固氮施氏假单胞菌亚硝酸盐还原酶基因nirS转录特性及功能鉴定

【目的】研究固氮施氏假单胞菌(Pseudomonas stutzeri)A1501亚硝酸盐还原酶结构基因nir S的转录调控机制及其在反硝化过程中的功能。【方法】构建nir S-lac Z融合载体,利用三亲本结合法将其导入野生型A1501,通过β-半乳糖苷酶活性的测定,分析不同供氧状况、不同浓度的硝酸盐、亚硝酸盐对nir S基因表达的影响;同时将该载体导入rpo N突变株中,研究氮代谢调控因子Rpo N对nir S基因转录影响。通过同源重组方法构建nir S突变株,通过生化表型测定明确nir S在反硝化过程中的功能。【结果】启动子活性测定表明,nir S基因厌氧条件下高水平表达,是好氧条件下表达水平的4倍;nir S的表达受硝酸盐诱导,但不受亚硝酸盐的诱导;Rpo N突变株中,nir S的表达活性为野生型的1/4,nir S启动子未发现Rpo N的保守结合位点,表明nir S的表达受Rpo N间接调控。表型测定显示以硝酸盐为电子受体时Δnir S的反硝化能力降低了约20%;以亚硝酸盐为电子受体时Δnir S仅有微弱的反硝化能力,并且nir S的突变使得菌体在反硝化条件下利用亚硝酸盐的能力显著减弱。nir S突变提高了菌体在亚硝酸为电子受体的反硝化条件下的固氮酶活。【结论】A1501中nir S基因的转录受外界氧及硝酸盐的影响,同时受氮代谢Sigma因子Rpo N的调控。nir S在A1501菌反硝化过程中起关键作用,参与了亚硝酸盐的转化。     

Physiological Functions of the COPI Complex in Higher Plants

COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed α-, β-, β′-, γ-, δ-, ε-, and ζ-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions has hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The β′-, γ-, and δ-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of β′-, γ-, and δ-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of β′-COP caused aberrant cell plate formation during cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.     

Isolation and characterization of a <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> strain capable of degrading zearalenone

The worldwide contamination of cereals, oilseeds, and other crops by mycotoxin-producing moulds is a significant problem. Mycotoxins have adverse effects on humans and animals that result in illnesses and economic losses. Reduction or elimination of mycotoxin contamination in food and feed is an important issue. This study aimed to screen soil bacteria for degradation of zearalenone (ZEN). A pure culture of strain CK1 isolated from soil samples showed most capable of degradation of ZEN. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, CK1 was identified as Bacillus licheniformis. Addition of 2 ppm of ZEN in Luria–Bertani (LB) medium, B. licheniformis CK1 decreased 95.8% of ZEN after 36 h of incubation. In ZEN-contaminated corn meal medium, B. licheniformis CK1 decreased more than 98% of ZEN after 36 h of incubation. In addition, B. licheniformis CK1 was non-hemolytic, non-enterotoxin producing, and displayed high levels of extracellular xylanase, cellulase, and protease activities. These findings suggest that B. licheniformis CK1 could be used to reduce the concentrations of ZEN and improve the digestibility of nutrients in feedstuffs simultaneously.     

Structure and mechanism of Escherichia coli glutathionylspermidine amidase belonging to the family of cysteine; histidine-dependent amidohydrolases/peptidases

The bifunctional Escherichia coli glutathionylspermidine synthetase/amidase (GspSA) catalyzes both the synthesis and hydrolysis of Gsp. Its amidase domain (GspA), which catalyzes the hydrolysis of Gsp into glutathione and spermidine, plays an important role in redox sensing and protein S-thiolation. To gain insight of the regulation and catalytic mechanism of and further understand the recycling of the Gsp dimer and Gsp-S-protein adducts, we solved two crystal structures of GspA and GspSA both with the C59A mutation and bound with the substrate, Gsp. In both structures, Cys59, His131, and Glu147 form the catalytic triad, which is similar to other cysteine proteases. Comparison of the GspA_Gsp complex and apo GspSA structures indicates that on binding with Gsp, the side chains of Asn149 and Gln58 of the amidase domain are induced to move closer to the carbonyl oxygen of the cleaved amide bond of Gsp, thereby participating in catalysis. In addition, the helix-loop region of GspA, corresponding to the sequence (30)YSSLDPQEYEDDA(42), involves in regulating the substrate binding. Our previous study indicated that the thiol of Cys59 of GspA is only oxidized to sulfenic acid by H(2)O(2). When comparing the active site of GspA with those of other cysteine proteases, we found that limited space and hydrophobicity of the environment around Cys59 play an important role to inhibit its further oxidation. The structural results presented here not only elucidate the catalytic mechanism and regulation of GspA but also help us to design small molecules to inhibit or probe for the activity of GspA.     

新疆蓝刺头化学成分研究

以新疆蓝刺头(Echinops ritro L.)全草为研究材料,通过硅胶柱色谱,Sephadex LH-20柱色谱,重结晶等技术对新疆蓝刺头化学成分进行分离纯化,通过理化性质分析及1H-NMR,13C-NMR等技术对化合物结构进行鉴定.结果表明,共分离出5个化合物,分别是三萜类化合物蒲公英甾醇乙酰酯(化合物1)、蒲公英甾醇(化合物2)、黄酮苷类化合物金丝桃苷(化合物3)、胡萝卜苷(化合物4)与β-豆甾醇葡萄糖苷(化合物5).其中化合物1,2,5首次从该种植物中分离,化合物3为首次从该属植物中分离.     

宽窄行栽植模式下三倍体毛白杨根系分布特征及其与根系吸水的关系

采用剖面法对宽窄行栽植模式下三倍体毛白杨(triploid Populus tomentosa)的根系分布特征进行了研究;采用管式TDR系统对土壤剖面含水率变化动态进行了连续观测,并据此计算林木根系吸水速率,以探讨土壤含水率、根系分布和根系吸水分布之间的相关关系。研究结果表明:毛白杨的总平均根长密度在林带两侧和不同径向距离处非常接近(P>0.05);但在不同土层间变化很大(P<0.01),其中0-20和60-150 cm土层为根系主要分布区域,其根系所占比例共达86%;不同径阶间的根长密度差异显著(P<0.01),且其比例关系会随空间位置的改变而发生变化。不同栽植方位下,林带东侧毛白杨根系分布的浅层化程度高于西侧,且在径向240-280 cm内其0-0.5 mm的极细根显著多于西侧(P<0.05)。因此,宽窄行栽植模式下,深度和径阶是毛白杨根系分布的主要影响因子,而栽植方位会对其形态构型产生影响。毛白杨根系吸水模式受细根分布的影响,但会随土壤剖面水分有效性分布的变化而变化:当表土层水分有效性增加时,根系吸水主要集中在表土层;当表土层水分有效性降低时,深层土壤根系的吸水贡献率会逐渐增加;当土壤剖面水分条件异质性较高时,根系吸水主要集中在根系密度与水分有效性均较高的区域;当土壤剖面水分分布均匀且不存在水分胁迫时,根系吸水分布与细根分布最为一致。     

Time-dependent botulinum neurotoxin serotype A metalloprotease inhibitors

Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to treat the deadly flaccid paralysis resulting from BoNT intoxication. Among the seven BoNT serotypes, the development of therapeutics to counter BoNT/A is a priority (due to its long half-life in the neuronal cytosol and its ease of production). In this regard, the BoNT/A enzyme light chain (LC) component, a zinc metalloprotease responsible for the intracellular cleavage of synaptosomal-associated protein of 25 kDa, is a desirable target for developing post-BoNT/A intoxication rescue therapeutics. In an earlier study, we reported the high throughput screening of a library containing 70,000 compounds, and uncovered a novel class of benzimidazole acrylonitrile-based BoNT/A LC inhibitors. Herein, we present both structure–activity relationships and a proposed mechanism of action for this novel inhibitor chemotype.