Prokaryotic Expression, Purification and Characterization of a Novel Rice Seed Lipoxygenase Gene OsLOX1

Lipoxygenase (LOX, EC1.13.11.12) is a key enzyme during the degradation of lipids in animals and even plants, and also the first key enzyme responsible for the biosynthesis of jasmonate. To purify and characterize the OsLOX1 gene from rice seeds, the entire coding region of the OsLOX1 gene was inserted into an expression vector pET30a(+) and transformed into Escherichia coli BL21 (DE3). Expression of the fusion protein was successfully induced by isopropyl-β-D- thiogalactopyranoside (IPTG) and the purified ...     

四川不同产区烟叶脂氧合酶活性及基因表达水平的差异

为烟叶香气物质形成机制的研究提供参考,以云烟87和红花大金元2个烤烟品种为试验材料,在四川6个产区采用田间试验研究烤烟旺长期、现蕾期和成熟期烟叶的脂氧合酶活性、基因表达量差异及其相关性。结果表明:在四川6个产区2个烤烟品种不同生育期的脂氧合酶活性及其基因相对表达量存在差异,但变化趋势相同,并多在现蕾期达最高,且各产区云烟87和红花大金元烤烟不同生育期烟叶的LOX酶活性差异多达显著水平。在旺长期和现蕾期云烟87烟叶LOX基因的相对表达量与LOX酶活性呈极显著正相关,相关系数分别为0.96和0.97;在成熟期呈不显著正相关,相关系数为0.14。在旺长期、现蕾期和成熟期红花大金元烟叶的LOX基因相对表达量与LOX酶活性均呈极显著正相关,相关系数分别为0.98、0.95和0.97。烟叶脂氧合酶活性与其基因相对表达量呈正相关关系,且在旺长期、现蕾期和成熟期其相关性均达到极显著水平。     

水稻种子脂氧合酶基因OsLOX1的原核表达、纯化及鉴定

脂氧合酶是动植物体内催化脂质降解的关键酶,也是茉莉酸合成途径的第一个关键酶。以先前克隆到的水稻种子脂氧合酶基因OsLOX1全长cDNA为模板,用含有特异酶切位点的P1、P2为引物,通过PCR方法将它构建到大肠杆菌表达载体pET30a(+)上并转化到大肠杆菌菌株BL21（DE3）中,获得相应的重组工程菌。经过20℃条件下的IPTG诱导,OsLOX1重组蛋白在BL21（DE3）菌株中得到表达,经生化特性分析,发现该重组蛋白具有LOX催化活性,其最适pH和温度分别为4.8和30℃。该重组子可进一步应用于体外生产茉莉酸和研究植物种子LOX结构与功能的关系。     

Changes of the lipid catabolism in potato tubers from cultivars differing in susceptibility to autolysis during the storage

Summary The activities of lipolytic acyl-hydrolases (LAH) and lipoxygenases (LOX) were compared in stored tubers of potato cultivars resistant (Acresta, Eba, Pentland Envoy) and susceptible (Kastor, Pana, Tasso) to post-wounding autolysis. In most cultivars, LAH activities had reached a maximum by the end of December but in cv. Kastor activity continued to increase throughout the storage period. LOX activities increased during most of the storage period except in cv. Tasso. The level of the fatty acid hydroperoxides, assumed from determinations of the malonaldehyde level, also increased during storage. This work was supported by Project CPBP 05.02.2.10 financed by the Polish Academy of Science.     

紫果西番莲果实发育过程中挥发性香气物质及相关酶活性的变化

以西番莲香气物质较丰富的紫果‘台农1号’为试材,选取未成熟（T₁）、近成熟（T₂）和完全成熟（T₃）3个时期果实,采用顶空–固相微萃取–气质联用（HS-SPME-GC-MS）技术测定3个果实发育过程的挥发性香气物质组成及含量变化情况,同时检测对应果实中酯类香气物质合成相关的脂氧合酶（LOX）和醇酰基转移酶（AAT）活性,以期了解不同发育时期果实主要致香物质变化情况及物质与相关酶活性之间的相关性。结果表明：随着西番莲果实成熟度的增加,挥发性香气物质的种类和含量均有很大的提高,3个时期检测的香气物质总数分别为72种、95种和136种,总峰面积呈倍数增加;未成熟（T₁）和近成熟（T₂）2个时期萜烯类和酯类物质为主要的挥发性香气物质,在完全成熟（T₃）时期主要的挥发性香气为酯类,其峰面积占总峰面积达80.31%;随着果实成熟度的增加,酯类香气的种类和含量不断的提升,其中己酸乙酯,1-甲基己酯-己酸、丁酸乙酯、辛酸乙酯、1-甲基辛酯-丁酸、己酸己酯、1-甲基己酯-丁酸、丁酸己酯、(Z)-3-己烯基酯-己酸、乙酸己酯、2-甲基-辛酯-丙酸、辛酸己酯、3-羟基乙酯-己酸、(Z)-2-丁酸-乙酯等含量提升或占比较大,这些脂肪酸代谢途径产生的酯类在西番莲果实成熟过程中可能发挥了致香作用。LOX是酯类合成第一步的限速酶,AAT为酯类合成最后的关键酶。对不同发育期果实酯类合成相关酶活性测定结果表明,随着果实成熟度的增加,LOX、AAT酶活性呈上升趋势,尤其是T₃时期AAT酶活性显著的提升。由此推断,紫果西番莲果实发育过程中LOX和AAT酶的活性与酯类香气成分形成直接相关,酶活性的提升促进酯类香气的合成。     

Chitosan Stimulates Defense Reactions in Grapevine Leaves and Inhibits Development of Botrytis Cinerea

Chitosan (β-1,4-linked glucosamine oligomer) derived from crab shells conferred a high protection of grapevine leaves against grey mould caused by Botrytis cinerea. Under controlled conditions, it was shown to be an efficient elicitor of some defense reactions in grapevine leaves and to inhibit directly the in vitro development of B. cinerea. Treatment of grapevine leaves by chitosan led to marked induction of lipoxygenase (LOX), phenylalanine ammonia-lyase (PAL) and chitinase activities, three markers of plant defense responses. Dose-response curves show that maximum defense reactions (PAL and chitinase activities) and strong reduction of B. cinerea infection were achieved with 75–150 mg l⁻¹ chitosan. However, greater concentrations of chitosan did not protect grapevine leaves with the same efficiency, but inhibited mycelial growth in vitro. Present results underlined the potency of chitosan in inducing some defense responses in grapevine leaves which in turn might improve resistance to grey mould.     

稻瘟菌诱导的稻叶脂氧合酶的纯化及部分氨基酸序列测定

 作者对已报道的稻瘟菌诱导稻叶脂氧合酶CM-LOX 1和CM-LOX 2的纯化方法作了进一步改进和完善。改进后的纯化方法可缩短该脂氧合酶的纯化时间,并提高纯化倍数。利用改进的方法,作者从20 g非亲和性稻瘟菌小种接种稻叶中获得了电泳纯的酶蛋白CM-L OX 1 133 μ g和CM-LOX 2 208μg。在此基础上,采用特异性的蛋白酶endoproteinase Lys-C和溴化氰对该酶蛋白进行了裂解,回收其肽段,得到了CM-LOX 1的3个不同肽段共30个氨基酸和CM-L OX 2的7个不同肽段共87个氨基酸的序列,为最终克隆这2个酶的基因奠定了基础。     

去势公兔腥味物质及其脂氧合酶同工酶的研究

应用改进的NPT技术提得去以兔与正常公兔兔肉腥味物质，经气－质联谱比较分析，初步确定中级醛类是兔肉腥味物质的主导成分。用非变性聚丙烯酰胺凝胶电泳检测去势公兔与正常公兔体内脂氧合酶（LOX，亚油酸：氧氧化还原酶，EC1．33．11．12）同工酶的变化，结果显示，公兔去势后体内缺少了一种LOX同工酶，LOX同工酶Ⅰ可能与兔肉腥味的产生有关。     

光敏核不育水稻幼苗叶片脂氧合酶活性及其基因表达的昼夜变化

为探查光敏核不育(PGMS)水稻中脂氧合酶(LOX)活性的光暗期变化特性,以PGMS水稻N5088S和D38S、常规水稻中早25、盐粳7号和中嘉早17及三系不育水稻G2A和东B11A为材料,在不同光周期(12L/12D、14L/10D和10L/14D)和光/暗期温度(30℃/30℃和30℃/26℃)下,研究了其幼苗叶片中LOX活性的昼夜变化,并采用RT-PCR方法对PGMS水稻幼苗叶片进行LOX基因的昼夜表达分析。结果表明,在所有光周期和培养温度处理下,PGMS水稻LOX活性均表现出暗期高而光期低的昼夜变化规律,但常规水稻和三系不育水稻的LOX活性均无明显的光暗期差异;暗期PGMS水稻LOX活性显著高于常规水稻和三系不育水稻。在12L/12D光周期和30℃/26℃(L/D)的培养温度下,检测的13个LOX基因中,检测到8个Os LOX基因,OsLOX2、Os LOX3、Os LOX4、Os LOX8和Os LOX13等基因的表达丰度在N5088S和/或D38S幼苗叶片中呈现暗期高光期低的趋势。因此,光敏核不育水稻N5088S和D38S幼苗叶片LOX活性及Os LOX家族的部分基因的表达具有明显的暗期高光期低的变化特征。     

The 5‐lipoxygenase inhibitor tepoxalin induces oxidative damage and altered PTEN status prior to apoptosis in canine osteosarcoma cell lines

The 5‐lipoxygenase (5‐LOX) inhibitor tepoxalin has been shown to slow canine osteosarcoma (OSA) tumour xenografts growth, yet the mechanisms are poorly elucidated. Further examination of tepoxalin in canine OSA cell lines shows that tepoxalin treated cells undergo apoptosis through caspase‐3 activation and annexin staining. Interestingly, apoptosis is superseded by an increase in reactive oxygen species (ROS), as measured by activation of dihydrorhodamine 123 and mitosox. This increase in ROS appears to be related to the 5‐LOX inhibitor regardless of cellular 5‐LOX status, and was not observed after treatment with the tepoxalin metabolite RWJ20142. Additionally, 5‐LOX inhibition by tepoxalin appears to increase phosphatase and tensin (PTEN) homolog activity by preventing its alkylation or oxidation. PTEN modification or inhibition allows phosphoinositide‐3 (PI3) kinase activity thereby heightening activation of protein kinase B (AKT) phosphorylation. Our data suggest that off target oxidation and LOX inhibition play roles in the apoptotic response.