Bradyrhizobium japonicum工程菌株HN32中外源质粒pHN32增效作用的验证

将Bradyrhizobium japonicum HN32菌株中的外源质粒pHN32,通过三亲本杂交转入大肠杆菌HB101中,继而转入Bradyrizobium japonicum22—10中,得到一系列抗性杂交子HNF1,HNF3及HNF5,检测其中所含质粒,并以22—10,HNF5及HN32进行盆栽试验,pHN32的增效作用得到了证实。

慢生型大豆根瘤菌(Bradyrhizobium japonicum)USDA110菌株3-羟丁酸脱氢酶基因(bdhA)的克隆、序列及特性

通过功能互补试验 ,从慢生型大豆根瘤菌USDA110菌株基因文库中筛选到能互补广宿主根瘤菌NGR2 34的bdhA突变体菌株NGRPA2和苜蓿根瘤菌的bdhA突变体菌株Rm1110 7、使之恢复Hbu+ 表型的克隆 ;经酶活测定和Southern杂交证明该克隆含有bdhA基因。测定了bdhA基因全序列 ,并在GenBank登记 (登记号为 :AY0 775 81)。该基因由 789个碱基对组成 ,编码分子量为 2 7.5 9ku、含 2 6 2个氨基酸残基的 3 羟基丁酸脱氢酶。在该基因的开放阅读框内插入interposonΩKm ,并通过同源重组构建了BradyrhizobiumjaponicumbdhA突变体 (bdhA ::ΩKm)。植株试验未显示bdhA基因的突变对结瘤、固氮有明显影响。

慢生型大豆根瘤菌(Bradyrhizobium japonicum)聚羟丁酸合成酶基因(phbC)突变体的构建及其特性

通过转座子Tn5诱变和同源重组 ,构建了BradyrhizobiumjaponicumUSDA110聚羟丁酸合成酶基因 (phbC)突变体 .序列测定确定了转座子插入的精确位置 ,所获得的 4个转座子诱变的质粒其Tn5插在 phbC基因内两个相距仅 9bp的位点 .被Southern和PCR证实的突变体菌株仍能产生相当于野生型菌株 12 .97%～ 2 5 .10 %的PHB ,并且在突变体和野生型菌株总DNA杂交图上都呈现出一条约 5kb的阳性带 ,推测在B .japonicum基因组中存在不止一个聚羟丁酸合成酶基因 .图 3表 4参

Analysis of Two Bradyrhizobium japonicum Strains with Different Symbiotic Matching for Nodulation by Primary Proteomic

The symbiotic matching for nodulation of Bradyrhizobium japonicum strains is a synergy of multi-proteins and plays a key role in symbiotic nitrogen fixation in nature. Studies on mechanism of symbiotic matching are significant in both theory and practice. In this paper, B. japonicum USDA110-A with high symbiotic matching with high-oil content soybean cultivar Suinong 20 and B. japonicum 2178 with low symbiotic matching were selected for proteomic to reveal mechanism of different symbiotic nodulation. The results showed that the amount and categories of proteins identified in this test were different when the two strains were treated by symbiotic nodulation. There were 10 up-regulated proteins and 5 down-regulated proteins with significant difference for B. japonicum USDA110-A. Proteins associated with nodulation and metabolism of energy and material, which were propitious to symbiotic nodulation, were all up-regulated, such as PHDPS synthase, metal-dependent phosphohydrolase, glycosyl transferase family. In contrast, only 5 up-regulated and 7 down-regulated differential proteins were detected in B. japonicum 2178. Molecular chaperones and defensive proteins, which influence the folding of nascent polypeptide chains and the active of azotase were down-regulated. To a certain extent, the different responses of B. japonicum to daidzein were one of the most important reasons that cause varieties in symbiotic matching ability.

Bradyrhizobium japonicum基因工程菌株HN32中外源片段的来源检验

采用Southern转移,放射性同位素分子杂交等研究手段,对本课题组构建的一株高效固氮大豆根瘤菌HN32中的外源片段的来源进行了分析。研究结果证实:该外源片段确系来源于Sinorhizobium fredii B52菌株,并且在受体Bradyrhizobium japonicum 22—10菌株中本不存在。

Proteomic Study on Two Bradyrhizobium japonicum Strains with Different Competitivenesses for Nodulation

Competitiveness for nodulation of Bradyrhizobium japonicum strains plays a key role in symbiotic nitrogen fixation. In order to reveal the difference in competitiveness, B. japonicum 4534 with high competitiveness and B. japonicum 4222 with low competitiveness for nodulation were analyzed by proteomic technique. The results showed that differential proteins were fewer when two strains were treated with just daidzein. Only 24 and 10 differential proteins were detected with an up-regulated rate of 58 and 40% in B. japonicum 4534 and B. japonicum 4222, respectively. However, more differential proteins were detected upon treatment with daidzein and mutual extracellular materials simultaneously. There were 78 differential proteins detected in B. japonicum 4534 with 43 being up-regulated and 35 being down-regulated. These differential proteins, such as metabolism-related proteins, transporters, transcription-related proteins, translation-related proteins, and flagellin, were found to be associated with nodulation process. 25 up-regulated and 22 down-regulated proteins were detected in B. japonicum 4222. Some of these proteins were not related to nodulation. More differential proteins associated with nodulation in B. japonicum 4534 may be the reason for its high competitiveness. The results can provide a guide to the selection and inoculation of effective strains and are significant to biological nitrogen fixation.

Bradyrhizobium japonicum工程菌株HN32田间占瘤率的测定

应用特定根瘤菌株接种豆科植物与土著根瘤菌竞争结瘤,在竞争中特定根瘤菌结瘤数占总瘤数的百分数称为占瘤率。这是分析特定根瘤菌株产生增产效果的重要因素。测定占瘤率的方法,主要有抗性标记技术,荧光抗体技术,酶连免疫技术,分子杂交技术和红外线指纹图谱法等,每种方法都有其优缺点。本研究利用HN32菌株的四环素抗性测定技术和α—^(32)P标记的pHN32为探针进行的DNA/DNA杂交技术来考查田间接种大豆根瘤菌工程菌株HN32的占瘤率。

Soybean Seed Co-Inoculation with <i>Bradyrhizobium</i>spp. and <i>Azospirillum brasilense</i>: A New Biotechnological Tool to Improve Yield and Sustainability

Legume nodulation by rhizobia can supply crops with nitrogen and reduce environmental impacts caused by chemical fertilization. The soybean crop in Brazil is an impressive example of how biological N2 fixation can be employed with a plant species of high economic value. However, the development of more productive cultivars, along with the increasing global climatic changes demand agricultural practices to become more productive and yet more environmentally friendly. Plant growth-promoting rhizobacteria (PGPR) are highly beneficial to agriculture worldwide, acting in plant nutrition, protection, and growth stimulation. Azospirillum is, certainly, the most employed PGPR in the world, but little is known about its interaction with rhizobia, when both are applied to legume seeds. We have evaluated the co-inoculation of bradyrhizobia and azospirilla on soybean seeds under different soil and climate conditions in Brazil. Our results demonstrated that co-inoculation is efficient and beneficial to the crop, and promotes yield increases without adding any chemical N fertilizers even in soils where established populations of soybean bradyrhizobia exist. The strategy of co-inoculation thus represents a new biotechnological tool to improve soybean yield without adding any chemical N fertilizers, thus contributing to current practices of sustainability in agriculture.

Incompatible Nodulation of <i>Bradyrhizobium elkanii</i>Strains BLY3-8 and BLY6-1 with <i>Rj</i>3 Gene-Harboring Soybean Cultivars

Bradyrhizobia are known symbiotic partners of soybean. However, some soybean cultivars restrict nodulation by some Bradyrhizobium bacterial strains. These restrictions are related to compatibility between the Rj genes of soybean cultivars and nodulation types of inoculated bacteria. The objective of this study was to determine nodulation incompatibility of Type B strains with Rj3 soybean cultivars. Newly isolated B. elkanii strains BLY3-8 and BLY6-1 from Myanmar and specific strain Bradyrhizobium elkanii USDA33, which are incompatible with Rj3 soybean cultivars, and B. japonicum USDA110 were used as inoculants to check compatibility or incompatibility with Rj3 soybean cultivars. Nitrogen fixation activity was measured by the acetylene reduction method. Ethylene concentration (reduction of acetylene) was determined by flame ionization gas chromatography. According to the inoculation test results, USDA110 was compatible with all soybean cultivars because it formed effective nodules (Figure S1 in Appendix) and possessed nitrogenase activity. Similarly, B. elkanii strains BLY3-8, BLY6-1, and USDA33 were highly compatible with non-Rj and Rj4-gene harboring soybean cultivars because they had the ability to form functional nodules and possessed nitrogenase activity. Inversely, BLY3-8, BLY6-1, and USDA33 were incompatible with Rj3 soybean cultivars because they produced ineffective nodules. Consequently, the ratio of ineffective nodule number to total nodule number was >0.5. Therefore, nodule formation by the newly isolated B. elkanii strains BLY3-8 and BLY6-1 was restricted by the Rj3 soybean cultivars potentially making them useful as specific strains to detect the Rj3 gene in soybean cultivars.

慢生大豆根瘤菌(Bradyrhizobium japonicum)聚羟丁酸合成酶基因(phbC)的克隆和序列测定

以苜蓿根瘤菌Rm10 2 1的 phaC基因突变体菌株Rm1114 4 (phaC ::Tn5 - 2 33)为受体菌 ,通过功能互补 ,成功地从构建的Bradyrhizobium japonicum USDA110基因文库中 ,筛查到能与Rm1114 4互补 ,使之恢复在以乙酰乙酸为唯一碳源的M9培养基 (M9-AA)平板上 5d形成明显可见菌落 ,以及在MOPS平板上形成粘液型菌落的表型的重组粘粒 pDC2 ;经证实 ,该粘粒带有 phbC基因 .完成了该基因的全序列测定并已在GenBank登记 ,登记号为AY0 775 80 .B .japonicumphbC基因由 180 3碱基对组成 ,GC含量 6 1.8% ,AT含量 38.2 % ;编码 6 0 0个氨基酸 ,Mr=6 6 .95× 10 3 .图 3表 3参

大豆根瘤菌(Bradyrhizobium japonicum)苹果酸脱氢酶(mdh)基因的插入失活

本试验是用粘末端连接法把卡那霉素抗性基因(Kan^r)插入到大豆根瘤菌B.j110的苹果酸脱氢酶(mdh)基因中,使mdh基因失活。首先,通过EcoRI切点把Kan^r连接到pTZ18U载体上,经转化把这一重组DNA转入大肠杆菌XLI-Blue中,利用含有氨苄青霉素和卡那霉素的选择培养基选出转化菌株,提取pTZ18U-Kan^r质粒DNA;然后,用Sall分别切已克隆在pTZ19U中的B.j110mdh基因及pTZ18u-Kan^r,经粘末端连接把卡那霉素抗性基因连接到苹果酸脱氢酶基因中间,从而使其失活。再将所得到的这一重组DNA用电脉冲法(Electroporation)转入大豆根瘤菌B.j2143中,用以检验经插入失活后的苹果酸脱氢酶基因对大豆固氮作用的影响。

Cloning,Sequencing and Characterization of 3-Hydroxybutyrate Dehydrogenase Encoding Gene(bdh A)in Bradyrhizobium japonicum USDA110 Strain

The current study describes the molecular characterization of a clone which can restore the ability of bdh A mutant strains NGRPA2 and Rm11107 to utilize 3-hydroxybutyrate as a sole carbon source (Hbu+ ). This clone was screened out by complementation experiment from Bradyrhizobium japonicum US-DAI 10 genomic library, and the presence of bdhA gene in the clone was verified by Bdh assay and Southern blot analysis. Furthermore, the entire sequence of bdhA. gene was sequenced and the sequence was deposited in GenBank database under the accession number AY077581. bdhA gene comprises 789 base pairs and encodes Bdh with 262 amino acid of MW 27.59 kDa. Interposon JlKm was inserted into the bdhA ORF at EcoR I site and the bdhA mutant was constructed in B .japonicum by homologous recombination. Plant assay result did not show obvious effects of mutation of bdhA gene on nodulation and nitrogen-fixation.

Phylogenetic Diversity and Evaluation the Effectiveness of Indigenous <i>Bradyrhizobium</i>Strains for Myanmar Black Gram (<i>Vigna mungo</i>L. Hepper) Cultivars

Black gram (Vigna mungo L. Hepper) is one of the main leguminous crops that provide chief source of food. Several Bradyrhizobium species are able to induce effective nodules in black gram cultivars. In the present study, we characterized forty isolates of indigenous black gram bradyrhizobia from Myanmar based on the sequence analysis of the bacterial 16S rRNA gene. The sequence analysis confirmed that all isolates were categorized and identified as the genus Bradyrhizobium and they were conspecific with B. elkanii, B. sp., B. liaoningense, B. japonicum and B. yunamingense. Almost all the collected isolates from major black gram growing regions of Nyaunglebin Bago Regio, Chaungzon Mon State, Sittwe Rakhine State, Danubyu Ayeyarwady Region and Launglon Tanintharyi Region were identified as B. liaoningense. At Danubyu Ayeyarwady Region and Pyinmanar Nay Pyi Taw Region, most of the strains were identified as B. japonicum. On the other hand, more or less all the isolates from Launglon Tanintharyi Region and Hpa-an Kayin State were related to B. elkanii. However, all B. sp. strains were found in Salingyi Sagaing Region black gram growing region. This is the first report describing Bradyrhizobium strains that were isolated from soil samples of major black gram growing areas in Myanmar. Evaluation of the effectiveness of Myanmar Bradyrhizobim strains isolated from soil samples of major black gram growing areas of Myanmar for plant growth and nitrogen fixation was studied in pot experiments with completely randomized design and three replicates. The nodule dry weight, shoot dry weight and acetylene reduction activity of the plant inoculated with Bradyrhizobium elkanii LauBG38 were significantly higher in ARA per plant, nodule and shoot dry weights than the other tested isolates in both Yezin-4 and Yezin-7 black gram varieties. We expect that Myanmar Bradyrhizobium elkanii LauBG38 will be able to use as Biofertilizer for black gram cultivars.

Low-Density Co-Inoculation of Myanmar <i>Bradyrhizobium yuanmingense</i>MAS34 and <i>Streptomyces griseoflavus</i>P4 to Enhance Symbiosis and Seed Yield in Soybean Varieties

This study examined whether low-density co-inoculation of Myanmar Bradyrhizobium yuanmingense strain MAS34 and Streptomyces griseoflavus P4 would enhance nodulation, N2 fixation, and seed yield in two soybean varieties. A field experiment was conducted during the July to November 2012 growing season at Kyushu University Farm, Japan, using a split-plot design with three replications and the following four treatments: T1, an uninoculated treatment with peat moss (uninoculated);T2, a single inoculation with S. griseoflavus P4 (P4);T3, a single inoculation of B. yuanmingense MAS34 (MAS34);and T4, a dual inoculation of P4 with MAS34 (P4 + MAS34). Two varieties of soybean, Yezin-3 (Rj4) and Yezin-6 (non-Rj), were used. The N2 fixation activity of soybean was evaluated by the relative ureide method using xylem solute from root bled sap at the early pod-fill stage (R3.5). Dry matter production, N2 fixation, and seed yield were significantly (P 0.01) different between the inoculated treatments. The effect of variety was also significant (P 0.05) for nodule dry weight at the V6 stage, percentage of N derived from the atmosphere at the R3.5 stage, and seed yield at the maturity stage. The number of nodules on the tap roots was significantly higher in Yezin-3 than in Yezin-6. The single inoculation of P4 did not have a significant effect on dry matter production, N2 fixation, and seed yield in either soybean variety. The dry matter production, relative ureide index, percentage of N derived from the atmosphere, and seed yield were significantly (P 0.01) enhanced by a single inoculation of MAS34 in Yezin-3 and by dual inoculation of P4 + MAS34 in Yezin-6. These results indicate that low inoculum concentrations (105 cells seed-1) increase N2 fixation and seed yield in these soybean varieties under open field conditions. Myanmar B. yuanmingense MAS34 and S. griseoflavus P4 are expected to be useful biofertilizers for soybean production.

Dual inoculation of salt tolerant Bradyrhizobium and Glomus mosseae for improvement of Vigna radiata L. cultivation in saline areas of West Bengal, India

This study is aimed as to evaluate the interaction between salt tolerant Bradyrhizobium sp. and Glomus mosseae in the rhizosphere of legume crop Vigna radiata L. under pot culture and field conditions in different saline zones of West Bengal, India. Bradyrhizobium sp. when inoculated alone showed marked increase in number of nodules, root and shoot length, total plant biomass, arbuscular mycorrhizal fungal (AMF) colonization and population etc. when compared with plants inoculated only with AMF. However, when used in combination, the in oculants showed marked change in the above mentioned parameters over single inoculation of both salt tolerant AM fungi and Bradyrhizobium. These results suggest that AMF along with Bradyrhizobium can greatly help in establishment of V. radiata L. cultivation in the saline soils of West Bengal, India. The increased production of the legume crop could also lead to further benefit of the poor farmers by up lifting their socio-economic conditions with the net profit achieved by cultivating this crop in saline stress condition of West Bengal as a second crop during rabi season.

大豆慢生根瘤菌(Bradyrhizobium japonicum)的血清型研究

以自东北地区分离的 8株大豆慢生根瘤菌为供试菌用交叉凝集反应比较了它们与目前国内外已报道的 15株大豆慢生根瘤菌标准血清型菌株之间的血清学关系。研究结果表明 :(1)菌株 93H10F5和HJ15不与供试标准血清型菌株发生交叉凝集反应 ,分别命名为O14 和O15血清型。 (2 )菌株HJ19、HJ2 8、93F42与标准血清型菌株USDA94有交叉凝集反应 ,进一步的抗原吸收试验结果表明 :HJ19和HJ2 8的抗原组成完全相同 ,命名为O3bcd血清亚型 ;USDA94仅与HJ19有交叉反应 ,命名为O3ab血清亚型 ;93F42也与HJ19有部分共同抗原 ,命名为O3de血清亚型。 (3)菌株 93HA8与标准血清型菌株USDA135有部分共同抗原 ,分别命名为O12bc和O12ab血清亚型。 (4)标准菌株USDA12 7、USDA12 9与USDA12 3有交叉凝集反应 ,抗原吸收试验分别将其细分为O10ab,O10de和O10bcd等 3个血清亚型。 (5 )将USDA的 10个标准菌株重新命名为O1,O2 ,O4 ,O5,O6,O7,O8,O9,O11和O13

Identification of Rhizobia Isolated from Nodules of Mexican Commercial Soybean Varieties

Rhizobia, crucial for nitrogen fixation in leguminous plants, play a vital role in soybean cultivation. This study, conducted in Mexico, a major soybean importer, aimed to identify bacteria from nodules of five soybean varieties in high-production regions. Multilocus sequence analysis (MLSA) was employed for enhanced species resolution. The study identified six Bradyrhizobium species: Bradyrhizobium japonicum USDA 110, Bradyrhizobium japonicum USDA 6, Bradyrhizobium elkanii USDA 76, Bradyrhizobium neotropicale, Bradyrhizobium lablabi, and Bradyrhizobium icense. Bradyrhizobium japonicum USDA 110 predominated in the soils, displaying symbiotic preference for the Huasteca 400 variety. However, phylogenetic analysis didn't reveal a clear association between strains, soil, and soybean variety. This research sheds light on the diversity of rhizobia in Mexican soybean cultivation, contributing to the understanding of symbiotic relationships in soybean production systems.

大豆埃氏慢生根瘤菌Y63-1菌株培养基的筛选与优化

为了促进广谱高效大豆埃氏慢生根瘤菌Y63-1菌株的高效生产与推广应用,本研究对其培养基进行了筛选与优化。首先比较了Y63-1在YMA、TY、SM、PA和BSE 5种根瘤菌基本培养基中的生长速度,结果表明Y63-1在TY基本培养基中生长最快。以TY为基本培养基进行单因素碳源及无机盐利用试验,结果表明葡萄糖是Y63-1生长的最佳碳源,CaCl_(2)为必要的培养基成分,Rh微量元素对菌株的生长有很大的促进作用。进一步对蛋白胨、葡萄糖、酵母粉及Rh微量元素4种组分进行正交优化,获得了适宜Y63-1生长的最佳培养基,配方(1 L)为:8 g蛋白胨、10 g葡萄糖、3 g酵母粉、0.1 g CaCl_(2)·6H_(2)O、3 mL Rh微量元素,pH7.0。此培养基也能显著提升USDA110的生长速率,可以广泛应用于慢生根瘤菌菌剂的大规模生产。

Fungicide tolerant Bradyrhizobium japonicum mitigate toxicity and enhance greengram production under hexaconazole stress

Bacterial strain RV9 recovered from greengram nodules tolerated 2400 μg/mL of hexaconazole and was identified by 16 S rDNA sequence analysis as Bradyrhizobium japonicum(KY940048). Strain RV9 produced IAA(61.6 μg/mL), ACC deaminase(51.7 mg/(protein·hr)), solubilized TCP(105 μg/mL), secreted 337.6 μg/mL EPS, and produced SA(52.2 μg/mL) and 2,3-DHBA(28.3 μg/mL). Exopolysaccharides produced by strain RV9 was quantified and characterized by SEM, AFM, EDX and FTIR. Beyond tolerance limit,hexaconazole caused cellular impairment and reduced the viability of strain RV9 revealed by SEM and CLSM. Hexaconazole distorted the root tips and altered nodule structure leading thereby to reduction in the performance of greengram. Also, the level of antioxidant enzymes, proline, TBARS, ROS and cell death was increased in hexaconazole treated plants.CLSM images revealed a concentration dependent increase in the characteristic green and blue fluorescence of hexaconazole treated roots. The application of B. japonicum strain RV9 alleviated the fungicide toxicity and improved the measured plant characteristics. Also,rhizobial cells were localized inside tissues as revealed by CLSM. Colonization of B.japonicum strain RV9 decreased the levels of CAT, POD, APX, GPX and TBARS by 80%, 5%,13%, 13% and 19%, respectively over plants grown at 80 μg/(hexaconazole·kg) soil. The ability to detoxify hexaconazole, colonize plant tissues, secrete PGP bioactive molecules even under fungicide pressure and its unique ability to diminish oxidative stress make B.japonicum an attractive choice for remediation of fungicide polluted soils and to concurrently enhance greengram production under stressed environment.

花生根瘤菌Bradyrhizobium sp.MM6 Ⅲ型分泌系统的结构和功能

【目的】探究花生根瘤菌Bradyrhizobium sp.MM6的Ⅲ型分泌系统(T3SS)的结构及其在根瘤菌与不同宿主建立共生关系中的作用。【方法】同源比对分析菌株MM6的T3SS基因簇的结构特征,并采用三亲本接合转移的方法构建T3SS调节基因ttsI突变菌株;通过蛭石结瘤和石蜡切片实验,比较突变体与野生型的共生固氮表型差异。【结果】经预测,MM6的T3SS基因簇编码区长约34.1 kb,可分为3个区域,包含10个保守结构基因和8个效应蛋白基因,与B.diazoefficiens USDA110相应基因的序列相似性为83%–93%;成功构建了MM6的ttsI突变株;ttsI突变株与野生型分别与花生(S523和Y45)、野大豆和大豆中黄57结瘤,ttsI突变体在花生中的总瘤数显著增加(P<0.05),根瘤中含菌细胞更多;ttsI突变体在野大豆中平均每株植物增加4个根瘤,根瘤中含菌细胞更多,地上部干重相比野生型MM6显著增加(P<0.05);在大豆中黄57中,野生型MM6能形成红色的有效根瘤,ttsI突变体不结瘤,且植株叶片发黄,地上部干重相比野生型MM6显著降低(P<0.05)。【结论】MM6的T3SS在花生和野大豆共生体系中起着有害的作用,而在大豆中黄57的共生体系中起着有利的作用。