放线菌线型复制子新的生物学功能及其应用

原核生物的染色体和质粒DNA一般为环型结构。在过去的几十年里 ,人们以不同的材料为研究对象建立了原核生物环型染色体和环型质粒生物学功能的模式体系。近年来 ,在链霉菌属 (Streptomyces)中发现 1 2～ 1 70 0kb的线型质粒[1 ] 和约 80 0 0kb的线型染色体[2 ] ,有的巨大线型质粒上还带有完整的抗生素生物合成基因簇[1 ] 。与链霉菌属同属于放线菌目 (Actinomycetales)的诺卡氏菌属 (Nocardia)和红球菌属(Rhodococcus)中也发现了线型染色体和线型质粒 ,并揭示了降解多种工业上有毒化合物的基因常由线型质粒所携带[3,4 ] 。功能研究表明 ,…     

游动双孢菌线型质粒pPR2的全序列测定及分析

[目的]获得游动双孢菌线型质粒pPR2的全序列,并揭示新型的端粒复制蛋白和可能的中间复制位点.[方法]用分段克隆的方法和序列拼接获得pPR2的全序列,利用软件分析端粒DNA的二级结构和可能的端粒复制蛋白,利用链霉菌原生质体转化的方法检测可能的中间复制的位点.[结果]pPR2全长为15520 bp,(G C)含量为68.1%.其端粒末端反向重复序列的长度为329 bp,不能像多数链霉菌的线型质粒那样能形成保守的"折返"的二级结构.pPR2虽然没有参与链霉菌端粒复制的保守的tap/tpg基因,但是pPR2.3c基因编码了一个双结构域蛋白,分别同链霉菌的端粒复制相关蛋白Tap和嗜血杆菌的解旋酶具有相似性.pPR2缺少典型的链霉菌重复序列-复制基因(iteron-rep)区段,将几乎覆盖全长pPR2的两段DNA进行克隆后,不能转化变铅青链霉菌.此外,pPR2基因还编码可能参与线型DNA复制的调控的单链结合蛋白(SSB)和与放线菌质粒接合转移相关的主要蛋白(Tva).[结论]pPR2是链霉菌之外的放线菌中最小的线型质粒,其序列在游动双孢菌属的线型质粒中是首次报道.pPR2可能具有新型的端粒复制的机制,其中pPR2.2c和pPR2.3c编码可能的端粒复制蛋白.     

拟诺卡氏菌线型质粒pNPL1的测序和分析

【目的】检测和分析稀有放线菌中新的线型质粒。【方法】从植物内生菌中分离链霉菌之外的放线菌菌株,检测、测序和分析线型质粒。【结果】从中草药植物紫花前胡的叶片中分离到一株内生放线菌25L-1-1c,经过16S rRNA基因序列比对属于拟诺卡氏菌。从该菌株中检测到一个约25 kb的线型质粒pNPL1。克隆和测序了pNPL1新的端粒,含有多个小的回文序列。测序获得全长为24 621 bp的线型质粒pNPL1,预测编码22个基因,其中2个基因与链霉菌质粒的端粒复制基因同源,1个基因与链霉菌质粒主要的接合转移基因相似,其余19个基因为未知功能。携带pNPL1端粒复制基因的质粒不能转化变铅青链霉菌,暗示需要发展拟诺卡氏菌的遗传操作系统。【结论】这是首次在拟诺卡氏菌中发现和描述线型质粒。     

五个链霉菌线型质粒端粒的克隆和分析

本室从西藏采集的土壤样品中分离到了一批链霉菌,利用脉冲电泳确定了其中5株链霉菌含有较小的线型质粒。【目的】克隆、测序和分析5个线型质粒的端粒。【方法】采用改良的"在凝胶中进行DNA碱处理和限制性内切酶酶切"的方法来克隆线型质粒的端粒DNA。【结果】克隆和测序了5个线型质粒的端粒DNA。通过与链霉菌典型端粒进行比较,发现这5个新的线型质粒的端粒序列同样含有多个回文序列。但是有的端粒保守的回文序列I不一定能够"折返"与内部序列配对形成"超级发卡"结构,回文序列的"突出环"不一定都为3nt。【结论】采用改良的方法克隆和鉴定了5个线型质粒新的端粒序列,这些新端粒的特征暗示:回文序列I的"折返"和3nt的回文序列的"突出环"不是端粒复制必需的。     

西藏土壤中分离的链霉菌含有丰富的线型和环型质粒

【目的】研究极端自然环境对链霉菌线型和环型质粒分布的影响。【方法】从西藏高原采集了20份土壤样品,分离和初步鉴定链霉菌,提取和检测质粒DNA。【结果】从中分离到46株链霉菌,其中有23株菌含有1 4个线型质粒,大小在19 650 kb之间,8个菌株含有1 4个环型质粒,大小在4 80 kb之间。【结论】西藏土壤来源的链霉菌含有大量的、多样的线型质粒和环型质粒,暗示极端环境中诸如强紫外辐射等可能会引发DNA损伤和修复,进而造成质粒的多样性。     

链霉菌质粒的复制与进化

近年来, 随着大质粒提取和检测技术的发展, 尤其是高通量DNA测序技术的应用, 使得链霉菌大的环型质粒和线型质粒的研究取得了较快的进展。相比于研究透彻的细菌Theta型复制的质粒, 链霉菌Theta型质粒在复制区的结构、复制蛋白和调控蛋白作用的分子机理等方面具有多样性和新颖性。新鉴定的许多线型质粒的中心复制区表明中心复制的起始可以靠近端粒, 一个质粒也可以有2个以上的复制区。新分离的端粒序列显示端粒“折返”不是必需的, 而形成二级结构对于端粒复制是重要的。链霉菌环型和线型质粒的测序分析显示它们之间存在亲缘关系。环型质粒可以与噬菌体共整合, 实验证明它们在一定条件下可以相互转换。这些研究结果表明, 链霉菌环型、线型质粒和噬菌体从结构到功能到进化具有多样性、新颖性和亲缘关系。     

小葱植物内生链霉菌线型质粒pYY8L的端粒与复制区的克隆和鉴定

从小葱植物中分离到一株编号为36R-2-1B的链霉菌菌株,该菌株含有一个约为280kb的线型质粒pYY8L。【目的】克隆、测序和分析pYY8L新的端粒和复制区。【方法】采用改良的"在凝胶中进行DNA碱处理与酶切"的方法来克隆大的线型质粒pYY8L的端粒,通过构建基因组柯斯文库和次级克隆的方法来缩小和鉴定pYY8L的复制区。【结果】在小葱植物内生链霉菌36R-2-1B中检测到约为280kb的线型质粒pYY8L,克隆了pYY8L的端粒。其末端的152bp包含6个小的回文序列,可以形成复杂的二级结构。利用柯斯文库构建、次级克隆和测序获得了4891bp的pYY8L的复制区。该复制区含有6个基因,其中2个与天蓝色链霉菌线型质粒SCP1的复制基因非常相似,但是邻近的重复序列不同。【结论】采用新的改良的方法克隆和鉴定了pYY8L新的端粒和复制区。本文首次报道了植物内生链霉菌线型质粒的端粒和复制基因。     

高温对刺孢吸水链霉菌生长及抗菌物质合成的影响

自1970年梅泽等证明链霉菌中存在质粒并与抗生素合成有关以来,放线菌质粒的研究工作进展迅速。我们在工作中发现高温对刺孢吸水链霉菌(Streptomyces hygrospinosus)的生长及抗菌物质合成有显著影响。本文报告提示该菌中存在质粒的实验结果。     

酸性和碱性土壤新分离的放线菌中线型和环型质粒的检测

从湖南、云南采集的19份酸性土壤样品分离到50株放线菌,在pH4.5和pH7.0的培养基上都能生长.从新疆、青海、云南的14份碱性土壤样品中分离到38株放线菌,在pH10.0和pH7.0的培养基上均能生长.因此,这些菌株属于中度嗜酸或中度嗜碱放线菌.利用脉冲电泳技术和普通凝胶电泳在其中12株放线菌中检测到线型质粒,其中3株放线菌中检测到环型质粒.这是首次在中度嗜酸和中度嗜碱的放线菌中报道线型和环型质粒.     

水稻植物内生链霉菌中线型和环型质粒的检测

以广东番禺和五山地区水稻植株中分离到的内生链霉菌为对象,调查可能存在的内源质粒.利用脉冲电泳技术从8个菌株中检测到大小在60 kb～410 kb的线型质粒,其中4个菌株的线型质粒可能有保守的端粒复制基因.该结果与土壤链霉菌中检测到线型质粒和具有保守端粒复制基因的比例相似,表明水稻植物组织内部的独特环境不会造成链霉菌线型质粒的多样性分布产生大的变化.此外,从13个菌株中检测到6 kb～60 kb的环型质粒.     

Chromosome diversity and similarity within the Actinomycetales

Many chromosomes from Actinomycetales, an order within the Actinobacteria, have been sequenced over the last 10 years and the pace is increasing. This group of Gram-positive and high G+C% bacteria is economically and medically important. However, this group of organisms also is just about the only order in the kingdom Bacteria to have a relatively high proportion of linear chromosomes. Chromosome topology varies within the order according to the genera. Streptomyces, Kitasatospora and Rhodococcus, at least as chromosome sequencing stands at present, have a very high proportion of linear chromosomes, whereas most other genera seem to have circular chromosomes. This review examines chromosome topology across the Actinomycetales and how this affects our concepts of chromosome evolution.     

A self-ligation method for PCR-sequencing the telomeres of Streptomyces and Mycobacterium linear replicons

Actinomycete species from many genera often harbor linear plasmids and some contain linear chromosomes. A self-ligation and PCR-sequencing method was developed for identifying three novel telomere sequences of linear plasmids of Streptomyces and Mycobacterium. This and four previously described methods for actinomycetes telomere cloning and sequencing are discussed.     

贵州施秉云台山大型真菌生态调查

在贵州省施秉县云台山国家级自然保护区采集并鉴定了大型真菌55科,132属,261种。其中腐生菌195种,菌根菌59种,虫生菌7种。包括贵州省新记录种22种,食用菌85种,药用菌74种,有毒种类21种。     

A new beginning with new ends: linearisation of circular chromosomes during bacterial evolution

Bacterial circular chromosomes have sporadically become linearised during prokaryote evolution. Unrelated bacteria, including the spirochete Borrelia burgdorferi and the actinomycete Streptomyces, have linear chromosomes. Linear chromosomes may have been formed through integration of linear plasmids. Linear chromosomes use linear plasmid strategies to resolve the 'end-of-replication problem', but they have generally retained from their circular ancestors a central origin of replication. Streptomyces linear chromosomes are very unstable and at high frequency undergo amplifications and large deletions, often removing the telomeres. At least in Streptomyces, chromosome linearity is reversible: circular chromosomes arise spontaneously as products of genetic instability or can be generated artificially by targeted recombination. Streptomyces circularised chromosomes are very unstable as well, indicating that genetic instability is not confined to the linearised chromosomes. Bacterial linear chromosomes may contain telomere-linked regions of enhanced genomic plasticity, which undergo more frequent genetic exchanges and rearrangements and allow differential evolution of genes, depending on their chromosomal location.     

Parallel occurrence of asynaptic sex chromosomes in gray voles (Microtus Schrank, 1798)

In many eutherian species, pairing and recombination of X and Y chromosomes are indispensable for normal meiotic progression and correct segregation of sex chromosomes. The rodent subfamily Arvicolinae provides an interesting exception. The majority of arvicoline species with asynaptic sex chromosomes belong to the genus Microtus sensu lato. However, some vole species of the genus Microtus and other genera display normal X-Y pairing in meiosis. These observations indicate that synaptic condition was typical for the common ancestor of all voles, but the gaps in taxonomic sampling makes impossible to identify a lineage or lineages, in which the asynapsis occurred. The methods of electron and fluorescent microscopy were used to study the synapsis of sex chromosomes in males of some additional species of the subfamily Arvicolinae. This extended taxonomic list allowed us to identify asynaptic species in every large lineage of the tribe Microtini. Apparently, the ability of sex chromosomes to pair and recombine in male meiosis was lost in arvicoline evolution for at least three times independently. Our results indirectly suggest the unnecessity of sex chromosome pairing in male meiosis of arvicoline rodents, and presence of alternate molecular mechanism of sex chromosome segregation in this large mammalian tribe.     

Conjugative transfer of a plasmid from Escherichia coli to various strains of the order Actinomycetales

The conjugal transfer of autonomous and integrative plasmids from the donor strain Escherichia coli S17-1 to strains of genera Actinomadura, Arthrobacter, Kitasatoa, Micromonospora, Nocardia, Rhodococcus, Saccharopolyspora, and to 16 strains of the genus Streptomyces was demonstrated. The status of plasmids in recipient strains and the stability of their inheritance were analyzed. Plasmids constructed for strains of the genus Streptomyces were shown to function in a large number of strains belonging to the order Actinomycetales. The well-developed system of Streptomyces vector molecules and cloned genes of antibiotic biosynthesis allows their transfer to those microorganisms for which conventional techniques of plasmid transfer by regenerated protoplast transformation or electroporation have not been developed or are inefficient.     

A linear plasmid temperature-sensitive for replication in Streptomyces hygroscopicus 10-22

Streptomyces hygroscopicus 10-22 harbors a conjugative, autonomously replicating linear plasmid pHZ6 of ca. 70 kb, which shows no obvious homology with chromosomal DNA and is temperature-sensitive for replication, being stable in the host at 28 degrees C but easily lost at 37 degrees C. On a lawn of the wild-type S. hygroscopicus 10-22 cured of pHZ6, pHZ6 elicit pocks. Temperature sensitivity seemed to be a unique property for pHZ6 among six linear plasmids tested, including the well-known linear plasmids SLP2 in Streptomyces lividans 1326 and SCP1 in Streptomyces coelicolor A3(2). The distinct identity of pHZ6 from previously identified pHZ1-pHZ5 was demonstrated by the profile of relevant plasmids in six well-defined strains originated from S. hygroscopicus 10-22.     

Flavonoids of Lavoisiera, Microlicia and Trembleya (Melastomataceae) and their taxonomic meaning

Leaves of species of three closely related genera of Melastomataceae, Lavoisiera, Microlicia and Trembleya were analyzed for their flavonoid composition. A total of 116 compounds were obtained, comprising 69 flavonol and 47 flavone glycosides. The common occurrence of flavones, including 6-oxygenated derivatives, characterize Lavoisiera (which often yielded methoxylated flavonols and flavones). Flavonols predominate in species of Microlicia. The flavonoid patterns of Trembleya are rather confusing, some species being akin to Lavoisiera, others to Microlicia. UPGMA analysis using the flavonoid aglycones as characters and the samples analyzed as OTUs gives no complete resolution for the three genera, but provides clusters combining exclusively or preferentially species of either Lavoisiera and Microlicia. Species of Trembleya emerge from the phenogram interspersed among species of the other genera. The data suggest the recognition of Lavoisiera and Microlicia and the lumping of species of Trembleya with either of these genera. If greater flavone diversity is viewed as indicative of further evolutionary advancement, shrubby habits in Microliceae (Lavoisiera and Trembleya) should be regarded as derived from herbaceous ones (Microlicia).     

湖南野生木本观赏植物资源及利用

湖南有维管束植物5500余种(含变种,下同),其中野生木本观赏植物1200余种。笔者根据湖南木本观赏植物自然分布规律．将湖南野生木本观赏植物划分为4个类型．即中温型、低温型、耐寒型、广温型。对湖南主要的野生木本观赏植物如针叶类(39种)、茶属(31种)．冬青属(68种)、兰花类(86种)、杜鹃花属(54种)、槭树属(42种)等种类较多的类群进行了探讨,并对它们的观赏特性、抗寒性和选育潜力也进行了研究．提出了开发利用的建议。