链霉菌科分类的研究III．链霉菌科中的一个新属

从我国云南省西双版纳热带植物研究所的土壤中分离到二株菌,编号80—56、80 57。该菌株气丝形成非轮生的孢子链,基内菌丝体不断裂,形态与培养特征与链霉菌属基本相似。但由于细胞壁水解物中含有…o一二氨基庚二酸、甘氨酸。全细胞水解物中含有半乳糖,与链霉菌属胞壁组分含LL二氨基庚二酸、甘氨酸,不含特征性糖有明显区别。故菌株80—56、80一57不能归人到过去任何一个放线菌属中,因此建立新属——类链霉菌属(Streptomycoides n.gen．),此二菌株为该新属的代表种,定名为青黄类链霉菌(Streptomycoides glaucoflavus n.sp．),典型菌株80—56。     

链霉菌YIM69228发酵液中化学成分的研究

对链霉菌YIM69228发酵液进行了化学成分的研究,从中分离得到十个化合物。根据波谱数据分析,鉴定其结构分别为:(S)-2-羟基-3-(4-甲氧基苯基)-丙酸(1),异莨菪亭(2),N-苯乙基乙酰胺(3),肉桂酸(4),丁二酸单甲酯(5),3,4-二羟基苯甲酸(6),海藻糖(7),吐叶醇(8),Citroside A(9),苄基-β-D-吡喃木糖基-(1″-6’)-β-D-吡喃葡萄糖苷(10)。首次鉴定了天然产物化合物1的构型,其中化合物8～10为首次从链霉菌属放线菌中分离得到。     

链霉菌育种进展

链霉菌是放线菌中最重要的一属,它产生绝大部分抗生素和其它众多活性物质。随着分子生物学的发展,链霉菌育种技术也日益多样。本文从基因突变与育种,遗传重组与育种、基因表达调控与代谢调节育种、基因工程与定向育种等四个方面综述链霉菌育种的进展。     

链霉菌原生质体的再生

（续１９９８年第３３卷第１期第４１页）３原生质体的再生链霉菌原生质体的再生是指经去壁的原生质体在高渗的再生培养基上,一方面再生出原有的细胞壁,恢复菌丝原来的完整形态,另一方面恢复细胞的生理功能,保证细胞萌发、生长和分裂。原生质体再生过程有以下几个步骤...     

链霉菌原生质体的制备

原生质体是细胞去除了坚韧细胞壁后对渗透压极为敏感的球质体,最外层是裸露的细胞质膜,失去了细胞壁的原生质体,染色体ＤＮＡ在诱变剂作用下,更易引起死亡突变,敏感性提高。菌种的敏感性越高,诱发突变的机会越大。因此,用原生质体代替孢子、菌丝细胞作为诱变育种的...     

链霉菌属的两个新种

从陕西省武功县的土壤中分离到二株链霉菌,编号为78一113和78一118。其气生菌丝体分别为淡蓝及浅灰蓝,基内菌丝体为蓝色和棕色。螺旋形孢子丝随着培养过程逐渐盘卷成球形或亚球形的团状体,未观察到硬的孢囊壁,类似假孢囊,盘卷的分生孢子链清晰可见。经鉴定,认为是链霉菌属的两个新种,命名为蓝色假孢囊链霉菌(Streptomyces cyaneo-pseudosporangiusn.sp.)和微蓝灰假孢囊链霉菌(Streptomyres plumbeo-pseudosporangius n．Sp．)。     

粉红孢链霉菌的两个新种

从山东省和西安市的土样中,分离到3株气丝为粉红色调的链霉菌,编号为0769、01762和01 763。经形态、培养特征和生理生化特性的研究,它们与已知的近似种均不相同,因此定为新种,命名为玫瑰暗红链霉菌(Streptomyces roseoerythraeus n.sp.)(0769)和玫瑰肉色链霉菌(Streptomyces roseocarneus n.sp．)(01 762、01763,其中以01 762为标准株)。     

链霉菌分类研究进展

自从Waksman和Henrici[1]于1943年建立链霉菌属(Streptomyces)以来,链霉菌已成为细菌域中种的数量最大的一个属.目前,包括出现在专利中的种已经超过3000个,至1996年已有有效描述的种464个和亚种45个[2].由于链霉菌是产生抗生素等生物活性物质种类最多的一类微生物,因此至今各国学者仍在不断研究设计新的分离、培养、鉴定和分类方法,以指导新的天然生物活性物质的寻找.但长期以来,国际上因缺少可以接受的统一的分类鉴定标准,导致了链霉菌分类中的混乱.     

链霉菌ACCC40021的多基因系统进化分析

链霉菌ACCC40021是尹莘耘1953年分离筛选,用于农业生产的抗生菌肥。该菌株1979年经鉴定为泾阳链霉菌(Streptomyces jingyangensis),但该菌株名称一直没有在国际上得到有效发表。为了在分子水平上明确该菌株的分类地位,对ACCC40021的菌株进行了16SrRNA和gyrB,recA,rpoB和trpB等基因的进化分析,将ACCC40021鉴定为黄赭色链霉菌(Streptomyces silaceus)。     

Rapid identification of Rhizobium strains by targeted PCR fingerprinting

Numerous polymerase chain reaction (PCR) based procedures are routinely used to produce genomic fingerprints of prokaryotes. Many of them have drawbacks however such as sensitivity to experimental variation, lack of reproducibility, poor resolution and the inability to distinguish between closely related strains. To overcome these difficulties, we developed an alternative procedure, Targeted PCR Fingerprinting (TPF) which is based upon the amplification of few but carefully selected markers, followed by high resolution RFLP analysis of the amplified DNA fragments. In contrast to most fingerprinting protocols that use low resolution agarose gels, TPF patterns are produced on denaturing polyacrylamide gels which allow the precise recording of the genomic fingerprints. TPF analysis, which can simultaneously process 96 samples in less than 12 h and remains unaffected by slight experimental variations, is particularly adapted for the rapid identification of target strains amongst many field isolates. Using PCR primers specific for the nifH and recA genes, this procedure was also sufficiently sensitive to discriminate between Rhizobium species NGR234 and R. fredii USDA257, two closely related bacteria in which the symbiotic loci are 98% homologous. Interestingly, comparison of several of their symbiotic genes as well as the partial DNA sequences of their 16S rDNA and recA genes suggest that chromosomes and symbiotic plasmids did not co-evolve, but that symbiotic functions were acquired by lateral gene transfer long after NGR234 and USDA257 diverged from their common ancestors. In this respect, TPF fingerprints produced with distinct chromosomal and plasmid born markers, such as the recA and the nifH genes in NGR234 and USDA257, are probably more likely to detect lateral transfer of genes in bacterial field-populations than procedures relying on the amplification of numerous fragments of unknown genomic position and biological function.     

海洋放线菌Streptomyces sp.大片段DNA基因组文库的构建

目的:构建稀有海洋放线菌Streptomyces sp.基因组文库.方法:以稀有海洋放线菌Streptomyces sp.为实验材料,随机剪切提取的总DNA,5'-磷酸末端补平回收40kb左右的DNA片段,与pWEBTM载体连接,经包装蛋白包装成噬菌体后侵染宿主细胞E.coli EPI100,构建该菌株的基因组文库,并对该文库进行质量鉴定.结果:成功构建了稀有海洋放线菌Streptomyces sp.的基因组文库,效价达9.0×104CFU/mL,得到4000个阳性克隆子,远远大于按覆盖率为99%计算至少所需的837个阳性克降子数,且平均插入片段长度为36kb,重组率100%.阳性克隆子保存于96孔板中,-80℃保存.结论:所构建文库的各项指标均达到要求,为了进一步评估Streptomyces sp.所能合成的所有潜在天然产物,还需要进一步检测该文库中包含有生物合成基因簇的大肠杆菌的表达情况.     

山蛭基因组DNA的提取及其PCR扩增产物的分析

利用PCR技术从海南山蛭体内分离山蛭素（抗凝血蛋白）基因,首先需获得不带有山蛭体表色素且完整的山蛭基因组DNA,本试验通过结合使用SDS-蛋白酶裂解法和CTAB法,有效的去除了山蛭基因组DNA提取过程中难以去除的色素,得到的基因组DNA保持完整,无降解,以之作为模板,进行PCR扩增,获得一个长度约为237bp的特异性片段,此片段与印度山蛭蛭素的cDNA大小几乎一致。初步表明,这个序列没有内含子,而仅是海南山蛭蛭素的编码序列。     

Mammalian species identification by interspersed repeat PCR fingerprinting

Most DNA methods for species identification of animal tissues test the presence/absence of one species per assay, requiring several tests for a complete analysis and prior knowledge of the species that are potentially present in the sample. Here we demonstrate that PCR with fluorescently labeled MIR (mammalian-wide interspersed repeat) primers generate fingerprints that are suitable for rapid identification of known and unknown species on an automatic sequencing apparatus and with computer-assisted data processing. The method allows the analysis of processed meat samples and offers a convenient alternative to sequencing of mitochondrial DNA. Received 19 December 1997/ Accepted in revised form 15 June 1998     

快速PCR研究进展

PCR是最常用的分子生物学技术之一,通过变性、退火和延伸的循环来完成核酸分子的大量扩增。快速PCR就是基于普通PCR的工作原理,在保证PCR反应特异性、灵敏性、保真度的前提下,在更短时间内完成对核酸分子的扩增。近年来已经开展了许多有关方面的研究工作。本文将以DNA聚合酶的改进、添加剂的选择、热循环仪改进为重点内容,综述快速PCR技术的研究进展。     

实时定量PCR法检测生物技术药物中宿主基因组DNA残留

利用基于SYBR GreenⅠ荧光染料的实时定量PCR方法检测酵母表达生物技术药物产品中宿主DNA残留量。该方法检测灵敏度可达到1.0 fg/μL, DNA浓度在1.0 fg/μL～1.0 ng/μL范围内线性良好,其标准曲线的相关系数为099以上。应用该方法对3批不同实验样本进行测定,宿主DNA残留量分别为8.635×105 fg/μL、6.265×102 fg/μL和1436 fg/μL 。实验表明该方法操作简便、灵敏度高,可用于生物技术药物产品中酵母DNA残留的定量测定。     

Genomic and Phenotypic Analyses of Microorganisms Isolated from the Sediments of Lake Baikal

Genetic and biochemical methods and morphological examination were used to study microorganisms isolated from samples of deep drilling of the Lake Baikal bottom sediments. Based on blot hybridization patterns, the strains investigated were divided into several groups according to the degree of homology of their genomic DNA. Morphological, biochemical, and ultrastructural characteristics of bacterial strains are described, and their compliance with the genomic analysis data is demonstrated.     

Genomic fingerprints of Salmonella species generated with repetitive element sequence-based PCR

We describe the use of repetitive element sequence-based PCR (rep-PCR) on the two repetitive sequences, REP and ERIC elements, to distinguish members of closely related Salmonella species. Within the species, ERIC–PCR showed a higher discriminative potential than REP–PCR, but by using a combination of the two PCR methods it was possible to distinguish all the isolates examined. The rep-PCR fingerprints of Salmonella organisms were distinctly different from some Gram-positive bacteria, for example Staphylococcus, Bacillus megaterium, and even the closely related Escherichia coli and Serratia marcescens. Identical fingerprints were observed with whole-cell preparations. Rapid specimen preparation has enhanced the value of rep-PCR in timely analysis of epidemiological relationships.     

Identification, isolation and sequencing of the recA gene of Streptomyces lividans TK24

Abstract An internal fragment of the recA gene of Streptomyces cattleya was amplified by the polymerase chain reaction (PCR) employing degenerate oligonucleotide primers. Using this fragment as a hybridization probe, a recA homologous gene could be shown in each tested Streptomyces strain. A 4.4 kb Bam HI fragment which carried the complete recA gene was isolated from Streptomyces lividans TK24. Sequence analysis suggested that the coding region of the recA gene consists of 1122 bp. The highest similarity (∼78%) could be detected to the recA genes of Mycobacterium tuberculosis and Mycobacterium leprae . After fusion with an E. coli promoter the S. lividans recA gene could partially complement an Escherichia coli recA mutant.