鸭梨PPO与GFP融合基因的烟草转化及亚细胞定位观察

将鸭梨PPO基因与绿色萤光蛋白GFP基因相融合共同进行遗传转化的方式,对鸭梨多酚氧化酶开展细胞定位研究。通过克隆该酶基因除终止密码子TAA外长度为1 779bp的CDS序列,与绿色荧光蛋白基因重组构建了荧光表达载体pBI121-PPO-GFP,借助农杆菌转化烟草,转基因烟草叶片细胞经激光扫描共聚焦显微镜观察,绿色荧光蛋白荧光与叶绿体自发荧光相重合。结果表明鸭梨多酚氧化酶为叶绿体蛋白质。     

一种改进的烟草多酚氧化酶Ⅱ纯化方法

目的:选择不同的分离、纯化步骤并比对分析,筛选出纯化烟草中多酚氧化酶(PPO)的优化组合方案。方法:采用分段盐析、DEAE-SepharoseFastflow和SephadexG-150柱层析纯化PPO,通过测定和比较酶活性筛选最佳条件。结果:确定了最佳盐析浓度(40%)和柱层析条件,SDS-PAGE、FPLC以及动力学常数的检测结果表明,纯化出的蛋白质相对分子质量为42000,Km为1.2mmol/L,得到了纯化91倍的烟草多酚氧化酶Ⅱ。结论:优化方案减少了有机溶剂分级沉淀、阳离子交换层析等步骤,使纯化过程大大缩短。     

毛头鬼伞多糖对烟草酶活性和同工酶谱的影响

分析了毛头鬼伞(Coprinus comatus)真菌多糖诱导烟草对烟草花叶病毒(TMV)抗性过氧化物酶(POD)、多酚氧化酶(PPO)、苯丙氨酸解氨酶(PAL)、几丁质酶、-β1,3-葡聚糖酶活性的变化。结果表明,毛头鬼伞多糖可提高POD、PPO、PAL、几丁质酶和-β1,3-葡聚糖酶的活性,接种TMV后毛头鬼伞多糖处理的烟草酶活性显著高于不处理者。上述结果提示,毛头鬼伞多糖处理后烟草酶活性的增强可能与其诱导烟草获得抗性有关。     

采收方法对烤烟植株上部叶烘烤特性的影响

在烘烤过程中,烟草上二棚叶和顶叶中叶绿素（Chl）含量和多酚氧化酶（PPO）活性都有不同程度的下降,而淀粉酶活性和丙二醛（MDA）含量则呈上升趋势。一次性带茎采烤的烟叶中上二棚叶和顶叶中的PPO活性和MDA含量均下降,淀粉酶活性则增高。     

合欢硬枝扦插生根解剖及相关酶活性变化研究

以合欢1年生硬枝为试材,通过石蜡切片法对合欢插穗不定根发生进行解剖学研究,同时,利用比色法对扦插后不同时间的材料进行了过氧化物酶(POD)、多酚氧化酶(PPO)和吲哚乙酸氧化酶(IAAO)测定。结果表明:合欢属于诱导型生根,观察到其不定根原基起源于形成层;在整个生根过程中,处理组和对照组的POD、PPO和IAAO酶活变化趋势基本一致,但3种酶活均高于对照。     

机械性损伤与氨基丁酸的交互作用对烟草防卫性酶活性的影响

研究β-氨苯丁酸（BABA）和剪叶对烟草（Nicotiana tabacum L．cv．Yunyan 85）防卫性酶活性和两者间互作对其抗性激发的影响。结果表明：喷施BABA或剪叶都能明显地提高烟草的苯内氨酸解氨酶（PAL）和多酚氧化酶（PPO）的活性,且这些酶活性的变化与BABA的浓度和诱抗时间有关。然而存剪叶后使用BABA,会削弱对PAL和PPO活性的诱导。BABA＋剪叶处理后烟草花叶病（TMV）症状明显较BABA单独处理的严重,这表明BABA和机械性损伤呈现拮抗效应。BABA及其它以水杨酸（SA）为信号分子的化学诱抗剂可能不适宜用于剪叶后的烟草幼苗。     

丛枝菌根真菌对烟草香气相关物质代谢的影响

丛枝菌根(AM)真菌能够与植物共生,促进寄主植株营养元素的吸收,提高植株抗逆性,但鲜见其对香气物质代谢作用的报道。本试验研究了AM真菌摩西球囊霉对烟草叶片腺毛和香气相关物质代谢的影响。结果表明: 接种AM真菌能够增加烟草叶片腺毛的密度,并诱导烟草叶片腺体毛状体脂质分泌所必需的腺体特异性脂质转运蛋白基因(NtLTP1)表达量增加;提高香气相关物质绿原酸、茄尼醇和类胡萝卜素含量;同时促进了香气物质合成途径中关键酶苯丙氨酸解氨酶(PAL)和多酚氧化酶(PPO)活性;诱导香气相关物质代谢关键酶苯丙酰胺转氨酶和多酚氧化酶基因NtPAL和NtPPOE,以及黄酮醇合酶和角鲨烯合酶基因NtFLS和NtTSS表达上调。说明接种摩西球囊霉能够增加香气产生部位腺毛的数量和分泌活性,并促进烟草叶片香气物质的生物合成过程。     

植物遗传工程

961449 葡萄多酚氧化酶的分子分析及PPO表达的反义介导抑制的研究[英]/Martinez,M.V.…//Abstr.Pap.Am.Chem.Soc.-1995,209Meet.,Pt.1.-AGF022[译自DBA,1995,14(22),95-13271] 降低多酚氧化酶(PPO)表达的新方法是通过导入反义PPO基因构建物产生葡萄转基因植株。克隆并测序的红色葡萄品种中的PPO基因,并利用葡萄     

砀山酥梨石细胞发育过程中PPO酶学特性及其cDNA片段克隆

以砀山酥梨果实为材料,研究了影响石细胞形成的木质素合成酶-多酚氧化酶(polyphenol oxidase,PPO)的酶学特性,并对PPO基因进行了克隆.结果表明,在砀山酥梨果实发育的过程中,PPO活性在花后27 d和47 d出现高峰,早于木质素含量高峰(花后47 d、61 d)和石细胞团含量高峰(花后51 d),且P...     

莲藕多酚氧化酶同工酶的比较分析

采用垂直板聚丙烯酰胺凝胶电泳技术,对同一植株莲藕的不同部位、不同湖区野生莲藕以及不同品种的特定部位的多酚氧化酶(PPO)同工酶进行了分析。结果表明,同一植株莲藕不同部位PPO同工酶带有一定的特异性, 其主要表现在区带数目、迁移位置和区带染色深浅三方面;而不同湖区野生莲藕以及不同品种莲藕,处于同一生长周期、同一部位中的PPO同工酶遗传多样性则较低。     

Activation of tobacco leaf polyphenol oxidase by sodium dodecyl sulfate

The effect of sodium dodecyl sulfate (SDS) on purified tobacco leaf PPO (PPO II) was investigated at various pHs and temperatures. SDS increased the activity of PPO II due to the formation of SDS-PPO II complex, leading to conformational changes, thus making access to active center easier. The relationship between the activity and the molar ratio of SDS-PPO II to PPO II showed that the critical point reached a plateau of activity at the molar ratio of about 1.2. The pH had a significant effect on interaction between SDS and PPO II, as compared to PPO II. The optimum catalytic temperature of the complex rose by 10 degrees C, suggesting that stabilization of the structure had been improved by the formation of complex.     

Functional definition of the tobacco protoporphyrinogen IX oxidase substrate-binding site

PPO (protoporphyrinogen IX oxidase) catalyses the flavin-dependent six-electron oxidation of protogen (protoporphyrinogen IX) to form proto (protoporphyrin IX), a crucial step in haem and chlorophyll biosynthesis. The apparent K(m) value for wild-type tobacco PPO2 (mitochondrial PPO) was 1.17 muM, with a V(max) of 4.27 muM.min(-1).mg(-1) and a catalytic activity k(cat) of 6.0 s(-1). Amino acid residues that appear important for substrate binding in a crystal structure-based model of the substrate docked in the active site were interrogated by site-directed mutagenesis. PPO2 variant F392H did not reveal detectable enzyme activity indicating an important role of Phe(392) in substrate ring A stacking. Mutations of Leu(356), Leu(372) and Arg(98) increased k(cat) values up to 100-fold, indicating that the native residues are not essential for establishing an orientation of the substrate conductive to catalysis. Increased K(m) values of these PPO2 variants from 2- to 100-fold suggest that these residues are involved in, but not essential to, substrate binding via rings B and C. Moreover, one prominent structural constellation of human PPO causing the disease variegate porphyria (N67W/S374D) was successfully transferred into the tobacco PPO2 background. Therefore tobacco PPO2 represents a useful model system for the understanding of the structure-function relationship underlying detrimental human enzyme defects.     

Herbivore damage to sagebrush induces resistance in wild tobacco: evidence for eavesdropping between plants

Whether plants respond to cues produced by neighbors has been a topic of much debate. Recent evidence suggests that wild tobacco plants transplanted near experimentally clipped sagebrush neighbors suffer less leaf herbivory than tobacco controls with unclipped neighbors. Here we expand these results by showing evidence for induced resistance in naturally rooted tobacco when sagebrush neighbors are clipped either with scissors or damaged with actual herbivores. Tobacco plants with sagebrush neighbors clipped in both ways had enhanced activity levels of polyphenol oxidase (PPO), a chemical marker of induced resistance in many solanaceous plants. Eavesdropping was found for plants that were naturally rooted, although only when sagebrush and tobacco grew within 10 cm of each other. Although tobacco with clipped neighbors experienced reduced herbivory, tobacco that grew close to sagebrush had lower production of capsules than plants that grew far from sagebrush. When neighboring tobacco rather than sagebrush was clipped, neither levels of PPO nor levels of leaf damage to tobacco were affected. Eavesdropping on neighboring sagebrush, but not neighboring tobacco, may result from plants using a jasmonate signaling system. These results indicate that plants eavesdrop in nature and that this behavior can increase resistance to herbivory although it does not necessarily increase plant fitness.     

Linoleic acid-induced expression of defense genes and enzymes in tobacco

Linoleic acid (LA) is a naturally occurring fatty acid (FA) found to elicit induced systemic resistance (ISR) of tobacco against the bacterial soft rot pathogen, Pectobacterium carotovorum subsp. carotovorum (PCC). In this study, we examined effects of six doses of exogenous LA on the induction of defense genes and enzymes. The optimum ISR activity was observed in plants treated with 0.1 mM LA where the effect of LA on membrane permeability was minimal. The application of LA as a root drench enhanced the activity of defense enzymes such as phenylalanine ammonia-lyase (PAL), peroxidase (POD), and polyphenol oxidase (PPO) and induced the expression of β-glucuronidase (GUS). PAL and POD activities were increased in a concentration dependent manner while the maximum PPO activity was observed after treatment with 0.01 mM LA. An RT-PCR analysis of the defense-related genes, Coi1, NPR1, PR-1a and PR-1b, of tobacco plants treated with 0.1 mM LA revealed an association of LA with elicitation of ISR in tobacco.     

Distinct intrinsic and synaptic properties of pre‐sympathetic and pre‐parasympathetic output neurons in Barrington's nucleus

Barrington's nucleus (BN), commonly known as the pontine micturition center, controls micturition and other visceral functions through projections to the spinal cord. In this study, we developed a rat brain slice preparation to determine the intrinsic and synaptic mechanisms regulating pre‐sympathetic output (PSO) and pre‐parasympathetic output (PPO) neurons in the BN using patch‐clamp recordings. The PSO and PPO neurons were retrogradely labeled by injecting fluorescent tracers into the intermediolateral region of the spinal cord at T13‐L1 and S1‐S2 levels, respectively. There were significantly more PPO than PSO neurons within the BN. The basal activity and membrane potential were significantly lower in PPO than in PSO neurons, and A‐type K⁺ currents were significantly larger in PPO than in PSO neurons. Blocking A‐type K⁺ channels increased the excitability more in PPO than in PSO neurons. Stimulting μ‐opioid receptors inhibited firing in both PPO and PSO neurons. The glutamatergic EPSC frequency was much lower, whereas the glycinergic IPSC frequency was much higher, in PPO than in PSO neurons. Although blocking GABA_A receptors increased the excitability of both PSO and PPO neurons, blocking glycine receptors increased the firing activity of PPO neurons only. Furthermore, blocking ionotropic glutamate receptors decreased the excitability of PSO neurons but paradoxically increased the firing activity of PPO neurons by reducing glycinergic input. Our findings indicate that the membrane and synaptic properties of PSO and PPO neurons in the BN are distinctly different. This information improves our understanding of the neural circuitry and central mechanisms regulating the bladder and other visceral organs.     

A thermostable laccase from Streptomyces lavendulae REN-7: purification, characterization, nucleotide sequence, and expression

We found a polyphenoloxidase (PPO) in the cell extract of Streptomyces lavendulae REN-7. About 0.8 mg of purified PPO was obtained from 200 g of the mycelia with a yield of 9.0%. REN-7-PPO showed broad substrate specificity toward various aromatic compounds. Moreover, this enzyme was capable of oxidation of syringaldazine, which is a specific substrate for laccase. Interestingly, REN-7-PPO retained its original activity after 20 min of incubation at even 70 degrees C. The gene encoding the PPO was cloned. Four copper-binding sites characteristics of laccases were contained in the deduced amino acid sequence. We constructed a high-level expression system of this gene in Escherichia coli. The properties of the recombinant enzyme were identical that of wild-type. In conclusion, this PPO is a thermostable laccase.     

Changes in phenolic metabolism of tobacco plants during short-term boron deficiency

The effects of boron (B) deficiency on several phenolics and enzyme activities involved in the biosynthesis of these compounds were investigated in tobacco plants (Nicotiana tabacum L. cv. Gatersleben). The levels of phenylpropanoids (mainly the caffeic acid esters, chlorogenic acid and its isomers) as well as phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and polyphenoloxidase (PPO, EC 1.14.18.1) activities were determined in plants subjected to B starvation for 1–7 d. The results presented here show that a short-term B deficiency causes both quantitative and qualitative changes in the phenolic metabolism of tobacco plants, which are especially evident after 3 d of B starvation. Although the concentration of B decreased from the onset of B starvation, root B level was less affected than leaf B by a short-term B deficiency. The concentration of phenylpropanoids as well as PAL and PPO activities increased mainly in the leaves of tobacco plants during B starvation. Moreover, leaves starved of B for 7 d showed the accumulation of new compounds, one of which was identified as caffeoylputrescine. In addition, a positive correlation between PAL activity and phenylpropanoid concentration was observed in tobacco leaves, especially after 5–7 d of B starvation, suggesting that an increase in PAL activity during B starvation could be responsible for the enhancement in the levels of phenylpropanoids.     

Polyphenol oxidases in plants and fungi: going places? A review

The more recent reports on polyphenol oxidase in plants and fungi are reviewed. The main aspects considered are the structure, distribution, location and properties of polyphenol oxidase (PPO) as well as newly discovered inhibitors of the enzyme. Particular stress is given to the possible function of the enzyme. The cloning and characterization of a large number of PPOs is surveyed. Although the active site of the enzyme is conserved, the amino acid sequence shows very considerable variability among species. Most plants and fungi PPO have multiple forms of PPO. Expression of the genes coding for the enzyme is tissue specific and also developmentally controlled. Many inhibitors of PPO have been described, which belong to very diverse chemical structures; however, their usefulness for controlling PPO activity remains in doubt. The function of PPO still remains enigmatic. In plants the positive correlation between levels of PPO and the resistance to pathogens and herbivores is frequently observed, but convincing proof of a causal relationship, in most cases, still has not been published. Evidence for the induction of PPO in plants, particularly under conditions of stress and pathogen attack is considered, including the role of jasmonate in the induction process. A clear role of PPO in a least two biosynthetic processes has been clearly demonstrated. In both cases a very high degree of substrate specificity has been found. In fungi, the function of PPO is probably different from that in plants, but there is some evidence indicating that here too PPO has a role in defense against pathogens. PPO also may be a pathogenic factor during the attack of fungi on other organisms. Although many details about structure and probably function of PPO have been revealed in the period reviewed, some of the basic questions raised over the years remain to be answered.     

Transcriptional regulation of a pineapple polyphenol oxidase gene and its relationship to blackheart

Two genes encoding polyphenol oxidase (PPO) were isolated from pineapple (Ananas comosus[L.] Merr. cv. Smooth Cayenne). Sequence analyses showed that both contained a single intron and encoded typical chloroplast-localized PPO proteins, the sequences of which corresponded to two pineapple PPO cDNAs, PINPPO1 and PINPPO2, recently described by Stewart et al. (2001). Southern blot analyses suggested that pineapple contained only two PPO genes. Analysis of expression of PINPPO1 promoter GUS fusion constructs showed this promoter had a low basal activity and was cold- and wound-inducible, consistent with known mRNA expression profiles. Striking homologies to gibberellin response complexes (GARC) were observed in sequences of both the PINPPO1 and PINPPO2 promoters. Transient assays in mature pineapple fruit and stable expression in transgenic tobacco showed that PINPPO1 promoter-GUS fusions were indeed gibberellin (GA) responsive. A role for the element within the putative GARCs in mediating GA-responsiveness of the PINPPO1 promoter was confirmed by mutational analysis. PINPPO2 was also shown to be GA-responsive by RT-PCR analysis. Mutant PINPPO1 promoter-GUS fusion constructs, which were no longer GA-inducible, showed a delayed response to cold induction in pineapple fruit in transient assays, suggesting a role for GA in blackheart development. This was supported by observations that exogenous GA(3) treatment induced blackheart in the absence of chilling. Sequences showing homology to GARCs are also present in some PPO promoters in tomato, suggesting that GA regulates PPO expression in diverse species.