The use of elements of the E. coli Ntr-system for the design of an optimized recombinant expression system for high cell density cultivations.

The inducible glnA promoter 2 of the E. coli glutamine synthetase gene is suitable as an expression unit for the production of recombinant proteins at low and high cell densities. It is active when the concentration of ammonium as the sole nitrogen source in the culture medium is below 1 mM. This nitrogen regulatory system was optimized by introduction of expression cassettes consisting of additional elements of the ntr-system. These artificial constructions result in enhanced recombinant gene expression in the production phase. Furthermore, the basic recombinant protein level during the growth phase is reduced due to a tighter promoter control. A three- to four-fold higher accumulation of chloramphenicol-acetyltransferase (as reporter protein) and of anti-EGF-receptor miniantibodies was achieved by increasing the amount of the final regulator molecule NtrC approximately P via plasmidal co-expression of the ntrC gene. The introduction of a modified glnA promoter 1 inverse to glnAp2 lowered the basic activity of glnAp2 to about one half. It is assumed that under nitrogen excess conditions sigma 70-RNA polymerase binds at glnAp1 and thereby prevents most of the binding of sigma 54-RNA polymerase at glnAp2. The optimized expression systems were successfully applied in low and high cell density cultivations. In the fed-batch phase of high cell density cultivations recombinant protein formation was induced through external nitrogen limitation under FIA-controlled concentration of glucose as carbon source.     

Effect of sediment elutriate on the chemical composition of Gymnodinium sp. (Dinophyceae). II.

Abstract

The chemical composition of Gymnodinium sp. was studied in cells grown in batch cultures with sediment elutriate, enriched or unenriched with F/2 as the culture medium. The cellular protein, chlorophyll a, nitrogen, carbon and phosphorus content were determined in conjunction with cell density. For the enriched media, a high density of biomass was always measured; differences in cellular composition between control and elutriate cells were not observed and the atomic C:N:P ratios were typical of unstarved phytoplankton. Unenriched media produced different effects on the algal growth, which also affected the biochemical composition of the cells. Relative to the control, some elutriates reduced the algal growth, affected the chemical composition of the cells which showed very high levels of carbon, nitrogen and protein, and resulted in an aberrant morphology. The stimulating growth elutriate affected the chemical composition of the cell in another manner: the greatest effect was a decrease of carbon content, whereas the other compounds remained similar to the control; aberrant forms were not observed.     

Canopy structure and leaf nitrogen distribution in a stand of Lysimachia vulgaris L. as influenced by stand density

Summary A hypothesis that a dense stand should develop a less uniform distribution of leaf nitrogen through the canopy than an open stand to increase total canopy photosynthesis was tested with experimentally established stands of Lysimachia vulgaris L. The effect of stand density on spatial variation of photon flux density, leaf nitrogen and specific leaf weight within the canopy was examined. Stand density had little effect on the value of the light extinction coefficient, but strongly affected the distribution of leaf nitrogen per unit area within a canopy. The open stand had more uniform distribution of leaf nitrogen than the dense stand. However, different light climates between stands explained only part of the variation of leaf nitrogen in the canopy. The specific leaf weight in the canopy increased with increasing relative photon flux density and with decreasing nitrogen concentration.     

毕赤酵母工程菌高密度发酵中氨态氮含量的测定

首次尝试将钠氏法应用于毕赤酵母高密度发酵中,并与靛酚蓝检测法进行了比较,证实钠氏法在0~10 mg/L的范围内,相关性好(r2=0.996 5),精确度(RSD=2.14%~5.32%)和精密度(Prec ision=97%~105%)高,更适合于毕赤酵母发酵中氨态氮含量的检测。并对其检测条件进行优化,确定检测波长为400 nm,最佳的显色反应为10 min。该方法用于毕赤酵母高密度发酵表达重组人血清白蛋白胸腺肽的结果表明,在发酵液氨基氮含量低于1.5 mg/kg,重组目的蛋白的表达出现了明显的降解,改善并控制发酵液中氨态氮的含量后,可以明显控制目的蛋白的降解,提高产品的产率。结果证实该方法用于发酵液中氨态氮的检测时,准确可靠,切实可行。     

翅荚木人工林不同径阶间细根主要功能性状与根际土壤养分的关系

细根能敏感地感知土壤环境变化,对植物生长发育具有重要影响.以6年生翅荚木人工林为对象,对其不同径阶的细根主要功能性状与根际土壤养分特征及两者间关系进行分析.结果表明:细根生物量、根长密度与根体积密度均随径阶增加而增加,比根长与比根面积则随径阶增加呈先升高再下降后升高的趋势,根组织密度则与径阶大小不相关.不同径阶翅荚木根际土壤的pH值及含水率、全碳、全磷、铵态氮、硝态氮和总有效氮含量均存在显著差异,大径阶林木的根际土壤全碳、全氮、硝态氮、总有效氮含量相对较高,小径阶林木的根际土壤含水率、土壤全磷、铵态氮含量相对较高.土壤全氮、全碳、硝态氮和总有效氮含量与林木细根的生物量、根长密度、根体积密度呈显著正相关;土壤全磷与林木细根的根组织密度呈显著正相关,与比根长、比根面积呈显著负相关;土壤含水率与林木细根的生物量和根体积密度均呈显著正相关;根际土壤pH和林木细根的比根长、比根面积呈显著正相关,与根组织密度则呈显著负相关.研究结果可为翅荚木优良种质资源选育提供科学依据.     

Changes in nitrogen content in malformed inflorescence of mango caused by Fusarium moniliforme Sheld. var. subglutinans Wr. et Rg.

Abstract

Fusarium moniliforme Sheld. var. subglutinans Wr. et Rg. causes malformations in young inflorescences of mango and seriously affects fruit formation. Quantitative studies of total, soluble and protein nitrogen contents of malformed inflorescence of two cultivars, ‘Himsagar’ and ‘Bombai’ were made. Healthy inflorescences of the former cultivar showed slightly higher nitrogen content than that of the latter. After infection, considerable increases in both soluble and protein fractions of nitrogen were observed in both host varieties. The proportion of these two nitrogen fractions, however, showed some changes in ‘Bombai’ but in ‘Himsagar’ it remained more or less unaltered.     

Detection of intracellular forms of secretory aspartic proteinase in Candida albicans.

The extracellular proteinase (EPR) of Candida albicans was induced in a medium containing bovine serum albumin as sole nitrogen source. There were two intracellular forms in cells induced to produce EPR, a 43 kDa protein (EPR) and a 45 kDa protein (cross-reacting material of EPR; CRM-EPR); these were detected by immunoblotting using anti-EPR antiserum. The 43 kDa protein (EPR) may be the same as the extracellular form judging by molecular mass, and the 45 kDa protein (CRM-EPR) may be a precursor form of EPR. Many dense granules were observed by electron microscopy near the plasma membrane of the mother cells in EPR-producing cells. Both the 43 and 45 kDa proteins were recovered in a membrane fraction and were solubilized by Triton X-100. When the membrane fraction was further fractionated by sucrose density gradient centrifugation, the 43 and 45 kDa proteins were differentially fractionated. This suggests that they were located in different membrane-bound structures and is consistent with an assumption that the 45 kDa protein is a precursor for EPR.     

耕作方式及施氮量对砂姜黑土区小麦氮代谢及籽粒产量和蛋白质含量的影响

为明确砂姜黑土区小麦(Triticum aestivum)产量和品质形成的耕作方式及施氮量最优组合, 在大田试验条件下, 以深松、旋耕和常规耕作3种耕作方式为主区, 0、120、225、330 kg·hm^-2 4个施氮量为副区, 研究了不同耕作方式及施氮量组合对小麦拔节后氮代谢、籽粒产量和蛋白质含量的影响。结果表明, 随着生育期的推进, 叶片谷氨酰胺合成酶活性、游离氨基酸含量和可溶性蛋白含量均呈先升后降的趋势, 深松方式配合中高氮处理的峰值在花后10天, 而常规耕作和旋耕的4个施氮处理以及深松的低氮处理峰值多在开花期。与常规耕作和旋耕相比, 深松耕作显著降低了10-40 cm的土壤容重, 提高了土壤总空隙度和根干质量, 有利于中后期根系氮素吸收。耕作方式和施氮量对籽粒产量和蛋白质含量影响显著, 均以深松方式最高。3种耕作方式下小麦产量和蛋白质含量均随施氮量增加而增加, 籽粒产量以深松方式配合330 kg·hm^-2施氮量最高, 而常规耕作和旋耕方式的产量在施氮量为225 kg·hm^-2时达到最大。3种耕作方式下籽粒蛋白质含量均以施氮225 kg·hm^-2最高。因此, 在砂姜黑土区宜采用深松耕作方式配合适宜的施氮量, 以改善土壤条件, 促进根系氮素吸收, 延长叶片功能期, 达到产量与蛋白品质提升之目的。     

Effects of tillage and nitrogen addition rate on nitrogen metabolism,grain yield and protein content in wheat in lime concretion black soil region

Aims The purpose of this study was to determine a suitable combination of tillage method and nitrogen rate to improve wheat (Triticum aestivum) yield and protein content in lime concretion black soil.
Methods Under the field experimental conditions, three tillage methods (subsoiling and rotary tillage, rotary tillage, and conventional tillage) were used as the main treatments, and four nitrogen application rates (0, 120, 225 and 330 kg·hm^–2) were used as sub-treatments. Nitrogen assimilation after jointing stage, grain yield, and protein content were determined in wheat plants to study the effects of different tillage methods and nitrogen application rate on these variables.
Important findings Results showed that the glutamine synthetase (GS) activity, free amino acid content, and soluble protein content in wheat plants initially increased and then decreased during growth. The peaks of GS activity, free amino acid content, and soluble protein content occurred 10 days after flowering in the subsoiling treatment with 225 or 330 kg·hm^–2 nitrogen application rate, and at the flowering stage for other treatment combinations. Compared with the conventional tillage and rotary tillage, the bulk density of 10 to 40 cm soil in the subsoiling treatment was significantly reduced, and the soil total porosity and root dry weight were significantly increased. Tillage method and nitrogen application rate had a significant impact on grain yield and protein content in wheat plants. Grain yield and protein content were highest in the subsoiling treatment. Regardless of the tillage method, the grain yield and protein content both increased with increasing nitrogen application rate. The grain yield in the subsoiling treatment was highest with nitrogen application rate at 330 kg·hm^–2, whereas the outputs of conventional tillage and rotary tillage were peaked at nitrogen application rate of 225 kg·hm^–2. The grain proteincontent was highest at nitrogen application rate of 225 kg·hm^–2 under the three tillage methods. Thus, subsoiling with optimum nitrogen rate should be promoted in lime concretion black soil. Subsoiling increased grain yield and protein quality by improving soil conditions and the absorption of root systems for soil nitrogen.     

Regulatory Mechanisms of Reproductive Effort in Plants 1. Plasticity in Reproductive Energy Allocation and Propagule Output of Helianthus annuus L. (Compositae) Cultivated at Varying Densities and Nitrogen Levels

Abstract Plasticity in growth, reproductive energy allocation (RA), and reproductive output were investigated in Helianthus annuus L. cultivar. Russia (Compositae) grown under varying densities and soil nitrogen levels. The role and behaviour of pollinators in seed production was also examined.
Exceedingly marked plastic responses were detected in individual biomass, the patterns of resource allocation to total reproductive structures (RA) and also to propagules, fecundity, reproductive outputs, and propagule size and weight under changing densities and soil nitrogen levels. Plants cultivated at higher densities exhibited proportionately lower individual biomass, lower RA, lower fecundity, lower seed output, and smaller seed size in response to increasing density and decreasing soil nitrogen levels. However, differences due to different N-levels were not as great as those to changing density.
One of the most significant findings was that seed production under limited resource availability, i.e., lack of ample solar radiation and soil nutrients, due to strong interference at higher density plots, is exceedingly costlty , This was most clearly demonstrated by a very sharp increase in relative energy partitioning to a single propagule in response to the increased density and decreasing nitrogen levels, the relative energy cost to a single achcne (R_A) increasing from one to twenty-fourfold. Reproduction was also affected by pollinator-limitations, and seed size showed a marked position effect.     

玉米-大豆间作和施氮对红壤地中小型土壤动物群落特征的影响

通过2年田间定位试验(2013—2014),利用干漏斗法收集土壤中小型动物,探讨不同种植模式(玉米单作、玉米大豆间作、大豆单作)和施氮(0、150 kg·hm^-2)对红壤土壤动物数量和群落结构的影响.结果表明: 两年取样共捕获土壤中小型动物8349只,分属3门10纲29目,优势类群为蜱螨目和弹尾目.2013年间作和施氮均提高了玉米拔节期土壤动物的平均密度.2014年土壤动物类群数较2013年有明显的增加,施氮条件下的土壤动物平均密度较不施氮有所增加.两年多因素方差分析结果表明,种植模式和生育期对土壤动物平均密度、类群数和多样性指数都无显著影响.但在2014年,施氮显著影响了土壤动物类群数、Simpson指数和密度-类群指数;种植模式与玉米生育期的交互作用显著影响了土壤动物平均密度和类群数,同时对密度-类群和丰富度指数也有显著影响;施氮和生育期的交互作用显著影响了Simpson指数、密度-类群指数和Shannon指数.从两年的土壤动物群落去趋势分析结果来看,红壤地土壤动物群落在种植模式和施氮水平之间均没有明显的差异,群落组成的变化与玉米生育期相关.合理施氮条件下的玉米-大豆间作模式有利于提高红壤地土壤中小型动物平均密度和类群数,保持较好的多样性.     

Regulation of exocellular protease in Neurospora crassa: induction and repression under conditions of nitrogen starvation.

Exponential-phase cells of Neurospora crassa require the continued presence of a protein inducer and nitrogen starvation to induce exocellular protease under conditions where protein is the sole nitrogen source. The nature of the protein inducer appears relatively unimportant, since both soluble proteins (e.g., myoglobin) and insoluble proteins (e.g., corn zein) will effect induction. Nonstarved cells of N. crassa appear to have small nitrogen pools, since nitrogen starvation of exponential cells prior to transfer into a medium where protein is the sole nitrogen source effects starvation-time-dependent decreases in protease biosynthesis. Ammonium ion represses protease synthesis, with apparent specificity at low concentrations. The amino acids arginine, tryptophan, and threonine effect repression of protease biosynthesis under conditions of nitrogen starvation. Under conditions of sulfur starvation, the amino acids cysteine, methionine, and cystine repress protease biosynthesis. In carbon-starved cells, all of the above amino acids, plus histidine, isoleucine, leucine, lysine, phenylalanine, and valine, effect repression. Examination of amino acid pools formed when cells are grown on protein as the sole nitrogen source demonstrated that the amino acids which repress protease biosynthesis under conditions where protein is the sole carbon source accumulate in significant amounts during the course of protease induction, with kinetics consonant with the induction process.     

In vivo and in vitro kinetics of nitrogenase.

We measured some of the kinetic parameters of nitrogenase to intact systems of Clostridium pasteurianum and Klebsiella pneumoniae to compare them with the kinetics of the enzyme in vitro. We found that the enzyme showed multiple apparent Km values for acetylene reduction in vivo, as it does in vitro. Carbon monoxide was a noncompetitive inhibitor of acetylene reduction; azide was a noncompetitive inhibitor of acetylene reduction, and nitrogen was a partial inhibitor of acetylene reduction. Cyanide was a noncompetitive inhibitor of acetylene reduction in C. pasteurianum but it was a metabolic poison in K. pneumoniae, in addition to being an inhibitor of nitrogenase. The partial nature of nitrogen inhibition was apparent in assays where both nitrogen and CO were present. Nitrogen did not alter the apparent Ki for CO, nor did the presence of CO enhance the competitive effectiveness of nitrogen. By using recombined nitrogenase fractions, we found that the ability of nitrogen to inhibit hydrogen evolution or acetylene reduction varied with the ratio of protein components. The in vivo inhibition of acetylene reduction by dinitrogen was comparable to that obtained with an excess of the Fe protein in vitro. We conclude that there is an effective excess of the Fe protein available under active growth conditions in vivo.     

Hydrogen bonds between nitrogen donors and the semiquinone in the Qi-site of the bc1 complex

The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (approximately 9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the Nepsilon of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Ndelta in other quinone-processing sites. The experiments with 15N showed that the Nepsilon of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the Nepsilon of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38+/-0.13A. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.     

呼伦贝尔典型退化草原土壤理化与微生物性状

以内蒙古克鲁伦河流域呼伦贝尔典型草原为对象,设置了轻度、中度和重度退化3种类型样地,研究不同程度退化草原的物种组成、地上生物量、土壤理化性状、土壤微生物数量和酶活性,以及微生物生物量的变化.结果表明: 中度退化样地的群落物种丰富度最大,轻度退化样地的地上生物量显著高于重度退化样地.退化样地的土壤水分、养分(有机质、全氮),微生物量碳、氮,以及微生物数量和酶活性显著下降,土壤容重显著增加.退化样地的土壤微生物生物量碳、氮在128～185和5.6～13.6 g·kg^-1,土壤脱氢酶和脲酶活性均与土壤容重呈显著负相关,与土壤全氮、有机质、微生物数量以及微生物生物量碳、氮呈显著正相关,地上生物量与土壤细菌和真菌数量呈不同程度的正相关.     

Effect of nitrogen source on cyanophycin synthesis in Synechocystis sp. strain PCC 6308

Experiments were carried out to examine the effects of nitrogen source on nitrogen incorporation into cyanophycin during nitrogen limitation and repletion, both with or without inhibition of protein synthesis, in cyanobacteria grown on either nitrate or ammonium. The use of nitrate and ammonium, 14N labeled in the growth medium and 15N labeled in the repletion medium, allows the determination of the source of nitrogen in cyanophycin using proton nuclear magnetic resonance spectroscopy. The data suggest that nitrogen from both the breakdown of cellular protein (14N) and directly from the medium (15N) is incorporated into cyanophycin. Nitrogen is incorporated into cyanophycin at different rates and to different extents, depending on the source of nitrogen (ammonium or nitrate) and whether the cells are first starved for nitrogen. These differences appear to be related to the activity of nitrate reductase in cells and to the possible expression of cyanophycin synthetase during nitrogen starvation.     

Change in biochemical composition of amaranth leaves as a result of selection for increased level of amaranthine pigment

The composition and content of secondary compounds produced by the shikimate pathway and the contents of protein and cellulose were determined in leaves of amaranth (Amaranthus tricolor L.) K-99 and the cultivar Valentina raised from it by family selection and enriched in the pigment amaranthine. It was found that intense biosynthesis of amaranthine, tyrosine, and phenylalanine resulted in a decrease in the contents of lignin, protein, and cellulose in leaves of Valentina by comparison with K-99 and in changed the morphological traits: color deepening and a decrease in leaf density. It is concluded that amaranth biosynthesis is related to nitrogen metabolism and amaranthine is an intermediate involved in conversion of nitrogen compounds in the cell.