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1.
旨在建立阿扎霉素F产生菌链霉菌211726的基因转移系统,以便基因敲除和外源基因表达等遗传操作。以整合型质粒pSET152和pIB139为出发质粒,通过接合转移构建了阿扎霉素F产生菌链霉菌211726的基因转移系统。结果显示25μg/mL阿泊拉霉素可有效筛选接合子。经PCR验证,质粒成功整合到菌株链霉菌211726基因组中,接合子经多次传代后,导入的质粒pSET152和pIB139仍稳定整合于接合子基因组上。  相似文献   

2.
以经理化诱变选育的多杀菌素高产菌株刺糖多孢菌(Saccharopolyspora spinosa)CB11为受体菌,将具有安普霉素(apramycin)抗性标记的整合型载体pSET152作为质粒供体,研究了制备刺糖多孢菌感受态细胞时菌体的生长阶段、质粒DNA浓度、电场强度等因素对电转化效率的影响,结果表明,在菌体培养4...  相似文献   

3.
【目的】建立并优化链霉菌Fostriecin产生菌Streptomyces pulveraceus的遗传转化系统。【方法】以整合型质粒pSET152为出发质粒,通过供体菌E.coli ET12567/pUZ8002与受体菌Streptomyces pulveraceus进行接合转移。【结果】确定了链霉菌Streptomyces pulveraceus的最佳接合转移条件:培养基为终浓度含15%甘氨酸的MS培养基;孢子热激条件为50°C 10 min;阿伯拉霉素覆盖的时间为18 h,终浓度为20 mg/L。同时,把组成型启动子ermE+与绿色荧光蛋白基因(gfp)克隆到pSET152载体上,通过接合转移整合到该链霉菌中,gfp获得表达。【结论】建立Fostriecin产生菌的遗传转化系统,并发现甘氨酸能显著提高链霉菌的接合转移效率。  相似文献   

4.
小单孢菌40027菌株噬菌体的分离及其生物学特性的研究   总被引:1,自引:0,他引:1  
以福堤霉素A产生菌──小单孢菌40027菌株为指示菌,从土壤中分离得到三株噬菌体:ΦHAU7、ΦHAU9和ΦHAU11。三株噬菌体的寄主专一性较强,在测试的15株放线菌菌株中,三株噬菌体能感染小单孢菌40027菌株和A-M-01菌株,ΦHAU9和ΦHAU11还能感染蔷薇小单孢菌(Micromonospora purprea)。三株噬菌体都是由多面体的头部和尾部组成;形成噬菌斑时培养基中适宜的Ca2 、Mg2 浓度分别为32mM和30mM;ΦHAU7在储存液中适宜的pH范围为6~12,而其它两株噬菌体的适宜的pH范围为6~10;在储存过程中三株噬菌体适宜的温度范围为28~37℃,经60℃保温30min后,除ΦHAU7仍有53%活力之外,其它两株噬菌体全部失活。限制性内切酶酶切结果表明:三株噬菌体基因组DNA均为双链DNA;基因组大小分别约为60kb、58kb和55kb。高压脉冲电泳结果揭示:三株噬菌体基因组DNA均具有粘性末端。  相似文献   

5.
为了建立适合米根霉的遗传转化体系,应用重叠延伸PCR的方法构建了以潮霉素B抗性为选择标记的单交换整合型表达载体p BS-hygro-ldh A;分别采用PEG/Ca Cl2介导的原生质体转化、原生质体电转化及萌发孢子电转化的方法将表达载体p BS-hygro-ldh A转化入米根霉AS 3.819菌株中,并研究了菌丝酶解时间、孢子萌发时间以及电转化电场强度对于转化效率的影响;通过荧光定量PCR(q PCR)对米根霉转化子基因组中质粒整合拷贝数进行了检测,并研究了其对米根霉转化子抗性稳定性的影响。实验结果表明成功获得整合了表达载体p BS-hygro-ldh A的米根霉转化子。菌丝酶解140 min产生的原生质体其再生率和转化率最高,原生质体电转化最佳电场强度为13 k V/cm,孢子萌发2.5 h转化率最高,萌发孢子电转化最佳电场强度为14 k V/cm。萌发孢子电转化方法转化率要高于原生质体转化的方法。荧光定量PCR检测结果表明,在一定范围内,高质粒整合拷贝数的米根霉转化子比较稳定。研究建立了用于工业米根霉菌株的遗传转化体系,为米根霉代谢调控研究以及菌种改造工作提供了基础与支持。  相似文献   

6.
研究避免了繁琐的原生体制备过程,直接使用萌发的黑曲霉孢子进行电转化,以潮霉素B作为筛选标记,从孢子萌发时间、电场强度及质粒浓度等方面考察了电转化效率的影响因素。研究表明,针对A.nigerMGG029-ΔaamA,其理想的电转化条件:孢子龄为4d,孢子萌发时间为2h,电场强度为5kV/cm。在上述条件下分别使用1μg环状或线状pBC-Hygro质粒DNA进行转化,平均可以得到34个和51个转化子,而在同样条件下使用质粒pRS303H平均可以获得163个和258个转化子。  相似文献   

7.
小单孢菌属放线菌是许多生物活性物质的重要来源,但自然条件下小单孢菌的活性产物产率普遍偏低,基因编辑与改造对提高小单孢菌属放线菌活性产物的产率具有重要意义。然而,有效的遗传转化体系成为小单孢菌属放线菌基因编辑改造的瓶颈。炭样小单孢菌JXUN-1是实验室从南昌瑶湖农田土壤样品中分离到的一株具有广谱抗菌活性的放线菌,其基因组具有GC含量高的特点。本研究以敲除炭样小单孢菌JXNU-1中抗生素合成相关基因P450为例,以温敏型质粒pKC1139为模板,构建了炭样小单孢菌JXNU-1 P450基因打靶载体p FD306,然后通过电转化将pFD306导入炭样小单孢菌JXNU-1新鲜菌丝体内,通过双交换获得基因缺失株炭样小单孢菌JXNU-ΔP450,最后通过PCR验证了菌株P450基因的缺失,表明炭样小单孢菌JXNU-1基因打靶载体pFD306构建成功。本研究确证了质粒pKC1139可以用于炭样小单孢菌JXNU-1基因组的编辑,为炭样小单孢菌JXNU-1抗生素合成相关基因的筛选及其功能研究提供有效帮助。  相似文献   

8.
从我国各地的土壤、堆肥及粪肥中分离到一批嗜热小单孢菌,经52℃培养,其中’524菌株在固体培养基上有发育良好的基内菌丝体,在基内菌丝体上着生茄子形孢子,细胞壁组分II型,含内消旋二氨基庚二酸、甘氨酸。此菌株为罕见的嗜热菌株,区别于已报道的小单孢菌属中的种,故认为是个新种,命名为热茄孢小单孢菌(Micromonospora thermoauberginospora n.sp.)。  相似文献   

9.
小单孢菌属的一个新种   总被引:1,自引:1,他引:0       下载免费PDF全文
从云南省丽江地区的高寒山区采集的土样中分离到两株小单孢菌Y81—917和Y81一558。它们不产生气生菌丝体。基内菌丝体蓝色。产生蓝色可扩散色素。孢子单个着生,表面皱褶。细胞壁化学组分II型。它们与所有已知的小单孢菌都不同,认为是小单孢菌属中的一个新种,定名为玉龙小单孢菌(Micromonospora yulongensis n. sp.),菌株Y81-917为模式株。  相似文献   

10.
福堤霉素A产生菌——小单孢菌40027菌株含有两个质粒pJTU101和pJTU 112.[目的]对质粒pJTU 112复制区进行克隆,并对质粒pJTU 112复制区序列进行测定和分析.[方法]克隆质粒pJTU 112的不同DNA片段导入消除质粒的小单孢菌40027菌株,通过复制功能的测定,确定质粒pJTU 112的复制区,并进行测序和生物信息学分析.[结果]质粒pJTU 112的复制区定位在约4.7 kb的SacI-KpnI DNA片段上,测序和生物信息学分析表明:4.7 kb的SacI-KpnI DNA片段包含5个ORFs(open reading frames),其中pJTU 112.1和pJTU 112.2与质粒接合转移有关,pJTU112.3、pJTU112.4和pJTU112.5与质粒复制有关.[结论] 质粒pJTU112的复制区定位在约4.7 kb的SacI-KpnI DNA片段上.  相似文献   

11.
Vector systems allowing autonomous or site-specific integrative gene cloning were developed for Micromonospora sp. strain 40027, a producer of the antibiotic fortimicin A. The autonomous system depends on the discovery of a low-copy-number, self-transmissible covalently closed circular plasmid, pJTU112 (ca. 14.1 kb), which was shown to be present in the progenitor strain in both integrated and autonomous states. The copy numbers of both wild-type pJTU112 and three derivatives of it can be amplified at least sixfold by addition of streptomycin to the culture medium. The integrative system was developed by the use of a pBR322-derived Escherichia coli plasmid vector, pSET152, mediated by the attP site of the Streptomyces phage PhiC31. Both vectors can be transferred by conjugation from E. coli into Micromonospora sp. strain 40027. The heterologous cloning and expression of the dnd gene cluster originating from Streptomyces lividans 1326 into Micromonospora sp. strain 40027 demonstrated the use of the two systems.  相似文献   

12.
The integrase from the Streptomyces phage (phi)C31 is a member of the serine recombinase family of site-specific recombinases and is fundamentally different from that of lambda or its relatives. Moreover, (phi)C31 int/attP is used widely as an essential component of integration vectors (such as pSET152) employed in the genetic analysis of Streptomyces species. phiC31 or integrating plasmids containing int/attP have been shown previously to integrate at a locus, attB, in the chromosome. The DNA sequences of the attB sites of various Streptomyces species revealed nonconserved positions. In particular, the crossover site was narrowed to the sequence 5'TT present in both attP and attB. Strains of Streptomyces coelicolor and S. lividans were constructed with a deletion of the attB site ((Delta)attB), and pSET152 was introduced into these strains by conjugation. Thus, secondary or pseudo-attB sites were identified by Southern blotting and after rescue of plasmids containing DNA flanking the insertion sites from the chromosome. The sequences of the integration sites had similarity to those of attB. Analysis of the insertions of pSET152 into both attB(+) and (Delta)attB strains indicated that this plasmid can integrate at several loci via independent recombination events within a transconjugant.  相似文献   

13.
Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the phiC31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of phiC31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.  相似文献   

14.
Vector systems allowing autonomous or site-specific integrative gene cloning were developed for Micromonospora sp. strain 40027, a producer of the antibiotic fortimicin A. The autonomous system depends on the discovery of a low-copy-number, self-transmissible covalently closed circular plasmid, pJTU112 (ca. 14.1 kb), which was shown to be present in the progenitor strain in both integrated and autonomous states. The copy numbers of both wild-type pJTU112 and three derivatives of it can be amplified at least sixfold by addition of streptomycin to the culture medium. The integrative system was developed by the use of a pBR322-derived Escherichia coli plasmid vector, pSET152, mediated by the attP site of the Streptomyces phage ΦC31. Both vectors can be transferred by conjugation from E. coli into Micromonospora sp. strain 40027. The heterologous cloning and expression of the dnd gene cluster originating from Streptomyces lividans 1326 into Micromonospora sp. strain 40027 demonstrated the use of the two systems.  相似文献   

15.
The site-specific recombination system used by the Streptomyces bacteriophage phiC31 was tested in the fission yeast Schizosaccharomyces pombe. A target strain with the phage attachment site attP inserted at the leu1 locus was co-transformed with one plasmid containing the bacterial attachment site attB linked to a ura4+ marker, and a second plasmid expressing the phiC31 integrase gene. High-efficiency transformation to the Ura+ phenotype occurred when the integrase gene was expressed. Southern analysis revealed that the attB-ura4+ plasmid integrated into the chromosomal attP site. Sequence analysis showed that the attBxattP recombination was precise. In another approach, DNA with a ura4+ marker flanked by two attB sites in direct orientation was used to transform S. pombe cells bearing an attP duplication. The phiC31 integrase catalyzed two reciprocal cross-overs, resulting in a precise gene replacement. The site-specific insertions are stable, as no excision (the reverse reaction) was observed on maintenance of the integrase gene in the integrant lines. The irreversibility of the phiC31 site-specific recombination system sets it apart from other systems currently used in eukaryotic cells, which reverse readily. Deployment of the phiC31 recombination provides new opportunities for directing transgene and chromosome rearrangements in eukaryotic systems.  相似文献   

16.
The genome of the Streptomyces temperate phage phiC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes. Previously, it was demonstrated that, in an in vitro recombination assay, phiC31 integrase catalyses integration (attP/attB recombination) but not excision (attL/attR). The mechanism responsible for this recombination site selectivity was therefore investigated. Purified integrase was shown to bind with similar apparent binding affinities to between 46 bp and 54 bp of DNA at each of the attachment sites, attP, attB, attL and attR. Assays using recombination sites of 50 bp and 51 bp for attP and attB, respectively, showed that these fragments were functional in attP/attB recombination and maintained strict site selectivity, i.e. no recombination between non-permissive sites, such as attP/attP, attB/attL, etc., was observed. Using bandshifts and supershift assays in which permissive and non-permissive combinations of att sites were used in the presence of integrase, only the attP/attB combination could generate supershifts. Recombination products were isolated from the supershifted complexes. It was concluded that these supershifted complexes contained the recombination synapse and that site specificity, and therefore directionality, is determined at the level of stable synapse formation.  相似文献   

17.
PhiHAU8, a temperate Micromonospora phage, which is capable of infecting Micromonospora sp. strains 40027 and A-M-01, was isolated. The PhiHAU8 virion has a polyhedral head and a flexible tail and has a small genome (ca. 42.5 kb) with double-stranded DNA and cohesive ends. PhiHAU8 was most stable at 4 degrees C in Difco nutrient broth within a pH range of 6 to 12. PhiHAU8 plaque formation on Micromonospora sp. strain 40027 was optimal with 32 mM Ca(2+) and 30 mM Mg(2+). A lysogen, LXH8, was isolated from turbid plaques, and a phasmid derivative that functions as a lambda cosmid vector in Escherichia coli and as a phage in Micromonospora sp. strain 40027 was constructed. Pulsed-field gel electrophoresis of AseI-digested total DNA showed that PhiHAU8 DNA integrates into the 500-kb AseI fragment of Micromonospora sp. strain 40027.  相似文献   

18.
Streptomyces phage phiC31 integrase was found to mediate site-specific integration of foreign genes at pseudo attP sites of genomes in human, mouse, rat, and Drosophila. This paper reports that phiC31 integrase can also mediate homologous recombination between attB and pseudo attP sites in bovine cells and foreign gene integration was increased at least 2-fold in bovine fibroblasts or Madin-Darby bovine kidney (MDBK) cells. Two intrinsic pseudo attP sites named BpsF1 and BpsM1 located in the inter-gene regions on chromosome 28 and 19, respectively, were identified in bovine genome. These pseudo attP sites shared similar characteristics with those from other species as previously described. Our study demonstrated that the phiC31 integrase system provides a new potential for genetic engineering of the bovine genome and might be beneficial for the research on ruminant.  相似文献   

19.
Allen BG  Weeks DL 《Nature protocols》2006,1(3):1248-1257
Bacteriophage phiC31 produces the enzyme integrase that allows the insertion of the phage genome into its bacterial host. This enzyme recognizes a specific DNA sequence in the phage (attP) and a different sequence in the bacterium (attB). Recombination between these sites leads to integration in a reaction that requires no accessory factors. Seminal studies by the Calos laboratory demonstrated that the phiC31 integrase was capable of integrating plasmid with an attB site into mammalian genomes at sites that approximated the attP site. We describe the use of attB-containing plasmids with insulated reporter genes for the successful integration of DNA into Xenopus embryos. The method offers a way to produce transgenic embryos without manipulation of sperm nuclei using microinjection methods that are standard for experiments in Xenopus laevis. The method aims to allow the non-mosaic controlled expression of new genetic material in the injected embryo and compares favorably with the time that is normally taken to analyze embryos injected with mRNAs, plasmids, morpholinos or oligonucleotides.  相似文献   

20.
Groth AC  Fish M  Nusse R  Calos MP 《Genetics》2004,166(4):1775-1782
The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.  相似文献   

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