藤黄绿脓菌素的自诱导及假单胞菌M18抗生物质代谢相关性初步分析

假单胞菌M18的生防功能归功于其分泌吩嗪-1-羧酸和藤黄绿脓菌素。为了研究抗生物质合成代谢相关性及调控机制,分别构建了两种抗生物质合成基因簇插入突变株M18T和M18Z1。用翻译融合表达载体pMEAZ(pltA′-′lacZ)分别转化野生株和突变株M18T、发酵培养并测定β-半乳糖苷酶活性,结果显示,添加藤黄绿脓菌素使突变株M18T(pMEAZ)的β-半乳糖苷酶活性比野生株M18(pMEAZ)增加约6倍,表明藤黄绿脓菌素对自身基因簇具正向自诱导作用。抗生物质的测定结果显示,突变株M18T无藤黄绿脓菌素合成,而吩嗪-1-羧酸的合成量与野生株相同;突变株M18Z1与野生株相比,吩嗪-1-羧酸明显减少,藤黄绿脓菌素却显著提高。过量的吩嗪-1-羧酸又抑制藤黄绿脓菌素的合成。表明,假单胞菌M18中独有的代谢相关方式为:藤黄绿脓菌素不影响吩嗪-1-羧酸,但吩嗪-1-羧酸负调控藤黄绿脓菌素。     

绿针假单胞菌GP72中aurI/aurR系统调控功能

【背景】绿针假单胞菌(Pseudomonas chlororaphis) GP72是一株可生产吩嗪类抗生素吩嗪-1-羧酸(PCA)和2-羟基吩嗪(2-OH-PHZ)的生防根际促生菌。基因组比对发现GP72菌中存在aurI/aurR双元调控系统。【目的】研究该系统对GP72中吩嗪类物质的调控作用。【方法】将aurI基因在大肠杆菌中异源表达,用紫色杆菌CV026和根癌农杆菌NTL4做显色实验。构建基因敲除菌株和回补菌株,发酵测量突变株的生长曲线与总吩嗪产量。构建转录融合质粒,测定吩嗪合成基因启动子的转录水平。【结果】显色实验显示,aurI能产生多种信号分子,使CV026显紫色、NTL4显蓝色。分别单独敲除aurI和aurR基因,同时敲除aurI/aurR基因,吩嗪产量均会升高,而回补菌株吩嗪产量降为野生型水平。β-半乳糖苷酶活性测定结果显示,突变株的酶活比野生型高。【结论】aurI/aurR负调控GP72的吩嗪合成,通过抑制吩嗪合成启动子的转录而影响吩嗪类物质的产量。     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素 (Pyoluteorin ,Plt )和吩嗪 1 羧酸 (Phenazine 1 carboxylicacid ,PCA)并抑制多种植物病菌的生长。从M18中克隆双基因调控系统gacS gacA的组成基因gacA ,并构建了该基因抗性插入突变株M18G。在KMB培养基中 ,M18G合成Plt的能力受到完全抑制 ,而PCA的积累约比野生型提高 31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明 ,在M18G菌株中 ,Plt合成的抑制并不引起PCA的过量积累 ,PCA的过量积累也不引起Plt合成的抑制。由此推测 ,gacA在基因表达的水平上全局性地执行着调控功能     

Pip介导铜绿假单胞菌吩嗪基因簇phz2的表达

[目的]为了确定铜绿假单胞菌调控因子Pip对两个不同吩嗪合成基因簇(phz1和phz2)的具体调控方式与可能的调控机制.[方法]根据基因比对结果,采用同源重组技术构建Pip调控因子缺失突变株PA-PG以及克隆ip基因作互补分析;再以已构建的吩嗪基因簇缺失突变株PA-Z1G和PA-Z2K为受体菌,构建突变株PA-PD-Z1G和PA-PG-Z2K,测定并比较野生株及相关突变株的吩嗪-1-羧酸和绿脓菌素的合成量,推定Pip对两个不同吩嗪合成基因簇的调控方式.[结果]在GA培养基中,突变株PA-PG的吩嗪-1-羧酸和绿脓菌素都比野生型明显减少;互补分析显示,突变株PA-PG的吩嗪-1-羧酸和绿脓菌素都显著提高并恢复到野生株PAO1水平;突变株PA-Z1G的吩嗪-1-羧酸和绿脓菌素合成量因Pip缺失而显著减少;而突变株PA-Z2K的吩嗪-1-羧酸和绿脓菌素合成量在Pip缺失后仍保持不变.[结论]初步推定,转录调控因子Pip对铜绿假单胞菌吩嗪合成代谢的确具有促进作用;Pip通过正向调控吩嗪基因簇phz2的合成功能实现对吩嗪合成代谢的调控.     

GacA对假单胞菌株M18两个phz基因簇和菌群传感的调控

摘要:【目的】假单胞菌株M18（Pseudomonas sp. M18）是从甜瓜根际土壤中分离获得的一株对多种植物病原菌具有显著拮抗作用的菌株,在菌群传感（quorum sensing）系统的调控下,能分泌吩嗪-1-羧酸（PCA）以及多种吩嗪（phz）类衍生物的抗真菌物质。全局性因子GacA是M18菌株吩嗪类物质的合成与菌群传感系统的重要调控因子,本文将就GacA对上述两者的调控做进一步研究。【方法】PCR基因扩增和测序研究M18菌株中PCA合成基因簇,运用RT-PCR及构建phzA-lacZ转录融合手段     

RNA聚合酶σ^38亚基缺失对假单胞菌抗生物质合成代谢的影响

设计引物从假单胞菌M18基因组DNA中扩增并获得rpoS基因的378bp保守区段。以此为探针,从假单胞菌基因组文库中克隆了包括rpoS基因全序列及其相邻序列的3·1kbEcoRⅠ-XhoⅠ片段。通过抗性基因(抗庆大霉素基因)的定点插入构建了σ38亚基缺失突变株M18S。HPLC检测结果显示,σ38亚基缺失引起该菌株的抗生物质合成代谢的显著变化。与野生株相比,缺失突变株的吩嗪-1-羧酸在PPM和KMB中2种培养基中合成量由58μg/mL和10·2μg/mL分别减少到20·4μg/mL和0μg/mL;而缺失突变株的藤黄绿脓菌素则相反,在PPM和KMB两种培养基中合成量由0·5μg/mL和20·5μg/mL分别提高到75·4μg/mL和185·6μg/mL。表明σ38亚基可区别性调控假单胞菌M18的抗生物质合成代谢。rpoS基因的互补实验和两种抗生素基因与β-半乳糖苷酶基因的翻译融合表达实验进一步验证了上述的结果:σ38亚基正调控吩嗪-1-羧酸的表达,而负调控藤黄绿脓菌素的表达。     

转录调节因子σ~(38)介导铜绿假单胞菌绿脓菌素合成代谢调控

【目的】为了进一步鉴定铜绿假单胞菌转录调控因子σ~(38)对2个拷贝吩嗪合成基因簇(phz A1-G1和phz A2-G2)的具体调控方式并推定介导绿脓菌素合成代谢的可能调控机制。【方法】根据铜绿假单胞菌基因组信息,利用同源重组原理构建rpo S基因缺失突变株Δrpo S以及克隆全长rpo S基因作互补分析;再以单一吩嗪基因簇缺失突变株Δphz1和Δphz2为出发菌株,分别构建rpo S缺失突变株Δrpo Sphz1和rpo S插入突变株Δrpo Sphz2,测定并比较野生株及相关突变株的绿脓菌素合成量,初步推定σ~(38)因子对2个不同吩嗪基因簇表达的调控方式。【结果】在GA培养基中,突变株Δrpo S的绿脓菌素合成量比野生株显著增加;互补分析证实,σ~(38)可使突变株Δrpo S的绿脓菌素降低并接近野生株PAO1水平;与对照株Δphz1相比,突变株Δrpo Sphz1的绿脓菌素合成量因σ~(38)因子缺失而显著减少;而与对照株Δphz2相比,突变株Δrpo Sphz2的绿脓菌素合成量因σ~(38)因子缺失显著增加。【结论】转录调控因子σ~(38)对铜绿假单胞菌绿脓菌素的合成代谢的确具一定的负调控作用;结合已报道的研究结果,初步推定:σ~(38)因子通过负调控吩嗪基因簇phz1,正调控吩嗪基因簇phz2的表达实现对绿脓菌素合成代谢的调控。     

RNA聚合酶σ38亚基缺失对假单胞菌抗生物质合成代谢的影响

设计引物从假单胞菌M18基因组DNA中扩增并获得rpoS基因的378bp保守区段。以此为探针,从假单胞菌基因组文库中克隆了包括rpoS基因全序列及其相邻序列的3.1kb EcoRⅠ_XhoⅠ片段。通过抗性基因（抗庆大霉素基因）的定点插入构建了σ38亚基缺失突变株M18S。HPLC检测结果显示,σ38亚基缺失引起该菌株的抗生物质合成代谢的显著变化。与野生株相比,缺失突变株的吩嗪_1_羧酸在PPM和KMB中2种培养基中合成量由58μg/mL和10.2μg/mL分别减少到20.4μg/mL和0μg/mL;而缺失突变株的藤黄绿脓菌素则相反,在PPM和KMB两种培养基中合成量由0.5μg/mL和20.5μg/mL分别提高到75.4μg/mL和185.6μg/mL。表明σ38亚基可区别性调控假单胞菌M18的抗生物质合成代谢。rpoS基因的互补实验和两种抗生素基因与β_半乳糖苷酶基因的翻译融合表达实验进一步验证了上述的结果: σ38亚基正调控吩嗪_1_羧酸的表达,而负调控藤黄绿脓菌素的表达。     

绿针假单胞菌HT66中ompR基因的功能

【背景】植物根部存在大量对植物生长有促进或对病原菌有拮抗作用的有益细菌,是当前农业微生物研究的热点之一。其中,绿针假单胞菌HT66是一株可高效合成吩嗪-1-甲酰胺(PCN)的环境友好型生防菌株。【目的】探究在绿针假单胞菌HT66中ompR基因的生理功能,以及其对菌株生防作用的影响。【方法】通过基因无痕敲除的方法构建HT66菌株的ompR基因缺失突变株,对比研究突变株与野生株在生长速率、渗透压感应、生物膜的合成、pH耐受性、群集运动和PCN产量的变化。【结果】与野生株相比,ompR基因缺失突变株的细胞生物量微量减少,生物膜的合成减少31.5%,群集运动以及对渗透压和pH的耐受性明显下降,但是其PCN产量较野生株提高了57.8%。【结论】在HT66菌株中,ompR基因对其运动性、环境耐受性和生理生防功能均有一定程度的调控作用。本研究丰富了绿针假单胞菌的代谢通路,此报道将对后续PCN合成机制的研究和应用提供一定的理论依据。     

假单胞菌gacA插入突变对藤黄绿脓菌素和吩嗪-1-羧酸合成代谢的差异性调控

假单胞菌株M18分泌藤黄绿脓菌素(Pyoluteorin,Plt )和吩嗪1羧酸（Phenazine1carboxylic acid, PCA）并抑制多种植物病菌的生长。 从M18中克隆双基因调控系统gacS/gacA的组成基因gacA,并构建了该基因抗性插入突变株M18G。在KMB培养基中,M18G合成Plt的能力受到完全抑制,而PCA的积累约比野生型提高31倍左右。Plt合成基因簇突变株M18T和在M18G基础上构建的PCA合成基因簇突变株M18GA的Plt和PCA合成的动力学变化表明,在M18G菌株中,Plt合成的抑制并不引起PCA的过量积累,PCA的过量积累也不引起Plt合成的抑制。由此推测,gacA在基因表达的水平上全局性地执行着调控功能。     

酚酸类物质对三七幼苗的化感影响

该文研究了不同浓度的阿魏酸、对香豆酸、丁香酸、对羟基苯甲酸、香草酸5种酚酸类物质对三七幼苗生长和生理的影响。结果表明:处理后,三七幼苗的苗高、根长、可溶性蛋白质含量、根系活力、CAT以及POD活性均有所降低。其中,阿魏酸各处理组幼苗的苗高及POD活性均显著降低,50、100 mg·L~(-1)的对香豆酸以及100 mg·L~(-1)的香草酸处理组幼苗苗高也分别比对照显著降低16.19%、16.67%和29.29%;对香豆酸、丁香酸以及对羟基苯甲酸各处理组幼苗根长均显著低于对照;香草酸处理组幼苗的根系活力也显著低于对照,且幼苗的CAT活性在10、50、100 mg·L~(-1)丁香酸、对羟基苯甲酸以及香草酸处理下也达到了显著降低水平。此外,1 mg·L~(-1)阿魏酸以及100 mg·L~(-1)香草酸处理组幼苗的叶绿素含量也均显著降低;中高浓度的阿魏酸、对香豆酸、丁香酸、对羟基苯甲酸增加了三七幼苗的MDA含量,而香草酸在0.1、1、10、100 mg·L~(-1)浓度下显著降低幼苗的MDA含量;丁香酸、香草酸、对羟基苯甲酸以及中高浓度的对香豆酸增加了三七幼苗的SOD活性,且香草酸各处理组均达到了显著性水平。综上结果表明,5种酚酸类物质对三七幼苗均具有一定的化感抑制作用,但各酚酸物质的作用方式及强度并不完全一致,阿魏酸的化感影响较大,这为进一步研究三七的化感自毒作用提供了一定的理论参考。     

Effects of flaxseed,raw soybeans and calcium salts of fatty acids on apparent total tract digestibility,energy balance and milk fatty acid profile of transition cows

Oilseeds offer some protection to the access of ruminal microorganisms and may be an alternative to calcium salts of fatty acids (FA), which are not fully inert in the ruminal environment. This study aimed to evaluate the effects of different sources of FA supplementation on apparent total tract nutrient digestibility, milk yield and composition, and energy balance (EB) of cows during the transition period and early lactation. We compared diets rich in C18:2 and C18:3 FA. Multiparous Holstein cows were randomly assigned to receive one of the four diets: control (n=11); whole flaxseed (WF, n=10), 60 and 80 g/kg (diet dry matter (DM) basis) of WF during the prepartum and postpartum periods, respectively; whole raw soybeans (WS, n=10), 120 and 160 g/kg (diet DM basis) of WS during the prepartum and postpartum periods, respectively; and calcium salts of unsaturated fatty acids (CSFA, n=11), 24 and 32 g/kg (diet DM basis) of CSFA during the prepartum and postpartum periods, respectively. Dry cows fed WF had higher DM and net energy of lactation (NE_L) intake than those fed WS or CSFA. The FA supplementation did not alter DM and NDF apparent total tract digestibility, dry cows fed WF exhibited greater NDF total tract digestion than cows fed WS or CSFA. Feeding WS instead of CSFA did not alter NE_L intake and total tract digestion of nutrients, but increased milk fat yield and concentration. Calculated efficiency of milk yield was not altered by diets. FA supplementation increased EB during the postpartum period. Experimental diets increased long-chain FA (saturated and unsaturated FA) in milk. In addition, cows fed WS and CSFA had higher C18:1 trans-11 FA and C18:2 cis, and lower C18:3 FA in milk than those fed WF. Furthermore, cows fed CSFA had higher C18:1 trans-11 and cis-9, trans-11 FA than cows fed WS. Although supplemental C18:2 and C18:3 FA did not influence the milk yield of cows, they positively affected EB and increased unsaturated long-chain FA in milk fat.     

Identification of glutamine chain lengths of endogenous Folypoly-γ-glytamates in rat tissues

A simplified procedure for the determination of the glutamate chain lengths of endogenous tissue folate is described.Natural pteroylpoly-γ-glutamates in tissue extracts, irrespective of their one-carbon moiety, were converted by a reductive cleavage procedure to a homologous series of p-aminobenzoylpoly-γ-glutamates, differing only in glutamate chain length. Desalting and concentration of the extracts were achieved by absorbing the derivatives on active charcoal followed by their elution with ethanol: ammonia. Aminobenzoylpolyglutamates were separated by DEAE- cellulose chromatography and quantitated by a colorimetric procedure for primary aromatic amines.The major endogenous folates in rat liver and kidney were pteroylpentaglutamate derivatives with smaller amounts of pteroyltetra- and hexaglutamate.     

氨基酸L-和D-异构体对离体小麦胚植株生长的影响

5种氨基酸L-和D-异构体对离体小麦胚植株生长的影响结果表明,在1~7 mmol.L-1浓度范围内,脯氨酸异构体均不抑制植株生长;缬氨酸和蛋氨酸的L-型严重抑制生长,D-型不抑制或轻微抑制;丙氨酸则相反,L-型无抑制作用,D-型是严重抑制类型;丝氨酸的L-型轻微抑制生长,D-型严重抑制生长。生长结果也表明,DL-异构体的抑制作用介于D-型和L-型之间。     

Separation and simultaneous determination of nalidixic acid, hydroxynalidixic acid and carboxynalidixic acid in serum and urine by micellar electrokinetic capillary chromatography

Separation in capillary electrophoresis is governed by various factors, including buffer type, buffer concentration, pH, temperature, voltage and micelles. Through proper adjustment of these parameters, nalidixic acid and its two major metabolites, 7-hydroxynalidixic and 7-carboxynalidixic, could be separated by micellar electrokinetic capillary chromatography using an electrophoretic electrolyte consisting of 50 mM borate buffer (pH 9) containing 25 mM sodium dodecyl sulphate and 10% acetonitrile. A linear relationship between concentration and peak area for each compound was obtained in the concentration range 0.15–100 μg ml⁻¹, with a correlation coefficient greater than 0.999 and detection limits in the 0.2–0.7 ng ml⁻¹ range. Intra- and inter-day precision values of about 0.8–1.2% RSD (n=11) and 1.3–2.0% RSD (n=30), respectively, were obtained. The method has been applied to the analysis of nalidixic acid and its two major metabolites in serum and urine with limits of sensitivity lower than 0.8 ng ml⁻¹.     

氨基酸分析仪测定饲料添加剂——叶酸的方法

用６ｍｏｌ／Ｌ盐酸于１１０℃条件下水解饲料添加剂——叶酸,使之游离出谷氨酸,用氢氧化钠中和调节ｐＨ到２,氨基酸分析仪测定谷氨酸含量,经与标准叶酸水解样品比较,计算出叶酸的纯度。该方法重现性好,变异系数ＣＶ＝０．０８％,平均回收率为９８．３４％,浓度与峰面积呈线性相关,相关系数ｒ＝０．９９８７,可随氨基酸分析同时进行,不需改变任何分析条件。     

Characterization of Transgenic Rice Plants Expressing an Arabidopsis FAD7

Fatty acid -3 desaturase (FAD) is the key enzyme catalyzing the formation of trienoic fatty acids. We utilized an Arabidopsis FAD7 gene and the seven independent transgenic rice plants harbouring 1 to 3 copies of this gene were generated. The expression of FAD7 mRNA was different among independent transgenic lines regardless of the copy number. The total linolenic acid (18:3) contents reduced by about 7 – 32 % in transgenic rice plants but the linoleic acid (18:2) content increased accordingly. With or without wounding treatments, the jasmonate content was higher in transgenic lines than in wild-type rice plant. The transgenic lines overproducing jasmonate also showed increased expression of PR1b mRNA and allene oxide synthase inresponse to wounding.     

Determination of aromatic metabolites in ruminant urine by high-performance liquid chromatography

A method based on reversed-phase HPLC is reported for the separation and quantification of various urinary aromatic metabolites: hippuric, phenylaceturic, salicyluric, benzoic, phenylacetic, salicylic. 3-phenylpropionic and cinnamic acids and several phenols in ruminant urine. In this method, a Nova-Pak C₁₈ (4 μm) 150×3.9 mm I.D. column, two solvents [A: 15°b methanol in 20 mM acetic acid (pH 3.3); B: methanol] in a gradient mode at a flow-rate of 0.8 ml/min, and UV detection at 210 nm were used. Quantification of the total (free and conjugated) benzoic, phenylacetic and salicylic acids present in urine was achieved by hydrolysis of the samples in 3 M HCl at 100°C for 24 h prior to HPLC analysis. The lowest detection concentration was 50 μmol/I. This method is useful for scanning the profile of aromatic metabolites in urine of ruminants, which provides information on the diets the animals receive.