nifV is contiguous to nifHDK in Frankia strain FaC1

The organization of genes with the capacity to code for four proteins involved in nitrogen fixation in Frankia strain FaC1 was determined by restriction fragment mapping and nucleotide sequence analysis. Analysis of the 44-kb genomic cosmid clone pFAH 1. isolated from a cosmid library made from Frankia strain FaCl, resulted in the identification of a 7.2-kb Pst I fragment to which Klebsiella nif H, nif D and nif K probes hybridized. This nif -hybridizing fragment was subcloned and analyzed by restriction fragment mapping. Further subcloning of the 7.2-kb fragment and subsequent sequence analysis of approximately 6.8 kb revealed the presence of six open reading frames (ORFs). Four of these ORFs have the potential to code for nif V-, nif H-, nif D- and nif K-like gene products and the two others are unidentified ORFs. The organization of the structural genes for nitrogenase is the same in this Frankia strain as it is in most other nitrogen-fixing prokaryotes, but the positioning of the nif V-like gene relative to the nif HDK cluster differs, A consensus nif -promoter-like sequence, found 5'to nif H. was not detected upstream of the nif V-like gene. Nine copies of a 7-bp direct repeat were found 5'to ORFA.     

山西牛奶子群落优势种种间关联性研究

基于2×2联列表,应用方差比率法、χ2检验、Pearson相关系数及Spearman秩相关系数检验等数量分析方法对山西牛奶子群落的25个优势种,共300个种对间的关联性进行分析研究。结果表明,25个优势种群的总体种间关联性呈不显著关联,种的分布相对独立。χ2检验结果有121个种对呈正相关,155个种对呈负相关,正负关联比为0.78;Pearson相关系数检验有111个种对呈正相关,189个种对呈负相关,正负关联比为0.59;Spearman秩相关系数检验有128个种对呈正相关,172个种对呈负相关,正负关联比为0.74;Spearman秩相关系数比Pearson相关系数的检验方法灵敏度更高。山西牛奶子群落总体上呈不显著负关联,表明其处于演替过程的初期。     

牛奶子树不同部位多糖抗氧化活性比较研究

从牛奶子(Elaeagnus umbellata Thunb.)的叶片、枝条、种子中提取多糖,并研究其抗氧化活性.采用热水提取、Savage法除蛋白、80％乙醇沉淀得其粗多糖.以Vc为对照,用番红花红光度法测定其对羟基自由基的清除作用;用邻苯三酚自氧化法测定其对超氧阴离子的清除作用;用Fe3+还原力法测定其还原能力.结果表明:3个部位所含多糖对羟基自由基、超氧阴离子自由基都可起到有效的清除或抑制作用,其中对羟基自由基清除活性依次为:种子＞叶片＞枝条＞Vc,对超氧阴离子自由基清除活性依次为:种子＞Vc＞叶片＞枝条,3个部位所含多糖都有还原性,但效果都不如硫脲.     

Studies of Seedling Inoculation of Frankia in Elaeagnus mollis Diels

The research on the preparation of Frankia solution, seedling culture and inoculation was carried out. Results indicated that the collective amount of cells may be increased when the logarithm phase cells were used as the inoculum and regularly homogenizing and transplanting inoculum by the method of magnetic stirring were adopted during the cultural process of Frankia. The inoculative tests verified that the strain with the highest ability to infect Elaeagnus mollis Diels is SIB 1301118 isolated from Elaeagnus angustifolia L. other than SIB 1332281 isolated from Elaeagnus mollis Diels. The effectivities of inoculation for germinating seeds and the seedlings to be transplanted were seed soaking and root soaking in Frankia solution preparel respectively. Early inoculation was beneficial to the growth and nodulation of seedings. The fefectivity of late inoculation was bad. Sand-soil mixture (2:1) was properly suitable to the seed germination and seedling growth. The seedling growth and nodulative rate could be promoted when 1/4 Sileris-Yong nutrient solution was suppplimented regularly.     

Altitude-related shift of relative abundance from insect to sunbird pollination in Elaeagnus umbellata (Elaeagnaceae)

The evolution of floral traits has been thought to be influenced by local, effective pollinators. However, little attention has been paid to the possibility that altitudinal variation in floral traits could be mediated by local pollinator functional groups, particularly a shift from bees to birds. Plant size, floral traits, pollinators and their pollination roles were investigated in the spring-flowering shrub Elaeagnus umbellata (Elaeagnaceae) at three altitudes (1160, 1676, and 2050 m) in Minshan, Sichuan Province, on the northern rim of the Hengduan Mountains, southwest China. Compared to lower altitudes, higher-altitude plants were smaller but the floral tubes were longer, with a larger volume of nectar of lower sugar concentration but with a greater proportion of sucrose. The visitation frequency of bees decreased with altitude, whereas the sunbirds did the opposite. Birds and bees foraged for nectar but not pollen, and birds deposited more pollen grains per visit relative to bees and least were syrphid flies. Excluding birds decreased seed set at high but not at mid- or low altitude. Our study of E. umbellata revealed an association between altitudinal variation in floral traits and a change in the relative abundance of the major pollinators with altitude from majority bees to majority sunbirds. Although abiotic factors also tend to vary with altitude and can affect floral traits, nectar properties of “pro-bird” pollination were observed at high altitude.     

Molecular cloning of a cDNA encoding glutamine synthetase from root nodules ofElaeagnus umbellate

We analyzed a cDNA clone encoding cytosolic glutamine synthetase,EuNOD-GS1, isolated from a root nodule cDNA library ofElaeagnus umbellata. This clone has an insert size of 1359 bp and encodes a protein for 355 amino-acid residues, with a molecular weight of 39.2 kDa. Its expression is slightly higher in the root nodules than in the leaves or uninfected roots. Analysis of the deduced amino acid sequences and phytogeny revealed thatEuNOD-GS1 is clustered with cytosolic GS-α isoenzymes. Therefore, based on this and previous results, we propose that the main physiological role ofEuNOD-GS1 is the assimilation of ammonia from secondary and, in part, primary sources.     

Analysis of phylogeny and codon usage bias and relationship of GC content,amino acid composition with expression of the structural nif genes

Bacteria and archaea have evolved with the ability to fix atmospheric dinitrogen in the form of ammonia, catalyzed by the nitrogenase enzyme complex which comprises three structural genes nifK, nifD and nifH. The nifK and nifD encodes for the beta and alpha subunits, respectively, of component 1, while nifH encodes for component 2 of nitrogenase. Phylogeny based on nifDHK have indicated that Cyanobacteria is closer to Proteobacteria alpha and gamma but not supported by the tree based on 16SrRNA. The evolutionary ancestor for the different trees was also different. The GC1 and GC2% analysis showed more consistency than GC3% which appeared to below for Firmicutes, Cyanobacteria and Euarchaeota while highest in Proteobacteria beta and clearly showed the proportional effect on the codon usage with a few exceptions. Few genes from Firmicutes, Euryarchaeota, Proteobacteria alpha and delta were found under mutational pressure. These nif genes with low and high GC3% from different classes of organisms showed similar expected number of codons. Distribution of the genes and codons, based on codon usage demonstrated opposite pattern for different orientation of mirror plane when compared with each other. Overall our results provide a comprehensive analysis on the evolutionary relationship of the three structural nif genes, nifK, nifD and nifH, respectively, in the context of codon usage bias, GC content relationship and amino acid composition of the encoded proteins and exploration of crucial statistical method for the analysis of positive data with non-constant variance to identify the shape factors of codon adaptation index.     

Organization of nitrogen fixation genes in a nonheterocystous, filamentous cyanobacterium

Abstract The nonheterocystous, filamentous cyanobacterium, Plectonema boryanum fixes nitrogen only under microaerophilic conditions. The organization of nitrogen fixation genes ( nifH, D, K ) in Plectonema was determined by using cloned fragments from the Anabaena nif genes as probes in Southern hybridizations. Regions of Plectonema DNA were homologous to Anabaena nifH, nifD , and nifK genes, and the resulting pattern of hybridization was used to construct a map of nifH, D, K DNA isolated from Plectonema cells grown under non-nitrogen fixing conditions (combined nitrogen and O₂ present). The nifH and nifD genes are on the same 3 kbp Hin dIII fragment, and nifK is on a 1 kbp Hin dIII fragment. All three nif fragments are adjacent to one another on a 12 kbp Cla I fragment.     

Relationship between electroporation conditions, electropermeability and respiratory activity for Frankia strain ACN14a

The use of electroporation for introducing macromolecules into intact cells of the actinomycete Frankia was investigated. Electropermeability was demonstrated by the uptake of dextran (70 kDa) molecules labeled with fluorescein isothiocyanate (FITC) inside Frankia cells. Upon pulsation with an exponentially decaying electric field, the cell membranes became permeable. Loading increased with initial pulsed electric field strength and capacitance. Increased loading efficiency was inversely related to INT (2-(p-iodophenyl-3-(p-nitrophenyl)-5- phenyltetrazolium chloride) reduction activity (respiring bacteria) of the cell population. The presence of CaCl2 in the electroporation and resealing buffer raised INT-reduction activity but K2SO4 decreased this activity. Resealing of electropores was confirmed by a decreasing FITC-dextran loading through the recovery period. The use of FITC-dextran molecules and INT-reduction assay are two new approaches for the study of permeabilization and cellular activity of electroporated bacteria.     

Genetic heterogeneity among Frankia isolates from root nodules of individual actinorhizal plants

Abstract Genetic variations among selected Frankia isolates from nitrogen-fixing root nodules harvested from an individual actinorhizal plant ( Elaeagnus angustifolia L. or Shepherdia argentea Nutt.) were estimated by restriction fragment analysis of their total genomic DNA. The presence of plasmids and their restriction enzyme patterns were used as additional criteria. Certain isolates from separate nodules on the same plant were found indistinguishable, being probably clones of the same strain. An endophytic passage of a strain isolated from S. argentea on another host plant, Hippophaë rhamnoides L., did not modify the structural characteristics of the genome in the reisolates obtained. However, in some cases, especially when restriction endonucleases cleaving Frankia DNA into relatively small fragments were used, multiple infection of the actinorhizal plants with different Frankia strains and the presence of more than one strain in a nodule were demonstrated. Some aspects of variability in natural populations of Frankia are discussed.     

一株弗兰克氏菌的分离培养及特性研究*

用根瘤匀浆法,从粗枝木麻黄(Casuarina glauca)根瘤中分离到一株内生菌FCg77。生物学特性试验表明：该菌株的适宜分离培养基为BAP培养基,最佳碳源为吐温-80,最适氮源为牛肉膏,能耐5％的盐分,生理类型为AB型,细胞壁化学组分为Ⅲ型。结合回接试验结果,初步判定分离菌株FCg77应属于弗兰克氏菌(Frankia)的成员。     

Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from Arabidopsis thaliana and regulation of its expression.

The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected.     

猪苓菌丝形成菌核伴生菌的发现及应用

在野生猪苓 (Grifolaumbellata (Pers .)Pil偄ｔ)菌核生长穴中首次分离到猪苓菌丝形成菌核所必需的伴生菌(Grifolasp .)。实验证明 :纯培养的猪苓菌丝不能形成菌核 ,但其与伴生菌共培养 ,无论在实验室培养基上或用树棒栽培 ,猪苓菌核形成很快且发育正常 ;伴生菌是猪苓菌丝形成菌核的关键生物因子。另外 ,伴生菌菌丝和猪苓菌菌丝二者形态差别较大 ,前者多为细长的薄壁菌丝 ,后者多为细胞直径较大的薄壁和厚壁菌丝。     

猪苓菌丝形成菌核伴生菌的发现及应用

在野生猪苓(Grifola umbellata (Pers.) Pilát)菌核生长穴中首次分离到猪苓菌丝形成菌核所必需的伴生菌(Grifola sp.).实验证明:纯培养的猪苓菌丝不能形成菌核,但其与伴生菌共培养,无论在实验室培养基上或用树棒栽培,猪苓菌核形成很快且发育正常;伴生菌是猪苓菌丝形成菌核的关键生物因子.另外,伴生菌菌丝和猪苓菌菌丝二者形态差别较大,前者多为细长的薄壁菌丝,后者多为细胞直径较大的薄壁和厚壁菌丝.     

一株降解纤维素放线菌的产纤维素酶基因克隆与表达

【背景】纤维素在自然界中储量丰富,但天然纤维素的难降解性成为广泛应用纤维素资源的壁垒,近年来利用微生物来降解纤维素成为热点研究。【目的】筛选分离得到一株具有降解纤维素功能的放线菌菌株Lb1,通过全基因组测序确定其产纤维素酶关键基因5676,对基因5676进行克隆转化,使其在大肠杆菌中进行表达。【方法】通过基因工程技术将产纤维素基因连接到表达质粒上并导入表达菌株,对其降解纤维素生成葡萄糖的能力进行探究。【结果】将Lb1菌株的16S rRNA基因进行比对,确定菌株Lb1属于链霉菌属,命名为Streptomyces sp. Lb1。成功构建出纤维素酶表达载体,并且导入表达菌株大肠杆菌BL21(DE3),重组菌株的产纤维素酶能力大于空载菌株。【结论】通过基因工程技术成功克隆出产纤维素酶基因,从而表达纤维素酶,为今后利用微生物降解纤维素的大规模应用提供参考。     

国家二级保护植物翅果油树传粉生物学的初步研究

对翅果油树进行了传粉生物学的野外观察和实验表明：（1）翅果油树的花具有自花传粉的结构特征,同时具有蜜腺,又具备异花传粉的特征。传粉者主要是一种家养的蜜蜂。（2）翅果油树花形态结构与传粉者的形态和传粉行为非常协调和适应,昆虫的传粉部位主要是头前部和胸部。（3）蜜腺的产蜜量大,开花后1～2天蜜腺分泌量最大。对传粉昆虫竞争中具明显优势。昆虫的访花频率明显高于同花期的其他植物。（4）翅果油树没有融合生殖现象。     

Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13.

The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate. The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27. A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27. In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence. The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp. strain B13 plasmid pWR1. A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.