菠菜叶片乙醇酸氧化酶的氧化甘油酸活性

首先从菠菜叶片中纯化了乙醇酸氧化酶（GO）。通过鉴定反应中氧的消耗以及反应产物H2O2的生成,证实菠菜GO具有氧化光呼吸途径中间代谢物甘油酸的活性。该氧化活性依赖于辅因子FMN和FAD,而不依赖核黄素和光黄素;其最适反应pH值为8．0,Km（甘油酸）值为7．14mmol／L,kcat值为1．04s^-1,活化能为17．29kJ／mol;草酸和丙酮酸对该氧化活性有明显的抑制作用,其中前者为典型的竞争性抑制。进一步通过两底物竞争作图表明：菠菜叶片GO氧化甘油酸反应和氧化乙醇酸反应为同一活性中心所催化。     

菠菜乙醇酸氧化酶基因的克隆及表达

采用RT-PCR技术从菠菜总RNA中分离扩增了乙醇酸氧化酶（GO）基因的cDNA序列,首先克隆到质粒pMD18T,进行了测序。然后将乙醇酸氧化酶的cDNA分别亚克隆至质粒pThioHisC、 pTIGTrx、pBV220和pET-2b(+),分别转化大肠杆菌DH5α和BL21（DE3）,并对重组乙醇酸氧化酶在大肠杆菌中的表达进行了研究。SDSPAGE和酶活分析表明,菠菜乙醇酸氧化酶在E.coli BL21 (DE3) （pTIGTrxGO）和E.coli BL21(DE3) （pET-22b(+)GO）里得到了高水平的表达,其中E.coli BL21(DE3) （pET-22b(+)GO）的乙醇酸氧化酶活性较高。     

Identification and Characterization of Glycolate Oxidase and Related Enzymes from the Endocyanotic Alga Cyanophora paradoxa and from Pea Leaves

Glycolate oxidase (GO) has been identified in the endocyanom Cyanophora paradoxa which has peroxisome-like organelles and cyanelles instead of chloroplasts. The enzyme used or formed equimolar amounts of O₂ or H₂O₂ and glyoxylate, respectively. Aerobically, the enzyme did not reduce the artificial electron acceptor dichlorophenol indophenol. However, after an inhibitor of glycolate dehydrogenase, KCN (2 millimolar), was added to the assay medium, considerable aerobic glycolate:dichlorophenol indophenol reductase activity was detectable. The leaf GO inhibitor 2-hydroxybutynoate (30 micromolar), which binds irreversibly to the flavin moiety of the active site of leaf GO, inhibited Cyanophora GO and pea (Pisum sativum L.) GO to the same extent. This suggests that the active sites of both enzymes are similar. Cyanophora GO and pea GO cannot oxidize d-lactate. In contrast to GO from pea or other organisms, the affinity of Cyanophora GO for l-lactate is very low (K_m 25 millimolar). Another important difference is that Cyanophora GO produced sigmoidal kinetics with O₂ as varied substrate, whereas pea GO produced normal Michaelis-Menten kinetics. It is concluded that there is considerable inhomogeneity among the glycolate-oxidizing enzymes from Cyanophora, pea, and other organisms. The specific catalase activity in Cyanophora was only one-tenth of that in leaves. NADH-and NADPH-dependent hydroxypyruvate reductase (HPR) and glyoxylate reductase activities were detected in Cyanophora. NADH-HPR was markedly inhibited by hydroxypyruvate above 0.5 millimolar. Variable substrate inhibition was observed with glyoxylate in homogenates from different algal cultures. It is proposed that Cyanophora has multiple forms of HPR and glyoxylate reductase, but no enzyme clearly resembling leaf peroxisomal HPR was identified in these homogenates. Moreover, no serine:glyoxylate aminotransferase activity was detected. These results collectively indicate the possibility that the glycolate metabolism in Cyanophora deviates from that in leaves.     

百合ACC氧化酶基因全长cDNA的克隆及序列分析

以亚洲百合Polyanna(Liliumspp.)花瓣为材料,根据已报道的百合ACC氧化酶基因片段设计1对末端扩增特异引物,采用RACE方法,获得百合ACC氧化酶基因的全长cDNA(GenBank登录号为EU296623).该cDNA全长1 152 bp,具有一个954 bp的开放阅读框,编码318个氨基酸.Blast搜索结果显示,百合ACC氧化酶基因核苷酸序列与其它植物已报道的ACC氧化酶基因具有71%~82%的相似性,氨基酸序列有70%~87%的相似性,聚类分析表明,与单子叶植物百合科郁金香首先聚类,其次与双子叶植物聚类,最后与单子叶禾本科和兰科植物聚类.     

菜心乙醇酸氧化酶的纯化和催化特性分析

用改进后的方法,从菜心绿叶中分离纯化得到一个亚基分子量为42kD的乙醇酸氧化酶,用氧电极法测定该酶同时能催化乙醇酸和乙醛酸的氧化。     

光和底物对乙醇酸氧化酶的基因表达的诱导

从菠菜中提纯了乙醇酸氧化酶并制备其抗体,经免疫双扩散、Ｗｅｓｔｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ证实水稻和豌豆黄化苗中不存在乙醇酸氧化酶。在黑暗中,底物可促进该酶基因的表达,而在黄化苗光照初期,推测光可能是不经过底物促进该酶基因的表达。     

兵豆甘油-3-磷酸酰基转移酶基因的克隆和序列分析及蛋白质二级结构的预测

克隆了云南强抗冷性植物兵豆 (Lensculinaris)GPAT基因的编码区完整的cDNA片段 ,该片段长 1374bp ,编码 4 5 7个氨基酸残基。与豌豆 (Pisumsativum)、蚕豆 (Viciafaba)和长柔毛野豌豆 (Viciavillosa)相比较 (从剪切点起 ) ,氨基酸序列的同源性分别为 96 2 1%、95 6 6 %和 95 6 6 %。应用PSIPREDHFORMAT预测了这 4种植物GPAT酶的二级结构。     

菜心中高等电点高活性的乙醇酸氧化酶同工酶的纯化和特性

乙醇酸氧化酶 (EC 1 1 3 15 ,GO)被认为只含4 0kD一种碱性亚基 ,是因为从多种植物中获得了具GO活性的蛋白 ,SDS PAGE后呈约 4 0kD单带[1] .菠菜GOcDNA编码约 370个氨基酸 ,即 4 0kD多肽 ,其碱 酸性氨基酸的比例高达 0 96 ,富含碱性氨基酸[2 ] .已克隆的GOcDNA在E .coli中表达     

Furanoacetylene and isoflavonoid phytoalexins in Lens culinaris

The ability of two Lens species to synthesise the furnaoacetylenes wyerone and wyerone epoxide, as well as the pterocarpan variabilin, has been demonstrated. This links Lens more close to Vicia than to the remaining genera of the tribe Vicieae.     

玉米苹果酸脱氢酶基因的分离与结构分析

以一个玉米（ＺｅａｍａｙｓＬ．）杂种一代超亲表达的ｃＤＮＡ片段为探针,从玉米幼苗期ｃＤＮＡ文库中筛选到一个全长１２８７ｂｐ的ｃＤＮＡ克隆。序列分析表明,该ｃＤＮＡ编码细胞质苹果酸脱氢酶,推导的氨基酸序列与龙须海棠（Ｍｅｓｅｍｂｒｙａｎｔｈｅｍｕｍ　ｃｒｙｓｔａｌｌｉｕｍ　Ｌ．）及拟南芥（Ａｒａｂｉｄｏｐｓｉｓ　ｔｈａｌｉａｎａ（Ｌ．）Ｈｅｙｎｈ．）同一编码基因的氨基酸序列同源性分别为９０％和８４％。这是禾谷类作物中首次克隆的编码细胞质苹果酸脱氢酶的完整基因。     

Molecular Cloning and Characterization of Stored mRNA in Cotyledons of Vigna unguiculata

From a gt1O cDNA library constructed from the total polyadenylatedRNA (poly A⁺ RNA) of mature cowpea cotyledons, we selected recombinantphages that hybridized with a cDNA probe complementary to polyA⁺ RNA from cotyledons collected 24 h after the onset of imbibition(cDNA-B) but that did not hybridize with a probe from cotyledonsat developmental stage II (13 to 15 days after flowering; cDNA-A).cDNA inserts of two of these phages were subcloned in pUC18plasmids and the resultant plasmids were designated pSAS5 andpSAS1O, respectively. We also selected two recombinant phagesthat hybridized with cDNA-A but not with cDNA-B, and one ofthem was subcloned in pUC18 to generate pSASC1. We then characterizedthe pSASlO cDNA insert as the cDNA of a putative stored mRNAof cowpea seeds and the pSASC1 insert as a reference cDNA. TheRNA-blot hybridization after gel electrophoresis, with pSAS10as probe, indicated that an essentially full-length cDNA hadbeen cloned, and the hybrid-select translation of pSAS10 mRNAgave a 10-kDa product. pSASC1 mRNA was shown to code for a 46-kDapolypeptide, which corresponds in size to one of the major storageproteins of the cowpea. pSAS10 mRNA was detectable only in cotyledonsat developmental stage III (17 to 19 days after flowering) orlater, and the level of the mRNA began to decline when seedsgerminated. The mRNA was also present in cowpea embryonic axes.mRNA hybridizable to pSAS10 cDNA was found in extracts of someother plants. (Received July 17, 1989; Accepted October 17, 1989)     

Phytochrome-Mediated Control of Diamine Oxidase Level in the Epicotyl of Etiolated Lentil (Lens culinaris Medicus) Seedlings

Diamine oxidase (DAO; EC 1.4.3.6) levels are strongly reduced in epicotyls of 3-day-old etiolated lentil (Lens culinaris Medicus) seedlings upon exposure to continuous red and blue light, as compared to etiolated controls. Far-red light inhibits DAO activity to a lesser extent. A less marked effect can also be obtained by short (5-10 min) daily exposures. Phytochrome involvement in this light-mediated response has been demonstrated by red/far-red reversibility experiments. These findings provide the first evidence that mechanisms underlying the photoregulation of DAO level in the Leguminosae are related to photomorphogenesis and are essentially unrelated to the photosynthetic capacity of the seedling.     

Isolation and Characterization of a cDNA Encoding Maize Cyto solic Malate Dehydrogenase

A cDNA fragment derived from a gene over-expressing in hybrid maize ( Zea mays L. ) was isolated with RT-PCR and used as probe to screen cDNA library of hybrid maize seedlings. A positive cDNA clone ZH02 corresponding to the full-length mRNA sequence was obtained, which was shown to have an open reading frame encoding 332 a.a. DNA and proteinase database search revealed that the deduced amino acid sequence of ZH02 has high similarity with the cMDH of Mesembryaathemum crystallium L. and Arabidopsis thaliana (L.) Heynh. up to 90% and 84%, respectively. This is the first report of the full-length gene coding for the cereal cMDH.     

三疣梭子蟹Profilin基因全长cDNA的克隆与表达分析

三疣梭子蟹（Portunus trituberculatus）是我国沿海重要养殖品种之一,近年来养殖病害呈逐年上升趋势,制约了三疣梭子蟹养殖产业的健康可持续发展。克隆三疣梭子蟹免疫相关基因,研究免疫基因的功能和作用机制,可为三疣梭子蟹养殖病害的防治奠定基础。本研究从三疣梭子蟹血细胞全长cDNA文库中克隆了742 bp 的profilin基因全长cDNA。Profilin全长cDNA中开放阅读框长375 bp,编码125 aa。推导的三疣梭子蟹profilin理论等电点pI 5.87,氨基酸序列与冈比亚按蚊（Anopheles gambiae）profilin同源性最高,序列一致性为42.9%。荧光定量RT-PCR分析结果显示在正常的三疣梭子蟹机体中,血细胞profilin表达水平最高,其次为肝胰脏;在致病菌副溶血弧菌（Vibrio parahaemolyticus）诱导后,血细胞中profilin表达量显著上升（P<0.01）,表明profilin可能参与了三疣梭子蟹的免疫防御反应,是一个免疫相关因子。     

Differential Expression of Alternative Oxidase Genes in Soybean Cotyledons during Postgerminative Development

The expression of the alternative oxidase (AOX) was investigated during cotyledon development in soybean (Glycine max [L.] Merr.) seedlings. The total amount of AOX protein increased throughout development, not just in earlier stages as previously thought, and was correlated with the increase in capacity of the alternative pathway. Each AOX isoform (AOX1, AOX2, and AOX3) showed a different developmental trend in mRNA abundance, such that the increase in AOX protein and capacity appears to involve a shift in gene expression from AOX2 to AOX3. As the cotyledons aged, the size of the mitochondrial ubiquinone pool decreased. We discuss how this and other factors may affect the alternative pathway activity that results from the developmental regulation of AOX expression.