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。     

山蚂蝗慢生根瘤菌的遗传多样性及系统发育

摘要:【目的】研究我国亚热带和温带地区与山蚂蝗共生的慢生根瘤菌遗传多样性和系统发育。【方法】采用BOX-PCR 和多位点基因序列分析(nifH,nodC 和recA 基因) 方法对分离自我国不同地区的29 株山蚂蝗慢生根瘤菌进行遗传多样性和系统发育分析。【结果】BOX-PCR 分析表明供试的山蚂蝗慢生根瘤菌形成25个基因遗传型,具有丰富的基因组多样性。多位点基因序列分析发现代表菌株位于慢生根瘤菌的3 个分支上,分别与埃氏慢生根瘤菌(Bradyrhizobium elkanii) ,大豆慢生根瘤菌( Bradyrhizobium japonicum) 和圆明慢生根瘤菌(Bradyrhizobium yuanmingense) 亲缘关系近。【结论】我国山蚂蝗慢生根瘤菌具有丰富的遗传多样性,共生基因系统发育分析表明它们多与持家基因共同进化,并以垂直进化为主。     

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．结果表明这一方法能很好区分、鉴别慢生根瘤菌,也支持该技术是一种快速、简单、准确及重复性好的微生物鉴定手段．     

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     

超慢生型大豆根瘤菌的生理生化和共生特性的研究

超慢生型大豆根瘤菌(ESG,extra—slow-gfowing soybean rhizobium)是不同于大豆另两类共生体——慢生型大豆根瘤菌(Bradyhizobium,japonicum)和快生型大豆根瘤菌(Sin-orhizobium fredii)的新类群。它们在生长速率和生理学特性等方面均表现出较大的差异。根瘤类菌体的扫描结果表明,ESG的类菌体形态为杆状,与另外两群相近,但发现有“Y”形类菌体。ESG利用碳源范围较窄,抗生素自然耐受性比慢生型低,在柠檬酸盐培养基上不生长;代时超长,已测定的7个菌株代时为23．3-41．9h。细胞成分N,c分析结果表明,ESG在三个类群中N含量最高,C含量最低。温室盆栽试验证明ESG中大部分菌株的固氮酶活性和植株含氮量与生产用菌株相当。ESG菌株可以在绿豆上结瘤并有固氮酶活性。     

沙冬青根瘤菌遗传多样性和系统发育分析

利用16S rDNA RFLP、16S-23S rDNA RFLP和16S rDNA序列分析方法,对分离自宁夏和内蒙古阿拉善地区的沙冬青根瘤菌进行了遗传多样性和系统发育分析.结果表明,分离自不同地区沙冬青根瘤菌的44株测试菌株分别归属于中慢生根瘤菌属(Mesorhizobium)、叶杆菌属 (Phylobacterium)、中华根瘤菌属(Sinorhizobium)、根瘤菌属(Rhizobium)、土壤杆菌属(Agrobacterium)5个属种.受寄主和地理环境因素的影响,沙冬青根瘤菌具有丰富的遗传多样性.     

超慢生大豆根瘤菌2062菌株的部分16SrRNA序列测定

采用ＰＣＲ（聚合酶链式反应）的方法扩增并克隆了超慢生大豆根瘤菌（ＥＳＧ,ｅｘｔｒａ－ｓｌｏｗｌｙ－ｇｒｏｗｉｎｇ ｓｏｙｂｅｎ ｒｈｉｚｏｂｉａ）２０６２菌株的１６Ｓ ｒＲＮＡ的部分区段,然后进行核苷酸序列测定和分析。比较了测定的２６４个碱基序列与已经发表的其他相关根瘤菌序列的差异,并通过计算机遗传距离进行聚类分析。结果表明,ＥＳＧ与Ｂｒａｄｙｒｈｉｚｏｂｉｕｍ ｊａｐｏｎｉｃｕｍ、Ｂ．ｅｌｋａｎ     

单株根系来源的土著大豆慢生根瘤菌多样性

本文对由１株大豆根瘤分离、鉴定的１２个土著大豆慢生根瘤菌（Ｂｒａｄｙｒｈｉｚｏｂｉｕｍｊａｐｏｎｉｃｕｍ）菌株进行了种内多样性分析。在ＹＥＭ琼脂培养基上观察菌落大小不同（直径≥１０ｍｍ至≤０５ｍｍ）的菌株,有形成粘稠而半透明的Ｍ型菌落和稀薄而透明的Ｗ型菌落。以ＥＬＩＳＡ鉴定血清类型,分为ＬＬ１９血清族（包括Ｓｚ１１２ｓ）血清组和Ｓｚ１１９ｓ血清型及其他血清组）和非ＬＬ１９血清族（包括Ｓｚ１１３ｓ和Ｓｚ１２１ｓ两血清型及未知血清型）。以Ｄｉｇｏｘｉｇｅｎｉｎ标记的ｐＭＪＳ１２为探针检测菌株染色体ＤＮＡ的ＥｃｏＲＩ消化产物的分子杂交谱,出现称为Ⅰ型的９６ｋｂ和Ⅱ型的６４ｋｂ两种类型的单一分子杂交带。ＨＰ５８９０Ａ气相色谱分析细胞脂肪酸,检出８种成分。其中,Ｃ１４∶１、Ｃ１８∶１和Ｃ１４∶０存在于所有供试菌株中,为不同于快生根瘤菌的３种主要成分,以此可将供试菌株分成６组不同菌株     

花生根瘤菌群体遗传多样性和系统发育研究

利用16S rRNA RFLP,16S rRNA序列分析和16S－23S IGS PCR RFLP技术对43株花生根瘤菌和来自其他种属的15个参比菌株进行了群体遗传多样性和系统分析。16S rRNA PCR RFLP分析结果表明,所有供试花生根瘤菌均属于慢生根瘤菌属,在系统发育上与B.japonicum的亲缘关系最近,具有相同的16S rRNA RFLP基因型,而与B.elkanii相对较远。16S rRNA 序列分析结果表明,供试花生根瘤菌在系统发育上更接近于B.liaoningense,序列间差异小于1％,而B.liaoningense在系统发育上与B.japonicum相距很近,其序列间差异小于1％,16S－23S rRNA IGS RFLP分析结果表明,尽管花生根瘤菌与B.japonicum和B.elkanii的亲缘关系很近,但在71％的相似性水平上供试花生根瘤菌仍各自聚为一群,并可进一步分为A、B、C和D4个亚群,该分群还明显反映了地理因素对群体遗传多样性和系统发育的影响。     

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。     

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.     

华北及西北地区岩黄芪根瘤菌的表型及遗传多样性

选用分离自河北、内蒙古等5省区的岩黄芪根瘤菌30株及17株已知参比菌株,进行了营养利用、抗生素抗性、耐逆性及生理生化反应等12 5项表型性状研究,所得数值分类树状图表明不同宿主及不同地域的岩黄芪根瘤菌的表型多样性。通过对其中部分菌株进行16 S r DNA PCR- RFL P及BOX- PCR指纹图谱分析,聚类结果表明供试岩黄芪根瘤菌具有遗传多样性。     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

Bradyrhizobium elkanii, Bradyrhizobium yuanmingense and Bradyrhizobium japonicum are the main rhizobia associated with Vigna unguiculata and Vigna radiata in the subtropical region of China

Cowpea (Vigna unguiculata) and mung bean (Vigna radiata) are important legume crops yet their rhizobia have not been well characterized. In the present study, 62 rhizobial strains isolated from the root nodules of these plants grown in the subtropical region of China were analyzed via a polyphasic approach. The results showed that 90% of the analyzed strains belonged to or were related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense and Bradyrhizobium elkanii, while the remaining represented Rhizobium leguminosarum, Rhizobium etli and Sinorhizobium fredii. Diverse nifH and nodC genes were found in these strains and their symbiotic genes were mainly coevolved with the housekeeping genes, indicating that the symbiotic genes were mainly maintained by vertical transfer in the studied rhizobial populations.     

桂西北喀斯特常见豆科植物根瘤菌的遗传多样性

调查了桂西北喀斯特24种常见豆科植物的结瘤情况及特征,并从15种宿主植物上获得39份根瘤样品,提取根瘤基因组DNA,扩增16S rDNA和nifH基因,构建系统发育树,对根瘤菌遗传多样性进行了研究.结果表明: 有15种豆科植物是结瘤的,其中14种为蝶形花亚科,1种为含羞草亚科,而云实亚科未发现结瘤.一些本应结瘤的植物未发现根瘤,可能与喀斯特土壤的保水性差有关.BLAST和系统发育分析结果均显示,来源于多种豆科植物的根瘤菌均归属于慢生根瘤菌属,仅有2个亮叶崖豆藤样品的根瘤菌归属于中慢生根瘤菌属.在系统发育树上,来源于同一地点或同一宿主植物的根瘤序列均表现出一定的聚集性,说明共生根瘤菌的种类可能受宿主植物及所处生态环境的共同影响.     

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。     

Bacterial phylogeny based on 16S and 23S rRNA sequence analysis

Abstract: Molecular phylogeny increasingly supports the understanding of organismal relationships and provides the basis for the classification of microorganisms according to their natural affiliations. Comparative sequence analysis of ribosomal RNAs or the corresponding genes currently is the most widely used approach for the reconstruction of microbial phylogeny. The highly and less conserved primary and higher order structure elements of rRNAs document the history of microbial evolution and are informative for definite phylogenetic levels. An optimal alignment of the primary structures and a careful data selection are prerequisites for reliable phylogenetic conclusions. rRNA based phylogenetic trees can be reconstructed and the significance of their topologies evaluated by applying distance, maximum parsimony and maximum likelihood methods of phylogeny inference in comparison, and by fortuitous or directed resampling of the data set. Phylogenetic trees based on almost equivalent data sets of bacterial 23S and 16S rRNAs are in good agreement and their overall topologies are supported by alternative phylogenetic markers such as elongation factors and ATPase subunits. Besides their phylogenetic information content, the differently conserved primary structure regions of rRNAs provide target sites for specific hybridization probes which have been proven to be powerful tools for the identification of microbes on the basis of their phylogenetic relationships.