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。     

小克银汉霉核糖体DNA PCR扩增产物的限制性片段长度多型性

应用一对寡核苷酸引物ITSl与ITS4对核DNA G+C百分数小于30％的小克银汉霉(Cunninghamella)属的核糖体DNA(rDNA)内转录间区(ITS)进行了扩增。测试的属于10个种和变种的22株菌都得到了扩增产物。在同属不同种之间扩增的ITS片段长度有巨大差别。据此可分为三组。这三组所含分类群数及其DNA长度分别为：第一组4种1变种,小于 764碱基对(bp);第二组2种,765～824 bp;第三组2种l变种,大于825 bp。所研究的许多种中,特别是第三组,单凭其PCR产物的长度就能区分开来。但在第一、二组就需借助限制性内切酶的分析才能予以区分。我们选用了Rsa I,Tru 9 I,和Hinf I 4种限制性内切酶,对所有扩增产物进行了限制性片段长度多型性(RFLPs)的分析。在雅致小克银汉霉(C elegans),巴西玉蕊小克银汉霉(C.bertholletiae)、和布拉氏霉(C. blakesleeana )各种的限制性酶切图谱,种内非常一致而种与种之间有差别。反之,在某些种其限制性酶切图谱不仅种间互不相同,在种内不同株之间也出现1～3种酶切图型的差异。在这种情况下,只能综合各种资料才能将它们区分开来。本研究肯定了PCR-RFLP在区分小克银汉霉种和变种上的意义,并发现种内个体的差异。这在我们后来所进行的序列分析研究中得到了进一步的证实。     

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.     

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.     

小克银汉霉核糖体DNA PCR扩增产物的限制性片段长度多型性(英文)

应用一对寡核苷酸引物ITS1与ITS4对核DNAG＋C百分数小于30％的小克银汉霉（Cunninghamella）属的核糖体DNA（rDNA）内转录间区（YTS）进行了扩增。测试的属于10个种和变种的22株菌都得到了扩增产物。在同属不同种之间扩增的ITS片段长度有巨大差别。据此可分为三组。这三组所含分类群数及其DNA长度分别为;第一组4种1变种,小于764碱基对（bp）;第二组2种,765～824bp;第三组2种1变种,大于825bp。所研究的许多种中,特别是第三组,单凭其PCR产物的长度就能区分开来。但在第一、二组就需借助限制性内切酶的分析才能予以区分。我们选用了RsaI,TaqI;Tru9I,和HinfI4种限制性内切酶,对所有扩增产物进行了限制性片段长度多型性（RFLPs）的分析。在雅致小克银汉霉（Celegans）、巴西玉蕊小克银汉霉（Cbertholletiae）、和布拉氏霉（Cblakesleeana）各种的限制性酶切图谱,种内非常一致而种与种之间有差别。反之,在某些种其限制性酶切图谱不仅种间互不相同,在种内不同株之间也出现1～3种酶切图型的差异。在这种情况下,只能综合各种资料才能将它们区分开来。本研究肯定了PCR－RFLP在区分小克银汉霉种和变种上的意义,并发现种内个体的差异。这在我们后来所进行的序列分析研究中得到了进一步的证实。     

西藏沙棘5种不同组织内生细菌多样性

西藏沙棘(Hippophae tibetana)是分布于高寒高海拔地区的一类特殊的放线菌结瘤植物, 弗兰克氏菌能够侵染其根部形成根瘤, 因共生固氮等作用而增强其生态适应性。在西藏沙棘的根瘤中, 除了弗兰克氏菌之外还有其他内生菌, 而弗兰克氏菌又不仅仅在根瘤中有分布。为了探究弗兰克氏菌在西藏沙棘不同组织中的定殖及可能的迁移规律, 分析不同组织中内生细菌的群落结构及多样性, 本研究以生长在甘肃省天祝藏族自治县抓喜秀龙金强河河滩地的西藏沙棘为材料, 应用16S rRNA扩增子高通量测序技术, 对西藏沙棘根瘤、茎、枝、叶和种子等不同组织的内生细菌多样性进行了分析。研究结果表明, 西藏沙棘根瘤内生细菌群落丰富度及多样性最高, 种子内生细菌群落丰富度最低, 茎内生细菌群落多样性最低。西藏沙棘5种不同组织中的弗兰克氏菌和其他内生细菌多样性都具有一定差异, 变形菌门均为优势门, 弗兰克氏菌属(Frankia)为根瘤内生细菌群落的优势属, 弗莱德门菌属(Friedmanniella)为茎内生细菌群落的优势属, 寡养单胞菌属(Stenotrophomonas)为枝、叶和种子内生细菌群落的优势属。研究结果还表明, 弗兰克氏菌属不仅仅存在于西藏沙棘的根瘤, 还能够分布于其他组织, 且在同一种组织中存在弗兰克氏菌属的不同“种”; 而在西藏沙棘不同组织中, 也分布有弗兰克氏菌属的相同“种”。此外, 对西藏沙棘5种不同组织内生细菌中的功能菌株的分析表明, 不同组织中均存在着具有固氮、促生和抑菌功能的内生细菌, 但具有固氮作用的内生细菌主要分布于根瘤, 具有促生作用以及抑菌功能的内生细菌主要分布于枝和叶。综上, 西藏沙棘5种不同组织内生细菌具有丰富的多样性, 但各组织内生细菌的群落结构和优势种群有所不同, 且不同组织也能够定殖具有多种功能的内生细菌。     

高黎贡山旱冬瓜Frankia的IGS PCR-RFLP分析

在云南省高黎贡山自然保护区海拔1310～2400m的范围内,采集30个旱冬瓜根瘤样品,直接从根瘤中提取Frankia DNA,对其,nifD-nifK基因间隔区(intergenic spacer,IGS)和16S-23S rDNA IGS进行PCR—RFLP分析．结果表明,nifD-nifK IGS的PCR产物长度差异很大,经HaeⅢ和Afa I双酶切后,得到15种酶切带型,检测到多种基因型的菌株同时与同一株宿主植物共生;16S-23S rDNA IGS的PCR产物长度相似,酶切后亦区分出15种酶切带型．通过对两个基因间隔区的PCR-RFLP联合分析,发现高黎贡山旱冬瓜Frankia存在20种基因型．     

利用PCR-RFLP技术鉴定传粉榕小蜂隐种混合样品的物种组成

隐种(cryptic species)是指形态上几乎完全相同但遗传组成存在显著分化的物种。在榕树–榕小蜂一对一共生系统中, 传粉榕小蜂隐种的发现对协同进化、物种共存等重要的生态和进化理论提出了严峻的挑战。因为很难从形态上直接区分隐种, 所以, 相关研究中的一个迫切需要解决的问题就是如何快速而准确地鉴定隐种。本文采用PCR-RFLP方法分析了mtDNACOI基因片段, 对木瓜榕(Ficus auriculata)和鸡嗉果榕(F. semicordata)的传粉榕小蜂隐种进行了区分。结果表明为木瓜榕传粉的大果榕小蜂(Ceratosolen emarginatus)存在两个隐种(A和B), 分别包含1个XhoI和1个BssSI酶切位点。将两个隐种的样品按不同比例混合, 提取基因组DNA, PCR扩增mtDNA COI片段, 经XhoI和BssSI分别酶切, 均能通过酶切图谱准确检测出混合样品的隐种组成。鸡嗉果榕小蜂(C. gravelyi)两个隐种的mtDNA COI基因序列也存在较大差别, 分别包含1个BmrI和1个AvaI酶切位点, 隐种混合样品经BmrI和AvaI分别酶切的结果也能准确鉴定混合样品的物种组成。我们的结果表明基于PCR和DNA酶切技术能快速而准确地区分传粉榕小蜂的隐种。     

深港两地工程土壤细菌多样性的T-RFLP分析

目的:建立一种高效的末端限制性片段长度多态性(T-RFLP)方法,检测工程土壤细菌。方法:以深圳和香港两地不同土层(0.5,10,20和30 m)的工程土壤为研究对象,提取并纯化总DNA,利用细菌16S r DNA基因通用引物8f-FAM/1492r进行PCR扩增,扩增产物分别用HhaⅠ、HaeⅢ和MspⅠ限制性内切酶酶切,酶切产物经毛细管电泳测序,序列上传至数据库进行分析。结果:经过比对分析,香港段土壤中大约存在细菌141属,235种;深圳段大约存在132属,206种;32个细菌种属只在香港土壤中有分布,3个细菌种属只在深圳土壤中有分布;植物病原细菌有14个种属。结论:建立的T-RFLP方法能够高效、快速地分析工程土壤细菌。     

多重环介导核酸等温扩增技术检测血液中常见病原体

背景：血液安全性筛查是输血前必要检测项目。目前临床采用血清学检测技术,存在较长的检测窗口期,易产生假阴性检测结果,造成输血交叉感染。目的：建立多重环介导核酸等温扩增技术,实现在一管反应体系内同时检测四种病原体：乙肝病毒,丙肝病毒,艾滋病毒和梅毒螺旋体。方法：通过限制性酶切处理多重环介导核酸等温扩增产物,利用酶切产物的长度分析扩增产物的种类,从而分析待测样本中含有何种血液病原体。结果：检测164例临床样本,其检测结果可以通过琼脂糖电泳,聚丙烯酰胺凝胶电泳及芯片电泳分析,且均可实现对多重扩增产物的酶切片段进行区分和鉴别。结论：多重环介导核酸等温扩增技术可以同时单管检测多种待测血液病原体,可以为临床提高简单、快速、高灵敏和高特异的检测技术。     

Transformation of Saussurea involucrata by Agrobacterium rhizogenes: Hairy root induction and syringin production

Agrobacterium rhizogenes-mediated genetic transformation of Saussurea involucrata was investigated. Four bacterial strains, A4, LBA 9402, R1000 and R1601 and three explant types, leaf blade, petiole and root, were examined. Over 100 hairy root lines were successfully established with strains R1601, R1000 and LBA9402, but none with A4. The highest transformation efficiency of 67% was achieved by using strain R1601 with root explants. One hairy root line isolated from this combination, HR1601-1, produced up to 43.5 ± 1.13 mg syringin g⁻¹ dw, which is about 50-fold higher than that in the wild type plants.Two other lines, HR1000-1 and HRLBA9402-1, isolated from R1000- and LBA9402-transformed roots, respectively, also displayed high capacity of syringin production, being 32.5 ± 3.08 and 39.7 ± 1.37 mg syringin g⁻¹ dw. These three lines were characterized in detail. Polymerase chain reaction analyses confirmed these root lines were of A. rhizogenes origin.     

The tropical legume Sesbania rostrata: Tissue culture, plant regeneration and infection with Agrobacterium tumefaciens and Rhizogenes strains

Conditions were examined for callus induction and in vitro morphogenesis of Sesbania rostrata. A protocol for organogenesis from different S. rostrata explants (cotyledons, hypocotyls, immature embryos) was established and used to regenerate plants. The cytokinin BAP was found to be essential for shoot formation at concentrations of 0.2–1.0 mg/l. SH medium, free of hormones or supplemented with 0.1 mg/l naphthaleneacetic acid (NAA), was found to stimulate root development of the regenerated plantlets. The susceptibility of S. rostrata to Agrobacterium mediated infection/transformation was tested using different wild type A. tumefaciens (C58 and B6S3) and A. rhizogenes (15834) strains. An extensive systemic infection of S. rostrata by the agrobacterial strains was observed, presumably occurring via spread of the bacteria in the vascular bundles.     

Characterization of transgenic root cultures of Trifolium repens, Trifolium pratense and Lotus corniculatus and transgenic plants of Lotus corniculatus

Hairy root cultures of three species of legume, Lotus corniculatus, Trifolium repens and T. pratense were established using a wild-type strain (C58C1 with pRi 15834) of Agrobacterium rhizogenes. Southern hybridization analysis confirmed that the lines were genetically transformed. Copy numbers of TL-DNA in different lines varied from one to eight. Examination of the transformed root cultures revealed changes in anatomy, morphology and cytology. Plants that had regenerated from hairy roots of L. corniculatus showed changes in morphology, physiology and cytology but no change in several parameters of nitrogen fixation activity.     

Phenotypic and genomic characteristics of rhizobia isolated from Genista tinctoria root nodules

Forty three rhizobial strains isolated from root nodules of Genista tinctoria growing in England, Ukraine, and Poland were compared with 21 representatives of the recognized rhizobial species and two unclassified Bradyrhizobium sp. (Lupinus) strains by performing a numerical analysis of 102 phenotypic features and with the reference bradyrhizobia by simplified AFLP analysis with one restriction enzyme PstI and one selective primer PstI-A. All Genista tinctoria microsymbionts were slow-growing bradyrhizobia with generation time of 10–14 h, acid tolerant, salt sensitive, and antibiotic resistant. Cluster analysis based on the phenotypic properties of all bacteria included, grouped dyer's broom rhizobia together with Bradyrhizobium strains, and classified them into three major phena according to their geographic origin. Genista tinctoria nodule isolates were separated into three clusters with the strain composition as in a phenogrouping by AFLP patterns. The presented results, suggest the relationship of G. tincoria microsymbionts to Bradyrhizobium species and show the usefulness of AFLP analysis for differentiation and classification of the studied rhizobia.     

刺槐核糖体蛋白同源基因RpRPL22在共生结瘤过程中功能研究

目的: 核糖体蛋白(RPs)属于多功能蛋白,能够参与调控细胞生长和响应胁迫条件。RpRPL22是一个从豆科植物刺槐中分离得到的结瘤相关基因,通过序列比对发现其与核糖体大亚基蛋白RPL22高度同源。对其如何通过调控根瘤菌侵染而在共生结瘤过程中发挥重要作用进行了较为深入的探索。方法: 利用实时荧光定量PCR技术(qRT-PCR)分析RpRPL22在接菌后不同时间及不同植物组织的表达变化。利用cDNA末端快速扩增技术(RACE)获得目的基因cDNA全长。通过GFP报告基因进行RpRPL22亚细胞定位分析。通过Gateway BP重组技术构建RNA干扰(RNAi)重组载体,借助电转化法将重组载体转至农杆菌K599,利用农杆菌介导植物根部,接菌后观察和测量植株表型。首先从宏观水平统计观察目的基因是否对结瘤过程有影响,其次从分子水平揭示目的基因在共生结瘤过程的重要功能。结果: 不同接菌时期、不同植物组织目的基因qRT-PCR相对表达量结果显示,几乎在所有取样的接菌时间,目的基因RpRPL22在接菌根中的相对表达量都低于未接菌对照根,只有接菌后第25天除外。在成熟的根瘤中,接菌后第25天该基因的表达量也最高。洋葱表皮和毛状根亚细胞定位结果均显示在椰菜花叶病毒(CaMV)的35S启动子控制下,RpRPL22融合绿色荧光蛋白GFP的荧光信号在细胞核和细胞质有明显的表达。RNAi转化植株的表型统计观察结果,比如植株鲜重、植株的有效结瘤数目较对照组均有明显的降低;同时RNAi转化植株在根瘤菌侵染过程形成的侵染线数目和根瘤原基数目较对照均显著降低。根瘤切片实验用于观察根瘤显微超微结构,结果显示RNAi植株根瘤中固氮区的受菌侵染细胞数目与对照相比明显减少。电镜观察根瘤单个受菌侵染细胞中类菌体形态显示,RNAi根瘤中类菌体侵染细胞胞体多呈不规则形状,皱缩变形严重,环类菌体周间隙空间增大,多共生体融合,表现出细胞凋亡的迹象。对照根瘤中的受菌侵染细胞胞体多呈圆形椭圆形,胞质饱满丰富且分布均匀,细胞发育正常,表明RNAi植株根瘤发育过程明显受阻。结论: 核糖体蛋白(RP)能够参与调控豆科植物共生结瘤过程,相关同源基因RpRPL22可能在起始根瘤菌侵染植物和阻止类菌体降解过程中起重要作用。     

The sporadic occurrence of a group I intron-like element in the mtDNA rnl gene of Ophiostoma novo-ulmi subsp. americana

The presence of group I intron-like elements within the U7 region of the mtDNA large ribosomal subunit RNA gene (rnl) was investigated in strains of Ophiostoma novo-ulmi subsp. americana from Canada, Europe and Eurasia, and in selected strains of O. ips, O. minus, O. piceae, O. ulmi, and O. himal-ulmi. This insertion is of interest as it has been linked previously to the generation of plasmid-like mtDNA elements in diseased strains of O. novo-ulmi. Among 197 O. novo-ulmi subsp. americana strains tested, 61 contained a 1.6 kb insertion within the rnl-U7 region and DNA sequence analysis suggests the presence of a group I intron (IA1 type) that encodes a potential double motif LAGLIDADG homing endonuclease-like gene (HEG). Phylogenetic analysis of rnl-U7 intron encoded HEG-like elements supports the view that double motif HEGs originated from a duplication event of a single-motif HEG followed by a fusion event that combined the two copies into one open reading frame (ORF). The data also show that rnl-U7 intron encoded ORFs belong to a clade that includes ORFs inserted into different types of group I introns, e.g. IB, ID, IC3, IA1, present within a variety of different mtDNA genes, such as the small ribosomal subunit RNA gene (rns), apo-cytochrome b gene (cob), NADH dehydrogenase subunit 5 (nad5), cytochrome oxidase subunit 1 gene (coxI), and ATPase subunit 9 gene (atp9).

We also compared the occurrence of the rnl-U7 intron in our collection of 227 strains with the presence of the rnl-U11 group I intron and concluded that the U7 intron appears to be an optional element and the U11 intron is probably essential among the strains tested.     

费氏中华根瘤菌HN01DL在大豆根圈的定殖动态与结瘤研究

以发光酶基因luxAB为标记,在根盒缩影条件下研究了费氏中华根瘤菌HN01DL在大豆根圈的定殖动态、分布范围及其结瘤情况.结果表明,HN01DL在根盒灭菌土壤和非灭菌土壤缩影中的大豆全根系定殖动态与水平明显不同,前者在第12d时达到最高定殖密度(8.65logcfu·g^-1),而后者的早期定殖数量下降较快,且于第15d时达到最高定殖密度(6.88logcfu·g^-1).HN01DL在大豆播种5d后在大豆根部的A(0～4cm)区根段上达到最高定殖密度(7.05logcfu·g^-1),然后开始缓慢下降,至第19d时仍维持相对稳定,在第33d时又开始回升.至播种后第46d时HN01DL可散布至种子下方16cm处的根段部位.HN01DL在A区根段的定殖密度持续较高,所形成的发光根瘤总数(16.3个)及发光比例(68.8%)最高,且发光根瘤主要集中于该区段主根上.发光根瘤比例沿A-E区段逐渐下降,在E区段未检测到发光根瘤.     

广西葛根内生细菌的分离鉴定及其促生特性

【背景】植物内生菌长期与宿主共生,对宿主生长发育产生影响。葛根作为重要的药食两用作物,葛根内生菌的研究具有重要实践意义。【目的】对广西葛根根部内生细菌进行分离、鉴定及促植物生长特性分析,旨在了解该药食同源植物内生细菌种群结构及其促生特性,为分析内生菌群体在药食同源植物产量和品质形成的作用及其内生细菌资源的开发利用提供参考。【方法】采用6种不同的培养基从广西葛根的根瘤、根系和根愈伤组织分离内生细菌,16S rRNA基因测序和系统发育分析内生细菌的分布特征和遗传多样性,采用生理生化方法测定分离菌株的固氮活性、溶磷特性、产生嗜铁素、分泌吲哚乙酸(indole-3-aceticacid,IAA)等促生特性。【结果】从葛根根瘤、根系和根部愈伤组织中共分离得到223个菌株,16S rRNA基因测序鉴定这些菌株隶属于2门4纲10科19属,其中芽孢杆菌属、假单胞菌属、土壤杆菌属、肠杆菌属为葛根优势菌群;内生细菌数量和群落组成存在明显的组织特异性,其数量表现为根瘤>根系>根愈伤组织,但其种群多样性表现为根愈伤组织>根系>根瘤。不同培养基分离出的细菌种群丰富度有差异。从供试菌株中筛...     

Application of lat gene disruption to increase the clavulanic acid production of Streptomyces clavuligerus

A 1.7 kb fragment of lat was obtained from Streptomyces clavuligerus NRRL 3585, and recombinant plasmid pKC1139-lat, which was used to disrupt the lat gene was constructed. pKC1139-lat was introduced into S. clavuligerus by bi-parental conjugation from Escherichia coli ET12567 to S. clavuligerus. The apramcin-resistant transformants were obtained and through homogeneous single-crossover between recombinant plasmid pKC1139-lat and the S. clavuligerus chromosome lat disrupted mutant strains were obtained. The genome of S. clavuligerus NRRL 3585 and the lat disrupted mutants were analyzed by PCR technique, the bioactivity of cephamycin C in the two kinds of strains were also tested. Both results proved that lat was disrupted by the insertion of pKC1139 in the lat disrupted mutants. And the production of clavulanic acid of these two kinds of strains were analyzed by HPLC with different incubation time interval (96 and 120 h), and the yield in the lat mutants was approximately 2.6 fold higher at their highest production point.