我国南北大豆产区慢生大豆根瘤菌的遗传多样性和系统发育研究

利用16S rRNA基因RFLP、16S rRNA基因序列分析以及16S-23S rRNA IGS PCR RFLP技术对分离自我国南北大豆产区的慢生大豆根瘤菌进行了群体遗传多样性和系统发育研究。16S rRNA基因PCR RFLP分析以及16S rRNA基因序列分析结果表明:所有供试慢生大豆根瘤菌可分为B.japonicum和B.elkanii两个类群,其中属于B.japonicum的为优势种群,占供试菌株的91%,属于B.elkanii的仅占9%,多样性水平较低。16S-23S rRNA IGS PCRRFLP研究结果表明:属于B.japonicum的慢生根瘤菌具有较丰富的遗传多样性,在69%的相似性水平上可分为群Ⅰ和群Ⅱ两大类群。群I的菌株以分离自黑龙江和河北等北部区域的菌株为代表,群Ⅱ的菌株以分离自广西和江苏等南部地域的菌株为代表,反映出明显的地域特征。两群菌株在系统发育上均与USDA6、USDA110和USDA122等B.japonicum的模式或代表菌株有差异。     

黄土高原地区大豆根瘤菌的遗传多样性和系统发育

【目的】研究黄土高原地区大豆根瘤菌的遗传多样性和系统发育。【方法】采用BOX-PCR、16S rDNAPCR-RFLP、16S-23S IGS PCR-RFLP和16S rRNA基因序列分析方法对分离自我国黄土高原地区4个省的15个地区的130株大豆根瘤菌及部分参比菌株进行了遗传多样性和系统发育分析。【结果】BOX-PCR反映的菌株多样性最丰富,形成的遗传群最多,16S rDNA PCR-RFLP方法在属、种水平上聚群较好,16S-23S IGSPCR RFLP反映的多样性介于BOX-PCR和16S rDNA PCR-RFLP之间,能够较好地反映出属、种和亲缘关系很近的菌株间的差异,3种方法聚类分析结果基本一致,可将所有供试菌株分为两大类群,中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium)。从系统发育来看,供试的快生大豆根瘤菌为费氏中华根瘤菌(Sinorhizobium fredii),慢生大豆根瘤菌为日本慢生大豆根瘤菌(Bradyrhizobium japonicum)和辽宁慢生根瘤菌(Bradyrhizobium liaoningense)。【结论】我国黄土高原地区大豆根瘤菌具有较丰富的遗传多样性,S.fredii优势种,慢生大豆根瘤菌仅占10%,同时,分离到2株B.liaoningense。     

江汉平原及其周边地区花生根瘤菌的遗传多样性

采用RAPD分析技术和16S－23S rRNA间隔区段（IGS）RFLP分析,分别对分离自江汉平原及其周缘地区的花生根瘤菌进行了遗传多样性和系统发育研究。结果表明,全部供试验菌分别在48％和50％的相似性水平分为Ⅰ、Ⅱ两群,供试花生根瘤菌与参比菌株B.japonicum和B.elkanii聚在群I,参比菌株Rhizobium Sinorhizobium,Mesorhizobium和Agrobacterium聚在群Ⅱ。供试花生根瘤菌的遗传多样性及其在系统发育中的地位主要受地域因素的影响,来自江汉平原中心地带天门和潜江的菌株在76％以上的相似性水平上聚在一起,处于周边地带的武汉和荆州,由于其特定的地理因素的影响。菌株的多样性更为丰富,部分菌株在分类上与其它地域的菌株相互融合,并在较高的相似水平存在一定摆动性,来自外缘随州的菌株,表现了明显的地理分隔作用,其在系统演化中的地位相对独立,总体上从平原腹地到外缘地区。根瘤菌地理分隔作用逐渐明显,在平原外缘的交接地带,根瘤菌的多样性最为丰富。     

铜绿假单胞菌M18 rsmY突变株的构建及其对PCA和Plt的区别性调控

【目的】在假单胞菌中,小RNA(sRNA)参与初级和次级代谢产物、多种毒素因子以及菌群传感系统的调控,通过在植物根际促生铜绿假单胞菌M18中研究RsmY对吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的调控作用,深入了解假单胞菌中次级代谢的途径并为构建高产抗生素工程菌株提供了一定的理论基础。【方法】运用同源重组技术,构建了铜绿假单胞菌M18株的rsmY突变菌株M18RY,通过基因过表达、lacZ报告基因融合分析实验,进一步验证了RsmY对抗生素合成基因的调控作用。【结果】比较野生型M18和突变株M18RY中PCA和Plt在同一培养条件下的生物合成量,突变菌株M18RY中PCA的产量显著增加,为野生型菌株的5倍左右,而Plt的产量降为野生型的1/8。LacZ报告基因融合分析进一步证明了RsmY对PCA的负调控作用主要是通过phz2基因簇来实现的。【结论】结果表明,rsmY基因区别性调控PCA和Plt的生物合成。     

假单胞菌株M18 psrA突变株的构建及其对吩嗪-1-羧酸和藤黄绿菌素合成的调控

【目的】假单胞菌M18是一株能同时合成吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的植物根际促生细菌。PsrA为细菌TetR家族转录调控因子。为了研究PsrA对PCA与Plt生物合成的影响,从M18菌株基因组中扩增psrA基因。【方法】通过同源重组技术,构建庆大霉素抗性片段置换psrA的突变菌株M18psrA。利用基因互补、lacZ报告基因融合分析实验,验证PsrA对抗生素合成基因的调控作用。【结果】在PPM和KMB培养基中,分别比较野生型菌株M18和突变菌株M18psrA的PCA与Plt产量,突变菌株M18psrA的PCA产量显著下降;Plt产量显著升高,为野生型菌株的10-15倍。基因互补、lacZ报告基因融合分析,进一步证明了psrA正调控PCA的phz2合成基因簇,负调控Plt的合成基因簇。【结论】PsrA区别性调控抗生素PCA与Plt的生物合成。     

假单胞菌M18rsmA-突变株的构建及其对Plt和PCA合成的区别性调控作用

假单胞菌(Pseudomonas sp．)M18是促进植物生长的根际细菌,能产生吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种不同的抗生素抑制植物病原菌,保护植物免受病害。运用PCR方法,从M18基因组中,扩增出rsmA基因部分片段,并以该片段为探针,从M18的基因组柯斯文库中筛出阳性克隆,切取带有rsmA基因及两侧序列的1．5kb片段,中间插入编码Km‘的DNA片段,获得rsmA^-体外突变体。运用同源重组剔除技术,构建了M18菌株的rsmA突变株M18R^-。突变株M18R^-生物合成Plt的能力比野生型M18提高4倍,但是,PCA产量仅为野生型的20％。研究结果表明,全局性调控基因rsmA可能通过不同的机制区别性地影响Plt和PCA的生物合成。     

Genetic diversity of Acacia tortilis ssp. raddiana rhizobia in Tunisia assessed by 16S and 16S-23S rDNA genes analysis

AIMS: In order to understand the genetic diversity of Acacia tortilis ssp. raddiana-rhizobia in Tunisia, isolates from nine geographical locations were obtained and analysed. METHODS AND RESULTS: Characterization using restriction fragment length polymorphism analysis (RFLP) of PCR-amplified 16S rRNA gene and the intergenic spacer (IGS) between the 16S and 23S rRNA genes was undertaken. Symbiotic efficiency of the strains was also estimated. Analysis of the 16S rRNA by PCR-RFLP showed that the isolates were phylogenetically related to Ensifer ssp., Rhizobium tropicii-IIA, and Rhizobium tumefaciens species. Analysis of 16S-23S spacer by PCR-RFLP showed a high diversity of these rhizobia and revealed eleven additional groups, which indicates that these strains are genetically very diverse. Full 16S rRNA gene-sequencing showed that the majority of strains form a new subdivion inside the genera Ensifer, with Ensifer meliloti being its nearest neighbour. Nodulation test performed on the plant host demonstrated differences in the infectivity among the strains. CONCLUSION: Rhizobial populations that nodulate specifically and efficiently Acacia tortilis ssp. raddiana in representative soils of Tunisia is dominated by E. meliloti-like genomospecies. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the first clear characterization and symbiotic efficiency data of rhizobia strains nodulating A. tortilis in Tunisia.     

Genetic diversity and population connectivity of the Asian green mussel Perna viridis in South China Sea,inferred from mitochondria DNA markers

The Asian green mussel Perna viridis is ecologically and economically important in the coastal regions of China. In order to characterize the genetic diversity and population connectivity of P. viridis in South China Sea, a 664 bp region of mitochondrial COI gene and a 293 bp region of 16S rRNA gene were sequenced and analyzed for 78 and 92 individuals from four populations in South China Sea, respectively. A total of 15 haplotypes were defined by 14 variable nucleotide sites in COI gene, and 7 haplotypes by 6 variable nucleotide sites in 16S rRNA gene. High haplotype diversity and low nucleotide diversity were observed in COI gene, while moderate haplotype diversity and low nucleotide diversity were observed in 16S rRNA gene. Pairwise F_ST values of COI gene were all negative and those of 16S rRNA gene ranged from −0.01409 to 0.10289. The results showed that no significant genetic divergence (or shallow genetic structure) and high levels of population connectivity among the four populations of P. viridis in South China Sea.     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素 (Pyoluteorin ,Plt )和吩嗪 1 羧酸 (Phenazine 1 carboxylicacid ,PCA)并抑制多种植物病菌的生长。从M18中克隆双基因调控系统gacS gacA的组成基因gacA ,并构建了该基因抗性插入突变株M18G。在KMB培养基中 ,M18G合成Plt的能力受到完全抑制 ,而PCA的积累约比野生型提高 31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明 ,在M18G菌株中 ,Plt合成的抑制并不引起PCA的过量积累 ,PCA的过量积累也不引起Plt合成的抑制。由此推测 ,gacA在基因表达的水平上全局性地执行着调控功能     

Spatial-temporal survey of Microcystis oligopeptide chemotypes in reservoirs with dissimilar waterbody features and their relation to genetic variation

The ability of cyanobacteria to produce toxins and other secondary metabolites is patchily distributed in natural populations, enabling the use of cellular oligopeptide compositions as markers to classify strains into ecologically-relevant chemotypical subpopulations. The composition and spatiotemporal distribution of Microcystis chemotypes within and among waterbodies was studied at different time scales by analyzing (i) Microcystis strains isolated between 1998 and 2007 from different Spanish reservoirs and (ii) individual Microcystis aeruginosa colonies collected from pelagic and littoral habitats in Valmayor reservoir (Spain) during a bloom. No agreement between chemotypes and both morphotypes and genotypes (based on cpcBA-IGS, 16S–23S rRNA ITS and mcyB genes) was found, suggesting that oligopeptide profiles in individual strains evolve independently across morphospecies and phylogenetic genotypes, and that the diversity of microcystin variants produced cannot be explained by mcyB gene variations alone. The presence of identical chemotypes in spatially-distant reservoirs with dissimilar trophic state, lithology or depth indicate that waterbody characteristics and geographical boundaries weakly affect chemotype composition and distribution. At smaller spatiotemporal scales (i.e. during bloom), M. aeruginosa populations showed high number of chemotypes, as well as marked differences in chemotype composition and relative abundance among the littoral and pelagic habitats. This indicates that the factors influencing chemotype composition, relative abundance and dynamics operate at short spatial and temporal scales, and supports emerging hypotheses about interactions with antagonistic microorganisms as possible drivers for widespread chemical polymorphisms in cyanobacteria.     

Phylogenetic diversity of fluorescent pseudomonads in agricultural soils from Korea

AIMS: To identify and compare the relative diversity and distribution of genotypes of culturable fluorescent pseudomonads from soils. METHODS AND RESULTS: Analysis of 160 isolates from seven soil samples using randomly amplified polymorphism DNA methods revealed 53 genotypes, which were subsequently identified by their 16S ribosomal DNA sequences. Phylogenetic analyses of the 53 genotypes along with 43 fluorescent pseudomonad type strains separated the genotypes into 10 distinct clusters that included two phylogenetic groups that were not represented by previously described type strains. CONCLUSIONS: The diversity of genotypes that was obtained from the soil samples was highly variable among the different soils and appeared to be associated with different soil management practices that also influence plant yields. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification and phylogenetic analysis of these genotypes offers opportunities for study of phenotypic traits that may be associated within taxonomically related groups of fluorescent pseudomonad species and how these groups vary in relation to soil management practices.     

基于16S-23S rDNA 间隔区序列（ITS）、REP序列的基因指纹图谱及UPGMA聚类法的拟诺卡菌属分类方法

拟诺卡菌属（Nocardiopsis）是拟诺卡菌科（Nocardiopsaceae） 的唯一属．该属内进行物种鉴别时通常是在多相分类方法基础上,以全基 因组杂交同源性在70%以下的为不同物种,此为国际公认的定种标准;但在 进行大量菌株的比对时操作比较复杂,于是多种基于DNA的基因图谱技术发 展起来．本实验利用适宜引物,对拟诺卡菌属15株基准株基因组DNA的16S -23S rDNA 间隔区序列（ITS）和REP序列进行了扩增,获得了两种基因指 纹图谱,同时根据UPGMA聚类法构建了相应的进化距离树图．结果表明,对 于拟诺卡菌属中不同物种的区分,两种基因图谱技术的分辨力相当,均可 以较好的呈现物种间差异,可以作为拟诺卡菌属菌株多相分类的组成部分 ,应用于物种水平的分类与鉴定．     

荧光假单胞菌M18的rpoS基因克隆及其功能分析

从荧光假单胞菌 (Pseudomonasfluorescentsp .)M1 8基因组中克隆了RNA聚合酶的稳定期σs 因子编码基因rpoS ,推测其氨基酸序列与铜绿假单胞菌、荧光假单胞菌和恶臭假单胞菌的同源性分别为 99 1 %、87 35 %和87 8%。利用体外定点插入突变和同源重组技术 ,构建了M1 8的rpoS突变株M1 8R- 。对突变株M1 8R- 合成抗生素吩嗪 1 羧酸 (PCA)和藤黄绿菌素 (Plt)的动力学分析结果表明 ,在KB或PPM培养基中 ,突变株合成PCA的能力比野生型分别提高了 2 5或 5 78倍 ,但Plt的积累量不受影响。与野生型相比 ,突变株对碳源饥饿的耐性下降。同时 ,在碳源饥饿条件下对过氧化氢、乙醇和和氯化钠等环境胁迫的交叉保护性减小 ,存活率显著降低     

cpcHID操纵子序列用于钝顶节旋藻品系分类与鉴定的研究

克隆并测定7株钝顶节旋藻品系的cpcHID操纵子序列,以及16SrRNA和16S-23SrRNA转录单元内间隔区(ITS)序列,进一步通过生物信息学和分子系统学等研究发现:(1)7株品系的cpcHID序列,以及16SrRNA和ITS序列具有很高的相似性。(2)基于7株品系cpcHID序列的GC含量绝对偏差平均值、碱基变异率和遗传距离系数普遍比基于16SrRNA和ITS序列的大。(3)基于cpcHID序列的分类结果与基于16SrRNA和ITS序列的十分相近。因此,cpcHID可作为节旋藻等蓝细菌分类与鉴定的一种新的分子标记,特别是以其丰富的信息量而在品系水平的分类鉴定中占有优势。     

几种经济作物根际拮抗细菌的多样性

采用抗菌谱测定以及BOXAIR-PCR、生理生化特征和16S rDNA序列分析等方法对分离自10种作物根际的55株拮抗细菌的多样性及主要拮抗菌类群进行了分析.结果表明： 根际拮抗细菌的拮抗作用具有丰富的多样性;BOXAIR-PCR分析中供试菌在721％相似性水平上可以聚为7个群,850%相似性水平上聚为25个群;所有供试根际拮抗菌分别属于芽孢杆菌属（Bacillus）芽孢杆菌属（Paenibacillus）、短芽孢杆菌属（Brevibacillus）、假单胞菌属（Pseudomonas）和产碱菌属（Alcaligenes）.作物根际拮抗菌具有丰富的遗传多样性和拮抗性能多样性.     

我国主要生态区域绿豆慢生根瘤菌的遗传多样性和系统发育研究

利用16SrRNAPCR-RFLP、16SrRNA序列分析以及16S-23SrRNAIGS(IntergeneticSpacer)PCR-RFLP技术对分离自中国主要生态区域的44株慢生型绿豆根瘤菌和5株参比菌株进行了遗传多样性和系统发育研究。16SrRNAPCR-RFLP分析表明:在76%的相似水平上,所有供试菌株可分为三大类群:群I由LYG1等13株慢生根瘤菌组成,该群在系统发育上与B.japonicum和B.liaoningense的参比菌株存在一定的差异;群Ⅱ由XJ1等21株供试菌株、B.japonicum和B.liaoningense的代表菌株组成;群Ⅲ由10株来自广东和广西的菌株和B.elkanii的代表菌株组成。16S-23SrRNAIGSPCR-RFLP分析将供试菌株分为A、B两大群。群A由34株供试菌株、B.japonicum和B.liaoningense的代表菌株组成。在85%的相似性水平上,可再分为AⅠ、AⅡ和AⅢ3个亚群。群B由10株分离自广西和广东的菌株和B.elkanii的代表菌株组成。在85%的相似性水平上,可再分为BI和BⅡ两亚群,表现出一定的多样性。与16SrRNAPCR-RFLP相比,16S-23SrRNAIGSPCR-RFLP具有更高的解析度,供试菌株表现出更加丰富的遗传多样性。分离自中国新疆、广东和广西等地的菌株在分群上具有较为明显的地域特征。     

Brasilonema lichenoides sp. nov. and Chroococcidiopsis lichenoides sp. nov. (Cyanobacteria): two novel cyanobacterial constituents isolated from a tripartite lichen of headstones

Cyanolichens are an assemblage of fungi and cyanobacteria from diverse, cosmopolitan habitats. Typically composed of a single species of cyanobacterium, with or without another eukaryotic alga, here we present two novel cyanobionts isolated from an undescribed tripartite lichen. This endolithic lichen was isolated from a granite cemetery tombstone from Jacksonville, FL, and contains two potentially nitrogen‐fixing cyanobionts. Employing a total evidence approach, we characterized the cyanobionts using molecular (the 16S rDNA and ITS gene region), morphological, and ecological data. Phylogenetic analyses revealed two novel taxa: Brasilonema lichenoides and Chroococcidiopsis lichenoides, both of which fell within well‐supported clades. To our knowledge, this represents the first instance of a tripartite lichen with two cyanobacterial and no eukaryotic members. These types of lichens may well represent an unexplored reservoir of cyanobacterial diversity. The specific epithets are proposed under the provisions of the International Code of Nomenclature for algae, fungi, and plants.     

Heterogeneity of Strains Assigned to Gluconobacter frateurii Mason and Claus 1989 Based on Restriction Analysis of 16S-23S rDNA Internal Transcribed Spacer Regions

Twenty-three strains, which were assigned to Gluconobacter frateurii and maintained at Culture Collection NBRC, were re-identified at the species level on the basis of restriction analysis of 16S-23S rDNA ITS regions by digestion with six restriction endonucleases: Bsp1286I, MboII, AvaII, TaqI, BsoBI, and BstNI. The strains examined were divided into six groups, Group III-1, Group III-2, Group III-3, Group III-4, Group III-5, and Group IV. Group III-1 and Group III-4 respectively were divided into two subgroups, Subgroup III-1a, Subgroup III-1b and Subgroup III-4a, Subgroup III-4b. Gluconobacter frateurii NBRC 3264^T was included in Group III-2, along with strains NBRC 3265 and NBRC 3270, and G. thailandicus BCC 14116^T was included in Group III-3, along with strains NBRC 3254, NBRC 3256, NBRC 3258, NBRC 3255, and NBRC 3257. These groupings were supported by a phylogenetic tree based on 16S-23S rDNA ITS sequences. Strains of group III-2 and Group IV were unequivocally re-identified as G. frateurii, but strains of Group III-3, Group III-4, and Group III-5 were not necessarily re-identified as G. frateurii. The results obtained indicate that the 23 strains have a taxonomically heterogeneous nature, and they are referred to as the G. frateurii complex.     

Genetic diversity and biological activity of Curcuma longa ecotypes from Rapa Nui using molecular markers

Curcuma Longa (CL) has been used for hundreds of years by native people from Rapa Nui for the treatment of different illness. Despite this plant was introduced from Polynesia or India, there is still scarce information about its origin. The objective of this study was to analyze the genetic variation of three CL ecotypes based on molecular phylogenetic and genotypification using internal transcribed spacer 2 (ITS2) and simple sequence repeats (SSR). Antioxidant and anti-inflammatory properties of rhizomes and leaves extracts of three CL plants were analyzed by spectrophotometric methods and cyclooxygenase 2 (COX-2) inhibition assay. Complementarily, we predicted the potential binding mode and binding energy of curcuminoids and nonsteroidals anti-inflammatory drugs (NSAIDs) into COX-2 via molecular docking. The ITS2 sequence shows two major clusters (I and II), group I consisted of Curcuma haritha and group II consisted of different species of Curcuma and Rapa Nui samples (MR-1, MR-2 and RK-2). Results of SSR markers show that genotype MR-2 was similar to MR-1 and RK-2 with 70.8 and 42.9% similarity, whereas genotype was similar to RK-2, MR-1 and MR-2 with 63.9, 43.2 and 42.9% similarity, respectively. MR-1 have better antioxidant and autoinflammatory activity than rest of CL samples due to its high concentration of polyphenols (33.68 mg/g) and curcumin (29.69 mg/g). Furthermore, docking results show that three curcuminoids of CL and selective NAIDs, as celecoxib, etodolac and meloxicam, share the same binding pocket into COX-2. However, three curcuminoids have a lower ΔG_binding than other COX-2 selective NAIDs as etodolac and meloxicam, except for Coxib family as valdecoxib, celecoxib and rofecoxib. Our findings suggest MR-1, MR-2 and MK-2 from Germplasm Bank (Mataveri Otai of CONAF) are closely related to Curcuma amada and Curcuma montana even though they have genetic variability.     

西北部分地区苦马豆根瘤菌的遗传多样性

苦马豆(Sphaerophysa salsula)是荒漠区重要的豆科植物。为了研究其共生根瘤菌的多样性, 本试验采用16S rDNA PCR-RFLP和16S rDNA全序列分析方法, 对西北部分地区的苦马豆根瘤菌进行了遗传多样性及系统发育分析。结果表明, 57株供试菌株共产生了9种遗传图谱类型, 对每种图谱类型的代表性菌株进行16S rDNA全序列分析的结果表明, 它们分别归属于中慢生根瘤菌属(Mesorhizobium)、根瘤菌属(Rhizobium)、中华根瘤菌属(Sinorhizobium)、土壤杆菌属(Agrobacterium)、叶杆菌属(Phyllobacterium)和Shinella kummerowiae。不同地域的菌株在多样性方面也有明显差异: 分离自银川的苦马豆根瘤菌的Jaccard相似性系数较低; 而来自民乐县和临泽县的菌株有着非常丰富的遗传多样性, 其Simpson指数分别为0.826和0.710, Shannon-Wiener指数分别为1.831和1.530。以上结果为进一步确定西北地区豆科植物根瘤菌的系统分类地位提供了依据。