ARDRA对植物根瘤内共生放线菌Frankia多样性的研究

应用原核生物16SrDNA特异性引物rD1和fD1,通过ARDRA（amplified ribosomal DNA restriction analysis）法直接扩增自中国云南、东北地区赤杨属3种植物和沙棘属1种植物根瘤内FrankiaDNA,得到一长约1500bp的扩增产物,选用两种内切酶HaeⅢ、AfaⅠ联合对扩增产物进行酶切,得到稳定的酶切图谱,将所测48个感染赤杨的Frankia样本区分为3个不同的组,所测43个感染沙棘的Frankia样本区分为3个不同的组,显示根瘤内Frankia存在丰富的遗传多样性。     

应用ARDRA技术研究Frankia菌多样性

应用原核生物16SrDNA特异性引物rD1和fD1,对分自4个分类接种群的12株纯培养Frankia菌总DNA进行扩增,得到1条长约1500bp的扩增产物.选用2种内切酶HinfI,MspI对扩增产物进行酶切,得到稳定的酶切图谱.对图谱的分析结果表明,Frankia菌间存在极其丰富的遗传多样性.     

马衔山中国沙棘根瘤内共生细菌多样性研究

采集甘肃省兰州市马衔山中国沙棘(Hippophae rhamnoidoes)根瘤样品,采用高通量测序和纯培养方法,分析中国沙棘根瘤内定殖的内共生细菌的组成、相对丰度和物种多样性,并比较两种方法研究结果的差异。在不同的微生物分类单元,高通量测序检测到中国沙棘根瘤内共生细菌24门50纲90目167科215属;而纯培养方法仅检测到3门5纲7目8科8属。进一步分析沙棘根瘤内共生细菌主要类群的相对丰度,发现高通量测序与纯培养方法的结果有明显差异,在科和属的分类单元上差异尤其显著。两种方法都表明中国沙棘根瘤内共生细菌具有丰富的多样性,但高通量测序能够较为全面的反映中国沙棘根瘤内共生细菌的群落组成,而纯培养方法仅能够分离到部分可培养的中国沙棘根瘤内共生细菌,在很大程度上极可能低估中国沙棘根瘤内共生细菌的物种组成并高估其丰度。     

长白山三种赤杨根瘤内Frankia基因多样性的REP分析

利用REP PCR方法研究了长白山不同海拔处 3种赤杨根瘤内Frankia菌基因多样性。结果表明 ,赤杨根瘤内Frankia菌存在丰富的基因多样性。宿主和海拔因素共同影响长白山赤杨属Frankia菌的基因多样性 :东北赤杨Frankia菌的宿主专一性很强 ,西伯利亚赤杨和色赤杨的Frankia菌的有较近的亲缘关系。不同海拔的同种宿主共生的Frankia菌基因类型不同 ,相同海拔的西伯利亚赤杨和色赤杨的Frankia菌出现宿主混乱现象     

沙棘根瘤内生菌的多型性

用透射电子显微镜观察了春、夏、秋、冬四个季节的沙棘根瘤,以及瘤瓣上、中、下三个部位。结果表明,不同季节,不同部位的瘤瓣内,根瘤内生菌有7种不同形态。即侵染菌丝体、繁殖菌丝体、营养菌丝体、春孢子及春孢子囊、泡囊,冬孢子及冬孢子囊和类菌体。在多年生珊瑚状的根瘤中,它们的世代交替是:春夏季以侵染菌丝、繁殖菌丝、营养菌丝、春孢子囊及春孢子、泡囊为主;秋冬季以衰退的营养菌丝、衰老泡囊、冬孢子囊和冬孢子、类菌体为主。冬孢子和类菌体是休眠体。     

甘南高寒地区不同海拔西藏沙棘根瘤内生菌多样性

为了研究甘南高寒地区3个不同海拔西藏沙棘根瘤中内生菌的多样性,采用Illumina MiSeq高通量测序技术对3种不同海拔的西藏沙棘根瘤内生菌的菌群组成和多样性进行了分析。实验结果表明,3个不同海拔西藏沙棘根瘤中的内生菌主要包括6大门,分别为蓝藻门(Cyanobacteria),放线菌门(Actinobacteria),变形菌门(Proteobacteria),拟杆菌门(Bacteroidetes),梭杆菌门(Fusobacteria)和厚壁菌门(Firmicutes)。其中占主导地位的微生物是蓝藻门和放线菌门,蓝藻门在3个海拔的西藏沙棘根瘤中的丰度分别为68.1%,64.7%和66.0%,放线菌门在3个海拔的西藏沙棘根瘤中的丰度分别为28.6%,30.2%和29.5%。放线菌门弗兰克氏菌属(Frankia)为共同的优势菌属之一,在3个海拔的丰度分别为28.2%,29.8%和29.1%。3个不同海拔西藏沙棘根瘤中的内生菌除了能与沙棘共生固氮的弗兰克氏菌外,还存在其他的微生物群落,可能存在一些潜在的有价值的内生菌种资源。     

微生物的ARDRA检测

ARDRA（扩增性rDNA限制性酶切片段分析）是新发展起来的一项生物检测技术，可在原位下获取其有关生物性状。本文阐述了ARDRA技术的原理和方法，介绍了该技术在微生物多样性和系统发育研究中的应用，并对ARDRA技术的应用前景提出展望。     

兰坪铅锌尾矿区豆科根瘤菌遗传多样性ARDRA分析

通过16S rDNA扩增产物限制性片段长度多态性分析(ARDRA),对兰坪铅锌尾矿区豆科植物根瘤菌的遗传多样性进行了研究。采用限制性内切酶Hae Ⅲ、Hind Ⅲ、Hinf Ⅰ和Taq Ⅰ对16S rDNA扩增产物进行了酶切分型,根据ARDRA酶切图谱的不同,进行树状聚类。结果表明:49株根瘤菌在40%的相似水平上按氮含量不同及铅锌含量的采集地不同分别聚为OTU1、OTU2和OTU33个群,说明根瘤菌的遗传多样性及分布与土壤中的氮含量和铅锌含量有关。代表菌株的16S rDNA测序结果分析表明,它们在系统发育树上属于Rhizobium sp.、Sinorhizobium sp.和Bradyrhizobium sp.3个系统发育分支,进一步说明兰坪铅锌尾矿区豆科植物根瘤菌多样性较丰富。     

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。     

欧洲赤杨根瘤及其内生菌的形态学研究

对在云南生长的欧洲赤杨自然发生的根瘤及其内生菌──Ｆｒａｎｋｉａ进行了光学显微镜和扫描电镜的观察,结果表明,这种根瘤与原产地生长的欧洲赤杨所结根瘤十分相似;与原产地在云南的蒙自桤木的根瘤结构也很相近。从这种根瘤中分离到的内生菌的纯培养具有典型的Ｆｒａｎｋｉａ的特征。     

Correlations between the ages of Alnus host species and the genetic diversity of associated endosymbiotic Frankia strains from nodules

Nodule samples were collected from four alder species: Alnus nepalensis, A. sibirica, A. tinctoria and A. mandshurica growing in different environments on Gaoligong Mountains,Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS between nifD and nifK genes was directly applied to uncultured Frankia strains in the nodules. A total of 21 restriction patterns were obtained. The Frankia population in the nodules of A. nepalensis had the highest genetic diversity among all four Frankia populations; by contrast, the population in the nodules of A. mandshurica had the lowest degree of divergence; the ones in the nodules of A. sibirica and A. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated that Frankia strains from A. sibirica and A. tinctoria had a close genetic relationship. Frankia strains from A. nepalensis might be the ancestor of Frankia strains infecting other Alnus species. From these results and the inference of the ages of Alnus host species, it is deduced that there was a co-evolution between Alnus and its microsymbiont Frankia in China.     

Correlations between the ages of Alnus host species and the genetic diversity of associated endosymbiotic Frankia strains from nodules

Nodule samples were collected from four alder species: Alnus nepalensis, A. si-birica, A. tinctoria and A. mandshurica growing in different environments on Gaoligong Mountains, Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS between nifD and nifK genes was directly applied to uncultured Frankia strains in the nodules. A total of 21 restriction patterns were obtained. The Frankia population in the nodules of A. nepalensis had the highest genetic diversity among all four Frankia populations; by contrast, the population in the nodules of A. mandshurica had the lowest degree of divergence; the ones in the nodules of A. sibirica and A. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated that Frankia strains from A. sibirica and A. tinctoria had a close genetic relationship. Frankia strains from A. nepalensis might be the ancestor of Frankia strain     

Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.     

Flavonoid-like compounds from seeds of red alder (Alnus rubra) influence host nodulation by Frankia (Actinomycetales)

Nitrogen-fixing root nodules are formed by Frankia spp. (Actinomycetales) on dicotyledonous hosts such as alders ( Alnus spp.). Flavonoid-containing preparations from seed washes of red alder ( Alnus rubra Bong.), and individual compounds isolated from such preparations, influenced nodulation of A. rubra by Frankia. Nodulation was enhanced by one flavonoid-like compound, and apparently inhibited by two other such compounds. Four flavonoid-like compounds had no significant effect on nodulation. The seven individual compounds purified from the seed washes were characterized spectrally as possible flavanones and isoflavones. Both the enhancer and the inhibitors appeared to be possible flavanones.     

Purity of Frankia preparations from root nodules of Alnus incana

Frankia vesicle clusters were prepared from Alnus incana (L.) Moench root nodules by a homogenization-filtration procedure. The preparation was examined by transmission electron microscopy and computerized picture analysis to quantify contamination from the host plant. Special attention was paid to plant mitochondria. Mitochondria were only found in 30% of the 50 sections of clusters examined. In sections containing mitochondria the mean number of mitochondria per cluster section was 1.5. The relative volume of all objects found in the vesicle clusters was calculated. More than 98% of the volume of a preparation consisted of Frankia vesicles and hyphae, while only 0.4% of the volume was host plant mitochondria. The frequency of mitochondria in a preparation could be further decreased by osmotic shock. It is concluded that Frankia vesicle clusters, prepared from Alnus incana by the homogenization-filtration technique used here, are sufficiently pure to be used for studies of Frankia metabolism.