GPX5在成年绵羊附睾中的表达与蛋白定位

【目的】研究谷胱甘肽过氧化物酶-5（glutathione peroxidase type-5, GPX5）在成年绵羊附睾的表达特征以及GPX5蛋白在附睾中的定位,为绵羊精子在附睾中抗氧化机制的研究提供理论依据。【方法】采取年龄相近的成年蒙古绵羊的附睾、睾丸和输精管。每一只公羊的附睾分别按照附睾头、附睾体和附睾尾进行分割。样品保存于-80 ℃冰箱,采用实时荧光定量PCR和Western blotting的方法,对成年绵羊的附睾、睾丸和输精管的GPX5 表达量进行分析。将新鲜的组织样品各部分切取适合大小浸泡于4%多聚甲醛溶液中24h,按照石蜡切片的方法制做组织切片。用GPX5特异性抗体孵育组织切片,利用DAB显色试剂盒对阳性信号进行标记。【结果】实时荧光定量PCR结果显示,成年绵羊附睾头GPX5基因的表达量极显著高于附睾体和附睾尾（P＜0.01）,而在输精管和睾丸几乎不表达。Western blotting的结果显示,GPX5蛋白在成年绵羊附睾头高表达,在附睾体和附睾尾有微量蛋白存在,睾丸和输精管中未发现GPX5蛋白。这表明GPX5主要在成年绵羊附睾头表达;经过免疫组化染色分析,GPX5蛋白主要位于附睾头上皮细胞质以及静纤毛,附睾体和附睾尾中的GPX5蛋白主要集中在静纤毛。在附睾头、附睾体和附睾尾中的精子上以及附睾腔都可以观察到GPX5蛋白,表明GPX5蛋白从附睾上皮分泌到附睾腔中,随着精子在附睾中的运输与精子结合。【结论】在成年绵羊附睾中,GPX5主要由附睾头上皮细胞合成和分泌,在附睾管腔中与精子结合,为精子功能的正常发挥提供保护。     

富硒酵母对蛋鸡生产性能、全血GPX活性和鸡蛋硒含量的影响

将300羽罗曼蛋鸡随机均分成3组,1组为对照组,饲以基础蛋鸡日粮;2组和3组为试验组,分别添加富硒酵母和亚硒酸钠,其剂量以硒计算,均为0．2mg／kg。试验期为35d。试验过程中,记录蛋鸡的生产性能,于试验的14d和35d采取全血测定GPX活性,于试验的14d、28aN35d采取鸡蛋测定硒含量。结果表明,日粮中添加富硒酵母或亚硒酸钠对蛋鸡的生产性能均无显著影响（P〉0．05）;日粮中添加富硒酵母或亚硒酸钠均能显著提高蛋鸡全血GPX活性（P〈0．05）,而亚硒酸钠提高蛋鸡全血GPX活性的能力优于富硒酵母（P〈0．05）;日粮中添加富硒酵母或Ⅱ硒酸钠均能显著提高鸡蛋硒含量（P〈0．05）,而富硒酵母提高鸡蛋硒含量的能力优于亚硒酸钠（P〈0．05）。     

玉米谷胱甘肽过氧化物酶生物信息学分析

谷胱甘肽过氧化物酶(GPX)是生物体内重要的活性氧自由基清除剂,它能够清除生物体内的过氧化氢和脂质过氧化物,阻断活性氧自由基对机体的进一步损伤,保证生物体能正常进行生命活动。以玉米谷胱甘肽过氧化物酶基因家族的11个成员为研究对象,对其编码的蛋白质的结构和功能进行分析,包括等电点、分子量、亲水性值、二级结构和亚细胞定位等,并建立了分子系统进化树。结果发现,玉米谷胱甘肽过氧化物酶基因家族的11个成员的等电点和相对分子量存在差异,而二级结构存在相似特征,其中,二级结构包括α-螺旋、β-折叠、β-转角和无规则卷曲。以上分析为全面解析玉米谷胱甘肽过氧化物酶的功能奠定了基础,并可为植物抵御氧化胁迫研究提供理论依据。     

饲料维生素E水平对黑鲷鱼抗氧化能力和鱼肉货架期的影响

探讨了饲料维生素E水平对黑鲷抗氧化酶活力和肉品质的影响.300尾平均初重为(350±12)g黑鲷随机分成3组,放入9个网箱.饲料中添加3个水平的维生素E(250、500和1 000 mg/kg α-生育酚乙酸酯),饲养8周.结果发现:(1)随着饲料中维生素E添加量的增加,黑鲷血清SOD、CAT酶活力明显上升,其中VE 1 000组最高,VE 1 000和VE 500组显著高于VE 250组(P＜0.05);(2)饲料维生素E含量对血清中谷胱甘肽过氧化物酶(GPX)活力没有显著影响(P＞0.05);(3)鲜活黑鲷肌肉氧化代谢产物丙二醛(MDA)的含量3个组间没有显著差异(P＞0.05);(4)黑鲷冷藏第3、6、9天,高水平维生素E(VE 500和VE 1 000)组肌肉MDA含量显著低于低水平维生素E组(VE 250)(P＜0.05).     

Comparison of the responses to NaCl stress of two pea cultivars using split-root system

Two pea (Pisum sativum L.) cultivars were compared: cv Lincoln and cv Douce de Provence. Seedlings grown for 14 d on standard medium were challenged for 21 d with salt using a split-root system. This protocol allowed salt-treated plants to absorb nutrients through a part of their root system maintained in control medium (C), the other part of the root system being placed in medium added with 75 mM NaCl (S). Full salt treatment (S/S) resulted in severe but non-lethal growth inhibition, high concentration of Na⁺ and Cl⁻ in leaves, and decrease in leaf K⁺ and chlorophyll contents. The two latter effects were more pronounced in Lincoln than in D. Provence. Growth inhibition was partially (Lincoln) or totally (D. Provence) alleviated in S/C configuration, and K⁺ content was less diminished than in full salt treatment. S/C treatment mitigated Na⁺ and Cl⁻ accumulation in Lincoln, but not in D. Provence. Thus, in the latter cultivar, growth inhibition by salt in S/S condition likely did not result from excessive Na⁺ and Cl⁻ accumulation in leaves. Increased electrolyte leakage from leaf tissues evidenced damages to leaf cell plasma membrane of both cultivars in S/S condition. However, damages to chloroplasts, as inferred from chlorophyll loss, were much pronounced in Lincoln than in D. Provence. Antioxidant enzymic activities in leaves were measured as proxies for oxidative stress. Catalase activity was stimulated by S/S treatment in both cultivars, but superoxide dismutase (Fe and Cu/Zn isoforms) and gaiacol peroxidase activities were augmented only in Lincoln. The absence of superoxide dismutase activity stimulation by salt in D. Provence could signify either that constitutive activity was sufficient to ensure protection against oxidative stress, or that intrinsic salt tolerance of this cultivar mitigated cellular oxidative stress. Thus, intraspecific variability for salt response exists between pea cultivars presenting similar growth sensitivity to salt.     

Avermectin exposure induces apoptosis in King pigeon brain neurons

The effect of avermectin was studied on King pigeon brain nerve cells by cytotoxicity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, MTT] and apoptosis [acridine orange/ethidium bromide (AO/EB) assay, transmission electron microscope (TEM) evaluation, measurement of mitochondrial membrane potential (Δψm), phosphatidylserine (PS) exposure, caspases activities, DNA fragmentation, reactive oxygen species (ROS) and caspase-3 mRNA expression] within the 2.5–10 μg L⁻¹ concentration-range. The results revealed that within the concentrations of 2.5–10 μg L⁻¹, avermectin showed obvious cytotoxicity and induced apoptosis in a dose-dependent manner to neurons of King pigeon in vitro. Cell viability were 99.93 ± 8.52%, 82.02 ± 4.99% and 78.23 ± 5.67% after 24 h of treatment with avermectin at the concentrations of 0, 2.5 and 5 μg L⁻¹, which decreased to 56.36 ± 2.17% of 10 μg L⁻¹. Treated cells showed typical apoptosis morphological changes including cytoplasmic vacuolation, chromatin condensation, unclear nuclear membrane and decreased/swollen mitochondria. Typical biochemical hallmarks of apoptosis including Δψm loss, PS exposure, activations of caspase-3, caspase-8 and caspase-9, DNA fragmentation were observed too. Moreover, the levels of ROS in the avermectin treatment groups increased significantly compared to control group. Furthermore, the caspase-3 mRNA levels increased significantly following AVM treatment. In conclusion, our experimental results show that avermectin has cytotoxicity to brain neurons of King pigeon in vitro and the mechanism of neurotoxicity induced by avermectin is closely related to apoptosis.     

应用RACE法分离和克隆猪GPX2基因研究

[目的]运用分子生物学技术克隆猪GPX2基因。[方法]以猪十二指肠总RNA为模板,根据人、大鼠、小鼠、牛、狗GPX2基因同源序列分析,设计兼并引物对,采用RT-PCR技术扩增获得了1段330bp的猪GPX2基因序列。根据获得的已知序列分别设计引物,采用3-′RACE和5-′RACE技术手段分离、克隆猪GPX2基因。并对所得基因进行基因序列分析。[结果]该试验成功克隆分离了1段长924 bp的mRNA序列,该序列包含完整3′-末端,与人、鼠、牛、狗GPX2基因具有较高的序列同源性,并在基因第114~116位置具有编码硒代半胱氨酸残基(Sec)的密码子TGA。[结论]序列分析比对的结果表明克隆的基因就是猪GPX2基因(NCBI GeneBank数据库序列号为DQ98982)。     

香菇GPX基因克隆及其在采后贮藏中的表达分析

以香菇（Lentinula edodes)为材料,对谷胱甘肽过氧化物酶（Glutathione peroxidase,GPX）进行基因的鉴定、编码序列克隆、蛋白生物信息学分析以及基因表达模式分析。结果表明：成功鉴定并克隆1个LeGPX基因,该基因开放阅读框（Open reading frame,ORF） 621 bp,所编码蛋白包含206个氨基酸残基;系统发育分析显示LeGPX蛋白与小皮伞（Marasmius fiardii）和灰色果腐菌（Moniliophthora roreri）的GPX亲缘关系较近,同源比对结果显示LeGPX蛋白包含GPX特征序列和催化位点。生物信息学分析表明：LeGPX为亲水性不稳定蛋白,蛋白分子质量22.81 kD,理论等电点8.83;二级结构以无规则卷曲为主,不含信号肽和跨膜结构。通过原核表达和纯化成功获得了LeGPX可溶性蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-polyacrylamide gel electrophoresis,SDS-PAGE）显示蛋白大小符合预期。荧光定量PCR分析表明,香菇褐变发生过程中LeGPX基因表达量快速上升,并且与菌盖相比,LeGPX在香菇菌柄部位表达更为活跃。     

Selenium‐enriched Saccharomyces cerevisiae improves growth,antioxidant status and selenoprotein gene expression in Arbor Acres broilers

One hundred and fifty 7‐day‐old Arbor Acres broilers were randomly assigned into five groups: group 1 served as a control that was fed a basal diet without selenium (Se) supplementation; groups 2, 3 and 4 were fed the basal diet supplemented with 0.15, 0.5 and 1.5 mg Se as Se‐enriched Saccharomyces cerevisiae (SSC) per kg of diet; and group 5 was fed the basal diet supplemented with 0.15 mg per kg of Se as sodium selenite (SS). Growth performance, glutathione peroxidase (GP_X) and superoxide dismutase (SOD) activities, total antioxidant capacity (T‐AOC), and malondialdehyde (MDA) content in plasma and liver, and cellular glutathione peroxidase (GP_X‐1) and phospholipid hydroperoxide glutathione peroxidase (GP_X‐4) mRNA levels in liver were determined. Compared with group 1, groups 2–4 exhibited higher body weights (p < 0.05), lower feed/gain ratios, and higher GP_X activities in plasma (p < 0.05) and GP_X and SOD activities and GP_X‐1 and GP_X‐4 mRNA levels in liver (p < 0.05). Compared with group 5, group 2 exhibited higher GP_X activity in plasma on day 21 (p < 0.05). Compared with group 2 and 5, group 3 exhibited lower MDA content in plasma on day 7 (p < 0.05), higher GP_X activity in plasma, SOD activity and GP_X‐1 mRNA levels in liver on day 14 and 21 (p < 0.05), and higher GP_X‐4 mRNA levels on day 14 (p < 0.05). Compared with group 4, group 3 exhibited lower MDA contents in plasma on day 14 (p < 0.05) and in liver on day 21 (p < 0.05), higher T‐AOC in plasma and higher GP_X‐1 mRNA levels on day 14 and 21 (p < 0.05), and higher SOD activity in plasma and higher SOD and GP_X activities in liver on day 21 (p < 0.05). Thus, SSC improves growth and antioxidant status of broilers; the short‐term bioavailability of SS was faster than that of SSC, but the long‐term bioavailability of SSC was greater than SS.