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。     

大豆—根瘤菌共生固氮氢代谢的研究

检测了四株大豆根瘤菌在不同的大豆品种上形成根瘤的放氢、吸氢、固氮活性及豆血红蛋白的含量;同时测定了植株干物质的积累。结果表明,所有固氮根瘤都放氢,自生条件下Hup~-根瘤菌形成的根瘤仍不具吸氢活性,相对固氮率在0.75左右。而Hup~(?)菌株根瘤的相对固氮率在0.91～1之间。寄主植物对Hup~(?)菌株的吸氢活性有影响。相关分析表明,根瘤的豆血红蛋白与吸氢活性呈负相关。干物质积累与固氮酶活性关系最密切,氢酶活性的影响是次要的。     

接种S.fredii WGF03及突变体对大豆土壤微生物群落结构的影响

为了研究接种S.fredii WGF03及其exo D基因突变体对大豆结瘤及土壤的微生物群落影响,进一步了解exo D基因的功能,在大豆盛花期摘取每株大豆的根瘤并计数,利用PCR-DGGE电泳结合测序技术分析土壤的微生物群落。结果表明,大豆接种S.fredii WGF03后,根瘤数比不接种组增加191.67%。而大豆接种驻exo D突变体的根瘤数最少,比不接种组减少了16.67%。与空白相比,种植大豆后土壤细菌的种类和数量明显增加;接种不同根瘤菌后,细菌种类及细菌多样性也有变化;测序结果显示,土壤中细菌以Proteobacteria为主,占45.5%,土壤中土著根瘤菌为Bradyrhizobium。总之,S.fredii WGF03能够促进大豆结瘤,种植作物比接种根瘤菌对土壤细菌群落的影响更大。     

百脉根小G蛋白Rac1基因的克隆与功能分析

小G蛋白Rop在植物细胞信号转导中发挥着重要的分子开关功能。该实验通过RT-PCR方法克隆了百脉根的一个Rop编码基因LjRac1,并对LjRac1基因序列进行生物信息学分析,然后采用半定量RT-PCR检测LjRac1基因在百脉根不同组织中的表达,用荧光实时定量PCR方法检测百脉根接种根瘤菌后LjRac1基因在不同阶段根系中的表达,构建过表达重组质粒,利用发根农杆菌介导的遗传转化法对LjRac1基因功能进行分析。结果表明:(1)序列分析显示,LjRac1完整编码区的cDNA序列长度为594bp,编码197个氨基酸,其编码蛋白具有典型的Rop家族保守结构域;同源分析显示,百脉根LjRac1与大豆GmRac1、野大豆GsRac1的一致性最高(94.42%)。(2)LjRac1基因在百脉根的根、茎、叶、根瘤和花中均有表达,且在根和根瘤中的表达水平较高;接种根瘤菌0.5h后,LjRac1基因在根系中的表达量呈显著升高趋势。(3)过表达转基因植株中LjRac1mRNA的表达水平为对照植株的14.3倍,且过表达植株的结瘤数目较对照明显增加。研究认为,LjRac1基因是一个受根瘤菌诱导增强表达的基因,过表达LjRac1基因可以引起植株结瘤数目的增加,说明LjRac1基因可能参与早期结瘤信号转导途径,从而在根瘤的发育中发挥一定作用。     

磁场对大豆共生固氮的效应

恒定磁场处理慢生大豆根瘤菌“005”和接种后的大豆植株,发现磁场可以提高根瘤的固氮活性。在一定的磁场强度(70—100mT)下,固氮活性平均可以提高4—5倍,植株的结瘤数和根瘤重量平均提高2—3倍。从这样的根瘤中所分离出的根瘤菌,由慢生型转变成快生型,在100植株中有17株的根瘤分离出快生菌。生长世代时间和培养溶液中的pH值与慢生型不同,而与快生型相同。     

大豆根瘤菌SCAUs8的接种效果、促生性及系统发育研究

【目的】大豆是我国重要的农作物,利用大豆-根瘤菌共生体系能有效减少化学氮肥的用量。将初筛的大豆根瘤菌SCAUs8在四川两个重要的大豆种植生态区进行田间接种验证试验,同时对该菌株进行分类地位研究。【方法】在四川丘陵区和攀西地区,采用大田试验调查接种大豆根瘤菌SCAUs8对大豆的增产效果。采用点接种法研究SCAUs8的抗逆性,用Salkowski比色法检测供试菌分泌吲哚乙酸(IAA)的能力。用多位点基因(16S r RNA,atp D,rec A,glnⅡ,nod C,nif H)序列分析对供试菌株SCAUs8进行分类地位的确定。【结果】大田试验表明,接种大豆根瘤菌SCAUs8后,大豆植株的产量、鲜重、干重等指标明显高于不接种对照(CK)处理,其中大豆植株产量比不接种CK增产21.4%-29.7%,其差异达显著水平。对供试菌株SCAUs8进行的耐酸碱性、耐盐性、生长温度范围以及分泌IAA能力测定的结果表明,供试菌株SCAUs8能在p H5.0-10.0正常生长,可耐受Na Cl浓度为0.5%,生长温度范围是10-40°C,能分泌IAA。综合16S r RNA、atp D、rec A、glnⅡ、nod C、nif H基因的序列分析,发现供试菌株SCAUs8与Bradyrhizobium diazoefficiens USDA110T相似性高达100%。【结论】供试菌株SCAUs8是与四川主栽大豆品种共生匹配性较好的广谱菌株。该菌株耐盐性较差,但具有较强的耐酸碱性、较宽的生长温度范围及分泌IAA的能力。系统发育研究将供试菌株SCAUs8确定为B.diazoeffciens。     

导入额外拷贝nifA基因对费氏中华根瘤菌共生固氮作用的影响

以广宿主、稳定性质粒pTR102为载体构建重组质粒pHN306,其上克隆有来自肺炎克氏杆菌（Klebsiellapneumoniae）的nifA基因和来自pDB30所含的发光酶标记基因（luxAB）。经三亲本接合转移,将pHN306导入费氏中华根瘤菌（Sinorhizobiumfredii）HN01,GR3和YC4。与出发菌相比较的盆栽试验结果表明：HN01（pHN306）和GR3（pHN306）分别在大豆渝豆一号和黑龙33上能显著提高瘤数,瘤重,植株地上部分干重和总氮量,YC4（pHN306）在大豆渝豆一号上也能显著提高瘤数,癌重和总氮量,对植株地上部分干重表现出一定的促进作用。结果表明：nifA基因对固氮效率和结瘤能力的促进作用与受体根瘤菌和大豆品种等因素有关。以luxAB为报告基因进行的菌落和根瘤发光检测结果表明：pHN306可在供试根瘤菌中稳定遗传。     

应用GUS基因研究弗氏中华根瘤菌的结瘤及效果

应用GUS(葡萄糖苷酶 )基因标记技术将标记基因GUS导入受体菌S .fredii8855,标记菌株形成的根瘤可被GUS染色缓冲液染成蓝色 ,而土著菌形成的根瘤不能着色 .由此即可十分简便地确定土著菌的影响程度 .盆栽试验表明 ,S .fredii 8855的结瘤抗酸碱能力高于土著菌 ,能在土壤中较大范围内迁移 .当它的根瘤占有率不小于 43%时 ,接种能显著提高大豆产量 ,大豆产量与根瘤占有率呈正相关 (r=0 .98) ,而与总瘤数关系不大 (r=0 .1 3) .土壤N素显著抑制其结瘤 ,补加P能缓解这种抑制作用 .     

接种不同大豆根瘤菌株的根瘤放氢和吸氢的研究

本实验测定和比较了七株大豆根瘤菌与三个大豆品种共生时的放氢和固氮效率。证明了大多数菌株接种不同寄主,其根瘤的放氢、吸氢和固氮效率的差异均明显。但USDA110在三个品种上所结根瘤不放氢或极少放氢;它的固氮量最高。根瘤的能量利用率与植株的氮积累及产量的相关性尚需进一步验证。     

OsPT6基因过表达对低磷条件下菜用大豆结瘤及固氮的影响

采用砂培方法,以转OsPT6基因的菜用大豆(T3株系)与其非转基因(NT)受体品种为实验材料,研究了两者在低磷条件下的生长发育指标,植株有效磷、全磷、全氮、豆血红蛋白和籽粒蛋白质含量以及谷氨酰胺合成酶活性的差异,并对植株结瘤及固氮相关基因表达进行检测,为阐明转OsPT6基因菜用大豆在低磷条件下结瘤及固氮相关机理提供理论依据。结果显示:(1)转基因植株的株高、茎粗、花数和荚数、根瘤数均显著高于NT植株。(2)转基因植株根、茎、叶及根瘤中有效磷,全株总磷、总氮含量,根瘤中的豆血红蛋白含量、功能叶片中谷氨酰胺合成酶的活性和籽粒蛋白质含量均显著高于NT植株。(3)相关性分析表明,豆血红蛋白含量、谷氨酰胺合成酶活性、总磷、总氮含量4个指标间均呈显著正相关关系。(4)GmENOD40a、GmENOD40b、GmGS1β1、GmGS1β2基因在转基因植株中的表达量显著高于NT植株。研究表明,OsPT6基因过表达增强了菜用大豆在低磷条件下的结瘤及固氮能力,该研究结果为进一步研究其调控机理奠定了基础。     

Expression of β-galactosidase in Rhizobium japonicum

Abstract The plasmid pGC91.14 was used to introduce via conjugation the Escherichia coli lac operon into fast-growing and slow-growing strains of Rhizobium japonicum . Exconjugants now expressed higher levels of β-galactosidase activity which was still inducible by isopropyl-β- d -thiogalactoside (IPTG). The presence of the lac operon allowed the slow-growing strain 61A76 to grow on lactose as the sole carbon source; the fast-growing strains grew poorly on lactose but growth was not inhibited by lactose as had been reported for Rhizobium meliloti . β-galactosidase could be detected in nodule extracts and bacteroid preparations from soybean plants ( Glycine max L. Merrill) infected with the strain 61A76 (pGC91.14).     

The Rhizobial hemA Gene Is Required for Symbiosis in Species with Deficient [delta]-Aminolevulinic Acid Uptake Activity

Most rhizobial hemA mutants induce root nodules on their respective legume hosts that lack nitrogen fixation activity and leghemoglobin expression. However, a Bradyrhizobium japonicum hemA mutant elicits effective nodules on soybean, and we proposed previously that synthesis and uptake of the heme precursor [delta]-aminolevulinic acid (ALA) by the plant and bacterial symbiont, respectively, allow mutant rescue (I. Sangwan, M.R. O'Brian [1991] Science 251: 1220-1222). In the present work, the B. japonicum hemA mutant MLG1 elicited normal nodules on three hosts, including cowpea, a plant that is not effectively nodulated by a hemA mutant of Rhizobium sp. These data indicate that B. japonicum rather than soybean possesses the unique trait that allows normal nodule development by a hemA mutant. Cowpea expressed glutamate-dependent ALA formation activity in nodules induced by B. japonicum strains I110 or MLG1 and by Rhizobium sp. ANU240. Exogenous ALA was taken up by B. japonicum bacteroids isolated from soybean or cowpea nodules, and the kinetics of uptake were biphasic. By comparison, Rhizobium sp. ANU240 had very low ALA uptake activity. In addition, ALA uptake was observed in cultured cells of B. japonicum but not in cultured cells of three other rhizobial species tested. We suggest that the differential success of legume-rhizobial hemA symbioses is due to an ALA uptake activity in B. japonicum that is deficient in other rhizobia, thereby further validating the ALA rescue hypothesis.     

Identification of “nodule-specific” host proteins (nodulins) involved in the development of Rhizobium-Legume symbiosis

Infection of legume roots with Rhizobium species results in the development of a root nodule structure in which the bacteria form an intracellular symbiosis with the plant. We report here that the infection of soybean (Glycine max L.) roots with Rhizobium japonicum results in the synthesis by the plant of at least 18–20 polypeptides other than leghemoglobin during the development of root nodules. Identification of these “nodule-specific” host polypeptides (referred to as nodulins) was accomplished by two-dimensional gel analysis of the immunoprecipitates formed by a “nodule-specific” antiserum with in vitro translation products of root-nodule polysomes that are free of bacteroidal contaminations. Nodulins account for 7–11% of the total ³⁵S-methionine-labeled protein synthesized in the host cell cytoplasm, and the majority of them are of 12,000–20,000 molecular weight. These proteins are absent from the uninfected roots, bacteroids and free-living Rhizobium, and appear to be coded for by the plant genes that may be obligatory for the development of symbiosis in the legume root nodules. Analysis of nodulins in ineffective (unable to fix nitrogen) nodules developed due to Rhizobium strains SM5 and 61A24 showed that their synthesis is reduced and their expression differentially influenced by mutations in rhizobia. Two polypeptides of bacterial origin were also found to be cross-reactive with the “nodule-specific” antiserum, suggesting that they are secreted by Rhizobium into the host cell cytoplasm during symbiotic nitrogen fixation.     

Adsorption and selection of rhizobia with ion-exchange papers

Ion exchange papers were used to study the adsorption of 32P-labelled rhizobia on defined surfaces. Two strains of Rhizobium japonicum and one each of R. leguminosarum and R. lupini were compared with Escherichia coli and Bacillus subtilis. The ratio of adsorption to strong and to weak acid papers/strong and weak basic papers was consistantly higher for all rhizobial strains compared to the other bacteria. The process of desorption by increasing the ion-concentration causes about 35% desorption between 0.02 and 0.1 M MgCl2, however, an increase to 1 M does not desorb more labelled Rhizobium japonicum or E. coli cells. The ratio of adsorbed cpm to colony formers, desorbed by 0.1 M NaCl was similar with Rhizobium japonicum for all six ion exchange papers. For E. coli this ratio varied widely for the different papers. The selection of Rhizobium against a more closely related bacterium by this adsorption/desorption procedure was demonstrated with mixed cultures of Rhizobium japonicum and Chromobacterium violaceum giving a more than 80 fold enrichment of the former. Rhizobium japonicum cells, ad/desorbed from all ion exchange papers kept their infectivity and formed nodules on Glycine max with an activity of 20-40 nM C2H4-hr(-1)-mg nodule(-1). A desorption of Rhizobium japonicum from soybean roots also occurred by increasing the ion concentration. 2-3 times as many cells were removed in this way compared to washing with water.     

Ineffective and non-nodulating mutant strains of Rhizobium japonicum.

Mutant strains of Rhizobium japonicum that were unable to allow the Corsoy cultivar of soybean to reduce acetylene or fix N2 were isolated. These strains grow as well as the wild type in a variety of media. Mutant strains SM1 and SM2 did not form nodules on the host plant; however, they reduced acetylene in the nonsymbiotic assay. Strains SM3 and SM4 produced nodules that did not have the characteristic pink pigment caused by leghemoglobin. The nodules formed by these strains also were small. One mutant strain, SM5, produced large pink nodules. The lesion in this strain seems to be in the gene that specifies nitrogenase component II.     

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).     

Identification and use of actinomycetes for enhanced nodulation of soybean co-inoculated with Bradyrhizobium japonicum

The utilization of actinomycetes as potential soybean (Glycine max (L.)) co-inoculants was evaluated. Soil samples from Carbondale and Belleville, Ill., were used to inoculate pre-germinated soybean plants to determine antibiotic sensitivity in the native Bradyrhizobium japonicum population. Sensitivity was in the order kanamycin > tetracycline > oxytetracycline > rifampicin > neomycin. Antagonism by five actinomycete cultures toward seven test strains of B. japonicum was also assessed. The ranking average inhibition (across all seven B. japonicum strains) by these actino mycetes was Streptomyces kanamyceticus = Streptomyces coeruleoprunus > Streptomyces rimosus > Streptomyces sp. > Amy colatopsis mediterranei. Ten antibiotic combinations were used to isolate antibiotic-resistant mutants of B. japonicum I-110 and 3I1B-110 via successive cycles of mutation. Eighty-one antibiotic-resistant strains were isolated and tested for symbiotic competency; nine of which were selected for further characterization in a greenhouse pot study. Few differences in nodule number were caused by these treatments. Nodule occupancy varied from 0% to 18.3% when antibiotic-resistant strains of B. japonicum were used as the sole inoculants. However, when three mutant strains of B. japonicum were co-inoculated with S. kanamyceticus, significant increases in nodule occupancy (up to 55%) occurred. Increases in shoot nitrogen composition (27.1%-40.9%) were also caused by co-inoculation with S. kanamyceticus.     

A dual-targeted soybean protein is involved in Bradyrhizobium japonicum infection of soybean root hair and cortical cells

The symbiotic interaction between legumes and soil bacteria (e.g., soybean [Glycine max L.] and Bradyrhizobium japonicum]) leads to the development of a new root organ, the nodule, where bacteria differentiate into bacteroids that fix atmospheric nitrogen for assimilation by the plant host. In exchange, the host plant provides a steady carbon supply to the bacteroids. This carbon can be stored within the bacteroids in the form of poly-3-hydroxybutyrate granules. The formation of this symbiosis requires communication between both partners to regulate the balance between nitrogen fixation and carbon utilization. In the present study, we describe the soybean gene GmNMNa that is specifically expressed during the infection of soybean cells by B. japonicum. GmNMNa encodes a protein of unknown function. The GmNMNa protein was localized to the nucleolus and also to the mitochondria. Silencing of GmNMNa expression resulted in reduced nodulation, a reduction in the number of bacteroids per infected cell in the nodule, and a clear reduction in the accumulation of poly-3-hydroxybutyrate in the bacteroids. Our results highlight the role of the soybean GmNMNa gene in regulating symbiotic bacterial infection, potentially through the regulation of the accumulation of carbon reserves.