Phylogeny of γ-proteobacteria inferred from comparisons of 3’ end 16S rRNA gene and 5’ end 16S-23S ITS nucleotide sequences

The phylogeny of γ-proteobacteria was inferred from nucleotide sequence comparisons of a short 232 nucleotide sequence marker. A total of 64 γ-proteobacterial strains from 13 Orders, 22 families, 40 genera and 59 species were analyzed. The short 232 nucleotide sequence marker used here was a combination of a 157 nucleotide sequence at the 3’ end of the 16S rRNA gene and a 75 nucleotide sequence at the 5’ end of the 16S-23S Internal Transcribed Spacer (ITS) sequence. Comparative analyses of the 3’ end of the 16S rRNA gene nucleotide sequence showed that the last 157 bp were conserved among strains from same species and less conserved in more distantly related species. This 157 bp sequence was selected as the first part in the construction of our nucleotide sequence marker. A bootstrapped neighbor-joining tree based on the alignment of this 157 bp was constructed. This 157 bp could distinguish γ-proteobacterial species from different genera from same family. Closely related species could not be distinguished. Next, an alignment of the 16S-23S ITS nucleotide sequences of alleles from same bacterial strain was performed. The first 75 bp at the 5’ end of the 16S-23S ITS was highly conserved at the intra-strain level. It was selected as the second part in the construction of our nucleotide sequence marker. Finally, a bootstrapped neighbor-joining tree based on the alignment of this 232 bp sequence was constructed. Based on the topology of the neighbour-joining tree, four major Groups, Group I to IV, were revealed with several sub-groups and clusters. Our results, based on the 232 bp sequence were, in general, in agreement with the phylogeny of γ-proteobacteria based on the 16S rRNA gene. The use of this 232 bp sequence as a phylogenetic marker presents several advantages over the use of the entire 16S rRNA gene or the generation of extensive phenotypic and genotypic data in phylogenetic analyses. First, this marker is not allele-dependant. Second, this 232 bp marker contains 157 bp from the 3’ end of the 16S rRNA gene and 75 bp from the 5’ end of the 16S-23S ITS. The 157 bp allows discrimination among distantly related species. Owing to its higher rate of nucleotide substitutions, the 75 bp adds discriminating power among closely related species from same genus and closely related genera from same family. Because of its higher percentage of nucleotide sequence divergence than the 16S rRNA gene, the 232 bp marker can better discriminate among closely related γ-proteobacterial species. Third, the method is simple, rapid, suited to large screening programs and easily accessible to most laboratories. Fourth, this marker can also reveal γ-proteobacterial species which may appear misassigned and for which additional characterization appear warranted.

利用16S-23S rDNA RFLP及16S rRNA基因序列分析方法对湖北省饭豆(Vigna umbellata L.)根瘤菌系统发育的研究

采用16S-23S rDNA间隔区段(IGS)PCR-RFLP与16S rRNA基因部分序列分析的方法对饭豆根瘤菌进行了遗传多样性及系统发育分析.由16S-23S rDNA IGS PCR-RFLP分析可知,所有菌株在52%的相似性水平上聚在一起,形成了慢生型菌群与快生型菌群这两大菌群.群Ⅰ中,在79%相似性的水平上分为ⅠA与ⅠB两个分支.群Ⅱ中,在62%相似性的水平上分为ⅡA与ⅡB两个分支,分支ⅡA在72%的相似性水平上进一步分为ⅡA1、ⅡA2和ⅡA3三簇;分支ⅡB中的饭豆根瘤菌与标准菌株USDA205T聚在一起,表现的差异并不大.由16SrRNA基因部分序列分析结果可知,供试的4个代表菌株分别位于不同的系统发育分支中.CYR4243与Sinorhizobium fredii的模式菌株USDA205T的序列相似性达到了99.87%.HCY1101与Rhizobium leguminosarum中的三叶草生物型(bv.trifolii)和豌豆生物型(bv.viceae)这两个生物型的参比菌株亲缘关系最近,序列相似性为100%.HCY5202与R.galegae亲缘关系最近,序列相似性为99.86%.CYY3302与Bradyrhizobium elkanii的参比菌株USDA86有最近的亲缘关系,序列相似性近似于100%.

基于16SrRNA和16S-23SrRNA基因间隔区序列探讨陆生和水生螺原体菌株的系统发生关系

采集江苏省养殖池塘内发病的中华绒螯蟹、克氏原螯虾和南美白对虾,利用已有螺原体16S rRNA和23S rRNA基因序列保守引物扩增并测序16S rRNA和16S-23S rRNA基因间隔区序列(ISRs).基于已获得的序列和GenBank中下载的螺原体序列分别进行同源性比对,比对的结果分别利用PAUP软件和MrBayes软件提供的最大似然法和贝叶斯法进行系统发生关系分析.分析结果是:所有淡水甲壳动物螺原体菌株Spiroplasma erio-cheiris(中华绒螯蟹)、Spiroplasmasp.CRAYFISH(克氏原螯虾)、Spiroplasmasp.SHRIMP(南美白对虾)严格聚为一支,并且与陆生兔扁虱螺原体(非凡螺原体)Spiroplasma mirum关系最近,而与中国报道的陆生植物螺原体菌株Spiroplasmasp.CR-1(油菜)、Spiroplasmasp.CNR-1(油菜)、Spiroplasmasp.CNR-2(油菜)、Spiroplasmasp.CAN-1(杜鹃花)、Spiroplasmasp.CRW-1(红花酢浆草)及昆虫螺原体菌株Spiroplasmasp.CH-1(蜜蜂)、Spiroplas-masp.M10(蜜蜂)关系较远,此外,3个淡水甲壳动物螺原体菌株与现今唯一报道的海水南美白对虾螺原体Spi-roplasma penaei系统发生关系也很远.因此,在螺原体属中,我国发现的淡水甲壳动物螺原体近缘种是非凡螺原体而不是我国陆生昆虫或植物螺原体及美洲的南美白对虾螺原体.

16SrRNA基因与16S-23SrRNA转录单元内间隔区序列分析及其在节旋藻和螺旋藻分类鉴定中的应用

测定了节旋藻属 3个品系和螺旋藻属 1个品系的全长 1 6SrRNA基因和 1 6S 2 3SrRNA转录单元内间隔区序列 (ITS) ,分析了已知的节旋藻、螺旋藻和相关品系的相应序列的同源性 ,构建了系统发生树 ,并评价了这两段DNA序列在节旋藻、螺旋藻种属分类和种质鉴定中的意义。结果表明 :( 1 ) 1 6SrRNA基因序列和ITS序列均可用于节旋藻属和螺旋藻属的属间分类 ,以两序列为基础的系统学分析结果一致 ;( 2 )ITS序列变异程度高于 1 6SrDNA序列 ,适用于节旋藻和螺旋藻属内品系或种质鉴定 ;( 3)节旋藻属可明确界定 ,1 6SrRNA基因序列相似性大于 98% ,ITS序列相似性大于 88% ;( 4 )螺旋藻属某些品系间 1 6SrDNA序列和ITS序列相似性较低 ,与不同属间的序列相似性程度为同一水平。

常见分枝杆菌种内不同株之间16S rRNA基因和16S-23S rRNA ITS序列分析结果的比较

目的针对常见分枝杆菌不同株对其基因序列进行分析,比较分析结果。方法利用16S rRNA Gene和16S-23S rRNAITS(转录间隔区序列)分析法分别对97株共7种DSMZ/ATCC引进的常见分枝杆菌进行种内不同株之间基因差异性分析,对比两种分型结果的异同。结果 16S rRNA基因可将13株草分枝杆菌分为3个型别,18株偶发分枝杆菌分为6个型别,17株耻垢分枝杆菌分为4个型别,8株戈登分枝杆菌分为3个型别,9株龟分枝杆菌龟亚种分为3个型别,15株堪萨斯分枝杆菌分为2个型别,17株产鼻疽分枝杆菌分为1个型别;而16S-23S rRNA ITS可依次将上述分枝杆菌分为3个、15个、7个、3个、4个、3个、5个型别。结论 16S rRNA Gene分析和16S-23S rRNA ITS分析均是分枝杆菌基因型分析的可靠方法,此外,16S-23SrRNA ITS的种内多态性高于16S rRNA Gene。

16S rRNA、23S rRNA及16S～23S rRNA基因在细菌分离与鉴定中的应用

在细菌的进化过程中,rRNA结构既具保守性又具高变性。保守性反映生物物种的亲缘关系,高变性则揭示生物物种的特征核酸序列,rRNA是属种鉴定的分子基础。因此,可以利用保守区设计通用引物扩增细菌的相应靶序列,再利用可变区的差异鉴别菌种。文章就16S rRNA、23S rRNA和16S～23S rRNA基因在细菌分离与鉴定中的应用做以下综述。

山羊奇异变形杆菌分离鉴定及其16S-23SrRNAISR序列RFLP分析

2012年初,山东菏泽某羊场的羊群发病,从发病羊器官分离到2株病原细菌。对病原细菌进行鉴定,并对其与已知同种异源菌株相似性差异进行分析。从患病山羊内脏器官分离细菌,经形态特征、培养特性、生化试验、血清学试验及致病性试验进行鉴定;再通过设计通用引物扩增16S-23SrRNA ISR(intergenic spacer region)序列,将PCR产物经HinfⅠ单酶切获得3条可视条带,同时对扩增条带中的主带测序并进行系统发育分析。结果表明,分离菌株为奇异变形杆菌;分离菌株同本实验室保存的兔源与鸡源奇异变形杆菌PCR-RFLP结果一致;分离菌株PCR产物同GenBank收录的HI4320株奇异变形杆菌及本实验室保存的兔源与鸡源奇异变形杆菌进行序列比较,分离羊源菌株与兔源菌株相似性为94.8%、与鸡源菌株相似性为96.0%～98.2%,与人源HI4320株相似性为96.9%。研究证实发病羊致病病原为奇异变形杆菌,其与鸡源、兔源和人源奇异变形杆菌的亲缘关系较近。

奇异变形杆菌、洛菲氏不动杆菌16s rRNA3’-23s rRNA 5’间序列的克隆及测序

目的:对奇异变形杆菌、洛菲氏不动杆菌16s rRNA 3’~23s rRNA 5’间序列(16S^23S rRNA intergenic spacer region,ISR)进行克隆测序,为分子探针技术鉴定两菌奠定基础。方法:针对临床常见致病菌16s rRNA 3’~23s rRNA 5’间序列两端的16S及23S rRNA保守序列设计PCR扩增的通用引物,对两菌进行扩增,利用PMD-18TVector质粒对两菌的PCR产物进行T载体克隆构建,测序后申报Genbank。结果:两菌的通用引物扩增产物构建的T载体克隆,测序结果经Blast分析,确定为两菌的ISR序列,申报Genbank获得接收。结论:成功的对奇异变形杆菌、洛菲氏不动杆菌ISR序列进行了克隆测序,该序列可以用于两种菌的通用引物PCR扩增技术鉴定。

基于线粒体16S rRNA基因的鹅肉源性成分鉴别方法研究

研究旨在建立基于线粒体16S rRNA基因的鹅源性成分鉴别方法。试验以鹅源性DNA为阳性模板,以猪、牛、羊、鸽、鹌鹑、火鸡、鸡和鸭等8个物种DNA为干扰模板的混合模板,设计筛选出鹅特异性引物,进行PCR和荧光定量PCR(qPCR)反应,并将鹅肉DNA模板浓度按101~1088个梯度进行稀释,检测方法灵敏度。结果显示:所设计的引物仅对鹅肉DNA有特异性扩增,对鹅以外的其他8个物种均没有扩增;当鹅肉DNA模板稀释104倍,PCR扩增条带仍然清晰;当稀释倍数达到107时,仍有较好的扩增曲线,且Ct值小于35。研究表明,建立的畜禽肉中鹅源性成分PCR和qPCR鉴别方法不仅具有良好的特异性,而且具有较高的灵敏性,为食品中鹅源性成分的鉴别提供了新途径。

Non-monophyly of Rhacophorus rhodopus,Theloderma and Philautus albopunctatus Inferred from Mitochondrial 16S rRNA Gene Sequences

Mitochondrial gene fragments of 16S rRNA gene of four species （Rhacophorus rhodopus, R. reinwardtii, Philautus albopunctatus and P. rhododiscus ） from 11 populations were sequenced in this study. Homologous sequences of R. bipunctatus, Theloderma asperum, T. corticale and Buergeria japonica were obtained by screening the GenBank database. After excluding all gaps and ambiguous positions, aligned sequences were 500 bp in length with 115 variable sites and 92 parsimony-informative sites. Using B .japonica as an outgroup, phylogenetic relationships were analyzed using Bayesian inference, maximum parsimony and maximum likelihood methods. Our results indicated that neither of R. rhodopus and P. albopunctatus were monophyletic at the species level. The population of R. rhodopus from Hainan Island was more close to R. bipunctatus than to populations of R. rhodopus from Yunnan Province. Furthermore, the populations of R. rhodopus from Yunnan Province can be divided into two main lineages. Theloderma corticale and P.rhododiscus were clustered together and T. asperum was nested in P.albopunctatus. We considered that P. albopunctatus Liu and Hu, 1962, was the synonymy of T. asperum Boulenger, 1886, and suggested removing P. rhododiscus from Philautus into the genus Theloderma.

中国布鲁氏菌16s-23s rRNA间隔区多态性研究

目的利用16 s-23 s rRNA间隔区(ITS)的多态性,对中国布鲁氏菌种间或种内生物型进行鉴别,评价ITS作为基因标识物的意义,寻找适合布鲁氏菌分型研究的基因标识物。方法应用聚合酶链反应-单链构象多态性(PCR-SSCP)分析技术,对中国120株布鲁氏菌的ITS进行分析和筛选,测序结果与Genbank中的布鲁氏菌ITS序列进行比较分析。结果对16 s-23 s rRNA间隔区的SSCP结果分析,得到4种不同的带型(ⅠI、I、Ⅲ、Ⅳ),测序序列有3个位点的差异,达到99.87%的一致性。结论中国布鲁氏菌的ITS序列高度保守,具有一定探讨其作为布鲁氏菌属种内分型的基因标识的价值。

分支杆菌临床分离株的16S-23S rRNA基因转录间隔区的序列分析

目的 建立鉴定分支杆菌分离株的 16S - 2 3SrDNA转录间隔区 (internaltranscribedspacer ,ITS)序列分析的方法 ,用该方法对临床分离株进行鉴定。方法 对从重庆某医院分离的 2 9株分支杆菌菌株分别进行了PCR扩增 ,得到它们的16S - 2 3SrDNAITS片段 ,并测定所得到片段的DNA序列。用DNA分析软件将所获得的序列与GenBank的分支杆菌序列相比较 ,计算种间相似性 ,并用序列构建分支杆菌菌株的系统发育树 ,对分离株进行鉴定。结果 在ITS序列分析中 ,有 10株的序列分别与结核分支杆菌复合群 (Mycobacteriumtuberculosiscomplex ,MTC)、戈登分支杆菌 ,脓肿分支杆菌的序列完全一致。依据完全一致的序列可以准确鉴定这些临床分离株为相应的种 ;4株 16SrDNA序列分析不能准确鉴定的分离株中其中 3株(M 4、M 5、M 12 )鉴定为戈登分支杆菌 ,1株 (M 2 3)鉴定为脓肿分支杆菌。部分分离株的的ITS序列在GenBank序列检索中无完全一致的匹配序列 ,这些不同的序列之间的相似程度为 2 7.1%～ 99.2 %。用临床分离株的ITS序列与其最相似的已知分支杆菌的ITS序列构建的系统发育树 ,菌株分群与 16SrDNA序列构建的系统发育树的分群基本一致。结论 分支杆菌的16S - 2 3SrDNAITS序列比 16SrDNA序列有更大的多样性 。

基于16S-23S rRNA ITS AFLP对米酒发酵过程中原核微生物的演替分析

利用16S-23S rRNA ITS AFLP指纹图谱技术监控四川传统米酒发酵过程中原核微生物的演替,并结合米酒发酵过程中理化因子的动态变化对其演替过程进行了分析。研究结果表明,米酒发酵过程中,随着酒曲的接入,米酒醅中原核微生物伴随米酒理化因子的动态变化而发生群落演替。在适应期和发酵期,米酒醅中的原核微生物群落分别聚成群I和群II,二者相关系数为0.49。群II中,在相关系数0.638水平上又分为IIA和IIB两个分支。分支IIA又进一步分为IIA1和IIA2两簇,相关系数为0.73。簇IIA2中主要发生的是发酵旺盛期微生物的演替;簇IIA1中I,IA1-1和IIA1-2亚簇分别表示发酵前期、发酵后期米酒醅中的原核微生物群落演替,二者聚集于相关系数0.80。其中I,IA1-2中,发酵52 h和55 h的米酒醅中原核微生物群落几乎相似,二者群落相关系数为1.0。

16S-23S rRNA ITS-AFLP指纹图谱分析在白酒窖泥原核微生物多样性分析中的应用

利用非培养的分子生物学方法对位于多粮原料生产的窖壁和窖底的新窖和老窖窖泥中原核微生物的16S-23S rRNA ITS进行AFLP(amplified fragment length polymorphism)分析,利用NTSYSpc 2.10e软件构建聚类图,分析其关系。结果表明,来自不同窖池的不同部位的原核微生物在16S-23S rRNA ITS水平上呈现出差异,其中老窖窖底和新窖窖底的原核微生物多样性存在着明显差异,其相关系数为0.47;而老窖窖壁和新窖窖壁的原核微生物多样性却差异不大,其相关系数为1.00。

Cloning and Sequence Analysis of 16S rRNA and COI Gene in Mitochondrial DNA of Scortum barcoo

[Objective] The aim was to provide molecular biological basis for the researches on the genetic resources,genetic relationship among species and phyletic evolution of S.barcoo.[Method] PCR amplification and sequencing were used to study the 16S rRNA and COI gene fragments.[Result] As for 16S rRNA gene fragments,nucleotide sequences of 791 bp were obtained,and the A,T,G and C contents in this fragment were 31.6%,21.4%,20.4% and 26.7%respectively.As for the COI gene fragments,the size was 631 bp and the A,T,G And C contents were 27.7%,23.6%,29.8% and 18.9% respectively.Among these two gene fragments,the content of GC was lower than AT,and AT/GC of these two fragments was 1.13 and 1.05 respectively.[Conclusion] The genetic characteristics of gene fragments of 16S rRNA and COI of S.barcoo suggested that the variation in the same species was relatively low.The sequences of 16S rRNA gene in three samples the same,while the sequences of COI gene was also the same,indicating that these two gene of S.barcoo were conservative.

16S rRNA Gene-Based Analysis of Ileal Bacterial Community and Phylogeny in Nursing and Weaned Piglets

Weaning of piglets is generally considered as a stressor which changes intestinal ecosystem and leads to clinical implications. Microbiota inhabiting in small intestine （especially ileum） are assumed to promote health, but their functional properties are yet poody dascdbed. As indicated by the 16S rRNA gene sequences of ileal micrebiota in nursing piglets （at the age of 21 and 28 d） and 28-day-old weaned piglets （weaned at 21 d of age）, the microbiota were mainly comprised of gram-positive bacteria. There were 40 operational taxonomic units （OTUs） （from 171 clones） in the ileum of nursing piglets aged 21 d, 61 OTUs （from 194 clones） in the ileum of nursing piglets aged 28 d, and 56 OTUs （from 171 clones） in the ileum of weaned piglets aged 28 d. The flea of nursing piglets aged 21 d were dominantly occupied by Lactobacilli （87.7%） as well as Streptococ cus （ 3.5 % ）. Lactobacillus amy/ovorus （41.5 % ）, Lactobaci/lus sp. （ 19.3 % ）, Lactobaci/lus reuteri （ 12.3 % ）, Lactobacillus salivarius （ 9.4 % ） and L. mucosae （4.7%） were the predominant species among Lactobacil/L Similar results were obtained in the nursing piglets at 28 d of age ex- cept that Lactobaci/li decreased to 71.1% and Streptococcus increased to 21.1% significantly. Lactobacillus （52.0%） and Streptococcus （26.3%） were the two major groups in the ileum of weaned piglets aged 28 d. Lactobacillus amylovorus （31.6%） and Lactobaci/lus reuteri （ 16.4% ） was the two most important species in Lactobacillus. Therefore, Lactobacilli were predominant in the ileum of nursing and weaned piglets, and they had the highest diversity, followed by Streptococcus. The diversity of ileal microbiota was not different remarkably between the nursing piglets and the weaned piglets, but the composition changed significantly. These findings are helpful to understand ileal bacterial ecophysiology and further develop nutritional regimes to prevent or counteract complications during the weaning transition.

16S-23S rRNA间区基因RFLP用于病原菌区分

[目的]探讨16S-23SrRNA间区基因RFLP用于临床常见病原菌区分的可能性。[方法]183株临床分离来的细菌经荧光PCR扩增后进行不完全限制性酶切,产物进行毛细管电泳。[结果]所有细菌的酶切产物经毛细管电泳后,不同细菌产生不同的RFLP谱图,互相之间能够明显区分。[结论]该方法可以将临床常见病原菌准确区分到种的程度,极有应用价值。

关节液中细菌16srRNA与23s rRNA诊断膝关节置换术后感染的价值

目的探讨利用关节液中细菌16s rRNA与23s rRNA诊断全膝关节置换术后感染的效率及两种基因诊断方法的差异。方法对33例无菌性松动及19例假体周围感染行人工膝关节翻修的患者,通过RT-PCR检测关节液中细菌16s rRNA、23s rRNA保守基因片段诊断假体周围感染。比较两种诊断策略的敏感性、特异性、阳性预测值、阴性预测值及准确性。结果以国外关于假体周围感染诊断方法的文献判定假体周围感染,利用16s rRNA进行诊断的敏感性78.8%,特异性93.9%,阳性预测值88.2%,阴性预测值为88.6%,准确性为88.5%;而采用23s rRNA扩增方法诊断的敏感性、特异性、阳性预测值、阴性预测值及准确性分别为68.4%、78.8%、65.0%、81.2%和75.0%。两种基因诊断的各指标比较差异均无统计学意义(P>0.05)。结论通过检测关节液中细菌16s rRNA或23s rRNA诊断人工膝关节置换术后感染,具有较高的诊断效率,且两者差异无统计学意义。

基于16S rRNA测序的观赏桃根系微生物多样性研究

观赏桃作为重要的园艺植物,其根际微生物群落对其生长和健康具有重要影响。本研究通过16S rRNA测序技术,分析了不同观赏桃品种根际微生物群落的多样性和结构特征,旨在揭示其微生物群落的差异性和互作关系。研究发现,不同观赏桃品种的Shannon多样性指数存在显著差异,“紫叶垂枝”(ZYCZ)显示出最高的多样性,而“中二乔”(ZEQ)和“烟云”(YY)等品种的多样性较低。此外,“紫叶垂枝”(ZYCZ)在特定分类群中的相对丰度较高,表现出明显的优势地位。研究还揭示了根际微生物群落之间的显著互作关系,如变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)之间的正相关关系,以及有益微生物与病原菌之间的负相关关系。本研究为观赏桃根际微生物群落的多样性和互作关系提供了新的见解,有助于优化观赏桃的种植和管理策略。未来将进一步研究微生物群落在根际环境中的具体功能、微生物-植物互作机制、环境因素的影响以及群落的长期动态变化,旨在提升观赏植物的健康生长和农业生产的可持续性。