The role of the beta-subunit of RNA-polymerase in specific recognition of promoters

The complex formation of T7 DNA with RNA polymerase from E. coli B/r WU-36-10-11-12 (E. coli W12) and its rifampicin resistant mutant with highly pleiotropic effect--rpoB409 was studied. As shown earlier rpoB409 RNA polymerase differs from the normal enzyme by the selection of RNA synthesis from early promoters of DNA from T7 and T4 phages. The change in the RNA specificity synthesis due to rpoB409 mutation was shown to occur at the stage of RNA polymerase interaction with DNA before open promoter complex formation. The data obtained together with the fact of highly pleiotropic effect of the rpoB409 mutation indicate that RNA polymerase beta-subunit takes part in specific recognition of promoters.     

利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*

目的: 利用CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) 系统构建玉米中心蛋白(Centrin)的表达载体,经转化后分析其对玉米生长发育的影响。方法: 针对ZmCen基因的第一个外显子设计sgRNA,将其连入pOMS01-Cas9-ZmCen-sgRNA表达载体,转化农杆菌GV3101后,侵染玉米自交系材料B104的愈伤组织,经继代、诱导、分化成苗,筛选出转基因后代。对T₀代和T₁代基因组DNA进行PCR验证、测序及表型分析。结果: 成功构建ZmCen的表达载体。侵染农杆菌后,PCR测序显示,T₀ 代和T₁ 代突变率分别为 20.13% 和 64.52%,其中T₁ 代的纯合缺失突变率为5%。序列分析表明,ZmCen基因的编辑靶点附近发生了碱基的替换、插入或缺失。经与野生型表型比对发现,ZmCen 突变体T₁代植株出现发育缓慢且雄花序不完全发育表型,纯合突变体植株雄花序则完全不发育。结论: 通过 CRISPR/Cas9技术成功地对玉米ZmCen基因进行了编辑,ZmCen突变体的获得为玉米雄性器官发育相关基因的研究奠定了基础。     

利用CRISPR/Cas9鉴定玉米发育相关基因ZmCen*

目的: 利用CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) 系统构建玉米中心蛋白(Centrin)的表达载体,经转化后分析其对玉米生长发育的影响。方法: 针对ZmCen基因的第一个外显子设计sgRNA,将其连入pOMS01-Cas9-ZmCen-sgRNA表达载体,转化农杆菌GV3101后,侵染玉米自交系材料B104的愈伤组织,经继代、诱导、分化成苗,筛选出转基因后代。对T₀代和T₁代基因组DNA进行PCR验证、测序及表型分析。结果: 成功构建ZmCen的表达载体。侵染农杆菌后,PCR测序显示,T₀ 代和T₁ 代突变率分别为 20.13% 和 64.52%,其中T₁ 代的纯合缺失突变率为5%。序列分析表明,ZmCen基因的编辑靶点附近发生了碱基的替换、插入或缺失。经与野生型表型比对发现,ZmCen 突变体T₁代植株出现发育缓慢且雄花序不完全发育表型,纯合突变体植株雄花序则完全不发育。结论: 通过 CRISPR/Cas9技术成功地对玉米ZmCen基因进行了编辑,ZmCen突变体的获得为玉米雄性器官发育相关基因的研究奠定了基础。     

Antimicrobial properties of berberines alkaloids in Coptis chinensis Franch by microcalorimetry

The growth thermogenic curves of Escherichia coli (E. coli) affected by berberine, coptisine and palmatine were determined quantitatively by microcalorimetry. The power–time curves of E. coli with and without the three berberines alkaloids (BA) were acquired, meanwhile the extent and duration of inhibitory effects on the metabolism were evaluated by growth rate constant (k), half-inhibitory ratio (IC₅₀), peak time of maximum heat-output power (t_p), total heat-production (Q_t) and so on. The inhibitory effects of BA on E. coli revealed that the sequence of their antimicrobial activity was berberine > coptisine > palmatine. The functional groups methylenedioxy at C2 and C3 on phenyl ring improve antimicrobial activity more remarkably than methoxyl at C2 and C3 on phenyl ring. However, the antimicrobial activity does not vary significantly with methylenedioxy or methoxyl at C9 and C10 on phenyl ring.     

Spectroscopic study and G-quadruplex DNA binding affinity of two bioactive papaverine-derived ligands

The interactions of G-quadruplex DNA with two oxidation products of papaverine, 6a,12a-diazadibenzo-[a,g]fluorenylium derivative (1) and 2,3,9,10-tetramethoxy-12-oxo-12H-indolo[2,1-a]isoquinolinium cation (2) were investigated. Their activity against telomerase was assessed using the conventional telomeric repeat amplification protocol (TRAP) assay. Effect of TRAP buffer and oligonucleotide length on the DNA-binding affinity of 1 and 2 were also studied. Three quadruplex-forming oligonucleotides with human telomeric sequence: dG₃(T₂AG₃)₃ (htel21), dAG₃(T₂AG₃)₃ (htel22), and d(T₂AG₃)₄ (htel24) were used in these investigations. Both ligands were capable of interacting with G4 DNA with binding stoichiometry indicating that two ligand molecules bind to G-quadruplex, which agrees with the binding model of end-stacking on terminal G-tetrads. Circular dichroism spectra revealed that preferences of quadruplex-forming oligonucleotide to adopt a particular topological structure may be also affected by the external ligand that binds to quadruplex. Telomerase activity was suppressed at very low ligand 1 and ligand 2 concentrations with an appreciable selectivity comparing with inhibition of Taq polymerase.     

RNA polymerase of a rifampicin-resistant mutant of Escherichia coli has an altered selectivity to phage T7 DNA promoters

The formation of complexes of RNA polymerases from E. coli W12 and its rpoB409 rifampicin resistant mutant with A1 and D promoters of T7 delta D111 DNA was studied by an abortive RNA synthesis technique. The mutation was shown to affect RNA synthesis initiation at these two promotors differentially so that the efficiency of D promotor utilization is enhanced but the use of A1 promotor is unchanged. The mutation does not interfere with the affinity of the enzyme for both initiating substrates. The results show that the change in RNA polymerase beta-subunit structure has a differential effect on the enzyme interaction with different promotors. The necessity of a classificatory approach to structure-functional analysis of promotors was proposed.     

An expeditious synthesis of 4-hydroxy-2E-nonenal (4-HNE), its dimethyl acetal and of related compounds

The facile one step synthesis of 4-hydroxy-2(E)-nonenal and its dimethyl acetal via a cross-metathesis reaction between commercially available octen-3-ol and acrolein or its dimethyl acetal is reported. The method was extended to the synthesis of C₆ and C₁₂ 4-hydroxy-2(E)-enals, their dimethyl acetal and of the 4-hydroxy-2(E)-nonenoic acid (4-HNA).     

Seasonal Thyroid Cycle of the Soft-shelled Turtle, Lissemys p. punctata (Bonnoterre)

The current study was undertaken to ascertain the seasonal influence of thyroid activity in female soft-shelled turtles, Lissemys p. punctata. Thyroid gland was studied month-wise throughout the year from relative gland weight, histology, epithelial height, glandular peroxidase activity, and RIA of T₃ and T₄ levels from blood serum and the thyroid gland. The values of all the parameters, except those of T₃ and T₄, were higher during March through May, decreased from June through August and began to rise thereafter (September through February). Whereas T₃ and T₄ levels were highest in May, lower during June to November and began to rise thereafter. The difference in the peaks between T₃ and T₄ levels and other parameters have been explained. The findings suggest that thyroid activity of Lissemys turtles varies seasonally and that seasonal factors like temperature play an important role in influencing thyroid activity in soft-shelled turtles.     

Increased enantioselectivity in the enzymatic hydrolysis of amino acid esters in the ionic liquid 1-butyl-3-methyl-imidazolium tetrafluoroborate

The initial rate and enantioselectivity of enzymatic asymmetric hydrolysis of amino acid esters were examined in methylimidazolium-based ionic liquids with anions including tetrafluoroborate, chloride, bromide and bisulfate and in typical organic solvents. Papain displayed much higher enantioselectivity but lower activity in phosphate buffer solution of 1-butyl-3-methylimidazolium tetrafluoroborate BMIM·BF₄ than in other media tested (i.e. E=100, V ₀=0.21 mM min^-1 in BMIM·BF₄, E=2, V ₀=0.43 mM min^-1 in phosphate buffer, E=14-92, V ₀=0.22-0.25 mM min^-1 in organic solvents for D,L-phenylglycine methyl ester). The influence of BMIM·BF₄ on enzyme activity and enantioselectivity also varied with the substrate and the enzyme used. All of the enzymes assayed showed no activity or low enantioselectivity in the ILs with anions including chloride, bromide and bisulfate.     

蘑菇渣堆肥对华北落叶松移植容器苗生长和营养积累的影响

为缓解草炭大量使用带来的环境问题,在华北落叶松容器育苗过程中添加蘑菇渣堆肥以替代草炭,添加蘑菇渣堆肥体积比例分别为0%(T₀)、15%(T₁)、18.75%(T₂)、25%(T₃)、37.50%(T₄)、50%(T₅)、56.25%(T₆)、60%(T₇),研究不同配比下基质的理化性质以及对华北落叶松1年生移植容器苗生长和营养积累的影响.结果表明: 当蘑菇渣堆肥替代草炭的比例≤50%时,苗木苗高、地径和生物量与常用草炭处理无显著差异,且处理T₂、T₄、T₅苗木体内养分浓度显著高于对照,基质pH值处于微酸性或中性范围,适宜苗木生长;当蘑菇渣添加比例＞50%时,基质pH值偏碱性,苗木生长受到不利影响,苗木质量下降.当蘑菇渣添加比例为15%时,苗木质量最好,苗高、地径和单株生物量达到最大.因此,蘑菇渣替代草炭培育华北落叶松移植容器苗是完全可行的,替代比例最高可达到50%,当添加比例为15%时,可培育出高质量的苗木.     

残次苹果发酵产物对连作土壤环境及‘平邑甜茶’幼苗生长的影响

在盆栽条件下,研究残次苹果发酵产物对连作土壤环境及平邑甜茶幼苗生长的影响,为减轻苹果连作障碍提供理论依据。试验以连作土壤为对照(CK),设置溴甲烷灭菌连作土壤(T₁)、连作土壤施加苹果发酵产物(T₂)、连作土壤施加灭菌苹果发酵产物(T₃)处理,测定了土壤和平邑甜茶幼苗的相关指标。结果表明: T₁、T₂、T₃能显著促进平邑甜茶幼苗的生长,其中以T₁和T₂的效果较好,其根系呼吸速率、株高、地径、鲜质量、干质量分别比CK提高了107.3%、50.6%、42.4%、171.7%、225.3%和104.4%、50.6%、42.3%、171.8%、225.5%。T₂和T₃提高了连作土壤中养分转化相关酶的活性,过氧化氢酶、脲酶、中性磷酸酶和蔗糖酶活性分别比CK提高了44.5%、169.5%、23.4%、169.3%和23.7%、72.6%、1.5%、121.5%;T₁处理的过氧化氢酶和蔗糖酶活性与CK无显著差异,脲酶和中性磷酸酶活性分别降低了40.8%和41.6%。T₂土壤铵态氮、硝态氮、速效磷和速效钾含量分别较CK提高了18.6%、50.6%、14.0%和36.7%;T₃只提高了速效氮的含量,铵态氮和硝态氮含量分别提高了7.0%和23.6%;T₁与CK相比速效养分含量有所降低。T₁和T₂显著降低了土壤放线菌和真菌数量,增加了细菌数量;T₃处理的细菌、放线菌和真菌数量均较CK显著降低。实时荧光定量PCR分析发现,T₁、T₂和T₃处理的层出镰孢菌、串珠镰孢菌、腐皮镰孢菌和尖孢镰孢菌基因拷贝数均较CK有不同程度的降低。表明苹果发酵产物处理能抑制连作土壤病原菌,改善土壤环境,促进平邑甜茶幼苗生长,可作为一种减轻苹果连作障碍的方法。     

Role of the beta-subunits of Escherichia coli RNA-polymerase in the regulation of gene activity

The DNA-dependent RNA-polymerase from E. coli B/r and its rif-r mutant rpoB409 with pleiotropic effect has been studied. It was shown, that multiple forms of promotor sites in T4- and T7-DNA "early" regions are recognized with different efficiences by RNA-polymerases from E. coli B/r and rpoB409. The rif-r rpoB409 mutation has been reported to affect the beta-subunit. Thus, the present data indicates that the selection of promoter sites can be controlled by the beta-subunit of RNA-polymerase.     

毛乌素沙地油蒿-杨柴灌丛生态系统蒸散组分分配及其影响因子

蒸散发(ET)是生态系统水分循环和能量流动的重要组成部分,准确估算ET及其各组分,对认识生态生理过程对水分平衡和植物水分利用策略的影响具有重要意义。本研究于2019年5月20日至9月15日,利用涡度相关技术和微型蒸渗仪对毛乌素沙地油蒿-杨柴灌丛生态系统ET、蒸发(E)和蒸腾(T)进行测定和估算,量化了油蒿-杨柴灌丛生态系统ET组分,并分析ET及其组分的季节特征及影响因素。结果表明: T为毛乌素沙地油蒿-杨柴灌丛生态系统生长季ET的主要组分,T/ET为53.1%。T/ET值随降水减少而升高,E/ET值随降水减少而减少,蒸散组分分配主要受降水调控。在季节尺度上,E与10 cm深处土壤含水量(SWC₁₀)和太阳净辐射(R_n)呈显著正相关,其中,SWC₁₀是E的主要影响因素;T随R_n和叶面积指数(LAI)的升高而升高,随30 cm处土壤含水量(SWC₃₀)的升高呈先升高后降低的单峰趋势,受到SWC₃₀、R_n和LAI的共同影响;水分是ET的主要影响因素。生长季蒸散/降水量(ET/P)为109.2%,5月ET/P为250.5%,表明生长季初期ET耗水部分来自非生长季降水。     

Design and synthesis of novel imidazole derivatives as potent inhibitors of allene oxide synthase(CYP74)

Allene oxide synthase (AOS) is a key enzyme in the oxylipin pathway in plants leading to jasmonic acid and other jasmonates (JAs), important signal mediators of defense signal networks in plants. AOS uses hydroperoxylinolenic acid as an oxygen donor as well as the substrate, thus the biochemical conversion of 13(S)-hydroperoxylinolenic acid to allene oxide can proceed in the absence of oxygen and NADPH. We have designed the synthesized of a series of novel imidazole derivatives and tested them in a bioassay as AOS inhibitors using a purified recombinant AOS enzyme isolated from Arabidopsis and expressed in E. coli. Among the derivatives prepared, heptyl 8-[1-(2,4-dichlorophenyl)-2-imidazolylethoxy]octanoate (k) was found to be the most potent inhibitor, with an IC₅₀ of 10±5 nM, which is 250,000-fold and 1,000,000-fold more potent than the known AOS inhibitors, acetylsalicyclic acid (2.5 mM) and ketoconazole (10 mM), respectively.     

Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.     

施用污泥等废料对稀土矿废弃地土壤中麻疯树生长和元素吸收的影响

城市污泥等废料可以用于调理稀土矿废弃地土壤,而能源植物麻疯树有望成为稀土矿废弃地的先锋植物。本研究通过向稀土矿废弃地土壤中添加污泥(T₁)、污泥+蔗渣(T₂)、污泥+蔗渣+钝化剂(T₃),并以矿区土壤为对照(CK),研究盆栽条件下各处理对麻疯树生长和元素吸收的影响。结果表明: 与CK相比,T₁仅显著提高麻疯树株高,T₂、T₃显著提高麻疯树株高、地径和生物量,其中总生物量提高184.7%以上;3个处理均显著促进麻疯树对N、P、K、Cu的吸收;T₁、T₂显著提高基质中可交换态Zn、Cd、Ni比例,T₃则相反,并显著降低Zn、Cd、Ni在基质中的迁移系数和活性系数,抑制麻疯树对Zn、Pb、Cd、Ni的吸收,抑制率达36.1%以上。隶属函数综合评价结果表明,各处理对麻疯树生长的促进顺序为T₂>T₃>T₁>CK,对麻疯树吸收Cu、Zn、Pb、Cd、Ni的抑制顺序为T₃>CK>T₂>T₁。混施污泥和蔗渣显著促进麻疯树生长和元素吸收,进一步加入钝化剂则显著抑制麻疯树对重金属的吸收,但不影响麻疯树生长。     

酸性土施用钙肥对花生产量和品质及相关代谢酶活性的影响

以花生(Arachis hypogaea)品种‘花育22号’为研究材料, 2013年在威海文登市、2014年在日照三庄镇的丘陵砂壤土上进行试验, 研究增施钙肥对酸性土花生的产量、品质的影响, 以及相关碳、氮代谢酶活性差异, 探讨酸性土花生钙肥最佳用量。试验设3个钙肥处理, 分别为每667 m²施CaO 0 kg (T₀)、14 kg (T₁)、28 kg (T₂)。结果表明: 酸性土增施钙肥显著增加了花生的荚果产量, 两个试验点T₁处理平均增产26.92%, T₂处理平均增产21.65%。增产原因是增施钙肥显著增加了花生单株结果数, 提高了双仁果率, 从而增加了单株荚果产量, 同时增加了籽仁的饱满度而显著提高了出仁率。钙肥处理均显著提高了花生籽仁蛋白质和脂肪含量, 提高了赖氨酸、总氨基酸含量和油酸/亚油酸(O/L)比值。酸性土增施钙肥显著提高了花生叶片的谷氨酰胺合成酶(GS)、谷氨酸合成酶(GOGAT)、谷草转氨酶(GOT)和谷丙转氨酶(GPT)活性, 其中T₁处理的GS活性显著高于T₂处理。钙肥处理显著提高了花生生育前期的叶片磷酸烯醇式丙酮酸羧化酶(PEPCase)、蔗糖合成酶(SS)和蔗糖磷酸合成酶(SPS)活性, 而生育后期的活性低于不施钙肥处理。不同钙肥施用量相比, 每667 m²施14 kg CaO的经济效益最好, 其产量最高, 品质最优。