中国沙棘(Hippophae rhamnoides ssp. Sinensis)根瘤内Frankia菌的遗传多样性

为了研究中国沙棘亚种共生菌Frankia的遗传多样性,利用PCR-RFLP分子标记方法,对从青海西宁到内蒙古库伦17个地点采集的106个中国沙棘根瘤样品进行遗传多样性分析.供试样品nifD-nifK基因间隔区(IGS)扩增产物分别用3种内切酶(HinfⅠ、HaeⅢ和MboⅠ)酶切,共产生21条酶切谱带,其中17条为多态性条带,多态位点百分比(PPL)为80.99%,所有样品可被划分为9个基因型.结果表明中国沙棘根瘤内的Frankia菌有丰富的遗传多样性,土壤质量较好地点的丰富度高于土壤质量较差地点,海拔较高地区的丰富度高于海拔较低地区,多数地点至少有2种不同基因型的Frankia 菌.聚类分析显示Frankia 菌不同基因型间的遗传距离在4.88%～55.96%之间,它们在不同地点的分布是不均匀的,没有发现不同基因型菌间的亲缘关系与地点有相关性.中国沙棘根瘤中Frankia菌可分为两个基因型组,组内基因型分布比较一致,而组间有明显差异.     

内蒙古沙棘共生菌Frankia的遗传多样性

利用RFLP分子标记方法,在自然条件下对内蒙古东、西部8个地点采集的24个沙棘根瘤样品进行沙棘共生菌Frankia的遗传多样性分析。结果表明,供试样品nifD-nifK基因间隔区(IGS)扩增片段的大小约1 100bp;不同样品的酶切图谱有明显差异;有些样品产生复合RFLP型,揭示在自然状态下不同基因型的Frankia菌株可共同侵染同一沙棘寄主。聚类分析显示,来源于相同地点和不同地点的根瘤样品内的Frankia菌株间均有遗传多样性;没有发现Frankia菌株遗传多样性的分布与采样地点有相关性。     

自然环境胁迫对旱冬瓜Frankia菌基因多样性的影响

利用rep-PCR方法,研究云南鸡足山及无量山不同生境下旱冬瓜根瘤内Frankia菌基因多样性及其变化,以了解不同自然环境胁迫对Frankia菌基因多样性的影响。结果表明,多样性随地域、海拔和坡向不同而变化,鸡足山Frankia菌基因类型比无量山丰富。鸡足山旱冬瓜根瘤内的Frankia菌在山底2300m处,Shannon指数平均为0．90;山顶海拔2650m以上,Shannon指数随之上升到1．33。南坡Frankia菌多样性高于北坡,表明多样性指数与环境胁迫大小成正相关,自然环境胁迫是产生和保持Frankia菌基因多样性的重要因子之一。     

二种淡水微囊藻rDNA16S-235基因间隔区的序列测定与分析

本研究采用PCR及序列测定的方法,对我国淡水铜绿微囊藻有毒株(M8641)和另一低毒的种类惠氏微囊藻(M574)rDNA16S-23S基因间隔区进行了序列的测定和分析,结果表明:rDNA16S-23S基因间隔区可以作为一个精细且稳定的指标,用于微囊藻的分类和鉴定。并从分子水平提出了铜绿微囊藻与惠氏微囊藻在种系发生上有较近缘的关系。本文首次对微囊藻属Microcystis rDNA基因间隔区全序列作了报道,为微囊藻属的鉴定及系统学研究提供了分子基础。       

3株杂色鲍致病菌——副溶血弧菌的16S-23SrDNA间区序列的分析

以细菌的16S rDNA 3′端和23S rDNA 5′端的高度保守区为引物,扩增了3株杂色鲍致病菌—副溶血弧菌（Vibrio parahaemolyticus）的16S23S rDNA 间区,克隆到pGEMT载体上,测序。用BLAST和 DNAstar软件对16S23S rDNA间区序列及其内的tRNA基因进行比较分析。结果表明副溶血弧菌ZSU008和ZSU009测出的16S23S rDNA 间区均有6条,间区类型也相同,分别为IGSGLAV、IGSGLV、IGSAG、IGSIA、IGSG和IGS0。其中IGSGLAV最大,包含tRNAGlu、tRNALys、tRNAAla和tRNAVal基因;IGSGLV包含tRNAGlu、tRNALys和tRNAVal基因;IGSAG包含tRNAAla和tRNAGlu基因;IGSIA,则为tRNAIle和tRNAAla基因;IGSG仅包含tRNAGlu基因;而IGS0最小,未包含任何tRNA。菌株ZSU010测出的16S23S rDNA IGS序列有5条,除缺少IGSAG外,其余的IGS类型均与ZSU008和ZSU009相同。与GenBank 内的副溶血弧菌IGS序列比较,发现副溶血弧菌所有类型的IGS的tRNA基因两端的非编码区具有较高的种内同源性。16S23S rDNA 间区结构的差异为建立一种新的副溶血弧菌检测方法奠定了基础。     

慢生根瘤菌16S—23S rDNA IGS的RFLP分析*

本文对慢生根瘤菌属（Bracyrhizobium）3个已知种及从10种豆科植物中分离的32株慢生根瘤菌进行了16S—23SrDNAIGS的RFLP分析。IGS的PCR产物电泳只出现一条rDNA片段,但表现在长度上菌株间有一定差异,大小在930～1050bp之间,可大致划分为IGSa、IGSb和IGSc3种。用4种四碱基识别序列的限制性内切酶AluI、HaeIII、HinfI和MspI酶解IGSrDNA,综合得到26种IGS－RFLP类型．每一种酶可产生6—12种不同的酶切图谱．结果表明这一方     

植物核基因组核糖体基因间隔区序列的结构特点及其在系统发育研究中的应用

目前,在植物系统发育研究中应用较多的核基因组的核糖体DNA基因间隔区序列主要有5S rDNA基因间隔区、内转录间隔区ITS和基因间间隔区IGS.虽然这些间隔区序列在长度、结构等方面各不相同,但都具有进化速率较快的特点,在植物属及属下分类水平的系统发育关系研究中非常有用.本文重点就核基因组的5S rDNA基因间隔区以及IGS在植物中的特点以及各自在植物系统发育研究中的应用进行了综述.     

油菜叶绿体基因组文库的构建和16S rRNA基因及16S-23S rDNA间隔顺序的分离

油菜ct-DNA经Bam H1酶解在0.7％琼脂糖上电泳呈现出27条DNA带,其中最大的为12.4kb,最小的为1kb。我们以pBR322为载体构建了油菜ct-DNA Bam HI片段文库。用烟草的16S和23S rRNA标记探针和油菜ct-DNA Bam HI带型杂交,证明F_5、F_(12)和F_(14)含有16S-23S rDNA。根据植物叶绿体rRNA基因的大小及排列,推算出F_(12)含有完整的16S rDNA及16S-23S rDNA的间隔顺序(spacer)。对携带F_(12)的杂合质粒pBN119做了一些初步的研究。     

阴道分离因肯毛孢子菌的分子鉴定和种内分型

分别采用rRNA基因内转录间隔区(ITS)和基因间隔区(IGS1)测序,ITS和IGS1区PCR限制性片段长度多态性分析(PCR-RFLP)和基因组DNA的随机扩增多态性DNA(RAPD)等方法,对三株因肯毛孢子菌Trichosporon inkin进行分子特征及种内分型研究。结果显示,不同菌株的rRNA基因ITS区和IGS1区的序列相似性均高达100%,RFLP酶切图谱具有较理想的种内一致性,而不同菌株的RAPD图谱不尽相同。研究表明:rRNA基因IGS1区测序及RFLP酶切可考虑用于因肯毛孢子菌的菌种分子鉴定,而基因组DNA的RAPD则较适合于菌种的种内分型。     

金针菇rDNA序列结构特征分析

rDNA序列中的ITS作为DNA barcoding广泛应用于真菌的系统发育与物种辅助鉴定,IGS被认为可以用于种内水平不同菌株的鉴别。食用菌中还没有完整的rDNA序列的报道。本研究采用二代和三代测序技术分别对金针菇单核菌株“6-3”进行测序,用二代测序的数据对三代测序组装得到的基因组序列进行修正,得到一个在基因完整性、连续性和准确性均较好的基因组序列,对比Fibroporia vaillantii rDNA序列,获得金针菇完整的rDNA序列。金针菇rDNA序列结构分析表明,它有8个rDNA转录单元,长度均为5 903bp,有9个基因间隔区,其长度有较大差异,3 909-4 566bp。rDNA转录单元中,各元件的序列长度分别为：18S rDNA 1 796bp、ITS1 234bp、5.8S rDNA 173bp、ITS2 291bp、28S rDNA 3 410bp。基因间间隔区中,IGS1 1 351-1 399bp、5S rDNA 124bp、IGS2 2 435-3 092bp。金针菇的5S、5.8S、18S、28S rDNA序列准确性得到转录组数据的验证,也得到系统发育分析结果的支持。多序列比对发现,不同拷贝的基因间间隔区序列（IGS1和IGS2）存在丰富的多态性,多态性来源于SNP、InDel和TRS（串联重复序列）,而TRS来源于重复单元的类型和数量。9个基因间间隔区之间,IGS1只有少量的SNP和InDel,IGS2不仅有SNP和InDel,还有TRS。本研究结果提示,在应用IGS进行种内水平不同菌株之间的鉴别时,需要选取不同拷贝之间的保守IGS序列。     

福建省放线菌结瘤植物共生菌Frankia遗传多样性研究

[目的]了解福建省放线菌结瘤植物共生固氮菌Frankia的遗传多样性.[方法]利用16S-23SrDNA间隔区(rrn)和nifD-K基因间隔区的PCR扩增和RFLP技术,分析了福建省木麻黄、杨梅、桤木、胡颓子等共生Frankia纯培养菌株的遗传差异.[结果]17个菌株获得rrn扩增片段,2个杨梅菌株和1个胡颓子菌株扩增未成功,酶切图谱经聚类分析表明6个地点的细枝木麻黄、短枝木麻黄、粗枝木麻黄12个共生Frankia菌株同源性高,属于一个类群,2个地点的4个杨梅菌株和1个四川桤木菌株亲缘关系近,为另一类群.25个Frankia菌株的,nifD-K基因间隔区PCR-RFLP分析结果显示,7个地点的3种木麻黄14个菌株聚类为一个类群,4个地点的7个杨梅菌株、2个地点的2个四川桤木菌株以及1个台湾桤木菌株聚类为另一个类群,胡颓子菌株则为独立的类群.[结论]研究结果表明福建省共生Frankia遗传多样性丰富.     

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.     

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。     

Molecular characterization of DNA encoding 16S-23S rRNA intergenic spacer regions and 16S rRNA of pectolytic Erwinia species

Sequences of 16S rDNAs and the intergenic spacer (IGS) regions between the 16S and 23S rDNA of bacterial strains from genus Erwinia were determined. Comparison of 16S rDNA sequences from different species and subspecies clearly revealed intraspecies-subspecies homology and interspecies heterogeneity. Phylogenetic analyses of 16S rDNA sequence data revealed that Erwinia spp. formed a discrete monophyletic clade with moderate to high bootstrap values. PCR amplification of the 16S-23S rDNA regions using primers complementary to the 3' end of 16S and 5' end of 23S rRNA genes generated two DNA fragments. The small 16S-23S rDNA IGS regions of Erwinia spp. examined in this study varied considerably in size and nucleotide sequence. Multiple sequence alignment and phylogenetic analysis of small IGS sequence data showed a consistent relationship among the test strains that was roughly in agreement with the 16S rDNA data that reflected the accepted species and subspecies structure of the taxon. Sequence data derived from the large IGS resolved the strains into coherent groups; however, the sequence information would not allow any phylogenetic conclusion, because it failed to reflect the accepted species structure of the test strains.     

Discrimination of Rhizobium tropici and R. leguminosarum strains by PCR-specific amplification of 16S-23S rDNA spacer region fragments and denaturing gradient gel electrophoresis (DGGE)

With the aim of detecting Rhizobium species directly in the environment, specific PCR primers for Rh. tropici and Rh. leguminosarum were designed on the basis of sequence analysis of 16S-23S rDNA spacer regions of several Rh. tropici, Rh. leguminosarum and Agrobacterium rhizogenes strains. Primer specificity was checked by comparison with available rDNA spacer sequences in databases, and by PCR using DNA from target and reference strains. Sequence polymorphisms of rDNA spacer fragments among strains of the same species were detected by denaturing gradient gel electrophoresis (DGGE). The specific PCR primers designed in this study could be applied to evaluate the diversity of Rh. tropici and Rh. leguminosarum by analysing the polymorphisms of 16S-23S spacer rDNA amplified from either whole-cell or soil-extracted DNA.     

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。     

油菜内生细菌16S核糖体DNA的RFLP分析

植物内生细菌定殖在植物组织内部但不引起明显的病害症状。从健康油菜植株的不同器官中分离到大量内生细菌,这些细菌菌落形态存在明显的差异,表明油菜组织中存在大量内生细菌,且类群丰富。分离到的122株内生细菌根据菌落形态可以划分为35类;利用细菌通用引物对16S核糖体DNA进行扩增, 获得约15 kb片段,分别用内切酶HaeⅢ和MspⅠ对扩增产物进行限制性酶切,产生不同的酶切图谱,根据酶切图谱聚类分析结果,所有供试菌株被归为39类,这一结果从遗传上显示油菜内生细菌类群的多样性。两种方法归类结果比较发现菌落特征所反映的信息量有限,只能作为初步的参考指标,核糖体DNA的限制性片段长度多态性分析快速、准确,可以作为油菜内生细菌多样性分析的一种有效方法     

2株创伤弧菌的16S-23S rDNA间区的克隆、测序及分析

根据细菌的16SrDNA3’端和23SrDNA5’端的高度保守区设计引物,PCR扩增了2株创伤弧菌（Vibrio vulnificus）的16S-23SrDNA间区（Intergenic spacer,IGS）,克隆到pGEM-T载体上,测序。用BLAST和DNA star软件对16S-23SrDNA间区序列及其内的tRNA基因进行比较分析。结果表明,2株创伤弧菌共测出9条16S-23SrDNA间区序列,其中ZSU006测出5条,间区类型分别为：IGS^GLAV、IGS^GLV、IGS^LA、IGS^A和IGS^G.其中IGS^GLAv最大,包含tRNA^Glu、tRNA^Lys、tRNA^Ala。和tRNA^Val基因;IGS^GLV包含tRNA^Glu、tRNA^Lys。和tRNA^Val基因;IGS^LA,则包含tRNA^Ile和tRNA^Ala基因;IGS^G包含tRNA^Glu基因;而IGS^A仅包含tRNA^Ala基因。菌株CG021测出的16S-23SrDNA IGS序列有4条,除缺少IGS^A外,其余的IGS类型均与ZSU006的相同。与GenBank内的创伤弧菌ATCC27562的IGS序列比较,发现创伤弧菌所有类型的IGS的tRNA基因两端的非编码区具有较高的种内同源性。16S-23SrDNA间区结构的差异为建立一种新的创伤弧菌检测方法奠定了基础。     

Heteroduplex structures in 16S-23S rRNA intergenic transcribed spacer PCR products reveal ribosomal interoperonic polymorphisms within single Frankia strains

AIMS: Detection of polymorphisms in intergenic transcribed spacer (ITS) 16S-23S rRNA within single Frankia strains. METHODS AND RESULTS: Polymorphisms in the 16S-23S rRNA ITS were investigated in single-colony subcultures of seven Frankia isolates. Multiple ITS-polymerase chain reaction (PCR) bands were detected solely in isolates BMG5.5 and BMG5.11. The slow-migrating bands in the ITS-PCR agarose gel electrophoresis profiles of the isolates were revealed to be heteroduplexes on the basis of their migration shift in different electrophoretic matrices, southern hybridization and the single-strand DNA mung bean endonuclease digestion. Laser-scanned capillary electrophoresis detected two ITS-PCR fragments differing in length by three and six nucleotide insertions/deletions in strains BMG5.5 and BMG5.11, respectively. Sequence analysis of the cloned ITS showed that in strain BMG5.5 the two ITS differed by the presence of three to four copies of the 3-bp tandem repeat 5'-TGG-3'. In strain BMG5.11, the two ITS differed by the presence of two to three copies of the 6-bp tandem repeat 5'-CTTGGG-3'. CONCLUSIONS: We demonstrate the occurrence of ITS 16S-23S rRNa polymorphisms within single Frankia strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We reported the occurrence of ITS 16S-23S rRNA polymorphisms within single Frankia strains from Elaeagnus host group recognized as the more flexible strains within Frankia genus. Furthermore, we underscored the applied interest of strains BMG5.11 and BMG5.5 in future ecological studies using ITS 16S-23S rRNA as molecular marker.     

Genotypic characterization of Bradyrhizobium strains nodulating small Senegalese legumes by 16S-23S rRNA intergenic gene spacers and amplified fragment length polymorphism fingerprint analyses

We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.