基于抗坏血酸调控氧化铈纳米酶催化活性的抗坏血酸氧化酶检测研究

纳米酶是一类既有纳米材料的独特性能又有催化功能的模拟酶,它既有天然酶的高催化活性,又有模拟酶稳定经济的特点,在生物传感检测领域的应用十分广泛。本研究采用水热法合成具有辣根过氧化物酶样活性的氧化铈纳米酶。该纳米氧化铈同时具有+4价和+3价两种形态,其中Ce^(4+)能与过氧化氢反应,使3,3’,5,5’-四甲基联苯胺(TMB)氧化,溶液显蓝色。由于抗坏血酸(AA)能还原Ce^(4+),抑制其催化活性以及TMB的氧化,从而使溶液的颜色较浅。而抗坏血酸氧化酶可催化氧化AA,阻碍了其对Ce^(4+)的还原,使纳米氧化铈的催化性能恢复。利用这种方法,实现对抗坏血酸氧化酶的定量检测,在12 U/L~400 U/L范围内呈良好的线性关系(R^(2)=0.9704),检测限为3.2 U/L。该研究为基于氧化铈纳米酶的比色方法在抗坏血酸氧化酶活性检测及相关应用提供基础。

Ascorbic acid synthesis and transportation capacity in old laying hens and the effects of dietary supplementation with ascorbic acid

Background: Laying hens over 75 weeks of age commonly show great declines in immunity and production performance.It is unclear whether these declines can be relieved by supplementing with ascorbic acid(AA) in feed.Two trials were conducted to investigate the synthesis and metabolism of AA in layers of different ages and the effects of dietary supplemental AA on the performance and the immune and antioxidant statuses of 78 weeks old hens.Methods: In Exp.1,equal numbers(24 hens) of 35 weeks old(Young) and 75 weeks old(Old) layers were fed the same diet without AA supplementation for 4 weeks.In Exp.2,360 healthy 78 weeks old laying hens were randomly assigned to 4 treatments(basal diet supplemented with 0,0.25,0.5,or 1 g AA/kg diet) in an 8-week feeding trial.Results: The old hens tended to have decreased L-gulonolactone oxidase(GLO) synthase activity in the kidney and liver than that of the young hens(P = 0.07 and P = 0.05,respectively).Compared with the young hens,the old hens had lower hepatic antioxidant capacity allowing for the lower thioredoxin(TXN),thioredoxin reductase(TXNR) and cytochrome b5 reductase(CYB5 R) gene expression(P < 0.05),whereas increased sodium-dependent vitamin C transporter(SVCT) 1 expression levels in the ileum and kidney and enhanced splenic and hepatic AA concentrations(P < 0.05).Dietary supplementation with AA significantly decreased GLO enzyme activity but increased splenic AA concentration and anti-bovine serum albumin IgG levels(P < 0.05) and tended to increase CD4+T lymphocyte numbers(P = 0.06) in serum.Supplementation of 0.25 g AA/kg diet significantly increased hepatic total antioxidant capacity(T-AOC,P < 0.05) relative to the control group.Conclusions: Laying hens could synthesize AA in both the kidney and the liver,though the GLO enzyme activities were 100 times greater in kidneys than in livers.The old laying hens had greater absorption and reabsorption capacity and higher AA retention in some tissues that did the young hens.Dietary supplementation of AA can improve the health of old layers by enhancing immunity and antioxidant capacity.

Ascorbate oxidase electrochemical biosensor based on the biocompatible poly(3,4-ethylenedioxythiophene) matrices for agricultural application in crops

The vitamin C（VC） in crops was successfully determined using ascorbate oxidase（AO） electrochemical biosensor based on the biocompatible poly（3,4-ethylenedioxythiophene）（PEDOT） matrices,which was easily prepared by one-step electrodeposition technique in ionic liquid microemulsions.The fabricated biosensor displayed excellent bioelectrocatalytic performance to the oxidation of VC,wide linear range,low detection limit,fast response time,good operational and storage stability,the good results of the determination of VC in vegetable crops indicated that the fabricated biosensor will be a good candidate for the physiological and biochemical studies of crops in near future.

Co-evolved plant and blast fungus ascorbate oxidases orchestrate the redox state of host apoplast to modulate rice immunity

Apoplastic ascorbate oxidases(AOs)play a critical role in reactive oxygen species(RoS)-mediated innate host immunity by regulating the apoplast redox state.To date,little is known about how apoplastic effectors of the riceblast fungus Magnaportheoryzaemodulate the apoplast redox state of rice to subvert plant immunity.In this study,we demonstrated that M.oryzae MoAo1 is an Ao that plays a role in virulence by modulating the apoplast redox status of rice cells.We showed that MoAo1 inhibits the activity of rice OsAO3and OsAO4,which also regulate the apoplast redox status and plant immunity.In addition,we found that MoAo1,OsAO3,andOsAO4 allexhibit polymorphic variations whosevaried interactions orchestrate pathogen virulence and rice immunity.Taken together,our results reveal a critical role for extracellular redox enzymes during rice blast infection and shed light on the importance of the apoplast redox state anditsregulation inplant-pathogeninteractions.

AN AMPEROMETRIC BIOSENSOR BASED ON COVALENT IMMOBILIZATION OF ASCORBATE OXIDASE ON BIOCOMPATIABLE AND LOW-TOXIC POLY(THIOPHENE-3-ACETIC ACID) MATRIX

The biocompatiable and low-toxic poly（thiophene-3-aeetic acid） （PTAA） matrix was successfully electrosynthesized in ionic liquid 1-butyl-3-metbylimidazolium hexafluorophosphate （BmimPF6） in comparison with the electrosynthesis of PTAA matrix in acetonitrile （ACN）. Ascorbate oxidase （AO） was used as a model for the development and application of biosensor. Vitamin C （VC） biosensors were facilely fabricated by the covalent immobilization of AO molecules on PTAA matrices electrosynthesized in ACN containing tetrabutylammonium tetrafluoroborate and BmimPF6, respectively. Electrochemical impedance spectroscopy, scanning electron microscopy and FTIR spectroscopy indicated that AO molecules were covalently immobilized on PTAA matrices. Parameters of the as-obtained biosensors such as working potential, pH and temperature have been optimized. The amperometric biosensor based on PTAA matrix electrosynthesized in BmimPF6 exhibited wider linear range, lower detection limit, higher sensitivity and bioaffinity, and better operational and storage stability than that electrosynthesized in ACN under optimal conditions. The as-obtained biosensor based on PTAA matrix electrosynthesized in BmimPF6 was employed for the detection of VC content in comme：rcial juices, and the result was close to the data given by manufacturers. Excellent results indicate that the PTAA matrix electrosynthesized in ionic liquid is a promising platform for the covalent immobilization of biologically-active species and the development of biosensors.

香蕉中ABA醛氧化酶基因家族的鉴定及其表达分析

ABA醛氧化酶(abscisic aldehyde oxidase,AAO)在植物生长发育和对生物/非生物胁迫的响应中起着重要作用。本研究从香蕉基因组中鉴定出3个MaAAOs基因,并对其遗传进化关系、结构域、理化性质及在不同组织、不同果实发育阶段和逆境处理下的表达情况进行了分析。结果表明,这3个MaAAOs分为两个亚族,相同亚族的基因呈现出类似的结构域组成和基因结构,与系统发育树的构建结果相一致。MaAAO1基因在果实的发育和成熟阶段都表现出显著的高表达水平,暗示着其在香蕉果实发育过程中可能发挥着重要功能。另外,MaAAO1和MaAAO2基因可能对渗透胁迫有响应,MaAAO2基因还可能对Foc4病菌的侵染有响应。基于以上结果推断MaAAOs基因在香蕉的生长发育调控以及逆境响应中具有重要作用。该结果不仅丰富了我们对MaAAOs基因家族的认识,也为进一步揭示其在香蕉生长发育和应激响应机制方面的功能提供了线索。

Fabrication of CeO2/rGO Nanocomposites with Oxidase-like Activity and Their Application in Colorimetric Sensing of Ascorbic Acid

Nanomaterial-based artificial enzymes（nanozymes） have shown great potential for application in colori- metric sensing. In this paper, the nanocomposites of CeO2 and reduced graphene oxide（rCK）） were fabricated via a one-pot hydrothermal process, in which CeO2 nanoparticles were well anchored on the surface of rGO. The prepared CeO2/rGO nanocomposites exhibited excellent catalytic activity for the oxidation of 3,3＇,5,5＇-tetramethylbenzidine（TMB） in the presence of 02. Compared with the individual CeO2 nanoparticles, the nanocomposites had a higher affinity and adsorptivity towards the substrate TMB, which was due to the combination of rGO. In addi- tion, based on the oxidase-like activity of CeO2/rGO nanocomposites, a facile and sensitive colormetric sensor for ascorbic acid（AA） was investigated. The detection limit of AA was low（0.15μmol/L） and the linear detection range was between 0.5μmol/L and 40μmol/L. Fttrthermore, the colorimetric system was used for the detection of AA in medicine and food analysis, such as tablets, beverage and milk powder.

刺梨果实中维生素C积累与相关酶活性的关系

刺梨果实发育过程中维生素C(AsA)积累呈“慢—快—慢”的模式,其中6月底至8月初是AsA积累最快和最重要的时期。半乳糖内酯脱氢酶(GalLDH)活性与AsA积累速率的变化趋势基本一致,二者间呈极显著的正相关关系。AsA分解酶抗坏血酸氧化酶(AAO)和抗坏血酸过氧化物酶(AAP)只在发育前期极短时间内表现活性,从而使AsA极少被氧化分解,这是刺梨果实能积累高水平AsA的重要原因之一;在果实发育过程中始终未能检测到单脱氢抗坏血酸还原酶(MDAR)和脱氢抗坏血酸还原酶(DHAR)的活性,表明它们不是刺梨果实积累高含量AsA的关键因素。

不结球白菜维生素C积累与相关酶活性研究

对不结球白菜‘常州乌塌菜’、‘矮脚黄’和‘二青’生长过程中维生素C(AsA)含量和L-半乳糖酸-1,4-内酯脱氢酶(GalLDH)、抗坏血酸氧化酶(AAO)、抗坏血酸过氧化物酶(APX)、单脱氢抗坏血酸还原酶(MDAR)、脱氢抗坏血酸还原酶(DHAR)的活性进行测定分析,结果显示,AsA含量的积累呈先升高后下降的模式,且‘常州乌塌菜’中的AsA含量显著高于‘矮脚黄’和‘二青’;GalLDH活性与AsA含量变化趋势基本一致,且3个品种中GalL-DH活性与AsA积累之间均呈极显著的正相关关系;AAO和APX活性的变化趋势则与AsA含量变化趋势相反,而‘常州乌塌菜’中AAO活性显著低于其它2个品种,但APX活性无显著差异;MDAR和DHAR的活性则主要表现在生长后期。结果表明,不结球白菜中AsA含量主要受GalLDH酶活性调节,同时AsA代谢酶AAO也对AsA积累起到了重要作用,而MDAR和DHAR酶活性对AsA积累的作用主要在生长后期。

烟草感染马铃薯Y病毒脉坏死株系后六种酶活性变化的研究

本文定期测定了感病品种ＮＣ８９接种ＰＶＹＮ后叶片内６种酶活性的动态变化，结果表明，接种后１４天内ＳＯＤ活性均较对照增高，其中第８天达高峰值，但接种后第１６天ＳＯＤ活性低于对照约２８％；ＡＡＯ活性在接种后２～６天明显高于对照，６～１２天，ＡＡＯ活性变化不定，但１２天后，ＡＡＯ活性又升高；ＰＯＤ活性在接种后２～４天比对照降低，４天后显著升高，至接种后第１４天达高峰值，接种后１６天又有下降趋势；ＣＡＴ活性仅在接种后第２天比对照略有升高，随后均低于对照，其中第６天和第１０天ＣＡＴ活性降低幅度较大；ＰＡＬ活性在接种后２～６天均比对照高，随后开始下降，第８～１０天ＰＡＬ活性低于对照，１０天后又升高，第１２天又达高峰值；ＰＰＯ活性在接种后２～１６天明显高于对照。本文还详细讨论了上述６种酶与烟草脉坏死病病程的相关性。

施钼对不同钼效率冬小麦叶片呼吸作用相关酶的影响

以冬小麦钼高效(97003)和钼低效(97014)品种为供试材料,采用土培方法,研究施钼对冬小麦分蘖期、拔节期、孕穗期和灌浆期功能叶多酚氧化酶(PPO)、抗坏血酸氧化酶(AAO)、乙醇酸氧化酶(GO)等呼吸作用相关酶类活性变化的影响。结果表明,施钼后,PPO活性在4个生育期均降低;AAO活性在分蘖期和拔节期降低,而在孕穗期和灌浆期上升;GO活性则在分蘖期、拔节期和孕穗期降低,而在灌浆期升高。钼对不同钼效率冬小麦叶片呼吸作用酶的影响存在着差异。施钼有利于促进冬小麦分蘖期和拔节期碳水化合物的积累,从而促进生物量的提高,而在孕穗期和灌浆期由于植株生长中心的转移,呼吸作用酶变化复杂。

抗坏血酸和亚硫酸钠在甘薯破碎中抗褐变的研究

甘薯破碎时多酚氧化酶(PPO)的作用,严重影响淀粉的白度。通过研究抗坏血酸对多酚氧化酶的抑制作用,确定了抗坏血酸在甘薯破碎中的最佳添加量为0.5‰。并分析了在甘薯破碎中抗坏血酸和亚硫酸钠混配时对PPO的作用效果,优选出抗坏血酸和亚硫酸钠混配时抗褐变的最佳添加量分别为0.1‰和0.5‰。

苹果多酚氧化酶的提取及其抑制作用的研究

以冷冻丙酮制备苹果多酚氧化酶(PPO),通过以邻苯二酚为底物测定氧化产物的方法,对苹果果肉中多酚氧化酶的pH值、温度、热稳定性及抗氧化剂抑制作用进行了研究。结果表明:用真空冷冻干燥得到的酶粉能更好的保持多酚氧化酶活性;酶氧化邻苯二酚的最适pH为6.0,最适温度为25℃;PPO在20-25℃时对热较稳定,期后随着温度升高后,酶活迅速降低;在100℃维持1min酶全部失活;抗坏血酸和黄酮类物质能明显地抑制酶促褐变。

开放式臭氧浓度升高对水稻叶片呼吸作用相关酶的影响

利用亚洲首个开放式臭氧浓度升高(Ozone-free air controlled enrichment)平台,以武运粳21(粳稻)和两优培九(杂交稻)两个耐性不同的水稻品种为材料,研究了大气臭氧浓度升高对水稻叶片乙醇酸氧化酶(GO)、多酚氧化酶(PPO)和抗坏血酸氧化酶(AAO)3种呼吸作用相关酶的影响,初步探讨了高臭氧浓度条件下水稻不同基因型对臭氧胁迫响应机制的差异性。结果表明:臭氧处理67天,促进了GO、PPO和AAO的活性,武运粳21升高的幅度(24.6%、9.25%和67.8%)大于两优培九的升高幅度(11.18%、7.26%和36.85%);在处理79天,臭氧胁迫抑制了水稻叶片GO、PPO和AAO的活性,品种间比较,武运粳21的3种酶降低的幅度(21.18%、6.14%和31.87%)小于两优培九(42.7%、33.93%和51.1%)。结果说明,武运粳21在通过提高3种酶的活力而进行较强的呼吸作用,起到耗散过剩光能并保护光合器官的作用,从而缓解臭氧对光合器官的损害缓解臭氧对水稻叶片造成的伤害,因此武运粳21比两优培九对臭氧耐受性强。

长白落叶松几种酶活力及在种源早期选择中的应用

不同季节测定了 8个种源长白落叶松 4种酶活力 ,分析了它们与生长的关系。结果表明 ,生长快的种源过氧化物酶、多酚氧化酶活力弱 ,抗坏血酸氧化酶、谷氨酸脱氢酶活力强 ;生长慢的种源则相反 ,4种酶活力与长白落叶松的生长速率密切相关。不同季节各种酶活力的聚类分析结果相似 ,且与生长性状的聚类分析结果相一致。回归分析表明 ,过氧化物酶活力与长白落叶松的生长呈显著负相关 ,抗坏血酸氧化酶、谷氨酸脱氢酶活力与长白落叶松的生长呈显著正相关。多酚氧化酶活力与长白落叶松生长的负相关性较低。初步研究表明 ,过氧化物酶、多酚氧化酶。

烤烟叶片多酚氧化酶和抗坏血酸氧化酶活性影响因素研究

研究了一些理化因素对烤烟叶片多酚氧化酶和抗坏血酸氧化酶活性的影响.结果表明,2种酶均较耐高温,但抗坏血酸氧化酶热稳定性好于多酚氧化酶.多酚氧化酶作用最适pH值为6,抗坏血酸氧化酶为5.Cu2+能增强多酚氧化酶活性,减弱抗坏血酸氧化酶活性.EDTA,NaHSO3,H3BO3,柠檬酸既是多酚氧化酶的良好抑制剂,又是抗坏血酸氧化酶的激活剂.

Nafion-甲基紫精修饰电极抗坏血酸氧化酶生物传感器的研究

本文用Nafion-甲基紫精修饰电极为基底,以牛血清白蛋白和戊二醛为交联剂,将抗坏血酸氧化酶固定在电极上,制成修饰电极抗坏血酸氧化酶生物传感器。用这种生物传感器测定人体血清中抗坏血酸,线性范围在7.5×10^(-4)～7.5×10^(-7)mol/L之间,响应时间为5s,检测下限为2.5×10^(-7)mol/L。该传感器具有选择性好、灵敏度高和响应时间短等特点。

聚邻苯二胺修饰电极抗坏血酸氧化酶生物传感器的研究

本文报道了聚邻苯二胺／抗坏血酸氧化酶生物传感器，用这种传感器测定人体血清中的抗坏血酸，线性范围在１．０×１０（－４）～２．５×１０（－７）ｍｏｌ／Ｌ之间，响应时间为７ｓ，检测限为１．０×１０（－８）ｍｏｌ／Ｌ。该传感器具有选择性好、灵敏度高和响应时间短等特点。

高温胁迫对不同黄瓜品种幼苗中抗坏血酸代谢的影响

对高温胁迫条件下黄瓜幼苗叶片中的抗坏血酸以及与之代谢相关物质的含量和酶活性变化进行了研究。结果表明,高温处理1 d,津春4号和农城3号的抗坏血酸总含量均急剧下降,降幅分别为50.8%和37.1%,处理2 d时总谷胱甘肽含量降至最低,降幅分别为36.9%和39.5%,之后逐渐上升,抗坏血酸过氧化物酶(Ascorbic peroxidase,AAP)活性经1 d胁迫后分别增加67.4%和39.6%,抗坏血酸氧化酶(Ascorbic ox-idase,AAO)活性胁迫4 d时分别降低44.0%和35.0%,另外,脱氢抗坏血酸还原酶(DHAR)和单脱氢抗坏血酸还原酶(MDAR)活性也有所增加。抗坏血酸含量与谷胱甘肽含量间呈显著的正相关关系,与AAP及AAO活性之间均呈显著负相关关系。耐高温品种抗坏血酸和谷胱甘肽含量的变化幅度较小,AAP,AAO,MDAR和DHAR等酶活性变化幅度较大。

柠檬酸和抗坏血酸对蝴蝶兰叶外植体褐变发生的影响

目的:探究柠檬酸和抗坏血酸对蝴蝶兰叶片外植体褐变发生的影响以及对PPO活性变化影响的作用机理。方法:以褐变率和褐变指数为参考数据,分析柠檬酸和抗坏血酸对外植体PPO活性和PPO反应产物积累的影响以及与外植体褐变发生的关系。结果:分别用100mg/L柠檬酸共培养和50mg/L抗坏血酸浸泡处理叶片外植体,经离体培养3d,褐变率分别比对照降低94.9%和54.9%,离体培养6d,褐变指数低于对照的0.53,分别为0.46和0.36,同时PPO活性降低。结论:推测柠檬酸抑制褐变的原因是直接与酶蛋白作用,抗坏血酸则与新生醌类物质结合。