马铃薯抗坏血酸含量及其代谢相关酶活性关系的研究

 为探讨马铃薯不同器官中抗坏血酸(AsA) 含量及其代谢相关酶活性关系, 研究了马铃薯幼叶、功能叶、老叶、茎和块茎中AsA和其氧化态脱氢抗坏血酸(DHA) 的含量与L - 半乳糖- 1, 4 - 内酯脱氢酶( GalLDH) 、脱氢抗坏血酸还原酶(DHAR) 、谷胱甘肽还原酶( GR ) 、抗坏血酸过氧化物酶(APX) 、抗坏血酸氧化酶(AO) 和单脱氢抗坏血酸还原酶(MDHAR) 等6种酶活性之间的相关性。结果表明, 马铃薯AsA在幼叶和块茎中含量很高。叶片和茎的抗坏血酸库(AsA与DHA之和) 水平与GalLDH活性显著相关, 而AsA含量与DHAR活性显著相关, DHA含量与APX活性显著相关。说明在马铃薯幼叶中高含量的AsA可能由于GalLDH和DHAR的高活性; 而块茎中AsA的积累, 主要来自于叶片的运输和DHAR催化的DHA再生。     

Ascorbate levels and the activity of key enzymes in ascorbate biosynthesis and recycling in the leaves of 22 Chinese persimmon cultivars

The objective of this study was to determine diversities of ascorbic acid (AsA) and activities of enzymes involved in AsA metabolism, and to determine their relationships to AsA content in the leaves of Chinese persimmon (Diospyros kaki) cultivars. Results showed that there were huge differences not only in the contents of AsA and glutathione (GSH) but also in the enzyme activities of l-galactono-1,4-lactone dehydrogenase (GLDH, EC 1.3.2.3), dehydroascorbate reductase (DHAR, EC 1.8.5.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) in the leaves of 22 Chinese persimmon cultivars. Control of AsA levels mainly depended on the capacity for biosynthesis and GLDH activity played a main role in determining AsA levels in the leaves of persimmon. Both MDHAR and DHAR, MDHAR in particular, also played an important role in maintaining AsA redox state by reducing oxidized AsA back to AsA. But contents of GSH and hydrogen peroide (H₂O₂) and activity of APX were not significantly correlated with AsA content in the leaves of persimmon.     

黄瓜叶面喷施硅肥对果实糖分和维生素C积累及相关酶的影响

研究了黄瓜叶面喷施硅肥对其生长、产量、果实糖分、维生素C及相关酶活性和基因表达量的影响。结果表明：叶面喷施硅肥显著促进了黄瓜生长,增加了单株产量,提高了果实中碱性转化酶（NI）、蔗糖磷酸合成酶（SPS）、蔗糖合成酶（SS）及半乳糖内脂脱氢酶（GaILDH）活性,提高了果实果糖、葡萄糖和维生素C含量。其中,1.420 g · L^-1单硅酸处理效应最显著,其果糖、葡萄糖和维生素C含量分别较对照高13.79%、18.08%和75.11%。实时荧光定量PCR分析表明,叶面喷施单硅酸的黄瓜果实碱性转化酶–3（NI-3）、蔗糖磷酸合成酶（SPS）和半乳糖内脂脱氢酶（GaILDH）基因表达量显著提高。     

Modulation of manganese toxicity in Pisum sativum L. seedlings by kinetin

In the present study, the effects of kinetin (KN; 10 and 100 μM) application under manganese toxicity (Mn; 50, 100 and 250 μM) were investigated, on growth, photosynthetic pigments, total protein, total nitrogen, ammonium (NH₄⁺) content, NH₄⁺ assimilating enzymes and antioxidant system in pea seedlings. The exposure of pea seedlings to Mn and 100 μM of KN alone and in combination, caused decrease in growth, photosynthetic pigments, total protein and total nitrogen contents, and an increase in NH₄⁺ content. However, application of 10 μM of KN together with Mn reduced the Mn toxicity symptoms, promoted the growth of seedlings and led to the decrease in NH₄⁺ content compared to Mn treatments alone. The root and shoot activities of glutamine synthetase (GS), glutamate oxoglutarate aminotransferase (GOGAT) and catalase (CAT) were decreased while glutathione reductase (GR) and dehydroascorbate reductase (DHAR) activities exhibited differential responses when pea seedlings were exposed to Mn and 100 μM of KN. However, under similar treatments, activities of glutamate dehydrogenase (GDH), superoxide dismutase (SOD) and ascorbate peroxidase (APX) in root and shoot were increased. It was noticed that addition of 10 μM of KN together with Mn, caused significant stimulation in activities of enzymes of NH₄⁺ assimilation and antioxidant defense system even over their respective control values. Non-enzymatic antioxidants (ascorbate and glutathione) in root and shoot of pea seedlings exposed to Mn stress were significantly increased by the addition of 10 μM of KN. Therefore, ameliorative effect of 10 μM of KN against Mn toxicity was observed. This study thus suggests that 10 μM of KN appreciably improves Mn tolerance of pea seedlings under Mn toxicity while reverse effects were exhibited by 100 μM of KN.     

中国野生葡萄醛脱氢酶基因在拟南芥中的过量表达

将质粒pWR306中包含ED35s启动子、Omega元件及TNOS终止子的一段核苷酸序列定向克隆到质粒pCAMBIA1300,构建了植物中间表达载体pWR-II;将重组质粒pGEM-T-ALDH的醛脱氢酶(ALDH)基因片段定向克隆到pWR-II,构建了葡萄ALDH基因的植物过量表达载体pWR-II-ALDH。采用改进冻融法将其导入农杆菌EHA105,采用花序浸蘸法转化拟南芥,获得了潮霉素抗性的转化拟南芥植株。PCR检测证明外源基因已整合进拟南芥基因组,Real-timePCR结果表明ALDH基因在转化植株的表达量远高于野生种。转化植株和野生植株在形态上有一定的差异,说明葡萄ALDH基因在拟南芥中表达对其生长发育有一定影响。     

阔叶猕猴桃果实GalDH cDNA 克隆及其在大肠杆菌中的表达

 以猕猴桃属植物中果实维生素C含量最高的阔叶猕猴桃(Actinidia latifolia Merr. ) 果实为试 材, 经RT2PCR扩增获得约110 kb的L - 半乳糖脱氢酶cDNA片段。生物信息学分析表明, 该cDNA片段长为997 bp, 最大开放阅读框为960 bp, 可编码319 个氨基酸残基, 命名为A lGalDH, GenBank登录号为EU525846, 核苷酸序列及其推导的氨基酸序列与已知其它植物GalDH核苷酸、氨基酸序列间的同源性分别在76%和70%以上, 且具有醛酮还原酶的保守结构域。构建了其原核表达载体pET-A lGalDH并转化大肠杆菌BL21, 经0.1 mmol·L ^{- 1} IPTG诱导, 获得具有较高活性的表达融合蛋白6 ×His-AlGalDH。经Ni-His亲和磁珠分离纯化, 获得单一的融合目的蛋白条带, 测定其酶活为120 pmol·min^{- 1} ·mg^{- 1}。     

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。     

猪链球菌2型次黄嘌呤核苷酸脱氢酶(IMPDH)重组蛋白的酶活性和免疫原性检测

通过PCR方法从猪链球菌2型(Streptococcus suis)05ZYH33分离株基因组中扩增出次黄嘌呤核苷酸脱氢酶基因(inosine 5-monophosphate dehydrogenase,impdh),长度1 064 bp.PCR产物和pET28a载体分别经过EcoR Ⅰ和Xho Ⅰ双酶切,连接,成功地构建了重组表达质粒pET28a-impah,并转入大肠杆菌(Escherichia coli) BL21(DE3)中,经过1 mmol/L IPTG诱导获得表达,蛋白大小35kD.表达蛋白具有良好的抗原性和酶活性.蛋白经过亲和层析纯化后,在37℃,pH7.0～9.0表现出较强酶活性,NADH A_(340)介于0.662～0.816之间.     

水稻白叶枯病菌对拌种灵抗药性分子机制研究

  通过紫外诱变获得了抗拌种灵水稻白叶枯病菌（Xanthomonas oryzae pv.oryzae）的突变体，这些突变体可以在含100 μg·ml-1拌种灵平板上生长，而敏感菌株在10 μg·ml-1浓度下则不能生长。敏感菌株琥珀酸脱氢酶活性受拌种灵强烈抑制，而抗药突变体的酶活性较低，且不受药剂抑制。应用6对引物，从水稻白叶枯病菌野生敏感菌株和室内诱导抗药性菌株中扩增到琥珀酸脱氢酶基因全序列。该基因全长3 616 bp，编码1 115个氨基酸，含有2个内含子。与柑橘溃疡病菌（Xanthomonas axonopodis pv. citri）琥珀酸脱氢酶核苷酸同源性93%，氨基酸同源性97%；与其它6种病原细菌琥珀酸脱氢酶基因编码的氨基酸序列同源性为43%～95%。敏感菌株和抗药菌株的琥珀酸脱氢酶序列分析表明，琥珀酸脱氢酶铁硫蛋白亚基中229位氨基酸由组氨酸（CAC）突变为酪氨酸（TAC）是导致X. oryzae对拌种灵产生抗药性的主要原因。