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Genome-wide expression analysis of the responses to nitrogen deprivation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. 总被引:5,自引:0,他引:5
A heterocyst is a terminally differentiated cell of cyanobacteria which is specialized in dinitrogen fixation. Heterocyst differentiation in Anabaena sp. strain PCC 7120 is triggered by deprivation of combined nitrogen in the medium. Although various genes that are upregulated during heterocyst differentiation have been reported, most studies to date were limited to individual or a small number of genes. We prepared microarrays in collaboration with other members of the Anabaena Genome Project. Here we report on the genome-wide expression analysis of the responses to nitrogen deprivation in Anabaena. Many unidentified genes, as well as previously known genes, were found to be upregulated by nitrogen deprivation at various time points. Three main profiles of gene expression were found: genes expressed transiently at an early stage (1-3 hr) of nitrogen deprivation, genes expressed transiently at a later stage (8 hr), and genes expressed when heterocysts are formed (24 hr). We also noted that many of the upregulated genes were physically clustered to form 'expressed islands' on the chromosome. Namely, large, continuous genomic regions containing many genes were upregulated in a coordinated manner. This suggests a mechanism of global regulation of gene expression that involves chromosomal structure, which is reminiscent of eukaryotic chromatin remodelling. The possible implications of this global regulation are discussed. 相似文献
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AIMS: The aim of the present investigation was to study the effects of different inorganic carbon and nitrogen sources on nitrate uptake and heterocyst differentiation in the culture of cyanobacterium Anabaena sp. PCC 7120. METHODS AND RESULTS: Anabaena was cultivated in media BG11 containing combined nitrogen and supplementary NaHCO3 or CO2. Cell growth, heterocyst differentiation, nitrate reductase (NR, EC 1.7.7.2), glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) and NO uptake were analysed. The cells cultivated in BG11(0) medium with aeration were taken as reference. Experimental results showed that the differentiation frequency of heterocysts when the cells were cultivated with elevated CO2 was higher than that of the cells grown with air or bicarbonate. Heterocysts appeared unexpectedly when CO2 was introduced into the medium containing nitrate. However, no heterocysts emerged when CO2 was added to medium containing NH or urea, or when NaHCO3 was supplied to the medium with nitrate. Both nitrate uptake rate and nitrate reduction enzyme activity were depressed by the supplement of CO2 to the culture. The activity of G6PDH was enhanced with the increase in heterocyst differentiation frequency. CONCLUSION: CO2 might compete with NO for energy and electrons in the uptake process and CO2 appears favoured. This led to a high intracellular C/N ratio and a relative N limitation. So the process of heterocyst differentiation was activated to supplement nitrogen uptake. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided an attractive possibility to form more heterocysts by rapid growth of Anabaena cells cultivated in the medium containing nitrate in order to increase nitrogen fixation and hydrogen production. 相似文献
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鱼腥藻7120遗传转化的研究进展 总被引:1,自引:0,他引:1
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。 相似文献
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生物反应器培养转基因鱼腥藻的研究 总被引:2,自引:0,他引:2
在反应器中研究了转人TNF-α基因鱼腥藻7120(Anabaena sp.PCC7120,pDC-TNF)的培养。结果表明气升式反应器适合于转基因鱼腥藻的培养。气升式反应器中通气量和光照是主要的影响因素,观察到1L罐中最适通气量为60~75L/h,最适光照强度为1200lx,此时在25℃混养,光照时间/黑暗时间为12h/12h,15d生物量干重大于3g/L,TNF表达水平约占总可溶蛋白的22%,达到了摇瓶培养水平。实验发现添加维生素B1 300μg/L、B12 200μg/L和生物素4μg/L时,生产周期为12d,缩短20%,表达水平相同。培养过程通入含有5%CO2的空气,能促进生长,缩短生产周期,但收获生物量不受影响。从添加维生素和通入CO2的培养结果证明反应器中培养时,光照是限制性因素,当反应器系统一定时,最终生物量有一个最大值,如需进一步提高产量,必须设法改变光照系统。 相似文献
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环境因子对转基因鱼腥藻培养的影响 总被引:6,自引:1,他引:6
摇瓶中对转TNF-α基因鱼腥藻7120(Anabaenasp.PCC7120,pDC-TNF)混合培养条件进行了研究,在含蔗糖9g/L,NaNO32.25g/L的BG-11培养基混合培养时,得到最适培养条件接种量5%,光照强度为1000Lux,光/暗周期(光照时间/黑暗时间)12h/12h,温度25-30℃,自然初始pH值,100mL摇瓶装液量40mL,转基因鱼腥藻15d生物量可达到3g/L以上,可溶性蛋白含量接近30%,TNF表达水平大于22%,与自养相比,生物量增加82.06%,表达水平提高38.79%,证明混合营养型培养是转rhTNF-α基因鱼腥藻7120实现高密度,高表达培养的途径。 相似文献
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为了提高小鼠金属硫蛋白-I(mMT-I)在鱼腥藻7120(Anabaena sp.PCC 7120)中的表达量、便于表达产物的分离纯化,构建了新的穿梭融合表达载体pKG-MT。通过pKG-MT,mMT-I cDNA在tac启动子的调控下,以与谷胱甘肽转硫酶(GST)C-末端相融合(GST-MT)的形式在鱼藻中表达。SDS-PAGE结果显示在异丙基硫代-β-D-半乳糖苷(IPTG)诱导下GST-MT在鱼腥藻中表达。经谷胱甘肽亲合层析,从转基因藻中分离、纯化得到GST-MT,利用GSTC-末端的凝血酶酶切位点,用凝血酶对GST-MT进行柱上酶切,经Sephadex G50除去凝血酶得到mMT-I。SDS-PAGE表明纯化得到所要的目标产物;ELISA测定结果显示从每克转基因藻(鲜重)中可纯化得到0.9 mg mMT-I;原子吸收测定表明纯化得到的mMT-I的镉离子结合能力接近于天然MT。 相似文献
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Abstract Several approaches have been followed to increase the nitrogenase level in filaments of Anabaena ATCC33047. In a nitrogen-free medium lacking added molybdate and supplemented with 10 mM tungstate, growth was impaired as a result of decreased nitrogenase activity level. Under these conditions, the filaments exhibited nitrogen starvation symptoms and a high heterocyst frequency, with heterocysts being up to 28% of the total number of cells in the filaments, while a regular pattern of heterocyst distribution was maintained. Normal nitrogenase level and nitrogen status were recovered upon molybdate addition, with resumption of growth and decrease of the heterocyst frequency with time until reaching a value of about 10%. The yield of ammonium photoproduction from N2 by filaments displaying different heterocyst frequencies and treated with l -methionine- d,l -sulfoximine (MSX) was determined. Maximal rates were obtained with filaments containing 16% of the cells differentiated as heterocysts. Results indicate that appropriate manipulation of the heterocyst frequency leads to an improvement in the efficiency of conversion of light energy into chemical energy through photoreduction of N2 to ammonium. 相似文献
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Abstract Certain strains of the nitrogen-fixing cyanobacterium Anabaena were found to release varying quantities of ammonia without any induction, both in the presence and absence of combined nitrogen (nitrate) in the medium, during the different phases of their growth. In general, growth and ammonia release were comparable in both media, although there were strain differences. 3 patterns of ammonia release were observed in different strains during the growth period. They were: (1) release pattern parallel to the growth curve; (2) a continuous increase in release; and (3) release showing a bimodal curve. 相似文献
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果糖-1,6-二磷酸醛缩酶和丙糖磷酸异构酶共表达对蓝藻光合作用效率的影响 总被引:1,自引:0,他引:1
以转高等植物ALD和TPI基因的鱼腥藻 7120为对象 ,研究了ALD和TPI两个酶表达量对细胞光合固碳效率的影响。考察了初始pH、NaHCO3浓度和CO2浓度对转基因藻和野生藻生长、光合活性及无机碳亲和力的影响。结果表明 ,转基因藻在较高碳源浓度下 ,其生长速率和光合放氧活性比野生藻有显著的提高 ,并且可以比野生藻耐受更高的pH。在含有2%CO2的空气中 ,转基因藻对外源无机碳的亲和力比野生藻提高了4.06倍. 相似文献
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hGM-CSF基因穿梭表达载体的构建及其在鱼腥藻7120中的克隆 总被引:5,自引:0,他引:5
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. 相似文献
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NtcA是鱼腥蓝细菌中一种重要的氮代谢调控蛋白质,参与异形胞的分化。许多受NtcA调控的基因都至少有一个保守的NtcA结合位点GTA-N8-TAC。经生物信息学分析,鱼腥蓝细菌PCC7120基因alr1390可能的转录起始位点的上游有一个保守的NtcA结合位点GTA-TAGTTTTC-TAC。【目的】为了鉴定alr1390和NtcA之间的关系,【方法】采用实时定量反转录PCR实验(Real-time RT-PCR)和电泳迁移率实验(Electrophoretic mobility shift assays,EMSA)对alr1390和NtcA之间的关系进行了分析。【结果】Real-timeRT-PCR结果显示,alr1390的转录水平在野生型鱼腥蓝细菌PCC7120中缺氮诱导后和诱导前持平,而在ntcA突变体中缺氮诱导12h后呈现上调趋势。但是EMSA实验中没有检测到明显的NtcA和alr1390启动子区片段结合的滞后带,却观察到一条拖带。【结论】这说明alr1390受到NtcA的调控。 相似文献
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在丝状蓝藻Anabaena sp.PCC7120细胞粗提液的碳酸酐酶(CA)分析中,发现了两种形式的CA活性.高CO_2下生长的细胞,在35μmol/L EZ(Ethoxyzolamide,碳酸酐酶的抑制剂)存在的情况下,CA总活性的85%左右被抑制,其半抑制浓度I_(50)为7.4μmol/L;随着EZ浓度的继续增加,CA活性在EZ浓度达到约150μmol/L处出现了第二个抑制峰,在250μmol/L处抑制程度达到最大,使CA总活性的15%被抑制,其半抑制浓度I_(50)为190μmol/L。在空气条件下生长的细胞中也出现了CA的两个抑制峰:低I_(50)为6μmol/L,高I_(50)为120μmol/L,对羧体的分离及体外测试表明,在羧体制备物中的CA活性只有一个EZ的抑制峰,而且在EZ浓度达到35μmol/L,正如所期望的那样,该CA活性全部被抑制。其半抑制浓度I_(50)为5.2μmol/L左右。这个值跟空气或高CO_2条件下生长的细胞粗提物中的低I_(50)(6μmol/L或7.4μmol/L)十分相似。说明低浓度的EZ可以特异性地抑制定位于羧体的CA活性。另外一种形式的CA,具有高I_50(120—190μmol/L),约占CA总活性的15—20%,则有可能定位于细胞质膜。 相似文献