DNA甲基化参与调控马铃薯响应干旱胁迫的关键基因挖掘

植物受到干旱胁迫时,会通过DNA甲基化做出快速反应以帮助其应对胁迫。为探究在干旱胁迫下,DNA甲基化是如何影响基因转录表达,本研究对甘露醇模拟干旱和5-azadC(去甲基化)处理下,抗旱性不同的2个马铃薯品种(抗旱型,青薯9号;干旱敏感型,大西洋)进行转录组学分析,以Fold-change>2和校正后P<0.01进行差异表达基因(DEG)的筛选。GO富集分析发现,2种处理都共同显著富集到氧化应激和碳水化合物代谢过程相关的GO term。说明不同耐旱性马铃薯在响应干旱胁迫时,与这些GO term相关的基因也受DNA去甲基化调控。对既响应干旱又响应DNA去甲基化的1345个DEG进行KEGG功能富集发现,与植物抗旱相关的通路有植物MAPK信号途径、植物激素信号转导途径、植物谷胱甘肽代谢通路、糖酵解与糖异生和磷酸肌醇代谢通路。说明这些通路相关基因在大西洋和青薯9号2个抗旱性不同的马铃薯品种中,响应干旱的敏感性受DNA甲基化调控。接着对DEG上游1500 bp启动子区域进行顺式作用原件和甲基化CpG岛分析发现,干旱胁迫下参与植物谷胱甘肽代谢的GST基因通过DNA去甲基化来降低启动子区ABRE和CAAT-box作用元件的甲基化水平,进而激活该基因的表达以应对干旱胁迫。因此,利用比较转录组学分析干旱和DNA去甲基化处理下的差异基因,可挖掘到DNA甲基化参与调控马铃薯响应干旱胁迫的相关基因,为研究马铃薯干旱胁迫响应的表观遗传学机理提供新的研究思路。     

茶树谷胱甘肽过氧化物酶编码基因CsGPX1功能分析

为明确茶树[Camellia sinensis(L.)O.Kuntze.]谷胱甘肽过氧化物酶(Glutathione peroxidases,GPX)编码基因CsGPX1的功能,本文利用茶树转录组数据克隆获得CsGPX1的编码区全长序列,进行序列比对与分析,在此基础上,将CsGPX1在烟草中进行过表达获得转CsGPX1基因烟草,并比较野生型烟草与转基因烟草耐旱性的差异,验证CsGPX1功能。序列分析表明,CsGPX1编码序列(CDS)长723 bp,编码240个氨基酸。BLAST比对发现,该基因与桃树(Prunus persica)的GPX基因具有高度同源性(>85%)。CsGPX1编码的氨基酸序列具有GPX蛋白保守特征序列和区别于其他家族成员的特有的结构域——磷脂氢谷胱甘肽过氧化物酶(PHGPX)。干旱胁迫处理结果显示,过表达CsGPX1烟草株系抗旱性强于野生型。GPX酶活性测定结果表明,转基因植株的GPX酶活性高于野生型植株。以上研究结果表明,过表达CsGPX1可提高转基因烟草的耐旱能力,说明CsGPX1可能与茶树的耐旱性能相关。本研究为提高茶树的耐旱性能研究提供了新的理论途径,并对降低茶园管理的成本具有一定的积极意义。     

NADP-malic enzyme and glutathione reductase contribute to glutathione regeneration in Fragaria vesca fruit treated with protective high CO2 concentrations

Treatment of harvested strawberries (Fragaria vesca L. Mara des Bois) with high concentrations of CO₂ is an effective means of limiting fungal decay and avoiding disorders caused by low temperature storage. In the present study, we investigated the role of NADP-ME gene expression and activity in lowering malic acid contents and in the provision of NADPH required for the regeneration of the reduced form of glutathione (GSH). We also measured glutathione reductase (GR: EC 1.6.4.2) activity in strawberries treated with different high CO₂ concentrations (0, 20 and 40% for 3 days) during storage at 0 °C. A decrease in malic acid content in fruit exposed to 20% CO₂ was primarily mediated by the stimulation of NADP-ME activity, rather than associated with changes in the expression of cytosolic NADP-ME genes. Moreover, malic acid decarboxylation was associated with a marked increase in GR activity, which may account for the increased levels of glutathione in fruit following exposure to 20% CO₂. This chain of events was not observed in untreated fruit stored in air or in fruit treated with 40% CO₂, suggesting that the unique cellular redox status of 20% CO₂-treated fruit plays an important role in detoxification and protection from damage during storage. Based on these findings, we propose that NADP-ME activation in fruit exposed to 20% CO₂ provides NADPH for glutathione regeneration by GR, thereby conferring protection against the cellular damage caused by low temperatures or excessive high CO₂ levels.     

外源谷胱甘肽对嫁接黄瓜砧穗种子萌发过程中自毒胁迫的缓解效应

为探讨外源谷胱甘肽(GSH)对自毒作用的缓解效果和生理作用机制,以连作嫁接黄瓜根际土壤浸提液模拟自毒胁迫,在100g·L~(-1)浓度土壤浸提液中分别添加0、25、50、100、200 mg·L~(-1)GSH,进行黄瓜、南瓜种子萌发试验,分别测定发芽率、主根长度、根系表面积、根尖数、α-淀粉酶活性、淀粉酶总活性和脯氨酸含量等指标。结果表明,土壤浸提液处理后,黄瓜和南瓜种子的发芽率、发芽指数、活力指数、主根长度、根尖数、根系表面积、α-淀粉酶活性、淀粉酶总活性得到显著抑制,脯氨酸含量升高。在自毒作用下,外施不同浓度GSH,可不同程度地提高种子发芽率、发芽指数、活力指数、主根长度、根系表面积、α-淀粉酶活性、淀粉酶总活性,降低脯氨酸含量。土壤浸提液能对黄瓜、南瓜种子产生自毒作用,添加外源GSH能诱导黄瓜和南瓜种子内淀粉酶活性增强,有效缓解自毒作用对黄瓜、南瓜种子萌发和幼苗生长的抑制作用,并且以100 mg·L~(-1)GSH缓解效果最显著。本研究结果为制定缓解嫁接黄瓜连作障碍措施提供了理论依据。     

绿盲蝽和中黑盲蝽解毒代谢酶活性的检测

害虫解毒代谢增强是对杀虫剂产生抗性的重要机制。为此,本研究建立了Bt棉田重要害虫绿盲蝽和中黑盲蝽成虫多功能氧化酶、酯酶和谷胱甘肽S转移酶3种解毒酶活性的测定方法。研究结果表明:以DCNB(1,2二氯4硝基苯)为底物,不能检测到棉盲蝽谷胱甘肽S转移酶的活性;棉盲蝽多功能氧化酶活性也不宜选用动力学法进行测定。同时2种棉盲蝽成虫的日龄对解毒酶活性影响不明显。因此,对由不同日龄成虫组成的棉盲蝽田间种群可以直接进行解毒酶活性的检测。     

Oxidative homeostasis in oocyte competence for in vitro embryo development

The objective of this study was to evaluate the levels of reactive oxygen species (ROS) and glutathione (GSH) in oocytes from follicles of different diameters and their relevance in the in vitro production of embryos (IVPE). Bovine ovaries were aspirated according to the diameter of the follicle [2–8 (general), 4–8 (large), and 2 < 4 mm (small)]. The oocytes were evaluated for levels of ROS, GSH, in vitro maturation, and IVPE. Higher levels of ROS and GSH were observed (p < 0.05) in oocytes of the large group (85.6 ± 7.2 and 140.0 ± 9.6) followed by those in the general (81.1 ± 10.5 and 134.3 ± 7.8) and small (73.5 ± 10.1 and 125.0 ± 10.6) groups. However, the proportion of ROS/GSH did not differ (p > 0.05) between the general, large, and small groups. The maturation was higher (p < 0.05) in the large group (87.8 ± 3.0%) than in the small group (72.2 ± 5.8%), but both were similar (p > 0.05) to that in the general group (82.2 ± 2.5%), whereas the IVPE of the large group (57.3 ± 3.0%) was higher (p < 0.05) than those in the general (44.7 ± 4.4%) and small (34.0 ± 4.0%) groups. We report that oocytes from large follicles are more competent for IVPE, whereas higher levels of ROS and GSH appear to be correlated with oocyte competence, as long as oxidative homeostasis is retained.     

Hepatic endogenous defense potential of propolis after mercury intoxication

Exposure to mercuric chloride (HgCl₂; 5 mg kg^?1 body weight; i.p.) induced oxidative stress in mice and substantially increased lipid peroxidation (LPO) and oxidized glutathione (GSSG) levels, decreased the level of reduced glutathione (GSH) and various antioxidant enzymes in liver and also increased the activities of liver marker enzymes in serum. Therapy with propolis extract, a resinous wax‐like beehive product (200 mg kg^?1 orally, after mercury administration), for 3 days inhibited LPO and the formation of GSSG and increased the level of GSH in the liver. Release of serum transaminases, alkaline phosphatase, lactate dehydrogenase and γ‐glutamyl transpeptidase were significantly restored after propolis treatment. The activities of antioxidant enzymes, that is, superoxide dismutase, catalase, glutathione‐S‐transferase and glucose‐6‐phosphate dehydrogenase, were also concomitantly restored towards normal levels after propolis administration. These observations clearly demonstrate that propolis treatment augments antioxidant defense against mercury‐induced toxicity and provide evidence that propolis has therapeutic potential as a hepatoprotective agent.     

Effects of dietary reduced glutathione on the growth and antioxidant capacity of juvenile Atlantic salmon (Salmo salar)

To investigate the effects of dietary reduced glutathione (GSH) on the growth performance and antioxidant capacity of juvenile Atlantic salmon (Salmo salar), 396 juvenile fish with initial body weight of 143.07 ± 6.56 g were randomly distributed into four groups fed four diets with graded supplementation levels of GSH (0, 100, 200 and 400 mg/kg diet) for 83 days. The results showed that the appropriate GSH supplementation (100 and 200 mg/kg diet) significantly increased the growth performance, activities and gene mRNA expression levels of glutathione peroxidase (GPx) and glutathione transferase (GST), and the content of GSH and total antioxidant capacity (TAOC), whereas it significantly decreased activities and gene mRNA expression levels of superoxide dismutase (SOD) and catalase (CAT), and the content of malondialdehyde (MDA; p < 0.05). However, the excess dietary GSH (400 mg/kg diet) had an adverse effect on the all above indexes. Interestingly, the dietary GSH had the opposite effect on GSH‐related antioxidant enzymes (GPx and GST) and other antioxidant enzymes (SOD and CAT). The results showed that the diet with 200 mg/kg GSH supplementation was optimal for the juvenile Atlantic salmon, which had a measured GSH content of 209.54 mg/kg.     

The effects of dietary selenium on growth performances,oxidative stress and tissue selenium concentration of gibel carp (Carassius auratus gibelio)

A 100‐day growth trial was executed to determine the dietary selenium (Se) requirement of juvenile gibel carp (Carassius auratus gibelio). Selenomethionine was supplemented to casein‐gelatin diets at 0, 0.1, 0.25, 0.5, 1.0, 2.5 and 5 mg Se kg^?1, respectively. Each of these seven semi‐purified diets containing 0.34, 0.47, 0.66, 0.82, 1.23, 2.77 and 5.13 mg Se kg^?1 was fed to triplicate groups of gibel carp (2.74 ± 0.02 g) in a flow‐through system. No behaviour abnormalities and no mortality were found in fish exposed to dietary Se concentrations. With the increasing dietary Se supplemented concentrations, weight gain of fish remarkably increased at the levels of ≤1 mg Se kg^?1 diet and then showed no significant difference above 1 mg Se kg^?1 levels. Although growth performances (weight gain, hepatosomatic index, condition factor and survival) were not impaired in gibel carp fed at above the levels of 2.5 mg Se kg^?1, indicators of oxidative stress were changed significantly. Serum superoxide dismutase (SOD) activities significantly declined, hepatic glutathione peroxidase (GPx) activities significantly increased and the tissue Se concentrations significantly raised at the highest supplemented level of 5 mg Se kg^?1. A clear linear relationship between Se‐depended GPx activities and hepatic Se concentrations was observed. The present results indicated that the dietary Se requirement for gibel carp is 1.18 mg Se kg^?1 diet based on weight gain, GPx activities and tissue accumulation.     

不同海拔地区牦牛心肌、骨骼肌GSH-PX的测定

在西宁屠宰场对来自青海省两个不同海拔县牦牛心肌和骨骼肌线粒体内谷胱甘肽过氧化物酶(GSH-PX)活性进行测定。结果表明,海拔4300m左右的玛多县牦牛心肌、骨骼肌GSH-PX分别为20.15U.mg-1±5.43U.mg-1和15.32U.mg-1±4.27U.mg-1,海拔3400m左右的刚察县牦牛心肌、骨骼肌GSH-PX分别为6.67U.mg-1±5.67U.mg-1和7.76U.mg-1±4.83U.mg-1;玛多牦牛的心肌、骨骼肌组织线粒体的GSH-PX活性显著高于刚察牦牛(P<0.05)。在高原低氧环境下,牦牛体内GSH-PX活性与海拔高度呈正相关。