Oxygen enrichment with magnesium peroxide for minimizing hypoxic stress of flooded corn

Flooding/waterlogging is a major factor responsible for hypoxic stress in agriculture. The aim of this study was to develop an effective oxygen buffer with magnesium peroxide (MgO₂) to generate hydrogen peroxide (H₂O₂) and release bioavailable oxygen. MgO₂ provided a relatively stable level (approx. 300 µM) of bioavailable oxygen. The oxygen‐buffer system is adjustable and controllable by adding Mg²⁺ or EDTA to the aqueous system. Regular H₂O₂ was also able to provide bioavailable oxygen but the system was poorly buffered with respect to oxygen release. The accessibility of plants to bioavailable oxygen was indicated by the activity of alcohol dehydrogenase (ADHase, EC 1.1.1.1), an anaerobically induced enzyme of flooded plants. The application of MgO₂ to flooded soil reduced ADHase activity in corn‐root tips by 91.3%. This application of MgO₂ presents a novel pathway to significantly (P < 5%) minimize adverse impacts of hypoxia on flooded corn seedlings. This finding may have broad implications for addressing hypoxicity problems in crop science and technology.     

Purification and Characterisation of Ascorbate Oxidase from Flour and Immature Wheat Kernels

White flour from wheat was shown to contain basic-ascorbate oxidase (AOX) enzymes (pI 7·6–9·6) and acidic-AOX enzymes (pI 5·1–6·6) in a ratio of 0·4:1, based on chromatography data. Immature wheat kernels (two weeks post-anthesis) contained about 12 times more AOX activity (units/g dry weight) than flour from mature grain, and the ratio of basic- to acidic-AOX was 5:1. Acidic-AOX was purified 90-fold from flour by hydrophobic interaction, gel filtration and anion exchange chromatography. Basic-AOX was purified 20 000-fold from immature wheat by hydrophobic interaction, anion exchange, cation exchange and gel filtration chromatography in a yield of 5%. The acid-AOX had a M of 140 k, was optimally active at pH 6·3 and 40 °C, and was stable in the pH range 5–9 and at 30 °C for 0·5 h at pH 6·2. The Km values were 0·26 m for L-ascorbic acid and 0·93 m for D-iso ascorbic acid. The basic-AOX had a M of 139 k and subunit M of 72 k. The enzyme was optimally active at pH 6·2 and 50 °C, and was stable in the pH range 5–9 and at 40 °C for 0·5 h at pH 6·2. The Km values were 0·30 m for L-ascorbic acid and 0·53 m for D-iso ascorbic acid. The absorption spectrum of basic-AOX had absorption maxima at 280 nm and 607 nm of similar magnitude to those measured in AOX fromCucurbita species (squash). This indicates that wheat AOX contains protein-bound copper similar to other plant AOX.     

转甜菜碱醛脱氢酶基因提高烟草抗旱及耐盐性

将甜菜碱醛脱氢酶（BADH）基因与组成型启动子CaMV 35S启动子融合,构建了植物表达质粒pBIBB。通过根癌农杆菌介导将BADH基因导入烟草,经PCR、Southern杂交、Northern杂交证明BADH基因已整合到烟草基因组中并在转基因植株中转录和表达。测定转基因植株叶片中甜菜碱醛脱氢酶活性,结果显示对照植株没有BADH酶活性,转基因植株的各个株系间甜菜碱醛脱氢酶比活力差异较大,范围在0.1~1.0 U mg-1间。转BADH基因的烟草在盐胁迫和聚乙二醇（PEG）胁迫条件下生长状态良好,生长势强于未转基因植株,说明BADH基因能在异源植物中正常翻译、表达和用于植物抗旱、耐盐基因工程的研究。     

五汁饮对急性酒精中毒大鼠肝脏的保护机制研究

目的 研究五汁饮对急性酒精中毒大鼠肝脏的保护作用。方法 SD大鼠分为5组,分别为空白组、模型组、葛花组、五汁饮低剂量组及高剂量组,每组10只。预防性给药 7 d后,空白组蒸馏水灌胃,其余各组按照1.5 mL/100 g的52度红星二锅头给予灌胃,每日1次,连续1周,建立急性酒精性肝损伤模型。测定血清谷丙转氨酶（ALT）、谷草转氨酶（AST）含量;测定肝脏匀浆中谷胱甘肽（GSH）、超氧化物歧化酶（SOD）、丙二醛（MDA）、乙醇脱氢酶（ADH）含量。结果 与模型组相比,葛花组、五汁饮高剂量组ALT含量降低（P<0.05）,五汁饮高、低剂量组AST含量降低（P<0.01）;五汁饮高剂量组ADH含量高于模型组（P<0.05）,葛花组、五汁饮低剂量组与模型组没有统计学差异（P>0.05）;葛花组、五汁饮高、低剂量组GSH、SOD含量高于模型组（P<0.01,P<0.05）,葛花组、五汁饮高、低剂量组均可显著降低MDA含量（P<0.01）。结论 五汁饮保肝作用可能通过激活ADH加快酒精代谢及SOD、GSH的抗氧化作用来实现。     

多粘芽孢杆菌中2,3-丁二醇脱氢酶的克隆和表达

将来自于多粘芽孢杆菌(Paenibacillus polymyxa)中的2,3-丁二醇脱氢酶基因pla C在大肠杆菌(Escherichia coli)BL21中进行了诱导表达,并对其酶学性质进行研究。结果表明,该基因的表达产物主要是生成内消旋型2,3-丁二醇,其氧化2,3-丁二醇的最适温度是50℃,最适pH为10,而还原乙偶姻的最适温度为60℃,最适pH为6。     

高温胁迫对棘胸蛙蝌蚪抗氧化酶活力的影响

实验利用水温从18~30℃骤升和缓升两种模式,高温胁迫处理棘胸蛙(Quasipaa spinosa)蝌蚪后,提取其尾部肌肉和内脏团的组织液,进行乳酸脱氢酶(LDH)、谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、碱性磷酸酶(AKP)等酶活力和丙二醛(MDA)含量测定。结果表明,棘胸蛙蝌蚪尾部肌肉提取液的LDH活力显著高于内脏团(P0.01);高温胁迫下棘胸蛙蝌蚪尾部肌肉和内脏团提取液的LDH活力显著提高(P0.01)。高温胁迫下棘胸蛙蝌蚪尾部肌肉和内脏团的GSH-Px活力均显著降低(P0.01,P0.05),且水温骤升模式下的内脏团GSH-Px活力显著高于缓升模式(P0.01)。在30℃高温胁迫时,棘胸蛙蝌蚪尾部肌肉和内脏团SOD活力均显著降低(P0.05,P0.01),且水温骤升模式的棘胸蛙蝌蚪尾部肌肉和内脏团SOD活力均高于缓升模式(P0.05,P0.01)。高温胁迫下CAT活力均显著下降(P0.01),且缓升模式下的棘胸蛙蝌蚪CAT活力显著高于骤升模式(P0.05,P0.01)。在两种升温模式下,高温胁迫对尾部肌肉的AKP活力均无显著影响;而在骤升模式下,高温胁迫的棘胸蛙蝌蚪内脏团的AKP活力显著提高(P0.05)。高温胁迫下的棘胸蛙蝌蚪尾部肌肉和内脏团MDA含量均显著升高(P0.01),骤升模式下高温胁迫的棘胸蛙蝌蚪内脏团的MDA含量显著高于尾部肌肉(P0.01)。     

The Suaeda liaotungensis kitag betaine aldehyde dehydrogenase gene improves salt tolerance of transgenic maize mediated with minimum linear length of DNA fragment

In this research we established a particular vector-free and marker-free plant transformation system of maize to overcome the obstacles of biosafety limits. The BADH gene was introduced into maize by pollen-tube pathway, using the principle of minimum linear length of the transformation element, which was composed of only the BADH gene, expression regulatory sequence (35S CAMV promoter, NOS terminator), and T-DNA border sequence at both sides. Twenty-seven of 2076 transformed samples were positive in PCR amplification and the PCR positive rate of T₁ generation was 1.3%. Further Southern blotting results indicated that the BADH gene was integrated into maize genome. Transgenic lines of progeny were examined for tolerance to NaCl by induced salt stress with 250 mM NaCl Hoagland solution. After 15 days of treatment, 73.9–100% of the transgenic seedlings survived and grew well, whereas most wild-type seedlings wilted and showed loss of chlorophyll. Only 8.9% of the wild-type plants survived but gradually died after salt stress. The electrical conductivity of the transgenic line of progeny after salt stress was lower than wild type. The transgenic progeny had higher glycinebetaine and Chlorophyll content than wild type after salt stress.     

牦牛乳酸脱氢酶A的分离纯化、酶学性质及其基因的克隆

 【目的】分离纯化牦牛骨骼肌中的乳酸脱氢酶-A（LDH-A）,并克隆其cDNA。通过比较牦牛与普通牛的LDH-A的酶学性质和cDNA序列,为研究牦牛适应高海拔、低氧环境,在分子水平上找到一些线索。【方法】采用染料亲和层析和DEAE-Sephadex离子交换层析等方法,分别从牦牛和普通牛骨骼肌中纯化LDH-A,并对其酶学性质进行比较;通过RT-PCR方法克隆牦牛LDH-A的cDNA序列,并与GenBank登录的普通牛LDH-A的cDNA序列进行比对。【结果】纯化的牦牛LDH-A比活力为103.9 U•mg-1蛋白,纯化倍数为18.2。SDS-PAGE和PAGE分析均显示一条带。酶动力学参数测定显示,牦牛LDH-A的Km NADH为0.097,Km 丙酮酸钠为1.897,均不同程度高于普通牛,其中对丙酮酸钠的Km大约是普通牛的2倍。根据牦牛LDH-A的cDNA序列预测的氨基酸序列与普通牛LDH-A的氨基酸序列比较,只有2个氨基酸残基的改变（257Val-Ala和315Tyr-Cys）。【结论】牦牛LDH-A对丙酮酸的高Km可避免骨骼肌中产生过多的乳酸,是适应进化的结果;这种升高可能与其氨基酸序列变化导致的空间结构微小改变有关。     

苎麻UDPGDH基因的cDNA克隆及表达分析

【目的】分离苎麻尿苷二磷酸葡萄糖脱氢酶（UDPGDH）基因。【方法】通过简并引物RT- PCR 结合 RACE 技术和半定量RT-PCR。【结果】成功克隆苎麻Boehmeria nivea (Linn.) Gaud.尿苷二磷酸葡萄糖脱氢酶（UDPGDH）基因cDNA序列,序列长1 837 bp,编码一480个氨基酸的蛋白质（GenBank No.EF178294）。半定量RT-PCR分析显示,UDPGDH在苎麻根、皮、茎和叶组织中均有表达,其表达量为茎＞皮＞叶＞根,相对表达量依次为0.7075,0.3051,0.2651,0.1490。【结论】苎麻UDPGDH与其它植物相应序列具有较高同源性,在苎麻茎中有较高的表达。     

转麻疯树甜菜碱醛脱氢酶基因提高烟草耐盐性

 【目的】研究麻疯树甜菜碱醛脱氢酶(JcBD1)的功能。【方法】通过RT-PCR和RACE的方法从麻疯树中克隆甜菜碱醛脱氢酶基因（JcBD1）的全长cDNA序列。将从麻疯树中克隆的JcBD1 cDNA与CaMV 35S组成型启动子融合,构建植物表达载体pBI-JB,利用根癌农杆菌介导转入烟草。对获得抗性的植株用PCR、RT-PCR及Western杂交检测分析,同时测定转基因烟草植株的电导率、JcBD1酶活性及抗盐性等特性。【结果】由JcBD1 cDNA序列推测的JcBD1氨基酸序列与已知的BADH具有很高的同源性,包括BADH家族绝对保守区域十肽“VSMELGGKSP”和半胱氨酸残基。这两部分区域在甜菜碱醛脱氢酶底物特异性结合过程中起着重要作用,与酶的催化活性有关。PCR及RT-PCR检测证明外源JcBD1已整合到烟草基因组中并成功表达,转化率为56.7%。转基因植株的电导率明显低于未转基因植株。盐胁迫处理后,在转基因植株的蛋白提取样中能检测到Western杂交信号,而在未转基因植株中没有检测到杂交信号;转基因植株的蛋白提取样中能检测到甜菜碱醛脱氢酶活性,而对照没有活性,表明JcBD1在转基因植株中得到了表达。转JcBD1烟草在盐胁迫下,生长势明显强于未转基因植株。【结论】JcBD1能在异源植物中正常翻译、表达;JcBD1是盐害胁迫相关的重要基因。