梨火疫病菌(Erwinia amylovora)双精氨酸运输系统基因(tatC)的功能分析

梨火疫病菌(Erwinia amylovora)可引起梨、苹果等蔷薇科植物的火疫病.双精氨酸运输系统(Tat)与致病相关蛋白的转运有特定的联系.本研究在梨火疫病菌全基因组中发现了同源基因,通过同源重组的方法,构建了梨火疫病菌的tatC基因突变体以及互补子.研究结果表明,tatC基因影响着梨火疫病菌的致病性、胞外多糖、鞭毛运动、游动性、趋化性、生长情况等多种生物学特性.然而,Ea△tatC仍能引起烟草过敏性反应,并且在胞外纤维素和生物膜的生成方面与野生型菌株相比没有明显差异.说明梨火疫病菌tatC基因对病菌的生长、游动性以及致病性方面具有关键作用.     

梨火疫病菌luxR转录调节因子的克隆与功能分析

梨火疫病菌(Erwinia amylovora)是一种具有毁灭性的植物病原细菌,能够侵染梨、苹果和其他蔷薇科植物引起火疫病,造成严重的经济损失。luxR家族调控因子具有重要的生理生化作用,是细菌群体感应机制中研究较多的一类重要的转录调节基因。本研究采用PCR技术从梨火疫病菌中扩增出一个luxR同源基因,命名为EaluxR,应用同源重组的方法构建了突变株(EaΔluxR),并对其功能进行初步研究。实验结果表明,EaΔluxR与野生型菌株相比产生的多烯大环内酯类代谢产物在抗氧化压力和生长情况方面有明显差异,对梨幼苗及幼果的致病能力下降;但是EaΔluxR仍能引起烟草过敏性反应,并且在抗生素耐受水平和生物膜的生成方面与野生型菌株相比没有明显差异。研究表明,梨火疫病菌对病菌的生长、次级代谢产物、抗氧化压力以及致病性方面具有关键作用,为深入认识梨火疫的群体感应系统提供了科学依据。     

西瓜嗜酸菌双精氨酸转运系统基因(tatB)功能分析

细菌双精氨酸转运系统(twin-arginine translocation system,Tat)可严重影响动植物病原细菌的致病性,为了研究Tat与西瓜嗜酸菌(Acidovorax citrulli)致病性的关系,本研究通过同源重组的方法构建了西瓜嗜酸菌的tatB基因缺失菌株(ΔtatB)以及互补菌株(CtatB),并从西瓜嗜酸菌致病能力、游动能力、生长能力、胞外多糖与胞外纤维素酶产生能力以及生物膜形成等方面阐明Tat系统对西瓜嗜酸菌的影响。实时荧光定量PCR探究tatB基因与Ⅲ型分泌系统相关基因(hrcN)和(hrpE)、Tat转运系统相关基因(Aave_1810)和(Aave_0034)以及细胞毒性相关基因(TrbC)的关系。结果表明,ΔtatB能够引起非寄主植物烟草(Nicotiana tabacum)的过敏性反应;tatB基因的缺失导致了嗜酸菌的致病力、游动能力和对数生长初期的生长能力下降;不影响西瓜嗜酸菌胞外多糖、胞外纤维素酶的产生以及生物膜的形成;导致了TrbC和hrcN基因表达量显著升高,Aave_1810、Aave_0034和hrpE基因的表达量显著降低。研究结果表明,Tat系统相关基因tatB的缺失会影响西瓜嗜酸菌一些生物学特性,并导致病菌致病能力降低。本研究为西瓜嗜酸菌Ⅲ型分泌系统外的其他分泌系统与致病性的关系研究提供了基础资料。     

西瓜噬酸菌吡哆醛磷酸(VB6)营养缺陷型与致病性相关

瓜类细菌性果斑病(bacterial fruit blotch,BFB)是由西瓜噬酸菌(Acidovorax citrulli,Ac)引起的一种病害,严重为害西瓜、甜瓜的叶片和果实,造成大量减产.迄今,对该病害的研究主要集中在病原菌鉴定、病原菌检测技术、病害防治方法、品种抗病性鉴定等方面,而对病原菌的致病机理仍然知之甚少.本实验以西瓜噬酸菌xjl12菌株为背景构建转座子(mini-Tn5)插入的突变体文库,以哈密瓜(Cucumis melo var.saccharinus)为实验材料,通过对哈密瓜种子浸种处理和子叶注射接种的方法筛选突变体,而后利用同源重组的方法获得插入突变体,并对所得的突变株及野生型、互补等各个菌株进行致病性、游动性等相关表型进行测定.研究结果表明,通过文库筛选得到的1株致病力明显下降的突变体,亚克隆鉴定可知该突变体△xj-25的Mini-Tn5插入位点为吡哆醛磷酸生物合成蛋白基因(pdxJ基因),其功能主要与吡哆醛磷酸(俗称VB6)生物合成蛋白相关.突变菌株△Pdx.J致病性显著下降,生长能力、游动性明显降低,无法正常形成鞭毛和产生生物膜.通过外源添加VB6可恢复其致病性和生长能力等主要表型.本研究证明VB6生物合成在致病性、生长能力以及鞭毛的合成等方面发挥着重要作用,为降低病原菌侵害提供了一个新的思路.     

EAsdiA基因的克隆和作为新靶标对梨火疫病菌的检测

基因sdiA属于与群体效应相关的LuxR家族,目前已证实存在于Escherichia coli和Salmonella typhimurium基因组中。本研究从梨火疫病菌(Erwinia amylovora)中克隆到了一个sdiA的同源基因,命名为EAsdiA(GenBank登录号:AY864839),该基因与E.coli和S.typhimurium的sdiA基因在氨基酸水平上分别有45.42%和43.33%的同源性,与其它细菌的luxR同源基因的同源性更低。根据梨火疫病菌EAsdiA和其它病原细菌的luxR基因的序列比对设计了1对特异引物F-EAluxR和R-EAluxR,能够特异地检测梨火疫病菌,检测灵敏度为10个菌体,在含有梨组织浸出液中可检测到102个菌体。本研究首次从梨火疫病菌中克隆sdiA基因并作为新靶标应用于该病菌分子检测。     

步双重PCR法检测梨火疫病原细菌(Erwinia amylovora)

由梨火疫病原细菌（Erwinia amylovora）导致的火疫病（fire blight）是梨、苹果及其它蔷薇科植物上的毁灭性病害,我国将其定为对外一类检疫性有害生物。该病随种苗、果实及包装材料传播。目前国际上所建立的E.amylovora分子检测技术主要基于该病菌所含有的pEA29质粒序列,研究发现失去质粒的人工突变菌株的致病力下降，认为质粒对于梨火疫病菌的致病力是至关重要的。虽然带有pEA29质粒的梨火疫病菌菌株在自然界中占有绝对的数量，但Llop et al．（2006）报道了自然侵染的植物中存在具有致病力但不含pEA29质粒的梨火疫病菌（IVIA1614-2a菌株）。     

梨火疫病菌分子检测新靶标——EAsdiA基因的克隆与应用

SdiA属于与群体效应相关的luxR家族基因，目前已证实存在于Escherichia coli和Salmonella typhimurium基因组中。本研究从梨火疫病菌中克隆到了一个sdiA的同源基因，命名为EAsdiA，该基因与Es. coli和S. typhimurium的sdiA基因在氨基酸水平上分别有45.42%和43.33%的同源性，与其他细菌的luxR同源基因的同源性更低。根据梨火疫病菌EAsdiA和其它病原细菌的luxR基因的序列比对设计了一对特异引物F-EAluxR和R-EAluxR，该引物能够特异地检测梨火疫病菌，检测灵敏度为 10个菌体，在含有梨组织浸出液的情况下可检测到102个菌体。本研究是首次从梨火疫病菌中克隆SdiA基因并应用于该病菌分子检测的报道。     

瓜类细菌性果斑病菌hpaP基因的功能分析

瓜类细菌性果斑病(bacterial fruit blotch of watermelon,BFB)是近年来发生在西瓜、甜瓜等葫芦科(Cucurbitaceae)植物上的重要细菌病害,由西瓜噬酸菌(Acidovorax citrulli,Ac)引起.本研究以xjL12菌株为背景构建了转座子(Tn5)插入文库.通过注射接种哈密瓜(Cucumis melo var.saccharinus)子叶和烟草叶片进行突变体的筛选,得到l株致病性完全丧失并失去激发烟草(Nicotiana tabacum)过敏反应的突变体△xj48.对△xj48中转座子插入基因的克隆和测序表明,其突变基因为西瓜噬酸菌Ⅲ型分泌系统(T3SS)中的保守基因hpaP.利用基因重组技术构建了hpaP基因的突变体,发现hpaP突变后影响了xjL12菌株的游动性、生物膜的形成能力及hrcV基因的表达.hpaP基因的互补菌株部分恢复其致病力.本研究证实了果斑病菌中的hpaP基因与该细菌的致病力相关,在侵染瓜类作物的过程中起着不可缺失的作用.     

瓜类细菌性果斑病菌过敏性反应和致病性(hrp)基因簇部分基因的克隆及功能分析

过敏性反应和致病性(hrp)基因存在于革兰氏阴性植物病原细菌中,决定病原细菌对寄主植物致病性和诱导非寄主及抗病植物过敏性反应(hypersensitive response,HR)。本研究从果斑菌(Acidovorax avenaesub sp.citrulli)的hrp基因簇中克隆了hpaA、hrcT、hrcC和hrpG基因,通过同源重组的方法,分别构建了其突变体。电镜观察发现,hpaA和hrpG基因突变体的鞭毛缺失且细胞形态发生显著变化,而hrcT和hrcC基因突变体的鞭毛和细胞形态未发生明显变化。在烟草(Nicotiana tabacam)和哈密瓜(Hami cantaloupe)叶片上的测定结果显示,hpaA、hrcT和hrcC的突变体均失去在烟草上的HR激发能力和在哈密瓜叶片上的致病性;hrpG在烟草上的HR激发能力和在哈密瓜上的致病性则显著减弱;进一步的生长曲线测定结果表明,hpaA、hrcT、hrcC和hrpG的突变体的定殖能力均显著下降。相应地,功能互补后突变体基本恢复至野生表型。证明瓜类细菌性果斑病菌hrp基因作为Ⅲ型分泌系统关键组份影响病原细菌对寄主植物的致病性和对非寄主及抗病植物的过敏性...     

梨火疫病菌的免疫检测技术（简报）

首次采用免疫分离和免疫鉴定相结合的方法对梨火疫病菌进行检测,检测样本中的目的的菌检测灵敏度达１０＾１－１０＾２ｃｆｕ／ｍＬ,回收率达３０％以上。检测人工接种材料,进口或国产的苹果,梨以及在江苏省内定点采集样品的结果,除实验室人工接种材料阳性外,所检测样品中均不带梨火疫病菌。     

Identification of oxidation products of (-)-epigallocatechin gallate and (-)-epigallocatechin with H(2)O(2)

(-)-Epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) are two important antioxidants in tea. They also display some antitumor activities, and these activities are believed to be mainly due to their antioxidative effects. However, the specific mechanisms of antioxidant action of tea catechins remain unclear. In this study are isolated and identified two novel reaction products of EGCG and one product of EGC when they were reacted separately with H(2)O(2). These products are formed by the oxidation and decarboxylation of the A ring in the catechin molecule. This study provides unequivocal proof that the A ring of EGCG and EGC may also be an antioxidant site. This study also indicates an additional reaction pathway for the oxidation chemistry of tea catechins.     

Excretion of (14)C-fumonisin B(1), (14)C-hydrolyzed fumonisin B(1), and (14)C-fumonisin B(1)-fructose in rats.

14C-Fumonisin B(1) (FB(1)) was produced by Fusarium proliferatum M-5991 in modified Myro liquid medium and purified to >95% purity with a specific activity of 1.7 mCi/mmol. Nine male and nine female F344/N rats were each dosed by gavage with 0.69 micromol of (14)C-FB(1), (14)C-hydrolyzed FB(1), or (14)C-FB(1)-fructose/kg body weight. Urinary excretion of (14)C-FB(1) and (14)C-FB(1)-fructose was 0.5% and 4.4% of the total dose, respectively, and was similar between male and female rats. Urinary excretion of (14)C-hydrolyzed HFB(1) was significantly greater (P > 0.05) in female rats as compared with male rats (17.3% vs 12.8% of the total dose, respectively). There were no significant (P > 0.05) differences in biliary excretion of the three fumonisin compounds with a mean of 1. 4% of the dose excreted at 4 h after dosing. Lesser amounts continued to be excreted up to 9.25 h after dosing. Although biliary excretion of the (14)C-FB(1), (14)C-hydrolyzed FB(1), and (14)C-FB(1)-fructose was similar, increased urinary excretion of the (14)C-hydrolyzed FB(1) as compared to (14)C-FB(1) and (14)C-FB(1)-fructose indicated a greater absorption of the hydrolyzed form.     

Chemoenzymatic synthesis of homochiral (R)- and (S)-karahanaenol from (R)-limonene

Terpinolene oxide, a monoterpene belonging to the p-menthane group, is easily derived from naturally abundant (R)-limonene. It was isomerized with montmorillonite clay catalyst to karahanaenone (2,2, 5-trimethylcyclohept-4-en-1-one) by ring enlargement. The enantiomers of the corresponding alcohol, karahanaenol (2,2, 5-trimethylcyclohept-4-en-1- ol), known for their individual organoleptic properties, were resolved through Pseudomonas cepacia lipase mediated enantiospecific alcoholysis of its acetate derivative.     

(三唑基-~(14)C-)粉锈宁的标记合成

本文报道了(三唑基-14C)-粉锈宁的制备。由14C-甲酸和重碳酸氨基胍形成(5-14C)-3-氨基-1,2,4-三唑,再经重氮化脱氨得到(5-14C)-1,2,4-三唑,最后再与对氯酚和二氯片呐酮反应得到(三唑基-14C)-粉锈宁。放化收率为26％(从甲酸-14C计),放化纯度大于95％。     

Theoretical speciation of ethylenediamine-N-(o-hydroxyphenylacetic)-N'-(p-hydroxyphenylacetic) acid (o,p-EDDHA) in agronomic conditions

The presence of ethylenediamine-N-(o-hydroxyphenylacetic)-N'-(p-hydroxyphenylacetic) acid (o,p-EDDHA) as the second largest component in commercial EDDHA iron chelates has recently been demonstrated. Here is reported the speciation of o,p-EDDHA by the application of a novel methodology through the determination of the complexing capacity, protonation, and Ca(2+), Mg(2+), Cu(2+), and Fe(3+) stability constants. The pM values and species distribution in solution, hydroponic, and soil conditions were obtained. Due to the para position of one phenol group in o,p-EDDHA, the protonation constants and Ca and Mg stability constants have different values from those of o,o-EDDHA and p,p-EDDHA regioisomers. o,p-EDDHA/Fe(3+) stability constants are higher than those of EDTA/Fe(3+) but lower than those of o,o-EDDHA/Fe(3+). The sequence obtained for pFe is o,o-EDDHA/Fe(3+) >/= o,p-EDDHA/Fe(3+) > EDTA/Fe(3+). o,p-EDDHA/Fe(3+) can be used as an iron chelate in hydroponic conditions. Also, it can be used in soils with limited Cu availability.     

Binding studies of oxovanadium(V) with ovalbumins (OA)

The effect of protein oxovanadium(V) ion concentration and pH on the ratio of diffusion current (id/id₀) was studied in vanadium(V) ovalbumin-S and denatured ovalbumin systems. In both the cases marked decrease in diffusion current was observed at the respective pH values, indicating that binding takes place with cationic groups of the proteins. The binding sites (n) were found to be pH dependent. The uniformity of logK and ΔG ⁰ value at all pH values indicated the involvement of same sites in interaction. Furthermore, the linear scatchard plots in both the systems supported the involvement of single class of independent sites in oxovanadium(V) anion interaction. The difference in binding sites (n) has been attributed to the folded structure of ovalbumin-S while unfolded one of denatured ovalbumin.     

Effect of pH on interaction between As(III) or As(V) and manganese(IV) oxide

The efficiency of As(III) oxidation by MnO₂, and retention of oxidation products varies with system pH. Maximum retention by hydrous Mn(IV) oxide occurs at pH < 5, declining at higher pH to about half total As at pH 10. The adsorption capacities of pyrolusite and cryptomelane at pH ～6.5 for As(V) species were 10 and 25 mmol kg^?1, respectively. HMO surface saturation (～10 mmol kg^?1) was reached with equilibrium As(V) levels of 5 to 8 × 10^?6 M but this was supplemented at higher levels by an absorption process where uptake increased linearly with concentration (e.g., 68 mmol kg^?1 with 2 × 10^?5 M As(V)). Added As(III) was avidly oxidized and most product retained at pH 3. At higher pH increasing amounts of As(III) remained unoxidized due to initial reactions apparently blocking access to internal pores. Added Na⁺ reduced the amount of As retained by the HMO, with the phosphate salt having a significant effect. Extraction studies confirmed that most As could be released by exposure to reducing agents or chelating agents (EDTA). The environmental significance of the results has been considered.