Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

The cycHJKL gene cluster plays an essential role in the biogenesis of c-type cytochromes in Bradyrhizobium japonicum

We present an extended genetic analysis of the previously identified cycH locus in Bradyrhizobium japonicum. Three new open reading frames found in an operon-like structure immediately adjacent to the 3 end of cycH were termed cycJ, cycK and cycL. A deletion mutant (cycHJKL) and biochemical analysis of its phenotype showed that the genes of the cluster are essential for the biogenesis of cellular c-type cytochromes. Mutations in discrete regions of each of the genes were also constructed and shown to affect anaerobic respiration with nitrate and the ability to elicit an effective symbiosis with soybean, both phenotypes being a consequence of defects in cytochrome c formation. The CycK and CycL proteins share up to 53% identity in amino acid sequence with the Rhodobacter capsulatus Ccll and Cc12 proteins, respectively, which have been shown previously to be essential for cytochrome c biogenesis, where-as cycJ codes for a novel protein of 169 amino acids with an M_r of 17857. Localisation studies revealed that CycJ is located in the periplasmic space; it is probably anchored to the cytoplasmic membrane via an N-terminal hydrophobic domain. Based on several considerations discussed here, we suggest that the proteins encoded by the cycHJKL-cluster may be part of a cytochrome c-haem lyase complex whose active site faces the periplasm.     

Identification and inter-relationship analysis of Bradyrhizobium japonicum strains by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD)

Genomic DNA of 13 Bradyrhizobium japonicum strains was prepared and analysed by restriction fragment length polymorphism (RFLP) with nif and nod probes, and by random amplified polymorphic DNA (RAPD) with 11 primers of arbitrary nucleotide sequence. Polymorphism was observed in both analyses. The RFLP and RAPD banding patterns of different strains were used to calculate genetic divergence and to construct phylogenetic trees, allowing studies on the relationships between the strains. RFLP with nif and nod probes permitted the separation of the strains into two divergent groups, whereas RAPD separated them into four main groups. RAPD allowed closely related strains to be distinguished.     

Genomic fingerprinting of Bradyrhizobium japonicum isolates by RAPD and rep-PCR

Genetic diversity of indigenous Bradyrhizobium japonicum population in Croatia was studied by using different PCR-based fingerprinting methods. Characteristic DNA profiles for 20 B. japonicum field isolates and two reference strains were obtained using random primers (RAPD) and two sets of repetitive primers (REP- and ERIC-PCR). In comparison with the REP, the ERIC primer set generates fingerprints of lower complexity, but still several strain-specific bands were detected. Different B. japonicum isolates could be more efficiently distinguished by using combined results from REP- and ERIC-PCR. The most polymorphic bands were observed after amplification with four different RAPD primers. Both methods, RAPD and rep-PCR, resulted in identical grouping of the strains. Cluster analysis, irrespective of the fingerprinting method used, revealed that all the isolates could be divided into three major groups. Within the major groups, the degree of relative similarity between B. japonicum isolates was dependent upon the method used. Our results indicate that both RAPD and rep-PCR fingerprinting can effectively distinguish different B. japonicum strains. RAPD fingerprinting proved to be slightly more discriminatory than rep-PCR.     

Identification of nifE-, nifN- and nifS-like genes in Bradyrhizobium japonicum

Summary The 17 kb region between the Bradyrhizobium japonicum nitrogenase genes (nifDK and nifH) was investigated for the presence of further nif or fix genes by site-directed insertion or deletion/replacement mutagenesis and interspecies hybridization. Mutant strains were tested for their ability to reduce acetylene in free-living, microaerobic culture (Nif phenotype) and in soybean root nodules (Fix phenotype). The presence of a gene, previously identified by hybridization with the Klebsiella pneumoniae nifB gene, was proved by isolation of a nifB insertion mutant which was completely Nif^- and Fix^-. Three other regions were found to be homologous to the K. pneumoniae genes nifE, nifN, and nifS, NifE and nifN insertion mutants were completely Nif^-/Fix^- whereas nifS mutants were leaky with 30% residual Fix activity. Taken together, the data show that the B. japonicum genome harbours a cluster of closely adjacent genes which are directly concerned with nitrogenase function.     

Jasmonates induce Nod factor production by Bradyrhizobium japonicum

Jasmonates are signaling molecules involved in induced systemic resistance, wounding and stress responses of plants. We have previously demonstrated that jasmonates can induce nod genes of Bradyrhizobium japonicum when measured by beta-galactosidase activity. In order to test whether jasmonates can effectively induce the production and secretion of Nod factors (lipo-chitooligosaccharides, LCOs) from B. japonicum, we induced two B. japonicum strains, 532C and USDA3, with jasmonic acid (JA), methyl jasmonate (MeJA) and genistein (Ge). As genistein is well characterized as an inducer of nod genes it was used a positive control. The high-performance liquid chromatography (HPLC) profile of LCOs isolated following treatment with jasmonates or genistein showed that both JA and MeJA effectively induced nod genes and caused production of LCOs from bacterial cultures. JA and MeJA are more efficacious inducers of LCO production than genistein. Genistein plus JA or MeJA resulted in greater LCO production than either alone. A soybean root hair deformation assay showed that jasmonate induced LCOs were as effective as those induced by genistein. This is the first report that jasmonates induce Nod factor production by B. japonicum. This report establishes the role of jasmonates as a new class of signaling molecules in the Bradyrhizobium-soybean symbiosis.     

Regulation of nod gene expression in Bradyrhizobium japonicum

Summary The best inducers of nod:: lacZ translational fusions in Bradyrhizobium japonicum are isoflavones, primarily genistein and daidzein. Upstream of the nodABC genes in B. japonicum is a novel gene, nodY, which is coregulated with nodABC. Measurements of the activity of lacZ fusions to the nodD gene of B. japonicum show that this gene is inducible by soybean seed extract and selected flavonoid chemicals. The induction of the nodY ABC and nodD operons appears to require a functional nodD gene, indicating that the nodD gene product controls its own synthesis as well as other nod genes.     

扇脉杓兰和无距虾脊兰的核型分析

为了解扇脉杓兰(Cypripedium japonicum Thunb.)和无距虾脊兰(Calanthe tsoongiana T. Tang et F. T. Wang)的核型,采用根尖压片法对扇脉杓兰和无距虾脊兰的染色体数目和核型进行了研究。结果表明,扇脉杓兰体细胞的染色体数为22,核型公式为2n=2x=22=16m+2sm+2st+2t,染色体相对长度组成为2n=22=2L+6M2+12M1+2S,核不对称系数为60.01%,核型分类为2B型;而无距虾脊兰体细胞的染色体数为40,核型公式为2n=2x=40=28m+10sm+2st,染色体相对长度组成为2n=40=8L+10M2+16M1+6S,核不对称系数为59.84%,核型分类为2B型;两者核型都较为对称。其中,无距虾脊兰的核型为首次报道。这为扇脉杓兰和无距虾脊兰的进化地位和种质保护提供了细胞学证据。     

In Bradyrhizobium japonicum the common nodulation genes, nodABC, are linked to nifA and fixA

Summary Using cloned Rhizobium phaseoli nodulation (nod) genes as hybridization probes homologous restriction fragments were detected in the genome of the slow-growing soybean symbiont, Bradyrhizobium japonicum strain 110. These fragments were isolated from a cosmid library, and were shown to lie 10 kilobasepairs (kb) upstream from the nifA and fixA genes. Specific nod probes from Rhizobium leguminosarum were used to identify nodA-, nodB-, and nodC-like sequences clustered within a 4.5 kb PstI fragment. A mutant was constructed in which the kanamycin resistance gene from Tn5 was inserted into the nodA homologous B. japonicum region. This insertion was precisely located, by DNA sequencing, to near the middle of the nodA gene. B. japonicum mutants carrying this insertion were completely nodulation deficient (Nod^-).     

Mutational analysis of the Bradyrhizobium japonicum common nod genes and further nod box-linked genomic DNA regions

Summary By insertional and deletional marker replacement mutagenesis the common nod region of Bradyrhizobium japonicum was examined for the presence of additional, essential nodulation genes. An open reading frame located in the 800 bp large intergenic region between nodD1 and nodA did not appear to be essential for nodulation of soybean. Furthermore, a strain with a deletion of the nodI- and nodJ-like genes downstream of nodC had a Nod⁺ phenotype. A mutant with a 1.7 kb deletion immediately downstream of nodD1 considerably delayed the onset of nodulation. This region carried a second copy of nodD (nodD2). A nodD1-nodD2 double mutant had a similar phenotype to the nodD2 mutant. Using a 22-mer oligonucleotide probe partially identical to the nod box sequence, a total of six hybridizing regions were identified in B. japonicum genomic DNA and isolated from a cosmid library. Sequencing of the hybridizing regions revealed that at least three of them represented true nod box sequences whereas the others showed considerable deviations from the consensus sequence. One of the three nod box sequences was the one known to be associated with nodA, whereas the other two were located 60 to 70 kb away from nif cluster I. A deletion of one of these two sequences plus adjacent DNA material mmutant 308) led to a reduced nodulation on Vigna radiata but not on soybean. Thus, this region is probably involved in the determination of host specificity.Dedicated to Prof. Giorgio Semenza on the occasion of his 60th birthday     

天竺桂凋落叶添加对凤仙花生理特性的影响

采用盆栽试验,通过向土壤(每盆8kg)中添加0(CK)、20(L20)、40(L40)和80g·pot-1(L80)天竺桂(Cinnamomum japonicum)凋落叶,模拟其自然分解对凤仙花(Impatiens balsamina)生理特性的影响。结果显示:(1)天竺桂凋落叶处理显著增加了凤仙花现蕾前期和现蕾期叶片H2O2含量,同时显著抑制了凤仙花现蕾前期、现蕾期和盛花期超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性。(2)凋落叶添加处理显著增加了凤仙花现蕾前期抗坏血酸(ASA)含量,但现蕾期和盛花期ASA含量却显著下降。(3)添加凋落物处理显著降低了现蕾前凤仙花叶片MDA含量,但显著增加了现蕾期MDA含量,而盛花期MDA含量在L20、L80处理下显著降低,在L40处理下则显著增加。(4)天竺桂凋落叶添加对可溶性蛋白和可溶性糖两种渗透调节物质含量影响不显著。(5)凋落叶处理显著抑制了凤仙花地径生长和地上生物量积累。(6)天竺桂凋落叶中主要化感物质可能有香豆素、桉叶油醇、肉桂醛、反式石竹烯和松油醇等。研究认为,非酶促活性氧清除系统可能在凤仙花抵御化感物质氧化胁迫过程中发挥着比酶促系统更为重要的作用,其中的ASA在氧化胁迫的早期过程扮演着重要角色;天竺桂凋落叶添加处理所造成的氧化胁迫可能超过了两类活性氧清除系统的清除能力,对凤仙花膜系统造成明显伤害,导致其地径和生物量积累显著降低。     

Comparison of 16S rDNA analysis and rep-PCR genomic fingerprinting for molecular identification of Yersinia pseudotuberculosis

16S rDNA sequence analysis and repetitive element sequence-based PCR (rep-PCR) genomic fingerprinting were evaluated on 11 type strains of the genus Yersinia and 17 recognized serotype strains of Y. pseudotuberculosis to investigate their genetic relatedness and to establish the value of techniques for the identification of Y. pseudotuberculosis. A phylogenetic tree constructed from 16S rDNA sequences showed that the type strains of Yersinia species formed distinct clusters with the exception of Y. pestis and Y. pseudotuberculosis. Moreover, Y. pestis NCTC 5923^T was found to be closely related to Y. pseudotuberculosis serotypes 1b, 3, and 7. Dendrograms generated from REP-PCR, and ERIC-PCR data revealed that members of the genus Yersinia differed from each other with the degree of similarity 62% and 58%, respectively. However, the BOX-PCR results showed that Y. pestis 5923^T clustered with the Y. pseudotuberculosis group with a degree of similarity 74%. According to these findings, 16S rDNA sequence analysis was unable to reliably discriminate Y. pseudotuberculosis from Y. pestis. However, REP-PCR and especially ERIC-PCR provided an effective means of differentiating between members of the taxa. This revised version was published online in June 2006 with corrections to the Cover Date.     

DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis

Forty isolates and five standard laboratory strains, representing serotypes c, e and f of Streptococcus mutans were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the genomic DNA with BssH II. The digestion patterns of standard laboratory strains were characteristic of serotypes c, e and f. Serotypes c and f generated diagnostic DNA fragments of approximately 145 kbp and of approximately 130-175 kbp in length, respectively. Serotype e generated a ladder of at least 14 fragments of 15-155 kbp in length. The digestion patterns of isolates were essentially similar to those of the standard laboratory strains. The patterns of almost all isolates obtained from a single individual were identical, but patterns of a few different types were also observed among isolates obtained from two individuals. Digestion with BssH II revealed differences among isolates obtained from different individuals. We used differences in banding patterns among isolates to construct a dendrogram. The dendrogram included two major clusters, one that consisted of isolates of serotypes c and f, and an other that consisted of isolates of serotype e. Our results indicate that BssH II is a useful enzyme for distinguishing among isolates of S. mutans and that digestion patterns obtained by PFGE can be used for chromosomal DNA fingerprinting.     

遮光对连香树幼苗光合特性及其叶片解剖结构的影响

以2年生连香树实生苗为材料,在田间通过黑色遮阳网设置全光照(L₀)及透光率55%(L₁)、25%(L₂)和10%(L₃)4种光环境,研究遮光对连香树幼苗光合作用及叶片解剖结构的影响。结果表明：(1)连香树幼苗叶片P_n在全光和L₁处理下呈非典型的“乁”形变化,未出现“午休”现象,中午14:00出现极值,而在L₂和L₃处理下变化相对缓和,极值出现在中午12:00;叶片G_s呈现与P_n类似的变化趋势,而C_i则呈基本一致的凹形变化。(2)各处理P_n、G_s和T_r的日均值均表现为L₀＞L₁＞L₂＞L₃,而C_i的日均值则呈相反的顺序;P_n与G_s、T_r、气温和光合有效辐射均呈极显著正相关关系(P＜0.01)。(3)全光照连香树幼苗的光补偿点(LCP)、光饱和点(LSP)、暗呼吸速率(R_d)均显著高于遮光处理,并维持较高的P_n而未出现明显的光抑制;遮光导致幼苗的LCP、LSP、R_d显著降低,有利于充分利用弱光,以满足低光环境下植株的正常生长。(4)与全光照相比,遮光下连香树叶片气孔密度显著变小,但气孔器长度、气孔器宽度、单个气孔器面积显著增加,气孔器面积百分比减少,影响幼苗细胞内外的水分和气体传递。(5)遮光条件下,连香树叶片明显变薄,表皮细胞厚度减小,栅栏组织(PT)厚度降低,排列变得疏松,海绵组织(ST)厚度增加,PT/ST相应减小。(6)与全光照相比,强度遮光下(L₂和L₃)连香树幼苗生长受阻,苗高(H)和基径(D)明显减小,生物量模型D²H下降;而轻度遮光(L₁)下幼苗H和D、H/D和D₂H均未出现显著变化。研究发现,连香树具有一定的光忍耐性和喜光性,对光照条件的生态幅较宽,轻度遮光影响较小,但强度遮光对连香树幼苗气体交换参数和光合响应特征产生了显著影响,同时影响了叶片的解剖结构和气孔分布特征,从而影响连香树幼苗的生长形态。在育苗生产中,适度遮光有利于降低气温、减小蒸腾,但遮光后田间有效辐射强度应保持在自然光强的55%以上。     

Oligonucleotide fingerprinting and RAPD analysis of Achillea species: Characterization and long-term monitoring of micropropagated clones

Two different DNA fingerprinting techniques were applied to a set of Achillea samples (Asteraceae), comprising ten taxa of the medicinally important A. millefolium group and six related species. Field-grown as well as in vitro-micropropagated plants were individually screened for abundance and polymorphism of target sequences recognized by oligonucleotide fingerprinting with 13 different microsatellite-complementary probes. While most probes revealed a high level of intra- and interspecific variability, fingerprints proved to be somatically stable in vegetatively propagated plant material. Analysis of the same samples by polymerase chain reaction with arbitrary 10-mer primers yielded less polymorphic patterns. Because of its higher discriminatory ability, oligonucleotide fingerprinting offers itself as the method of choice for the identification and discrimination of A. asplenifolia and A. roseoalba clones, as well as for monitoring their stability during micropropagation.Abbreviations BPA N-benzyl-9-[2-tetrahydropyranyl]-adenine - PCR polymerase chain reaction - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - TAE buffer 40 mM Tris acetate, 20 mM sodium acetate, 1 mM EDTA, pH 7.8 Part of the Ph.D. Thesis     

连香树水提物和乙醇提取物的体外抗氧化研究

为了探究连香树水提物和乙醇提取物的主要成分和抗氧化作用,该研究采用水提和醇提两种方法提取连香树叶片中的代谢物并测定其主要成分,通过体外抗氧化实验,即清除羟自由基(·OH)、DPPH自由基(DPPH·)、超氧阴离子(O_2~-·)和还原铁离子(Fe~(3+))的能力等四个指标来评价其抗氧化作用。结果表明:连香树水提物和乙醇提取物中均含有山萘酚。此外,水提物中还含有苜蓿素和异槲皮苷等黄酮类物质;乙醇提取物中还含有柚皮素和槲皮素3-O-β-D-葡萄糖苷等黄酮类物质。水提物和乙醇提取物均有清除羟自由基、DPPH自由基、超氧阴离子及还原三价铁离子的能力。抗氧化的作用随提取物浓度的增大而增强,其中清除超氧阴离子(IC50值分别为0.092、0.002 mg·mL~(-1))的能力强于阳性对照Vc(IC_(50)值为0.241 mg·mL~(-1))且铁离子还原力的IC_(50)值(水提物为0.014 mg·mL~(-1),乙醇提取物为0.001 mg·mL~(-1))相对较小,说明其总抗氧化活性较强。由此可见,连香树水提物和乙醇提取物均具有良好的抗氧化作用,可作为一种潜在的天然抗氧化剂。     

干旱胁迫对日本荚蒾幼苗光合生理特性的影响

为了探讨极小种群野生植物日本荚蒾(Viburnum japonicum)对干旱胁迫的响应,以日本荚蒾二年实生苗为材料进行盆栽控水实验,设置正常供水(CK)、轻度干旱(LS)、中度干旱(MS)和重度干旱(SS)4个水分处理,研究持续干旱处理对日本荚蒾幼苗光合生理特性的影响。结果表明:在CK、LS、MS水分梯度下,日本荚蒾幼苗生长正常,但在SS胁迫下受影响明显,植株全部死亡。各胁迫阶段叶片净光合速率(P_n)、SPAD值、最大净光合速率(P_(nmax))、表观量子效率(AQY)、暗呼吸速率(R_d)表现出随水分梯度的降低而减小,光饱和点(LSP)、光补偿点(LCP)则相反,各水分梯度下AQY随时间延长逐渐上升,SS处理幼苗在胁迫45d及以后P_n均为负值。最大光化学效率(F_v/F_m)在胁迫过程中变化范围较小;实际光量子效率(ΦPSII)、电子传递速率(ETR)、光化学猝灭系数(qP)随土壤水分的减少而降低,非光化学猝灭系数(NPQ)逐渐上升,LS、MS与CK间变化幅度较小,但SS与CK间差距逐渐增大。结果表明在轻度和中度干旱下日本荚蒾幼苗表现出一定的适应性和耐干旱能力,而长期重度干旱则对幼苗影响较为严重。     

Antioxidant defense system responses and role of nitrate reductase in the redox balance maintenance in Bradyrhizobium japonicum strains exposed to cadmium

In this work, we evaluated the effects of cadmium (Cd) on the antioxidant defense system responses and the role of nitrate reductase (NR) in the redox balance maintenance in Bradyrhizobium japonicum strains. For that, B. japonicum USDA110 and its NR defective mutant strain (GRPA1) were used. Results showed that the addition of 10 μM Cd did not modify the aerobic growth of the wild type strain while the mutant strain was strongly affected. Anaerobic growth revealed that only the parental strain was able to grow under this condition. Cd reduced drastically the NR activity in B. japonicum USDA110 and increased lipid peroxide content in both strains. Cd decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio in B. japonicum USDA110 although, a significant increased was observed in the mutant GRPA1. GSH-related enzymes were induced by Cd, being more evident the increase in the mutant strain. This different behavior observed between strains suggests that NR enzyme plays an important role in the redox balance maintenance in B. japonicum USDA 110 exposed to Cd.     

Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.