香蕉抗坏血酸过氧化物酶基因的克隆及表达分析

抗坏血酸过氧化物酶(Ascorbate peroxidase, APX)是植物重要的过氧化氢(Hydrogen peroxide, H2O2)清除酶之一。通过RACE(Rapid-amplification of cDNA ends)方法从香蕉根系cDNA均一化全长文库中获得一个抗坏血酸过氧化物酶基因,命名为MaAPX2基因。MaAPX2基因是香蕉APX基因编码框全长cDNA,包含一个750 bp的最大开放阅读框(Open Reading Frame, ORF),编码249个氨基酸的蛋白质。蛋白质序列结构域比对发现,在MaAPX2 N端具有典型的APX活性位点(APLMLPLAWHSA)和过氧化物酶近端血红素配体序列(DIVALSGGHTL)的保守结构域,属于典型的APX蛋白。系统进化树比对分析表明,MaAPX2与马蹄莲ZaAPX(AAC08576.1)的亲缘关系较近。组织特异性研究表明,MaAPX2基因组成型表达于香蕉各个组织,在叶片中表达量最大。在耐病和感病品种中,MaAPX2基因均上调表达,但在耐病品种中MaAPX2基因在所有时间点相对于对照增加的倍数均高于感病品种,表明该基因在香蕉的抗病性中起着重要作用。MaAPX2基因可以作为一个新的响应枯萎病侵染的标记基因。     

BTH-mediated antioxidant system responses in apple leaf tissues

BTH (S-methylbenzo-1,2,3-thiadiazole-7-carbothiate), an active compound of the commercial preparation Bion, has been studied as an elicitor of resistance to fire blight (Erwinia amylovora) in apple. However, the biochemical mechanisms of its action are not fully elucidated. Our study indicated that BTH at the best time of its protection activity (2–14 days after application) induced changes in prooxidant–antioxidant balance in the leaves of apple trees, but in different ways in the enzymatic and nonenzymatic antioxidants. Glutathione as low molecular antioxidant as well as superoxide anion radical and lipid peroxides as oxidants exhibited changes at the early phase of BTH action. Glutathione-dependent enzymes were strongly affected by the elicitor used. On the 2nd day glutathione transferase (GST) and glutathione peroxidase (GSH-Px) activities increased by about 70% and 30% above the control, respectively. GST activity normalized about the 14th day but GSH-Px at the same time showed 27% of the control value. Among enzymes utilising hydrogen peroxide only catalase showed increase (37%) at the early phase of experiment. Compared with the control, BTH-treated plants did not show changes in ascorbate peroxidase and phenylalanine ammonia-lyase activities. Tocopherol (TOC) level diminished starting from the 7th day after BTH treatment and on the 14th day it was only 28% of the control. It is proposed that extinguishing of BTH-mediated signal resulted from TOC and glutathione action. The diminished ascorbate level at all examined times may play a crucial role in BTH-mediated cell growth regulation. The direct influence of BTH on lipid metabolism should be also taken into consideration.     

枸杞抗坏血酸过氧化物酶基因的克隆与表达分析

为探索青海枸杞抗坏血酸过氧化物酶(APX)基因信息,并对枸杞APX基因进行生物信息学分析以及基因表达研究,通过RT-PCR和RACE方法克隆枸杞APX基因,利用生物信息学软件预测基因结构,采用实时荧光定量PCR方法分析基因表达的变化。结果显示:枸杞APX基因的全长cDNA序列为1 047bp(Genbank No.KX981601),命名为LcAPX基因。该基因含有1个753bp的完整开放阅读框(ORF),编码250个氨基酸,包含亚铁血红素结合位点、底物结合位点和K~+结合位点。枸杞LcAPX与茄科植物的APX蛋白聚为一类,说明两者的亲缘关系较近。LcAPX基因在枸杞的成熟叶中表达量最高,花中表达量最少。其在聚乙二醇(PEG)、氯化钠(NaCl)胁迫下诱导表达,显示LcAPX在枸杞抗干旱和抗盐逆境过程起一定作用。     

茶树鲜叶抗坏血酸过氧化物酶活性的变化规律及测定方法

探讨茶树鲜叶抗坏血酸过氧化物酶(APX)活性的测定条件,分析不同茶树品种以及茶树鲜叶不同叶位APX活性的变化.结果表明:聚乙烯聚吡咯烷酮(PVPP)加入量约为鲜叶重的1.5倍,酶提取液pH为7.8.反应液pH为7.O,抗坏血酸(AsA)浓度为0.50 mmol/L时茶树鲜叶APX活性最高;茶树鲜叶APX活性随冷藏时间的延长呈下降趋势;福建7个主要茶树品种以福鼎大白茶的APX活性最高,铁观音的APX活性最低;茶树鲜叶不同叶位APX活性变化趋势为:芽>第一叶>第二叶>第三叶>老叶>嫩茎.     

Effects of dietary ascorbic acid levels on cholesterol metabolism in rainbow trout (Oncorhynchus mykiss)

An 80‐day feeding trial was conducted to evaluate the effects of dietary ascorbic acid (AA) supplementation at different levels (0, 0.2 and 0.4 g/kg l ‐ascorbate‐2‐polyphosphate; 7.6, 77.2 and 146.7 mg/kg AA, respectively) on cholesterol metabolism in rainbow trout (Oncorhynchus mykiss). Dietary AA supplementation regardless of inclusion level increased the serum total cholesterol and low‐density lipoprotein (LDL) cholesterol levels and LDL cholesterol/high‐density lipoprotein cholesterol ratio. No significant differences were observed in the serum triiodothyronine and thyroxine levels, faecal cholesterol content, hepatic activity and mRNA expression of acyl‐coenzyme A:cholesterol acyltransferase among the dietary treatments. Dietary AA inclusion increased the faecal bile acid content, hepatic activity and mRNA expression of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase and hepatic cholesterol 7α‐hydroxylase mRNA expression, but decreased the hepatic LDL receptor content. High level of AA supplementation (0.4 g/kg) depressed the serum cortisol levels. These results suggest that dietary supplementation with 0.2?0.4 g/kg l ‐ascorbate‐2‐polyphosphate may increase the serum total cholesterol level in rainbow trout. The cholesterol‐raising effect of AA may be due to the increased hepatic cholesterol production and the depressed cholesterol clearance from serum. In addition, dietary AA inclusion also facilitates the hepatic conversion of cholesterol to bile acids.     

Cotton Leaf Senescence can be Delayed by Nitrophenolate Spray Through Enhanced Antioxidant Defence System

Leaf senescence is an oxidative process, and most of the catabolic processes involved in senescence are propagated irreversibly once initiated. An experiment was conducted to test the hypothesis that nitrophenolates (Atonik, a plant growth regulator) spray can delay the leaf senescence through reduced oxidative damage. Atonik 3.75 g a.i. ha^?1 was sprayed during boll filling stage on cotton, and the senescence process was evaluated by quantifying total chlorophyll contents, photosynthetic rate, Fv/Fm ratio, various reactive oxygen species (ROS) content, antioxidant content and antioxidant enzyme activity from 90 days after sowing (DAS) to 130 DAS. The result indicated that nitrophenolate spray reduced the hydrogen peroxide (H₂O₂), superoxide anion (O₂^?) accumulation, lipid peroxidation (malondialdehyde), lipoxygenase activity and membrane permeability over unsprayed control. The antioxidant enzyme activity (superoxide dismutase, SOD; ascorbate peroxidase, APX; peroxidase, POX; glutathione peroxidase, GSH‐Px) were significantly increased by the nitrophenolate spray. POX (118.1 %) and GSH‐Px (143.3 %) activities were enhanced to a higher level compared to APX (8.5 %) activity at 130 DAS. Enhanced accumulation of ascorbate (144.9 %), phenol (154.7 %) and proline (50 %) was seen in nitrophenolate‐sprayed plants compared with unsprayed control plants at 130 DAS. Ascorbate content is increased by higher dehydroascorbate reductase enzyme activity. Ascorbate was thus able to replenish reducing equivalents to phenoxyl radicals resulting in an increase in phenolic compounds. The increased phenolic acid content may be involved in scavenging the ROS produced during senescence process. The higher level of reduced ascorbate and low level of endogenous H₂O₂ in the leaves may be the prerequisite for delayed leaf senescence in the nitrophenolate‐sprayed plants. Based on the present work, it can be concluded that nitrophenolate‐sprayed plants can postpone the leaf senescence by peroxide/phenolic/ascorbate system which is involved in scavenging the ROS produced during leaf senescence.     

杂交小麦抗坏血酸过氧化物酶基因克隆及其在种子老化中的潜在功能分析

以BS型二系杂交小麦BS1453/11GF5135及其BS1453(母本)、11GF5135(父本)的种子为材料,在BS1453/11GF5135中通过同源克隆获得抗坏血酸过氧化物酶基因TaAPX。该基因包含一个832 bp的ORF,共编码277个氨基酸。通过进一步生信分析预测miRNA与TaAPX基因的互作关系,发现TaAPX基因可能受miR396等抗逆及种子活力相关miRNAs的调控。另外通过蛋白互作预测分析,发现APX蛋白主要与氧化还原相关的酶互作反应。通过对不同老化时间的父母本及杂交种的胚进行qPCR及酶活性分析,发现TaAPX基因在杂交种及亲本中的表达趋势是先被诱导上调表达,后下调表达,但杂交种子内TaAPX基因的表达量在第7天才开始下降,而亲本种子内TaAPX基因的表达量在第5天开始下降,且miR396与TaAPX互为拮抗作用。随着老化时间的延长,亲本种子内APX酶活性表现为下降趋势,杂交种子内APX酶活性在第3天呈短暂下降趋势,随后上升,第9天开始下降,表明在老化条件下杂交种子内APX酶清除体内过氧化物的能力高于亲本,即杂交种抗老化能力高于其父母本,且TaAPX基因对种子活力的调控是一个复杂的多因素参与的调控过程。本研究为进一步研究BS型杂交小麦种子活力的分子调控机理奠定了一定的基础。     

Overexpression of a Cytosolic Ascorbate Peroxidase Gene,OsAPX2, Increases Salt Tolerance in Transgenic Alfalfa

Alfalfa （Medicago sativa L.） is an important forage crop in the world and it is of great signiifcance for the improvement of its salt tolerance. To improve salt tolerance in alfalfa, a rice ascorbate peroxidase gene （OsAPX2） was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation with marker gene bar. The different T-DNA insertions in T1 transgenic alfalfa were identiifed by Southern hybridization. Three independent T2 transgenic lines were selected for stress analysis and the results showed that all of them were salt tolerant compared with wild-type plants. The transgenic plants had low levels of H2O2, malondialdehyde and relative electrical conductivity under salt and drought stresses. Moreover, the contents of chlorophyll and proline, and APX activity were high in transgenic plants under salt and drought stresses. Taken together, the overexpression of OsAPX2 enhances salt tolerance in alfalfa through scavenging reactive oxygen species.     

Photosynthetic characteristics and protective mechanisms against photooxidation during high temperature stress in two citrus species

Exposure of Satsuma mandarin (Citrus unshiu Marc.) and Navel orange (Citrus sinensis Osbeck) plants to high temperature (38 °C) led to reductions of the net photosynthetic rate (Pn), the photorespiration rate (Pr), the quantum efficiency CO₂ assimilation (ΦCO₂${\Phi }_{\text{C}{\text{O}}_2}$), the maximal photochemical efficiency of PS2 (Fv/Fm), the photochemical quenching (qP) and the quantum efficiency of PS2 photochemistry (Φ_PS2), whereas the minimal fluorescence yield (Fo) and the non-photochemical quenching (qN) increased. Increase in the value of Pr/Pn and ΦPS₂/ΦCO₂${\Phi }_{\text{PS}2}/{\Phi }_{\text{C}{\text{O}}_2}$ was attributed to the greater decrease in Pn and ΦCO₂${\Phi }_{\text{C}{\text{O}}_2}$ than Pr and Φ_PS2. In addition, the superoxide radical (O₂⁻) production, the H₂O₂ concentration and the activities of antioxidant enzymes such as the superoxide dismutase (SOD, EC 1.15.1.1), the ascorbate peroxidase (APX, EC 1.11.1.11), the dehydroascorbate reductase (DHAR, EC 1.8.5.1) and the catalase (CAT, EC 1.11.1.6) were raised. On the other hand, the chlorophyll concentration in leaves decreased during high temperature stress. These results suggest that decline in Pn related to inactivation of PS2 reaction centers may be due to the enhanced number of active oxygen species in the citrus leaves. The water–water cycle may play a role in limiting the degree of photodamage caused by high temperature. Lower O₂⁻ production rate, the H₂O₂ concentration and the antioxidant enzymes activity were observed in high temperature tolerant species of citrus. The exogenous active oxygen scavenger ascorbic acid (Asc) enhanced the ability to clear the O₂⁻ in citrus plants, and quicken the recovery of photosynthetic apparatus.