. A clone bank of pRDS1 constructed by ligating fragments from aXhoII digest of a pRDS1 cosmid clone into a mobilizable plasmid was used to locate an origin of replication of pRDS1. Because we were unable to cureA. variabilis of pRDS1, the clone bank was transferred by conjugation to another strain ofAnabaena sp., strain M-131. A 5.3 kb fragment of pRDS1 contained all of the sequences necessary for replication inAnabaena sp. strain M-131 as judged by the ability to rescue the hybrid vector from exconjugants in unchanged form after many generations. Hybrid plasmids derived from pRDS1, one bearing genes for luciferase, were also transferred by conjugation toA. variabilis, where they appeared to recombine with pRDS1.     

Respiratory terminal oxidases in the facultative chemoheterotrophic and dinitrogen fixing cyanobacterium Anabaena variabilis strain ATCC 29413: characterization of the cox2 locus

Upon nitrogen step-down, some filamentous cyanobacteria differentiate heterocysts, cells specialized for dinitrogen fixation, a highly oxygen sensitive process. Aerobic respiration is one of the mechanisms responsible for a microaerobic environment in heterocysts and respiratory terminal oxidases are the key enzymes of the respiratory chains. We used Anabaena variabilis strain ATCC 29413, because it is one of the few heterocyst-forming facultatively chemoheterotrophic cyanobacteria amenable to genetic manipulation. Using PCR with degenerate primers, we found four gene loci for respiratory terminal oxidases, three of which code for putative cytochrome c oxidases and one whose genes are homologous to cytochrome bd-type quinol oxidases. One cytochrome c oxidase, Cox2, was the only enzyme whose expression, tested by RT-PCR, was evidently up-regulated in diazotrophy, and therefore cloned, sequenced, and characterized. Up-regulation of Cox2 was corroborated by Northern and primer extension analyses. Strains were constructed lacking Cox1 (a previously characterized cytochrome c oxidase), Cox2, or both, which all grew diazotrophically. In vitro cytochrome c oxidase and respiratory activities were determined in all strains, allowing for the first time to estimate the relative contributions to total respiration of the different respiratory electron transport branches under different external conditions. Especially adding fructose to the growth medium led to a dramatic enhancement of in vitro cytochrome c oxidation and in vivo respiratory activity without significantly influencing gene expression.