葡萄糖氧化酶纳米粒的制备及其体内外抗菌效果

为了使葡萄糖氧化酶更加稳定,利用可逆加成-断裂链转移自由基聚合反应对葡萄糖氧化酶进行纳米包裹。动态激光散射仪测定显示,包裹后粒径为(12.4±1.0)nm, Zeta电位为(+4.2±0.5)mV。通过冻融循环、长期储存和胰蛋白酶试验证明了包裹后的稳定性。在体外,对大肠杆菌的最低抗菌浓度为0.01 mg/mL。对新西兰大白兔细菌肿包治疗显示,包裹后的葡萄糖氧化酶较包裹前有较好的稳定性和抑菌效果,可作为新型抗菌制剂。     

铜锌对华北落叶松多酚氧化酶活性的影响

以华北落叶松3年生幼苗为试验材料,对不同生育期4种处理(对照、锌、铜、铜 锌)下各器官(叶、枝、干、主根、侧根)多酚氧化酶(PPO)活性进行测定分析,结果表明:华北落叶松幼苗各器官内多酚氧化酶活性差异极为显著,干中PPO活性在3月达225.2 U·g-1,而叶中PPO活性在5月仅为57.3 U·g-1.叶子中PPO活性呈递增变化趋势,10月最高,枝、干、主根、侧根的PPO活性在年循环周期内呈规律性变化,7月均达高峰期.铜、锌对各器官内PPO活性有明显的影响,而且与生育期有关.铜对PPO活性的增强作用达显著水平,尤以枝最为突出,10月份铜处理后枝中PPO活性比对照提高95.1%;锌对主根PPO活性的提高也有较明显影响,对其他器官PPO活性有不同程度的抑制作用.10月份锌处理后干中PPO活性比对照下降35.5%;铜、锌互作对干和主根PPO活性的提高表现出较大的协同作用.华北落叶松幼苗体内铜元素含量与PPO活性呈显著的正相关,铜、锌元素通过影响PPO活性而影响幼苗的生长及抗寒性.     

草菇多酚氧化酶基因的克隆与表达分析

【目的】解析草菇多酚氧化酶基因家族序列信息,明确其特征和表达模式。【方法】通过转录组测序找出含有酪氨酸酶结构域的基因,并根据搜索得到的序列设计引物,以不同发育时期的草菇 v26 菌株,以及草菇蛋形期外菌膜、菌盖、菌柄等不同组织为材料提取 RNA,经反转录为 cDNA,扩增获得草菇多酚氧化酶基因序列。采用在线生物信息学网站预测草菇多酚氧化酶的理化性质。采用 RT-PCR 的方法,检测草菇不同发育时期和不同组织部位多酚氧化酶的表达模式。【结果】获得 4 个草菇多酚氧化酶基因家族的 CDS 序列,依次命名为 VvPPO1、VvPPO2、VvPPO3、VvPPO4,其中 VvPPO1 CDS 全长 1 596 bp、编码 531 个氨基酸,VvPPO2 CDS 全长 2 685 bp、编码 894 个氨基酸,VvPPO3 CDS 全长 1 593 bp、编码 530 个氨基酸,VvPPO4 CDS 全长2 067 bp、编码 688 个氨基酸。生物信息学分析表明,4 个基因编码的蛋白均含有 2 个铜离子结合区,为亲水性蛋白;RT-PCR 结果显示,4 个基因在草菇发育的菌丝期、原基期、蛋形期、成熟期均有表达,VvPPO1、VvPPO3 和VvPPO4 的表达量在子实体时期高于菌丝期,在蛋形期 VvPPO1 和 VvPPO4 在外菌膜的表达量比在菌柄和菌盖中的表达量高。【结论】在草菇中搜索得到 4 个多酚氧化酶基因,均含有 2 个铜离子结合区,4 个基因在各个发育时期均有表达。     

Effect of erythropoietin on expression of myocardial NADPH oxidase in pressure overload rats

AIM:To explore the effect of erythropoietin （EPO） on the expression of myocardial NADPH oxidase （Nox） in the pressure overload rats. METHODS:Male SD rats (n=36) were used to establish a pressure overload myocardial hypertrophy model by abdominal aorta ligation. The animals were divided into model group, control group (sham, without narrowing abdominal aorta, the rest of the operation was the same as the model) and recombinant human erythropoietin (rhEPO) treatment group (intraperitoneal injection of rhEPO postoperatively, 4 000 U/kg, twice a week). After 8 weeks, the cardiac ultrasound imaging and hemodynamic evaluation were conducted to determine the cardiac functions. Masson staining was used to observe the degree of myocardial fibrosis. The expression of Nox2 and Nox4 at mRNA and protein levels was detected by real-time quantitative PCR and Western blotting. The protein levels of myocardial inflammatory factors CD45, F4/80 and TGF-β were determined by Western blotting. RESULTS:Compared with model group, the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic pressure (LVESP) and left ventricular pressure maximum rising and falling rates (±dp/dtmax) increased significantly in EPO treatment group (P<0.01). At the same time, left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD) and left ventricular end-diastolic pressure (LVEDP) were decreased in EPO treatment group (P<0.01). EPO reduced the degree of myocardial fibrosis caused by pressure overload (P<0.01) and decreased the expression of Nox2 and Nox4 at mRNA and protein levels in the myocardium (P<0.05 or P<0.01), and reduced the protein expression of myocardial inflammatory factors CD45, F4/80 and TGF-β. CONCLUSION: EPO inhibits rat myocar-dial fibrosis induced by pressure overload, improves heart functions by decreasing NADPH oxidase activity and inhibiting myocardial oxidative stress levels and myocardial inflammatory reaction.     

重金属镉胁迫对大弹涂鱼肝脏黄嘌呤氧化酶活性的影响

研究了在重金属镉胁迫条件下,大弹涂鱼(Boleophthalmuspectinirostris)肝脏黄嘌呤氧化酶活性的变化。重金属镉胁迫12h时0·5mg/LCd2 组XOD酶活性和对照组比较显著升高(P<0·05);胁迫24h时,大弹涂鱼0·05mg/LCd2 组肝脏的XOD酶活性和对照组比较极显著升高(P<0·01),而0·5mg/LCd2 组酶活性结果却降低;恢复3d时0·5mg/LCd2 又极显著升高(P<0·01)。在镉胁迫解除5d时,3个浓度组的酶活性均极显著降低(P<0·01),其中0·05mg/LCd2 组和0·5mg/LCd2 组的酶活性和对照组酶活性比较无显著差异(P>0·05),5mg/LCd2 组的酶活性极显著低于对照组的酶活性(P<0·01)。实验结果表明,大弹涂鱼肝脏黄嘌呤氧化酶活性对水体中镉污染敏感,有可能用做海洋环境污染的生物标记。     

Ascorbic acid status as affected by dietary treatment in the Siberian sturgeon (Acipenser baeri Brandt): tissue concentration,mobilisation and L-gulonolactone oxidase activity

A study was conducted to evaluate tissue storage and mobilisation of L-ascorbic acid (AA) in the Siberian sturgeon (Acipenser baeri) fed three different experimental diets. The three treatments consisted of a diet devoid of vitamin C (diet A0) and two diets supplemented with equivalent of 300 mg AA kg^–1 in the form of either silicone-coated ascorbic acid (diet SC) or of ascorbyl-2-polyphosphate (diet AP). During the first phase (4 months) of the trial, six batches of 130 Siberian sturgeon (initial body weight: 25.5±0.5 g) each were fed one of the three diets in duplicate. During the second phase (3 months), fish from groups SC and AP were switched to diet A0 and those fed diet A0 during the first phase were switched to diet SC. Irrespective of the dietary treatment, growth rates were not significantly different from each other. At the end of phase I, in all tissues studied, total ascorbic acid (TAA) concentrations were higher in Siberian sturgeon fed diet AP than in the other two groups. During phase II, tissue ascorbate depletion was also higher in the AP group than in the other two groups. Transfer of the AA-free diet fed group onto a diet supplemented with 300 mg AA kg^–1 (diet SC) led to a slight increase in the TAA concentrations in all tissues. Blood plasma tyrosine concentrations were not significantly different between the three groups. Whole-body collagen levels were affected by dietary AA levels or forms at the end of phase I; the differences were not significant at the end of phase II. Muscle collagen levels were slightly affected. L-Gulonolactone oxidase activity was found in the kidney of Siberian sturgeon, but not in the liver. The ascorbyl-2-polyphosphate appears to be either better utilised by Siberian sturgeon, like in many other teleosts, or more stable than the silicone-coated AA during food processing and storage. Presence of L-gulonolactone oxidase activity in Siberian sturgeon kidney combined with the absence of gross scorbutic signs in AA-free diet fed groups expressing very good growth rates suggested no need of dietary AA byA. baeri.     

玉米弯孢叶斑病菌NADPH氧化酶生物信息学分析和ATMT敲除载体的构建

从JGI数据库玉米弯孢叶斑病菌(Curvularia lunata)基因组中获得2个编码NADPH氧化酶基因ClNox1和ClNox2。采用生物信息学方法对2个基因编码的蛋白质进行性质、结构和功能等方面的预测。结果表明,ClNox1和ClNox2的分子量分别为6.35和6.41 KD,等电点PI为8.87和8.93,不稳定指数为44.50和39.45;ClNox1是亲水性膜蛋白,包含6个明显的跨膜结构域;ClNox2也是膜蛋白,包含8个明显的跨膜结构域。在亚细胞水平上,ClNox1定位于线粒体上,而ClNox2定位于细胞质膜上;在氨基酸序列方面,ClNox1和ClNox2都含有多个苏氨酸、丝氨酸和酪氨酸激酶磷酸化位点;在功能方面,ClNox1和ClNox2都包含NADPH氧化酶特征结构域,并且与Cochliobolus heterostrophus的NADPH氧化酶基因具有较高的同源性。根据ClNox1和ClNox2序列设计特异性引物扩增目的片段,与标记基因NPTII和HphGFP分别连入骨架载体pPZP100,构建完成ClNox1和ClNox2基因的敲除载体pPZP100NPTIIClNox1和pPZP100HphGFPClNox2,为根癌农杆菌介导的遗传转化提供基础材料。     

104份甘肃小麦品种脂肪氧化酶和多酚氧化酶活性基因等位变异的检测

面粉和面制品的色泽是评价小麦品质的重要指标。小麦脂肪氧化酶(LOX)和多酚氧化酶(PPO)活性对面粉白度及面制品的色泽具有重要影响。为给甘肃省小麦品质育种提供参考依据,以104份甘肃省育成的小麦品种为材料,利用功能标记LOX16、LOX18、PPO18、PPO16和PPO29检测TaLox-B1、PpoA1及Ppo-D1位点的等位变异,分析甘肃小麦品种资源中LOX和PPO活性基因的组成和分布特点。结果表明,在甘肃小麦中,等位变异TaLox-B1a和TaLox-B1b的频率分别为22.12%和77.88%;其中,甘肃冬小麦品种高LOX活性等位变异TaLox-B1a的分布频率(30.56%)高于春小麦(3.13%)。等位变异Ppo-A1a、PpoA1b、Ppo-D1a和Ppo-D1b的频率分别为49.04%、50.96%、50.96%和49.04%;两个PPO基因的等位变异组合Ppo-A1a/Ppo-D1b、Ppo-A1a/Ppo-D1a、Ppo-A1b/Ppo-D1b和Ppo-A1b/Ppo-D1a的分布频率依次为28.85%、20.19%、20.19%和30.77%。说明在甘肃小麦品种中,低LOX活性等位变异(TaLox-B1b)品种比例较高;低PPO活性等位变异(Ppo-A1b、Ppo-D1a)品种分布比例略高于高PPO活性类型;其中,32份小麦品种在TaLox-B1、Ppo-A1和Ppo-D1三个位点同时含有高LOX活性和低PPO活性的等位变异。     

Bipolaris australiensis HD-1胆红素氧化酶纯化、鉴定及其酶学性质

[目的]为了获得B.bipolaris HD-1胆红素氧化酶纯品,确定酶的同源性及其常规酶学性质.[方法]将B.bipolaris HD-1发酵液依次通过硫酸铵盐析、阴离子交换层析和凝胶过滤层析确定蛋白的纯度、浓度和酶活力;通过基质辅助激光解析电离飞行时间质谱（MALDI-TOF）法获得氨基酸序列,并利用Mascot软件在NCBI蛋白库中的比对,以确定同源蛋白来源;用SDS-PAGE方法测其表观分子量;用等电聚焦方法测定等电点;用Linwerver-Burk双倒数作图法测定米氏常数;并对该酶的酶学性质进行了常规的测定.[结果]获得具有胆红素氧化酶酶活性的电泳纯单体蛋白,酶活为46 000 U/L,比活为1.15 U/mg;MALDI-TOF共获得135个氨基酸,该蛋白与来源于Hel-minthosporium tritici-vulgaris的漆酶前体蛋白序列同源性达100％;蛋白的表观分子量为68 kDa;pI为4.1;它对ABTS和胆红素的米氏常数分别为Km（ABTS） =1.8×10-5 mol/L（pH 3.0）和Km（bilirnbin）=2.7×10-5 mol/L（pH 7.5）;对胆红素的最适催化活性为36℃,酶在-40℃下可保持2年,酶活保持在90％以上,在pH 8 ～9.5下可保持相当高的催化胆红素活性,能够耐受高浓度的硝酸钠和尿素,低浓度的叠氮化钠对酶活有促进作用而高浓度则抑制,对氯化钠、溶解氧敏感.[结论]该蛋白具有典型的胆红素氧化酶特性,又存在明显的不同.     

木奈叶片乙醇酸氧化酶基因全长 cDNA的分离与表达研究

乙醇酸氧化酶（ glycolate oxidase,GLO）是植物光呼吸途径中的一种限速酶,催化羟乙酸盐氧化成乙醛酸盐和H2 O2,与植物的诱导抗病性密切相关。试验从已构建好的木奈（ Prunus salicina Lindl．var．cordata J．Y．Zhang et al．）5个不同生长发育时期叶片的均一化全长cDNA文库中分离得到了一个乙醇酸氧化酶（ GLO）基因,命名为PsGLO。序列分析表明,PsGLO基因cDNA全长为1531 bp,开放阅读框为1122 bp,编码374个氨基酸。氨基酸多重序列比对表明,该基因与陆地棉乙醇酸氧化酶基因相似性最高,为90％。荧光定量PCR分析表明, PsGLO基因在木奈叶中木奈叶芽、展开叶、幼叶、成熟叶、老叶5个不同生长发育时期中都有表达,其中,老叶中表达量最高,叶芽最低。