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.     

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。     

水稻内生链霉菌中的线型和环型质粒

植物内生放线菌的研究是一个近年来兴起的学科领域,在进一步探索和开发微生物资源方面,植物内生放线菌逐渐成为相关领域同行的关注热点.     

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 ,…     

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。     

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。     

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.     

尤马马杜拉放线菌染色体复制区的克隆及功能分析

尤马马杜拉放线菌(Actinomadura yumaensis)NRRL12515产生马杜拉霉素,用于防治禽类球虫病。试验以放线菌dnaA与dnaN基因保守区设计的简并引物进行PCR扩增,获得了包含尤马马杜拉放线菌的染色体复制区oriC的片段,并进行了序列分析和复制功能的研究。尤马马杜拉放线菌染色体的oriC全长为919碱基对,含有14个DnaA盒子和2个AT富含区,DnaA盒子的保守序列是(T/C)(T/C)GTCC(A/C)CA,与已发表的3个属的放线菌染色体oriC的序列特征不同。携带该oriC片段的大肠杆菌质粒可以在天蓝色链霉菌中复制并以低拷贝方式遗传,表明这是一段有复制功能的序列。比较来自放线菌4个属的oriC,发现以oriC序列和以16S rRNA基因序列构建的进化树十分相似,表明oriC序列也可以体现放线菌物种之间的关系。     

红球菌线型质粒pNSL1的克隆、测序和复制区的鉴定

从红球菌NS1中检测到两个线型质粒pNSL1和pNSL2。【目的】克隆、测序和分析pNSL1,并鉴定质粒的复制区。【方法】利用脉冲电泳方法从凝胶中回收大量的质粒DNA,进行鸟枪法克隆、测序和拼接,通过生物信息学分析和实验证明质粒的自主复制区。【结果】克隆、测序和拼接获得pNSL1全长为117252bp的序列,包括在红球菌中保守的1282bp端粒的序列。序列预测含有103个蛋白编码区,包括质粒的复制、分配、转移等功能基因。将pNSL1中一个与分枝杆菌质粒的复制基因同源的pNSL1.038及其上游的767bp非编码序列克隆到大肠杆菌质粒,电击转化珊瑚诺卡氏菌4.1037,获得了抗性转化子。【结论】克隆、测序了全长的线型质粒pNSL1,鉴定了质粒的复制区。     

Sporichthya polymorpha represents a novel line of descent within the order Actinomycetales

Abstract The 16S rDNA of Sporichthya polymorpha was PCR amplified from genomic DNA and almost completely sequenced using the dideoxy dye-terminator sequencing approach and automated DNA analysis. The primary structure was compared to the homologous molecule from 160 representatives of 37 genera of the order Actinomycetales . No higher similarity than 91.7% was found and the phylogenetic tree shows this morphologically unique organism to from an individual line of descent outside the main radiation of spore- and mycelium-forming organisms.     

双质粒系统共表达大肠杆菌肉碱脱水酶基因caiB及其辅酶合成基因caiE

caiB基因和caiE基因分别编码肉碱脱水酶及其辅因子合成酶 ,两者的共表达可以获得高活性肉碱脱水酶的重组菌。分别用两相容性质粒和不相容质粒共表达肉碱脱水酶及其辅酶合成基因 ,并对两种方法进行了比较。经IPTG诱导 ,相容性双质粒系统中两基因表达量分别占菌体总蛋白质的 17%和 10 % ;不相容性双质粒系统中两基因表达量分别占菌体总蛋白质的 39%和 2 0 %。共转化菌的酶活力比pET2 8caiB单质粒转化菌均提高 2 .3倍左右。两种双质粒转化系统相似 ,都需在外界抗生素选择压力下保持质粒稳定性     

Optimization,purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2°C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn²⁺ and strongly inhibited by Ba²⁺. All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.     

Two-component signal transduction systems in <Emphasis Type="Italic">Streptomyces</Emphasis> and related organisms studied using DNA comparative microarray analysis

Two component sensor-response regulator systems (TCSs) are very common in the genomes of the Streptomyces species that have been fully sequenced to date. It has been suggested that this large number is an evolutionary response to the variable environment that Streptomyces encounter in soil. Notwithstanding this, TCSs are also more common in the sequenced genomes of other Actinomycetales when these are compared to the genomes of most other eubacteria. In this study, we have used DNA/DNA genome microarray analysis to compare 14 Streptomyces species and one closely related genus to Streptomyces coelicolor in order to identify a core group of such systems. This core group is compared to the syntenous and non-syntenous TCSs present in the genome sequences of other Actinomycetales in order to separate the systems into those present in Actinomycetales in general, the Streptomyces specific systems and the species specific systems. Horizontal transfer does not seem to play a very important role in the evolution of the TCS complement analyzed in this study. However, cognate pairs do not necessarily seem to evolve at the same pace, which may indicate the evolutionary responses to environmental variation may be reflected differently in sequence changes within the two components of the TCSs. The overall analysis allowed subclassification of the orphan TCSs and the TCS cognate pairs and identification of possible targets for further study using gene knockouts, gene overexpression, reporter genes and yeast two hybrid analysis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

转基因抗虫作物鉴定质粒标准分子的构建与应用

苏云金芽胞杆菌基因是转基因抗虫作物中通用的外源功能基因,在绝大多数抗虫转基因作物中均有存在,然而Bt基因检测标准样品的缺乏却限制了我国转Bt基因抗虫作物检测工作的发展。为了弥补传统基体标准样品的缺失,首先将Cry1Ab、Cry1Ac、Cry3A 3种常用Bt外源基因克隆到pUC57质粒上,通过测序、酶切和qPCR等技术对质粒的序列和扩增功能进行了验证,然后对扩增效率和实际应用情况加以测试,评价其转基因检测的适用性,构建了质粒标准分子。结果显示,制备的质粒标准分子测序结果与靶标序列完全符合,酶切结果、qPCR扩增结果和扩增效率等均符合预期,在Cry1Ab、Cry1Ac、Cry3A基因特异性检测中的应用符合阳性对照要求,表明制备的阳性质粒标准分子能够作为转Cry1Ab、Cry1Ac、Cry3A基因qPCR基因特异性检测的阳性标准样品。     

In Situ Hybridization of Plant Meiotic and Mitotic Chromosomes: Differences in Signal Detection

The technique of in situ hybridization to both meiotic and mitotic chromosomes of Rumex acetosa is described. Differences in the efficiency of signal detection were observed between the two types of material. The implications of these results for in situ hybridization to other plant species are explored.     

Chromosomes and polyploidy in Lenophyllum (Crassulaceae)

Gametic chromosome numbers of 22, 32, 33, and 44 in five species of Lenophyllum suggest that they may be polyploids on a basic 11, but this number has not been found. Three species have 8-12 distinctively large chromosomes that do not pair with each other in their hybrids and probably belong to the same genome. In hybrids of many polyploid Mexican Crassulaceae preferential pairing occurs between corresponding chromosomes of their multiple genomes, which indicates that they are autopolyploids. However, little or no preferential pairing occurs between chromosomes of Lenophyllum in its hybrids, and its species appear to be allopolyploids. The putative parents are unknown.