紫云英根瘤菌基因文库的构建及含完整结瘤基因的重组质粒pRaZ15的分离

以紫云英根瘤菌菌株7653R为材料,制备总DNA,经EcoRⅠ限制酶部分酶解,通过10—50％蔗糖梯度离心,分离到20一30 kb的DNA片段。利用能在革兰氏阴性菌中转移和复制的广谱寄主载体——pLAFRl质粒,构建了紫云英根瘤菌基因文库。通过与苜蓿根瘤菌102l菌株中8．7kb的共同结瘤基因(作探针DNA)杂交,从基因文库中分离到紫云英根瘤菌共同结瘤基因片段。以紫云英根瘤菌不结瘤突变株7653R+1(7653R消除共生质粒)为受体、构建的7653R基因文库(E．Coli C600)为供体,通过协助转移质粒pRK2013(LE392)进行三亲交配,在含四环素的根瘤菌台成培养基(sM)上选择接合转移子。将得到的所有接台转移子混合在一起接种植物,通过植物结瘤试验,分离到含紫云英根瘤菌结瘤基因的重组质粒pRaz15。将该质粒用EcoRⅠ完全酶切,得到25kb左右的外源DNA片段,该片段携带完整的结瘤基因簇。     

紫云英根瘤菌共同结瘤基因nodA和nodBC的核苷酸序列

以~(32)p标记的苜蓿根瘤菌(Rhizobium meliloti)2.3kb nod DNA作探针,从紫云英根瘤菌(Rhizobium huakuii即R. astragali)159基因文库中分离到一株能与探针DNA呈阳性反应的克隆pRaN109。同源DNA-DNA杂交及DNA序列分析表明:pRaN109DNA的9kb EcoRI片段上携带了nodD_1BC基因,pRaN109 NDA的18kb EcoRI片段上携带了nodD_2A基因。共同结瘤基因nodA与nodBC两者相距6.7kb。在nodA基因和nodBC基因的上游都存在有结瘤盒(nod box)。与来自不同种属的菌株所报告的结果相比较,紫云英根瘤菌159中的共同结瘤基因有着明显不同的组合。     

中华根瘤菌Sinorhizobium sp.1128自体诱导物合成酶基因的克隆及对生理功能的影响

[目的]从中华根瘤菌Sinorhizobium sp.1128中克隆自体诱导物合成酶基因,从而研究该基因在Sinorhizobium sp.1128群体感应系统中的作用.[方法]利用基因序列同源性比对以及分子克隆的方法,从中华根瘤菌Sinorhizobium sp.1128中克隆自体诱导物合成酶基因;利用大肠杆菌异源表达、C18反相薄层层析(TLC)的方法研究该基因的特性;通过中间片段融合的方法缺失该基因,并通过结瘤实验研究该基因对Sinorhizobium sp.1128生理功能的影响.[结果]以草木樨中华根瘤菌Sinorhizobium medicae WSM419自体诱导物合成酶基因Smed_1560序列设计引物,通过PCR扩增在Sinorhizobium sp.1128中寻找到一新的自体诱导物合成基因,命名为traI2.该基因在大肠杆菌Escherichia coli DH5α中表达后能产生两种自体诱导物分子.在Sinorhizobium sp.1128中将该基因缺失,自体诱导物活性下降;回复突变后,自体诱导物活性得到恢复,结瘤实验结果表明该基因能影响根瘤菌的结瘤效率.[结论]中华根瘤菌Sinorhizobium sp.1128群体感应系统是一个复杂的交互系统,它对结瘤的生理功能具有一定的影响.     

豆科植物—根瘤菌共生固氮的基因调控和转移

豆科植物-根瘤菌共生固氮是由寄主与细菌双方基因共同参与完成的,在相互基因调控中形成特导的结构—根瘤。参与根瘤菌固氮(nif基因)和结瘤(nod基因)的基因已被克隆和分析。     

费氏中华根瘤菌042BS结瘤调节基因的克隆及功能检测

费氏中华根瘤菌 (Sinorhizobiumfredii) 0 4 2BS可以在大豆和苜蓿上结瘤。用费氏中华根瘤菌USDA2 5 7的nodD1和nodD2基因分别作为探针 ,与 0 4 2BS总DNA进行Southern杂交 ,发现其DNA经EcoRI酶切后分别在 3 0kb和 6 0kb处各有一条阳性带。回收这两条阳性带附近的DNA片段 ,建立部分基因文库 ,克隆到带有nodD1基因的 3 0kb片段 ,以及带有nodD2基因的 6 0kb片段。对nodD1和nodD2进行序列分析 ,结果表明 0 4 2BS的nodD1与费氏中华根瘤菌根瘤菌USDA2 5 7和USDA1 91的同源性高达 99% ,而nodD2与USDA2 5 7的同源性为1 0 0 %。再将nodD1的片段克隆到pBBRIMCS 5载体上 ,导入豌豆根瘤菌蚕豆生物变种 (Rhi zobiumleguminosarumbv.viciae)LPR5 0 5 4中进行功能检测 ,显示 0 4 2BS的nodD1均可被大豆分泌的类黄酮物质染料木黄酮以及苜蓿分泌的类黄酮物质毛地黄黄酮所诱导     

普通结瘤基因探针鉴定根瘤菌的研究

普通结瘤基因（ｎｏｄＡＢＣ）是所有根瘤菌所特有的、最为保守的基因,用苜蓿根瘤菌结瘤基因（ｎｏｄＡＢＣ）和豌豆根瘤菌的（ｎｏｄＣ）基因片段为探钉,与５２株包括常见土壤细菌、已知根瘤菌、根瘤未知分离物的总ＤＮＡ进行斑点杂交,探索用普通结瘤基因（ｎｏｄＡＢＣ）或（ｎｏｄＣ）探针鉴定根瘤菌的可能性。结果,未找到合适实验条件,使来自这两个种的结瘤基因只能与根瘤菌菌株杂交,而不与土壤细菌的菌株杂交。但在高温条件下,两种探针都专一性的和种内菌株杂交。此结果表明：在一定的实验条件下,普通结瘤基因探针用做根瘤菌的鉴定,只能     

慢生型大豆根瘤菌的结瘤基因转入快生型大豆根瘤菌中

通过三亲本杂交把慢生型大豆根瘤菌USDAllO的基因文库转移至Tn5诱变的不结瘤的快生型大豆根瘤菌321,338中,用链霉素和四环素平板选择大量接合子,接种大豆,通过结瘤基因功能互补,获得7个根瘤,从中分离出的每个菌株都仍具有链霉素和四环素抗性。分离其质粒,发现每个菌株都多了一条较载体质粒pLAFRl分子量大的质粒。将这些质粒转移至大肠杆菌HBl01中,分离其质粒,用32P标记的ncd探针进行DNA—DNA分子杂交,除载体质粒pLAFRl为阴性反应外,其他重组质粒均为阳性反应,即所获得pLAFRl克隆的DNA片段上确有nod结构基因。回收重组质粒pLAFRl：：nod,用限制性内切酶EcoR I进行酶切,从其琼脂糖凝胶电泳图上估计pLAFRl上克隆的DNA片段分子量为32kb。     

Rpfan37在刺槐共生结瘤过程中的功能探究

目的:研究刺槐中与磷脂酰肌醇转运蛋白有较高同源性的基因Rpfan37的功能,为探究相关基因参与豆科植物与根瘤菌共生结瘤过程提供新的思路。方法:通过前期研究,建立豆科植物刺槐与共生根瘤菌互作的抑制差减杂交反交文库,筛选疑似与共生结瘤相关的基因。利用PCR技术快速克隆经实时荧光定量PCR技术(qRT-PCR)分析基因在不同接菌时间及不同植物组织的表达。构建RNA干扰(RNAi)重组载体,转农杆菌介导转化植物根部,接种根瘤菌后验证该基因在刺槐共生结瘤过程的功能。结果:基因表达分析显示,在接菌与未接菌的刺槐根中,处理后第15天,Rpfan37表达均显著上调,但接菌与未接菌处理对该基因表达无显著影响;在成熟的根瘤中,该基因仅为低水平表达。RNAi转化植株的鲜重、株高、根长及结瘤数较对照组显著降低。在显微镜下观察到RNAi植株根毛发育异常;与对照相比,RNAi转化植株形成的根毛卷曲、根毛侵染线及根瘤原基数目均显著降低。根瘤石蜡切片结果显示RNAi植株根瘤中的侵染细胞与对照相比明显减少,分析豆血红蛋白表达发现,RNAi植株中根瘤发育成熟过程明显受阻。结论:在豆科植物刺槐中发现的相关基因Rpfan37能够参与刺槐共生结瘤过程,为研究磷脂酰肌醇转运蛋白在共生结瘤过程中的作用提供了新的理论基础。     

一株能在大豆上结瘤的苜蓿中华根瘤菌

苜蓿中华根瘤菌(Sinorhizobium meliloti)XJ96077分离自新疆的苜蓿根瘤中,其原宿主为紫花苜蓿(Medicago sativa)。交叉结瘤试验发现,它既可在苜蓿上又能在大豆上结瘤固氮。DNA(G C)mol％分析表明,XJ96077的DNA(G C)mol％为61．9％,与已报道的根瘤菌属的DNA(G C)mol％范围(59％-64％)相符。DNA同源性分析表明,XJ96077与苜蓿中华根瘤菌USDA1002^T和042BM的同源性分别达到93％和80％,说明XJ96077归属于苜蓿中华根瘤菌。应用绿色荧光蛋白基因标记XJ96077,得到重组菌株XJ96077(G)。将其接种普通紫花苜蓿,通过激光共聚焦荧光显微镜可以检测到标记基因的表达。接种北引1号大豆上,同样可以清楚地观察到标记基因在根瘤中的表达,从而确证了XJ96077能同时在苜蓿和大豆上结瘤。通过不同品种大豆的结瘤试验,发现XJ96077对大豆品种的结瘤能力不同。     

苜蓿中华根瘤菌042B共同结瘤基因nodABC的克隆与序列分析*

根瘤菌的nodABC和nodD在结构和功能上保守,在不同的菌种之间能够互换〔1〕,是目前所有供试的豆科植物结瘤所必不可少的,称为共同结瘤基因。另一类是寄生专一性结瘤基因,如苜蓿中华根瘤菌(Sinorhizobium meliloti)的nodPQ等〔2〕,这些基因决定根瘤菌能与哪些种属豆科植物结瘤〔3〕。根瘤菌的结瘤呈寄主专一性,例如苜蓿中华根瘤菌的寄主范围很窄,仅能在苜蓿、草木樨和葫芦巴3个属的豆科植物结瘤〔4〕。但在1995年。本实验室从新疆苜蓿分离到一株菌株042B,既能在大豆又能在苜蓿上结瘤．而且在大豆上具有较高的共生效率〔5〕。本文拟克隆其nodABC,并与其他菌株nodABC序列进行比较,以阐明042B能在大豆和苜蓿结瘤的分子机制。     

Isolation and characterization of the DNA region encoding nodulation functions in Bradyrhizobium japonicum.

The DNA region encoding early nodulation functions of Bradyrhizobium japonicum 3I1b110 (I110) was isolated by its homology to the functionally similar region from Rhizobium meliloti. Isolation of a number of overlapping recombinant clones from this region allowed the construction of a restriction map of the region. The identified nodulation region of B. japonicum shows homology exclusively to those regions of R. meliloti and Rhizobium leguminosarum DNA known to encode early nodulation functions. The region of homology with these two fast-growing Rhizobium species was narrowed to an 11.7-kilobase segment. A nodulation-defective mutant of Rhizobium fredii USDA 201, strain A05B-2, was isolated and found to be defective in the ability to curl soybean root hairs. Some of the isolated recombinant DNA clones of B. japonicum were found to restore wild-type nodulation function to this mutant. Analysis of the complementation results allows the identification of a 1.8-kilobase region as essential for restoration of Hac function.     

Cloning of nod gene regions from mesquite rhizobia and bradyrhizobia and nucleotide sequence of the nodD gene from mesquite rhizobia.

Nitrogen-fixing symbiosis between bacteria and the tree legume mesquite (Prosopis glandulosa) is important for the maintenance of many desert ecosystems. Genes essential for nodulation and for extending the host range to mesquite were isolated from cosmid libraries of Rhizobium (mesquite) sp. strain HW17b and Bradyrhizobium (mesquite) sp. strain HW10h and were shown to be closely linked. All of the cosmid clones of rhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite also supported nodulation of a Sym- mesquite strain. The cosmid clones of bradyrhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite were only able to confer nodulation ability in the Sym- mesquite strain if they also contained a nodD-hybridizing region. Subclones containing just the nodD genes of either genus did not extend the host range of Rhizobium (Parasponia) sp. to mesquite, indicating that the nodD gene is insufficient for mesquite nodulation. The nodD gene region is conserved among mesquite-nodulating rhizobia regardless of the soil depth from which they were collected, indicating descent from a common ancestor. In a tree of distance relationships, the NodD amino acid sequence from mesquite rhizobia clusters with homologs from symbionts that can infect both herbaceous and tree legumes, including Rhizobium tropici, Rhizobium leguminosarum bv; phaseoli, Rhizobium loti, and Bradyrhizobium japonicum.     

Cloning of the nodulation (nod) genes of Rhizobium phaseoli and their homology to R. leguminosarum nod DNA

In Rhizobium phaseoli strain 8002, a large indigenous plasmid, pRP2JI, had previously been shown to carry many of the genes necessary for the induction of nitrogen-fixing nodules on Phaseolus beans. A cosmid clone library was constructed using DNA from strain 8002. From this library, two overlapping recombinant plasmids (pIJ1097 and pIJ1098) were isolated which spanned about 43 kb of pRP2JI DNA. These plasmids could restore nodulation to some, but not all nodulation-deficient strains of R. phaseoli, indicating that the nodulation genes were not clustered within one small region of pRP2JI. The cloned R. phaseoli nodulation region shared extensive DNA homology with the nodulation genes of R. leguminosarum, and on the basis of DNA hybridization, the nitrogenase genes were found to be within 10 kb of the R. phaseoli nodulation genes. Close to the nodulation genes of R. phaseoli was located a sequence that was repeated on pRP2JI but which was not present elsewhere in the genome of strain 8002.     

Isolation and Characterization of Frankia sp. Strain FaC1 Genes Involved in Nitrogen Fixation

Genomic DNA was isolated from Frankia sp. strain FaC1, an Alnus root nodule endophyte, and used to construct a genomic library in the cosmid vector pHC79. The genomic library was screened by in situ colony hybridization to identify clones of Frankia nitrogenase (nif) genes based on DNA sequence homology to structural nitrogenase genes from Klebsiella pneumoniae. Several Frankia nif clones were isolated, and hybridization with individual structural nitrogenase gene fragments (nifH, nifD, and nifK) from K. pneumoniae revealed that they all contain the nifD and nifK genes, but lack the nifH gene. Restriction endonuclease mapping of the nifD and nifK hybridizing region from one clone revealed that the nifD and nifK genes in Frankia sp. are contiguous, while the nifH gene is absent from a large region of DNA on either side of the nifDK gene cluster. Additional hybridizations with gene fragments derived from K. pneumoniae as probes and containing other genes involved in nitrogen fixation demonstrated that the Frankia nifE and nifN genes, which play a role in the biosynthesis of the iron-molybdenum cofactor, are located adjacent to the nifDK gene cluster.     

Molecular biology of Diazepam Binding Inhibitor peptide

Complementary DNA (cDNA) clones containing the entire coding sequence for Diazepam Binding Inhibitor (DBI) peptide, a 10-kDa precursor of putative natural ligands of benzodiazepine recognition sites, were isolated from rat, human and cow libraries. The sequence of all these clones is highly conserved; however, the N-terminal sequence predicted by the human DBI clone differed from that of the other two clones. DBI cDNA, utilized as hybridization probe in Southern blot analysis, revealed that DBI of both human and rat might be encoded by a multiple family of 4–6 genes. Furthermore, we have used in situ chromosomes hybridization to map human DBI genes. The results indicate that a human DBI gene is localized on chromosome 2 and that three of the four hybridization signals detected by the human DBI probe are located on three other chromosomes. These findings raise a question as whether multiple DBI genes encode for different molecular forms of DBI. In the attempt to test this hypothesis, cow cDNA and human genomic libraries were screened with DBI cDNA. In this paper I report the isolation of clones from these libraries which, although hybridizing well to DBI cDNA, possess a low percentage of homology (46.7%), randomly distributed within the coding region of DBI cDNA. Whether or not these clones encode for peptides sharing the same physiological role as DBI is under investigation.Special issue dedicated to Dr. Erminio Costa     

Construction and characterization of the transformation-competent artificial chromosome (TAC) libraries of Leymus multicaulis

Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants.In order to clone genes highly tolerant to barley yellow dwarf virus(BYDV),Aphids,drought and salt from Leymus multicaulis,the two TAC genomic libraries I and II were constructed in vector pYLTAC17 and pYLTAC747H/sacB,which contain about 165000 and 236000 recombinant clones sepa-rately.The genome coverage of the two libraries was totally estimated to be about 3―5 haploid genome equivalents,as size selection of genomic DNA fragments was approximately from 9 to 300 kb.Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so,stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern using a GeneTAC?G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates.Nineteen positive clones were detected by using the probe glutahione reductase gene of L.multicaulis.TAC libraries constructed here can be used to isolate genomic clones containing target genes,and to carry out genome walking for positional cloning.Once the target TAC clones were isolated,they could be immediately transferred into plant genomes with the Agrobacterium system.     

Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene.

The presence of Frankia strains in soil samples collected from northern areas of Pakistan was detected by inoculating Coriaria nepalensis and Datisca cannabina plants. The abundance of compatible Frankia strains in some areas was indicated by profuse nodulation of the host plants, whereas soil samples from other localities failed to result in nodulation. An oligonucleotide probe (COR/DAT) directed against the 16S rRNA gene of the endophytes of Coriaria and Datisca spp. that did not cross-react with the RNA gene of Frankia strains isolated from other hosts was developed. Genetic diversity among Frankia strains nodulating D. cannabina was determined by sequence analysis of the partial 16S rRNA gene amplified from nodules induced by soil samples from different localities by PCR. Four types of Frankia sequences and one non-Frankia sequence were detected by hybridization with a Frankia genus probe and the COR/DAT probe as well as by sequence analysis of the cloned PCR products.     

Isolation and characterization of an ndvB locus from Rhizobium fredii

A gene (ndvB) in Rhizobium meliloti that is essential for nodule development in Medicago sativa (alfalfa), specifies synthesis of a large membrane protein. This protein appears to be an intermediate in beta-1,2-glucan synthesis by the microsymbiont. Southern hybridization analysis showed strong homology between an ndvB (chvB) probe and genomic DNA of R. fredii but not from Bradyrhizobium japonicum. A cosmid clone containing the putative ndvB locus was isolated from a Rhizobium fredii gene library. The cosmid clone which complemented R. meliloti ndvB mutants for synthesis of beta-1,2-glucans and effective nodulation of alfalfa was mapped and subcloned. Fragment-specific Tn5 mutagenesis followed by homologous recombination into the R. fredii genome indicated that the region was essential for beta-1,2-glucan synthesis and for formation of an effective symbiosis with Glycine max (soybean).