Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences.

Use of the sacB gene (J. L. Ried and A. Collmer, Gene 57:239-246, 1987) provides a simple, effective, positive selection for double recombinants in Anabaena sp. strain PCC 7120, a filamentous cyanobacterium. This gene, which encodes the secretory levansucrase of Bacillus subtilis, was inserted into the vector portion of a suicide plasmid bearing a mutant version of a chromosomal gene. Cells of colonies in which such a plasmid had integrated into the Anabaena chromosome through single recombination were plated on solid medium containing 5% sucrose. Under this condition, the presence of the sacB gene is lethal. A small fraction of the cells from initially sucrose-sensitive colonies became sucrose resistant; the majority of these sucrose-resistant derivatives had undergone a second recombinational event in which the sacB-containing vector had been lost and the wild-type form of the chromosomal gene had been replaced by the mutant form. By the use of this technique, we mutated two selected genes in the chromosome of Anabaena sp. strain PCC 7120. The conditionally lethal nature of the sacB gene was also used to detect insertion sequences from this Anabaena strain. Sucrose-resistant colonies derived from cells bearing a sacB-containing autonomously replicating plasmid were analyzed. Five different, presumed insertion sequences were found to have inserted into the sacB gene of the plasmids in these colonies. One of them, denoted IS892, was characterized by physical mapping. It is 1.7 kilobases in size and is present in at least five copies in the genome of Anabaena sp. strain PCC 7120.     

Isolation, sequence, and expression in Escherichia coli of an unusual thioredoxin gene from the cyanobacterium Anabaena sp. strain PCC 7120.

Two sequences with homology to a thioredoxin oligonucleotide probe were detected by Southern blot analysis of Anabaena sp. strain PCC 7120 genomic DNA. One of the sequences was shown to code for a protein with 37% amino acid identity to thioredoxins from Escherichia coli and Anabaena sp. strain PCC 7119. This is in contrast to the usual 50% homology observed among most procaryotic thioredoxins. One gene was identified in a library and was subcloned into a pUC vector and used to transform E. coli strains lacking functional thioredoxin. The Anabaena strain 7120 thioredoxin gene did not complement the trxA mutation in E. coli. Transformed cells were not able to use methionine sulfoxide as a methionine source or support replication of T7 bacteriophage or the filamentous viruses M13 and f1. Sequence analysis of a 720-base-pair TaqI fragment indicated an open reading frame of 115 amino acids. The Anabaena strain 7120 thioredoxin gene was expressed in E. coli, and the protein was purified by assaying for protein disulfide reductase activity, using insulin as a substrate. The Anabaena strain 7120 thioredoxin exhibited the properties of a conventional thioredoxin. It is a small heat-stable redox protein and an efficient protein disulfide reductase. It is not a substrate for E. coli thioredoxin reductase. Chemically reduced Anabaena strain 7120 thioredoxin was able to serve as reducing agent for both E. coli and Anabaena strain 7119 ribonucleotide reductases, although with less efficiency than the homologous counterparts. The Anabaena strain 7120 thioredoxin cross-reacted with polyclonal antibodies to Anabaena strain 7119 thioredoxin. However, this unusual thioredoxin was not detected in extracts of Anabaena strain 7120, and its physiological function is unknown.     

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。     

人肝金属硫蛋白-I_A基因在鱼腥藻中的克隆与表达

将人工合成的人肝金属硫蛋白（ｍｅｔａｌｏｔｈｉｏｎｅｉｎ,简称ＭＴ）－ＩＡ基因插入至中间载体ｐＲＬ－４３９上强启动子ｐｓｂＡ后,再将其与穿梭载体ｐＫＴ－２１０相连,得到大肠杆菌－蓝藻穿梭表达载体ｐＫＴ－ＭＴ,用三亲接合转移法将ｐＫＴ－ＭＴ转入丝状体蓝藻－鱼腥藻７１２０,经链霉素筛选,得到了稳定的转人肝ＭＴ－ＩＡ基因鱼腥藻．纯化单藻落,液体扩大培养．从鱼腥藻中提取的质粒经Ｓｏｕｔｈｅｒｎ印迹分析,确定人肝ＭＴ－ＩＡ基因已转入鱼腥藻７１２０中,Ｗｅｓｔｅｒｎ印迹分析表明,金属硫蛋白在转人肝ＭＴ－ＩＡ基因鱼腥藻中得到了表达．经原子吸收光谱法测定表达量约为７００μｇＭＴ／ｇ鲜藻,重金属耐受性实验表明,得到了能耐受重金属－镉的转人肝ＭＴ－ＩＡ基因鱼腥藻,它将在清除水域中重金属污染和医药研究方面发挥重要作用．     

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。     

Regulation of Anabaena sp. strain PCC 7120 glutamine synthetase activity in a Synechocystis sp. strain PCC 6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene.

The glnA gene from Synechocystis sp. strain PCC 6803 was cloned by hybridization with the glnA gene from Anabaena sp. strain PCC 7120, and a deletion-insertion mutation of the Synechocystis gene was generated in vitro. A strain derived from Synechocystis sp. strain PCC 6803 which contained integrated into the chromosome, in addition to its own glnA gene, the Anabaena glnA gene was constructed. From that strain, a Synechocystis sp. glnA mutant could be obtained by transformation with the inactivated Synechocystis glnA gene; this mutant grew by using Anabaena glutamine synthetase and was not a glutamine auxotroph. A Synechocystis sp. glnA mutant could not be obtained, however, from the wild-type Synechocystis sp. The Anabaena glutamine synthetase enzyme was subject to ammonium-promoted inactivation when expressed in the Synechocystis strain but not in the Anabaena strain itself.     

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.     

Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli.

The gene encoding thioredoxin in Anabaena sp. strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity. DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E. coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E. coli strain lacking functional thioredoxin. Transformants that complemented the trxA mutation in E. coli were identified by increased colony size and confirmed by enzyme assay. Expression of the cloned Anabaena thioredoxin gene in E. coli was substantiated by subsequent purification and characterization of the algal protein from E. coli. The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin. The E. coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.     

满江红鱼腥藻glnA启动区的克隆及在烟草中的活性表达

利用ＰＣＲ技术获得满江红鱼腥藻ｇｌｎＡ启动区,经克隆测序后构成谷氨酰胺合成酶基因启动子驱动的ＧＵＳ基因表达载体用基因枪经,将表达载体导入烟草中,在其叶片和茎中检测到ＧＵＳ活性,而在烟草根中未见表达。实验结果对真核生物与原核生物间基因表达调控、转录因子识别的研究以及构建衫载体具有一定价值。     

Excision of an 11-kilobase-pair DNA element from within the nifD gene in anabaena variabilis heterocysts.

The 3' region of the Anabaena variabilis nifD gene contains an 11-kilobase-pair element which is excised from the chromosome during heterocyst differentiation. We have sequenced the recombination sites which border the element in vegetative cells and the rearranged heterocyst sequences. In vegetative cells, the element was flanked by 11-base-pair direct repeats which were identical to the repeats present at the ends of the nifD element in Anabaena sp. strain PCC 7120 (Anabaena strain 7120). Although Anabaena strain 7120 and A. variabilis are quite distinct in many ways, the overall sequence similarity between the two strains for the regions sequenced was 96%. Like the Anabaena strain 7120 element, the A. variabilis element was excised in heterocysts to produce a functional nifD gene and a free circularized element which was neither amplified nor degraded. The Anabaena strain 7120 xisA gene is located at the nifK-proximal end of the nifD element and is required for excision of the element in heterocysts. The A. variabilis element also contained an xisA gene which could complement a defective Anabaena strain 7120 xisA gene. A. variabilis did not contain the equivalent of the Anabaena strain 7120 fdxN 55-kilobase-pair element.     

苏云金芽胞杆菌杀蚊基因在鱼腥藻中克隆和表达的初步研究

将苏云金芽胞杆菌以色列亚种的杀蚊晶体蛋白基因cry11A亚克隆到大肠杆菌－蓝藻的穿梭质粒载体pRL25C,然后用三亲本杂交的方法将重组质粒转移到一种具有固氮能力且可被蚊幼虫吞食的鱼腥藻（Anabaena）PCC7120中。Southernblot及Westernblot分析表明cry11A基因在鱼腥藻PCC7120中得以克隆和表达,但生物测定未能检测到转基因鱼腥藻对库蚊（Culex）的毒性,可能是因为带有苏云金芽胞杆菌自身启动子的Cry11A基因在鱼腥藻PCC7120中表达量不够高的缘故。     

Group 3 sigma factor gene, sigJ, a key regulator of desiccation tolerance, regulates the synthesis of extracellular polysaccharide in cyanobacterium Anabaena sp. strain PCC 7120.

The changes in the expression of sigma factor genes during dehydration in terrestrial Nostoc HK-01 and aquatic Anabaena PCC 7120 were determined. The expression of the sigJ gene in terrestrial Nostoc HK-01, which is homologous to sigJ (alr0277) in aquatic Anabaena PCC 7120, was significantly induced in the mid-stage of dehydration. We constructed a higher-expressing transformant of the sigJ gene (HE0277) in Anabaena PCC 7120, and the transformant acquired desiccation tolerance. The results of Anabaena oligonucleotide microarray experiments showed that a comparatively large number of genes relating to polysaccharide biosynthesis were upregulated in the HE0277 cells. The extracellular polysaccharide released into the culture medium of the HE0277 cells was as much as 3.2-fold more than that released by the control cells. This strongly suggests that the group 3 sigma factor gene sigJ is fundamental and conducive to desiccation tolerance in these cyanobacteria.     

Tn5 mutagenesis of Anabaena sp. strain PCC 7120: isolation of a new mutant unable to grow without combined nitrogen.

Methylation by Ava methylases in Escherichia coli increases the efficiency to transfer of Tn5 in pBR322bla:: Tn5 from E. coli to Anabaena sp. strain PCC 7120 by conjugation. Following conjugation, Tn5 but not pBR322 sequences were found at many different positions in the Anabaena chromosome. This procedure was used to mutagenize, tag, and clone a previously unrecognized gene required for nitrogen fixation in this Anabaena sp.     

Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen.

Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1,054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.     

Organization of the hupDEAB genes within the hydrogenase gene cluster of Anabaena sp. strain PCC7120

A 15-kb DNA fragment containing a cluster of hup genes has been identified and cloned from Anabaena sp. strain PCC7120. These genes are located upstream of the hupL gene in the adjacent fragment in the Anabaena chromosome. Sequence analysis of a 3.5-kb HindIII fragment showed the sequence of hupEAB and a part of the hupD gene, all of which showed high sequence similarity with hyp genes of Escherichia coli and hup genes of several nitrogen-fixing bacteria. These genes are oriented in one direction, as are the hup genes of other organisms. Although the Anabaena hupDEAB genes are in the same cluster as the hypABCDE cluster of E. coli, the relative positions of the genes differ and there is no hupC in Anabaena on either side of hupA or hupB. Unlike several other organisms, hupD and hupE are not closely linked or translationally coupled in Anabaena, but are separated by an intergenic space of 453 bp. RT-PCR analysis of RNA obtained from vegetative cells and heterocysts of Anabaena showed that the hupB gene is expressed only in heterocyst-induced cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

人表皮生长因子（hEGF）基因在蓝藻中的表达

人表皮生长因子（hEGF)是由53个氨基酸组成的蛋白,在临床上内服与外敷可促进内外表皮细胞的生长。将人工合成的hEGF基因连接到质粒pRL-489上,位于启动子psb下游。验证连接成功后,用三亲接合转移方法将载体pRL-hEGF导入聚球藻Synechococcus sp.PCC7002和鱼腥藻Anabeana sp.PCC7120。由于pRL-hEGF没有能在单细胞蓝藻中自主复制的复制子,通过筛选,hEGF在聚球藻7002中是整合到蓝藻染色体上进行表达的。用PCR扩增的方法在两种转基因藻中均检测到hEGF基因的存在。放射免疫分析证明,hEGF基因在两种转基因藻中均得到了表达。而且,在聚球藻7002中是采用分泌形式将表达产物分泌到培养液中。     

重组鱼腥藻脂肪氧合酶基因的克隆表达、分离纯化及活性分析

克隆鱼腥藻PCC7120基因组中脂肪氧合酶(ana-LOX)基因,对该功能基因进行了定点突变研究,确定了ana-LOX的最短功能基因长度,构建原核重组表达载体,对重组ana-LOX进行了分离纯化和性质研究。从GenBank中搜索到鱼腥藻PCC7120基因组中含有LOX基因,通过序列分析和比对,发现LOX功能基因位于双功能酶AOS(单加氧酶)-LOX的C端,通过定点突变研究,证实了ana-LOX活性中心位点为His197、His202、His369、Asn373和Ile455。通过逐步缩短基因长度的策略,获得ana-LOX基因的最短功能基因长度为1 254 bp。构建的表达载体pET-32a/ana-LOX转化入BL21(DE3)宿主内,在低温16℃条件下的诱导表达,重组脂肪氧合酶活力可达6 750 U/mL。表达产物通过Ni-NTA亲和柱进行分离纯化,比活达到11.4×104U/mg蛋白,酶活回收率为60.89%。重组ana-LOX最适反应温度45℃,最适反应pH 6.0,在常温下具有较好的稳定性,金属离子Fe2+、Mg2+、Ca2+对该酶存在明显的激活作用,而Fe3+和Cu2+对该酶有强烈的抑制作用。重组ana-LOX能够改善面团的显微结构。该研究获得了高效表达重组ana-LOX,为实现其在食品加工中的应用提供了参考。     

Expression of the Anabaena sp. strain PCC 7120 xisA gene from a heterologous promoter results in excision of the nifD element.

An 11-kilobase-pair element interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. The nifD element normally excises only from the chromosomes of cells that differentiate into nitrogen-fixing heterocysts. The xisA gene contained within the element is required for the excision. Shuttle vectors containing the Escherichia coli tac consensus promoter fused to various 5' deletions of the xisA gene were constructed and conjugated into Anabaena sp. strain PCC 7120 cells. Some of the expression plasmids resulted in excision of the nifD element in a high proportion of vegetative cells. Excision of the element required deletion of an xisA 5' regulatory region which presumably blocks expression in Anabaena sp. strain PCC 7120 vegetative cells but not in E. coli. Strains lacking the nifD element grew normally in medium containing a source of combined nitrogen and showed normal growth and heterocyst development in medium lacking combined nitrogen. The xisA gene was shown to be the only Anabaena gene required for the proper rearrangement in E. coli of a plasmid containing the borders of the nifD element.     

Infrequent cleavage of cloned Anabaena variabilis DNA by restriction endonucleases from A. variabilis.

A cosmid vector has been constructed, using a lambda replicon. A library of cosmids from Anabaena variabilis ATCC 29413 based on use of this vector is shown to be highly deficient in sites for the two type II restriction endonucleases found in that organism.