Overexpression of StRbohA in Arabidopsis thaliana enhances defence responses against Verticillium dahliae

Plant NADPH oxidases are key regulators of plant–microbe interactions and reactive oxygen species (ROS) are essential to plant defences against pathogens. A significant part in the role played by ROS has been ascribed to plant respiratory burst oxidase homologs (RBOHs). In potato (Solanum tuberosum), where RBOHs were previously shown to be involved in wound-induced oxidative burst, we assessed their expression after inoculation with Verticillium dahliae Kleb. and showed that StRbohA was the only homolog to be differentially induced in potato in response to inoculation. In order to investigate the potential role of this gene in plant protection against wilt diseases, we used Agrobacterium-mediated transformation of Arabidopsis to assess the effects of its overexpression on plant responses to V. dahliae. After inoculation with this pathogen, the transformed Arabidopsis line overexpressing StRbohA showed lower disease severity (percent damaged leaf area and vascular discoloration) as compared to the wild type. It also had higher ROS production and more cell death caused by hydrogen peroxide (H₂O₂), compared to the wild type. Suberization of root cells was also more pronounced in the line overexpressing StRbohA, and supports a possible role for StRBOHA in plant resistance to V. dahliae. Together, these findings indicate that overexpressed StRbohA in Arabidopsis enhances the ROS-mediated defence mechanisms against V. dahliae and can be a potential tool to improve plant resistance to this and other soilborne pathogens that cause wilts in economically important crops.     

Molecular characterization of Ascaridia galli from Bangladesh and development of a PCR method for distinguishing A. galli from Heterakis spp.

We analyzed the nuclear ribosomal internal transcribed spacer (ITS) 1 and ITS2 sequences for Bangladesh isolates of Ascaridia galli, and we determined that the sequences were unreliable as molecular markers for distinguishing A. galli from other Ascaridia species, because the sequences showed high identity with that of A. columbae. However, the ITS1 sequences were available for designing PCR primers distinguishable between Ascaridia galli and Heterakis spp. Bangladesh isolates of A. galli constituted a monophyletic clade along with other geographical isolates in the cytochrome c oxidase subunit I (COI) phylogenetic tree, however, we could not clarify the phylogenetic relationships between A. galli and other Ascaridia spp., because their available sequences in GenBank were very few. The developed PCR method using DNA from A. galli and Heterakis spp. eggs would enable differential diagnosis of the individual infections in the future.     

Green tea polyphenols supplementation alters immunometabolism and oxidative stress in dairy cows with hyperketonemia

Peripartal cows often experience negative energy balance, and are therefore prone to suffering from metabolic diseases such as hyperketonemia, which causes financial losses in dairy farms. This study aimed to investigate the effect of green tea polyphenol (GTP) supplementation during the periparturient period on production performance, oxidative stress and immunometabolism in dairy cows with hyperketonemia. One hundred Holstein cows were assigned to GTP (0.2 g/kg DM; n = 50) or control (without GTP; n = 50) group based on body weight, previous milk yield, and parity on d 15 before expected parturition. Subsequently, 10 cows with hyperketonemia were selected from each group, according to blood β-hydroxybutyric acid (BHBA) concentration between 1.2 and 2.9 mmol/L from 2 to 3 d postpartum. All cows were fed a close-up diet and a lactation diet with or without GTP supply from 15 d prepartum until 30 d postpartum. Milk and blood samples were obtained from 20 cows selected with hyperketonemia on 10, 20, and 30 d postpartum. Compared with control cows, greater milk yield and lower somatic cell count were observed in GTP cows. The GTP group had lower concentrations of BHBA, free fatty acids, cholesterol, triglyceride, reactive oxygen species, malondialdehyde, and hydrogen peroxide, greater concentrations of glucose, lower activities of aspartate aminotransferase, alanine aminotransferase, and glutamyl transpeptidase, alongside greater activities of superoxide dismutase, glutathione peroxidase, and total antioxidant capacity. Additionally, GTP supplementation up-regulated concentrations of interleukin-6 and interleukin-10, but down-regulated concentrations of tumor necrosis factor-α, interleukin-1β, interleukin-2, interleukin-8, and interferon-γ in plasma. Greater concentrations of plasma immunoglobulin G were also detected in the GTP group. Overall, the data suggested that GTP supplementation from 15 d prepartum to 30 d postpartum improved the milk yield and health status in cows with hyperketonemia during early lactation.     

红三叶多酚氧化酶特性的研究

红三叶(Trifolium pretense L.)多酚氧化酶仅作用于邻苯酚,与对苯酚、间苯酚和一元酚无作用,而且对不同的邻苯酚其活性也表现很大差异,其中以咖啡酸作底物时活性最大.红三叶多酚氧化酶反应的最适pH值为6.5～7.0,最适温度为25～30℃,50℃时热稳定性相对较好.     

软儿梨冷冻贮藏过程酚类物质与多酚氧化酶的区域化分布

为研究冷冻贮藏中不同部位酚类物质和多酚氧化酶的分布特征,以青海省地方梨优良品种‘软儿梨’果实为研究材料,采用高效液相色谱法和比色法测定软儿梨果实后熟(室温)、冻藏(-18℃)和解冻(室温)3个时期的果皮、果肉中总多酚、绿原酸的含量和多酚氧化酶活性。结果表明:软儿梨果实后熟后,酚类物质果肉?果皮,多酚氧化酶则果皮?果肉;冷冻贮藏过程中,不同部位的酚类物质和多酚氧化酶均呈现不同程度的升高,但绿原酸在果皮急剧下降、果肉中略有增加。解冻后总体呈下降趋势,2个部位总酚、多酚氧化酶下降明显,而果皮中的绿原酸变化不大。研究表明:软儿梨果实后熟后,酚类物质和多酚氧化酶分布呈现区域化,冷冻贮藏加剧了酚类物质和多酚氧化酶活性的积累,导致了软儿梨冷冻贮藏后果皮先于果肉发生了褐变。     

灰斑病菌对大豆叶片总多酚和总黄酮的诱导研究

利用大豆灰斑病菌不同致病力的生理小种接种抗感不同的大豆品种,结果表明:大豆叶片内总黄酮类物质的含量与品种的抗病性呈明显正相关;感病品种叶片内总多酚含量变化显著降低,抗病品种总体变化不大.接种致病力强的生理小种能够大幅度提高抗病品种体内总黄酮含量,这一结果可以作为筛选和鉴定抗病品种的一种生化指标;大豆灰斑病菌弱毒菌株能够诱导感病品种体内总黄酮含量提高,进而提高大豆的抗病性,这一结果为大豆灰斑病的生物防治提供了依据.     

不同种质李果实酚类物质组分及其含量分析

采用高效液相色谱（HPLC）技术对‘红布林’、‘黑宝石’、‘安哥诺’、‘07-68’、‘436’、‘北国红’、‘金红李’和‘晚熟红李’8个种质李果实酚类物质进行分析。结果表明,8个种质李果实总酚平均含量为2934.05mg/kg,总黄酮平均含量为2155.7mg/kg。原花青素B1、儿茶素、绿原酸、原花青素B2、表儿茶素、原花青素C、芦丁、槲皮素-半乳糖、槲皮素-葡萄糖、槲皮素-木糖醇和槲皮素-吡喃阿拉伯糖平均含量分别为51.94mg/kg、22.80mg/kg、9.85mg/kg、33.91mg/kg、17.08mg/kg、14.89mg/kg、19.79mg/kg、4.66mg/kg、31.39mg/kg、4.16mg/kg和19.99mg/kg。各组分变异系数为33.89%-266.65%,体现出丰富的多样性。不同试材中,‘北国红’富含原花青素B1、儿茶素,‘红布林’富含绿原酸、原花青素B2、表儿茶素和原花青素C,‘晚熟红李’富含芦丁和槲皮素-吡喃阿拉伯糖,‘安哥诺’富含槲皮素-半乳糖,‘436’富含槲皮素-葡萄糖,‘07-68’富含槲皮素-木糖醇。不同种质李果实酚类物质组成及含量体现出明显差异性。     

葡萄糖氧化酶基因植物表达载体的构建

为进一步开展转基因研究创造条件,用限制性内切酶将葡萄糖氧化酶(GOD)基因从pMD/GO载体上切下,定向连接到植物表达载体pROKⅡ的CaMV35S启动子下游和NOS终止子上游,成功地构建了GOD基因植物表达载体pROK/GO。利用冻融法将此表达载体导入根癌农杆菌LBA4404中,提取转化质粒,经PCR和酶切鉴定表明,GOD基因植物表达双元载体构建成功。     

Sulodexide protects against hyperglycemia-induced retinal vascular injury via suppressing NOX4/NLRP3 pathway

AIM To investigate the effect of sulodexide (SDX) on high glucose-induced damage in retinal microvascular endothelial cells. METHODS (1) High-fat diet combined with intraperitoneal injection of streptozocin were used to induce type 2 diabetes mellitus (DM) followed by injection of saline or SDX in C57BL/6J male mice. Retinal microvascular leakage and density, and the protein levels of NLRP3 inflammasome-related proteins, zonula occludens-1 (ZO-1) and NADPH oxidase 4 (NOX4) were measured. (2) Human retinal microvascular endothelial cells (HRMECs) were treated with normal glucose or high glucose with or without SDX, and were further transfected with siRNA to knock down NOX4, or infected by adenovirus to over-express NOX4. The protein levels of ZO-1, VE-cadherin (VE-Cad), NOX4 and NLRP3 inflammasome-related proteins as well as the level of reactive oxygen species (ROS) were detected. RESULTS Treatment with SDX increased the protein level of ZO-1, attenuated retinal leakage and NLRP3 inflammasome activation, and enhanced the density of microvasculature and the number of ganglion cells in diabetic retinas. The protein levels of ZO-1 and VE-Cad were decreased, while the levels of NOX4, NLRP3 inflammasome-related proteins and ROS generation were increased in high glucose-treated HRMECs. Silencing of NOX4 inhibited high glucose-induced increases in NLRP3 inflammasome and ROS generation, and decreases in the protein levels of ZO-1 and VE-Cad. Over-expression of NOX4 significantly increased the levels of NLRP3 inflammasome-related proteins and ROS generation in HRMECs, and reduced the protein levels of ZO-1 and VE-Cad. Treatment with SDX partly reversed NOX4 over-expression-induced changes. CONCLUSION SDX alleviates hyperglycemia-induced retinal microvascular endothelial injury via inhibiting NOX4/ROS/NLRP3 pathways.