通过同源重组在聚球藻7002中表达小鼠金属硫蛋白-Ⅰ的初步研究

用PCR方法从海洋单细胞蓝藻聚球藻7002（Synecohococcus sp.PCC7002)基因组DNA中扩增得到藻蓝蛋白β亚基基因（cpcβ)的上游序列（Pcpcβ),及编码谷氨酰胺合成酶的glnA基因片段,以Pcpcβ作为启动子,以glnA基因片段作为整合平台,构建含有小鼠金属硫蛋白-Ⅰ（mMT-Ⅰ)cDNA的同源整合表达载体pKGC-MT,通过自然转化法将整合表达载体导入聚球藻7002中,经氨苄青霉素筛选,得到遗传性状稳定的转基因藻,PCR检测证明mTM-Ⅰ基因已整合到蓝藻基因组DNA上;蛋白质印迹表明mMT-Ⅰ已在蓝藻中表达;ELISA结果显示mMT-Ⅰ在蓝藻中的表达量约为800μg/g。     

鱼腥藻7120遗传转化的研究进展

鱼腥藻7120作为模式生物被广泛用于光合、固氮、进化、代谢等基本生命现象的研究。近几年, 对其基因工程的研究使人们看到它在医药、环保、能源等方面的应用潜力, 但表达效率低是其发展的瓶颈。为了提高其表达效率, 研究者从鱼腥藻7120的载体(包括启动子、复制子、选择标记基因等)的改进、目的基因的优化(密码子和SD序列)、宿主的改善、转化方法的改变等方面进行了大量探索, 除了用于功能基因的研究, 已经有几十个外源基因在鱼腥藻7120中表达。除了研究载体, 诱变鱼腥藻7120形成有利于外源基因表达的突变体和摸索转基因蓝藻最佳生长条件和表达条件, 可能是新的发展方向。     

葡萄糖浓度对转人肿瘤坏死因子-α鱼腥藻IB02培养过程的影响

目的：通过在培养基中添加葡萄糖的方法,提高转基因鱼腥藻的产量和人肿瘤坏死因子（hTNF）-α的表达率。方法：在葡萄糖浓度为0～300mmol／L的范围内,进行了转hTNF-α鱼腥藻IB02的摇瓶混合营养培养,用比浊法和酶联免疫法测定转基因鱼腥藻的生长和hTNF-α的表达。结果：添加葡萄糖的藻液最高生长密度是未添加葡萄糖的3．5倍,且hTNFa的表达率也提高至4倍。结论：在各种葡萄糖浓度下,葡萄糖的利用都不明显。     

人肿瘤坏死因子α基因穿梭表达载体的构建和在鱼腥藻7120中的表达

报道了人肿瘤坏死因子α(hTNFα)基因穿梭表达载体的构建及其在丝状体蓝藻鱼腥藻 712 0中的表达和鉴定结果 .将质粒pRL rhTNF上hTNFα的cDNA连于pRL 43 9质粒上的PpsbA启动子下游 ,得到中间载体pRL TC .进一步与穿梭质粒pDC 8重组 ,得到可在大肠杆菌和蓝藻中均可表达的穿梭表达载体pDC TNF .pRL rhTNF ,pRL TC和pDC TNF三者在大肠杆菌中的表达量分别为 11.8% ,16 9%和 15 .0 % .通过三亲接合转移将pDC TNF引入鱼腥藻 712 0中并获稳定遗传的转基因株 .从转基因的鱼腥藻 712 0中检测到pDC TNF质粒的存在 ,且在和TNFα的cDNA探针进行的Southern杂交中呈阳性反应 .抽提转基因藻的蛋白样品进行检测 ,在Western印迹中和TNFα单克隆抗体呈阳性反应 .采用TNF对L92 9细胞的细胞毒性方法 ,证明转基因藻粗提液中 ,TNF确有细胞毒活性 .     

Comparative Studies on Photosynthetic Fluorescence Spectra and Fluorescence Kinetics of Bryophytes

Bryophytes are the transitional forms from water habitants to terrestrials, however, there have been only a few works on their photosynthesis. It was the first time to study on photosynthetic fluorescence spectra and fluorescence kinetics of primitive and advanced species comparatively. Both the primitive and advanced ones had the same fluorescence spectra at room temperature, which contained two maximum emissions: F_686-690 from the Photosystem II and F_736-740 from the Photosystem I. And then, there were three maximum emissions in the fluorescence spectra at 77K :F_687-689 and F_697-699 from Photosystem II, and F_723-734 from Photosystem I. The first two maximum emissions were the same for both the primitive and advanced species. According to the third maximum emission the bryophytes under study fell into two categories: The first one possessing the maximum emission around 725 nm, including Ditrichum flexicaule , Didymodon icmadophyllum , Didymodon rigidicaulis, Aloina obliquifolia, Plagiomnium confertidens and Marchantia polymorpha, which were primitive mosses and advanced liverwort. The second one possessing the maximum emission around 732nm, including Thuidium delicatulum , Pylaisia brotheri , Myuroclada maximowiczii , Taxiphyllum taxirameum, Gollania neckerella, Eurohypnum leptothallum, which were advanced mosses, and the primitive one Plagiomnium rostratum. The characteristics of fluorescence spectra implied that the Photosystem II was conservative and Photosystem I was changeable during bryophyte evolution. The primitive mosses possess mainly the PSI core complex (CPI) and then the advanced species contain both CPI and LHC-I. In analysis of photosynthetic fluorescence kinetics, Fv/(Fc+Fv) is a measure of the activity of the Photosystem II; Fv/Fm is dependent on efficiency of primary photoconversion in the Photosystem II; Fm/(Fo+Fv) is related to photosynthetic carbon assimilation; and Fd/Fs is a measure of the potential photosynthetic quantum conversion. The fluorescence kinetics of the bryophyte photosynthesis showed that the Photosystem II activity, the efficieiency of primary photoconversion in Photosystem II, the photosynthetic carbon assimilation and the efficiency of the potential photosynthetic quantum conversion in primitive species, such as Ditrichum flexicaule, Didymodon icmadophyllus, D. rigidicaulis, Plagiomnium rostratum and the liverwort Marchantia polymorpha, were lower than those in the advanced species, Myuroclada maximowiczii, Pylaisia brotheri , Gollania neckerella Taxiphyllum taxirameum , Thuidium delicatulum. However, the primitive Plagiomnium confertidens was of the high activities and efficiencies and the advanced Eurohypnum leptothallum was of low ones. It seemed that P. confertidens and E. leptothallum were an intermediatefrom the primitive to the advanced.     

砷对蓝藻光合作用和细胞生长的影响

在世界的许多地方, 砷污染是一个严重的问题, 尤其是水体中的污染程度更加严重。砷主要以As( V) 和As( ó ) 形式存在。由于AsO43- 在结构上与PO43-很相似, 它通过磷转运途径进入细胞, 因此砷的毒性主要是源于对磷代谢的干扰1 。砷对于人和动物的危害性是人所共知的, 它会引发细胞凋亡, 导致多种组织和器官发生癌变, 从而引起死亡。但是砷对于水体生态系统中的重要类群) 藻类的生理和生长的影响报道较少。       

丝状体蓝藻藻殖段的分化及其调节机制

本文介绍了丝状体蓝藻(亦称蓝细菌) 的藻殖段的分化及其调节机制。藻殖段与正常藻丝体的区别在于细胞形状、细胞内存有气囊和可移动的短而直的藻丝链等。本文对许多环境因子包括光和营养因素等促进或抑制藻殖段的分化进行了讨论;还介绍了念珠藻(Nostoc) ,单歧藻(Tolypothrix) 和眉藻(Calothrix)所具有复杂的细胞发育过程,即具气囊又可移动的藻殖段分化,异形胞分化以及营养细胞的补偿性色适应。这三种细胞类型的适应形成取决于两种不同的光受体系统。藻殖段和异形胞两者的分化可能取决于光合电子传递链;而营养细胞的补偿性色适应则受光敏色素的调节。此外,谷酰胺合成酶合成和活性调节的PII蛋白,在协同藻殖段分化、异形胞分化及营养细胞的补偿色适应中起重要作用。由于蓝藻藻殖段分化及其调节机制是一个新的研究领域,关于它的知识尚不完整,亟待人们加强研究。     

Expression of TNF-α Gene in Anabaena sp. PCC 7120 and Purification of Its Product by Affinity Chromatography

The effects of N (NaNO3) and C (NaAc) source in medium on the expression of tumor necrosis factor-α (TNF-α) gene in transgenic Anabaena sp. PCC 7120 were compared. The data showed that N source stabilized the expression of foreign protein and C source altered the synthesis of cell walls. Comparing several methods for breaking the cells, supersonic was able to extract TNF-α better than others. For purification of TNF-α, transgenic Anabaena cells were broken, the extracts were precipitated with ammonia sulfate, and the impure TNF-α was eluted from DEAE ion exchange chromatography. Electrophoresis (PAGE-SDS) showed a single band at 17 kD position.     

Homologous desensitization of ATP-stimulated mitogenesis: Mechanism involves desensitization of arachidonic acid release and cAMP elevation but not the activation of protein kinase A

Prolonged incubation of quiescent 3T3, 3T6, and A431 cells with the P_2Y purinoceptor agonists ATP, ADP, or AMPPNP reduced the mitogenic responses of target cells to a further challenge by these agonists, as measured by [³H]thymidine incorporation. The mitogenic desensitization was agonist-specific, for no effect was seen on DNA synthesis stimulated by epidermal growth factor, insulin, bombesin, 12-0-tetradecanoyl-phorbol-12 acetate (TPA), or adenosine. The desensitization was completely reversible, since after a 24 hr incubation in the absence of ATP, the cells responded fully to the mitogenic action of ATP. The presence of a low level of cycloheximide blocked recovery, suggesting that down-regulation of the P_2Y receptor may have occurred during desensitization. In Swiss 3T3 cells, stimulation of DNA synthesis occurs predominantly by activation of arachidonic acid release, followed by its oxidation to prostaglandin E₂ and stimulation of adenylyl cyclase. Interestingly, prolonged preincubation with ATP produced a similar degree of desensitization of DNA synthesis and of ATP-dependent arachidonic acid release and cAMP accumulation. Furthermore, this was true for both wild type cells and mutants with a defective cAMP-dependent protein kinase (PKA). We conclude that homologous desensitization is likely due to uncoupling of the P_2Y purinoceptor from phospholipase A₂, and this process does not require activation of protein kinase A. © 1995 Wiley-Liss Inc.     

在鱼腥藻7120中建立反义glnA系统

应用反义技术对鱼腥藻7120切的内源glnA基因的表达进行调控,首次获得了人工反义系统的蓝藻品系。先从编码谷酰胺合成酶（GS）的基因glnA中取得部分结构基因片段,与表达质粒载体pRL-439及穿梭质粒载体pDC－8相连接。通过酶切鉴定筛选出反向克隆的穿梭表达质粒pDC－AM,然后应用三亲接合转移法把它转入鱼腥藻对7120.通过新霉素筛选,酶谱鉴定,斑点杂交,质粒的交叉转化以及内源glnA基因表达的GS活性分析,GS相关的胞外泌氨分析及所获藻株的形态学变化,证明已在鱼腥藻7120中建立了人工反义glnA基因的品系。