Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae

The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD(+), PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M(-1) min(-1). A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M(-1) min(-1). Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the Location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.

Transformations of morphine, codeine and their analogues by Bacillus sp

A bacterial strain belonging to the genus Bacillus isolated by enrichment culture technique using morphine as a sole source of carbon transforms morphine and codeine into 14-hydroxymorphinone and 14-hydroxycodeinone as major and 14-hydroxymorphine and 14-hydroxycodeine as minor metabolites, respectively. When the N-methyl group in morphine and codeine are replaced by higher alkyl groups, the organism still retains its ability to carry out 14-hydroxylation as well as oxidation of the C-6-hydroxyl group in these N-variants, although the level of metabolites formed are considerably low. The organism readily transforms dihydromorphine and dihydrocodeine into only dihydromorphinone and dihydrocodeinone, respectively; suggesting that the 7,8-double bond is a necessary structural feature to carry out 14-hydroxylation reaction. The cell free extract (20,000 x g supernatant), prepared from morphine grown cells, transforms morphine into 14-hydroxymorphinone in the presence of NAD(+), but fails to show activity against testosterone. However, the cell free extract prepared from testosterone grown cells contains significant levels of 17 beta- hydroxysteroid dehydrogenase but shows no activity against morphine.

Renewable surface electrodes based on dopamine functionalized exfoliated graphite: NADH oxidation and ethanol biosensing

Exfoliated graphite (EG) was modified by covalently attaching dopamine (DA) (3,4-dihydroxyphenethylamine) through amide linkages, using -COOH groups introduced on the EG surface. The modified material was characterized by FT-IR spectroscopy, Xray photoelectron spectroscopy and electrochemical techniques. Composites of DA modified EG dispersed in organically modified silicates were prepared by a sol-get process. Electrodes were fabricated by casting the composites in glass tubes. The sol-gel based electrodes were found to be active for the electrocatalytic oxidation of NADH and biosensing of ethanol in presence of NAD(+) and alcohol dehydrogenase enzyme. The modified composite electrodes were found to be stable for several months. The surface of the electrode could be renewed just by mechanically polishing the electrode using emery sheets. The modified EG was also pressed and restacked in the form of a pellet and the use of this material as a binderless bulk-modified electrode was also demonstrated. The performance of sol-gel derived composite EG electrodes with binderless bulk-modified EG electrodes was compared. (C) 2002 Elsevier Science B.V. All rights reserved.

Microstructure evolution and metastable phase formation in laser-ablation-deposited films of Ti5Si3 intermetallic compound

Thin films of Ti62.5Si37.5 composition were deposited by the pulsed-laser ablation technique on single-crystal Nad substrates at room temperature and on ′single-crystal′ superalloy substrates at elevated temperatures. Both vapour and liquid droplets generated by pulsed-laser ablation of the target become quenched on the substrate. Amorphization had taken place in the process of quenching of vapour-plasma as well as small liquid droplets on NaCl substrates at room temperature. In addition to the formation of Ti5Si3, a metastable fcc phase (a 0 = 0.433 nm) also forms in micron-sized large droplets as well as in the medium-sized submicron droplets. The same metastable fcc phase nucleates during deposition from the vapour state at 500°C and at 600°C on a superalloy substrate as well as during crystallization of the amorphous phase. The evolution of the metastable fcc phase in the Ti-Si system during non-equilibrium processing is reported for the first time.

Comprehensive investigations on DNa center dot center dot center dot A (D = H/F) complexes show why `sodium bonding' is not commonly observed

A comprehensive study of D-Na center dot center dot center dot A (D = H/F) complexes has been done using advanced ab initio and atoms in molecule (AIM) theoretical analyses. The correlation between electron density at bond critical point and binding energy gives a distinguishing feature for hydrogen bonding, different from the `electrostatic complexes' formed by LiD and NaD. Moreover, the LiD/NaD dimers have both linear and anti-parallel minima, as expected for electrostatic dipole-dipole interactions. The HF dimer has a quasi-linear minimum and the anti-parallel structure is a saddle point. Clearly, characterizing hydrogen bonding as `nothing but electrostatic interaction between two dipoles' is grossly in error.

The position of a social economy in the Czech Republic at present

The Social Economy is a new phenomenon in the Czech Republic. It means the economy with its social effects that is being supported within public politics. Some analyses have been done at J.E:purkynI University in Ústí nad Labem during the last years. The topics of this research were the transformation of public administration, non-profit sector(respectively the civil mix sector), co-operatives, and social enterprises, and it is still ongoing.

Olefin cyclopropanation and carbon-hydrogen amination via carbene and nitrene transfers catalyzed by engineered cytochrome P450 enzymes

Synthetic biology promises to transform organic synthesis by enabling artificial catalysis in living cells. I start by reviewing the state of the art in this young field and recognizing that new approaches are required for designing enzymes that catalyze nonnatural reactions, in order to expand the scope of biocatalytic transformations. Carbene and nitrene transfers to C=C and C-H bonds are reactions of tremendous synthetic utility that lack biological counterparts. I show that various heme proteins, including cytochrome P450BM3, will catalyze promiscuous levels of olefin cyclopropanation when provided with the appropriate synthetic reagents (e.g., diazoesters and styrene). Only a few amino acid substitutions are required to install synthetically useful levels of stereoselective cyclopropanation activity in P450BM3. Understanding that the ferrous-heme is the active species for catalysis and that the artificial reagents are unable to induce a spin-shift-dependent increase in the redox potential of the ferric P450, I design a high-potential serine-heme ligated P450 (P411) that can efficiently catalyze cyclopropanation using NAD(P)H. Intact E. coli whole-cells expressing P411 are highly efficient asymmetric catalysts for olefin cyclopropanation. I also show that engineered P450s can catalyze intramolecular amination of benzylic C-H bonds from arylsulfonyl azides. Finally, I review other examples of where synthetic reagents have been used to drive the evolution of novel enzymatic activity in the environment and in the laboratory. I invoke preadaptation to explain these observations and propose that other man-invented reactions may also be transferrable to natural enzymes by using a mechanism-based approach for choosing the enzymes and the reagents. Overall, this work shows that existing enzymes can be readily adapted for catalysis of synthetically important reactions not previously observed in nature.

The Molecular Basis of Lysine Acetylation: Addition, Removal, and Recognition

Acetyltransferases and deacetylases catalyze the addition and removal, respectively, of acetyl groups to the epsilon-amino group of protein lysine residues. This modification can affect the function of a protein through several means, including the recruitment of specific binding partners called acetyl-lysine readers. Acetyltransferases, deacetylases, and acetyl-lysine readers have emerged as crucial regulators of biological processes and prominent targets for the treatment of human disease. This work describes a combination of structural, biochemical, biophysical, cell-biological, and organismal studies undertaken on a set of proteins that cumulatively include all steps of the acetylation process: the acetyltransferase MEC-17, the deacetylase SIRT1, and the acetyl-lysine reader DPF2. Tubulin acetylation by MEC-17 is associated with stable, long-lived microtubule structures. We determined the crystal structure of the catalytic domain of human MEC-17 in complex with the cofactor acetyl-CoA. The structure in combination with an extensive enzymatic analysis of MEC-17 mutants identified residues for cofactor and substrate recognition and activity. A large, evolutionarily conserved hydrophobic surface patch distal to the active site was shown to be necessary for catalysis, suggesting that specificity is achieved by interactions with the alpha-tubulin substrate that extend outside of the modified surface loop. Experiments in C. elegans showed that while MEC-17 is required for touch sensitivity, MEC-17 enzymatic activity is dispensible for this behavior. SIRT1 deacetylates a wide range of substrates, including p53, NF-kappaB, FOXO transcription factors, and PGC-1-alpha, with roles in cellular processes ranging from energy metabolism to cell survival. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an apo form and in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a beta-hairpin structure that complements the beta-sheet of the NAD^+-binding domain, covering an essentially invariant, hydrophobic surface. A comparison of the apo and cofactor bound structures revealed conformational changes throughout catalysis, including a rotation of a smaller subdomain with respect to the larger NAD^+-binding subdomain. A biochemical analysis identified key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain. DPF2 represses myeloid differentiation in acute myelogenous leukemia. Finally, we solved the crystal structure of the tandem PHD domain of human DPF2. We showed that DPF2 preferentially binds H3 tail peptides acetylated at Lys14, and binds H4 tail peptides with no preference for acetylation state. Through a structural and mutational analysis we identify the molecular basis of histone recognition. We propose a model for the role of DPF2 in AML and identify the DPF2 tandem PHD finger domain as a promising novel target for anti-leukemia therapeutics.

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding beta-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic beta-amylase encoding genes in pgi1 leaves, which was accompanied by increased beta-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P) H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.

浑善达克沙地优势豆科植物 光合生理特性研究

　　氮素是植物光合生产的决定性因素，尤其是在沙地草地生态系统中，氮素贫乏往往限制植物的生长发育。因此，研究沙地植物光合作用与叶片N含量之间的关系，以及不同植物功能型氮素利用效率，有助于理解不同植物资源利用效率的差异。以浑善达克沙地分布的80种植物为研究对象，对不同生境(固定沙丘、丘间低地和湿地)、不同生活型(乔、灌、草)、不同光合途径(C3和C4)以及豆科和非豆科植物等功能型进行研究，结果表明：无论在单位叶面积水平还是单位干重水平上的叶片氮含量，均与光合速率成极显著正相关，但单位氮素的光合利用效率在不同生境以及不同功能型之间差异很大;光合氮素利用效率表现为：湿地植物>沙丘>丘间低地植物;草本植物>灌木>乔木;C4草本>C3草本植物，非豆科植物>豆科植物。 　　为了验证浑善达克沙地豆科植物是否比非豆科植物具有更高的光合潜力，我们比较研究了3种优势豆科植物小叶锦鸡儿(Caragana microphylla)、木岩黄芪(Hedysarum fruticosum var. lignosum)、披针叶黄华(Thermopsis lanceolata)和2种非豆科植物羊草(Leymus chinensis)和黄柳(Salix gordejeviii)，结果表明并非所有豆科植物都比非豆科植物有着显著高的光合速率，仅木岩黄芪表现出较高的光合速率，其它两种豆科植物的光合速率和羊草、黄柳的差异并不显著(P>0.05)，甚至低于后者，这是因为氮素利用效率(PNUE)在其中起关键作用，通过对影响PNUE的几个主要因素进行分析得出：叶绿素对光能的吸收、光化学转换效率和CO2分压并不是构成豆科和非豆科植物PNUE差异的主要因素，而Rubisco羧化效率决定了所实验的5种植物对氮素利用效率的高低。 　　木岩黄芪在浑善达克沙地的沙丘上为优势种，甚至成为流动沙丘的先锋种。除了其显著高的氮含量外，对沙丘胁迫生境的光合适应性是我们关注的另一个重点。通过对木岩黄芪和其伴生种黄柳的光反应曲线以及光合日动态的研究，发现木岩黄芪具有显著高的光合速率、水分利用效率和PSII 光化学效率，其忍受中午强光和高温的能力较强(即“光合午休”现象不明显)。另外，该物种还表现出了显著高的光饱和点和低光补偿点。 对木岩黄芪的模拟降雨试验结果表明：气体交换参数以及叶绿素荧光参数均受到干旱和模拟降雨的影响，其中气孔因素和非气孔因素共同决定了干旱条件下木岩黄芪光合速率的降低;但降雨解除干旱后，气孔导度恢复较快，而PSII 潜在活性和PSII 光能转换效率的恢复却比较缓慢。在0-15mm的降雨量范围内，随降雨量的增加各项生理指标不断升高，但大于15mm的降雨量对木岩黄芪影响不大，因此木岩黄芪可被视为低耗水型植物。 　　对木岩黄芪光合酶的研究结果表明，其C4光合酶的活性很高，磷酸稀醇式丙酮酸羧化酶(PEPcase)、NAD-苹果酸酶(NAD-ME)、NADP-苹果酸酶(NADP-ME)、NAD-苹果酸脱氢酶(NAD-MDH)、NADP-苹果酸脱氢酶(NADP-MDH) 和丙酮酸磷酸双激酶(PPDK)等酶的活性，在整个生育期内为黄柳的5倍以上，但稳定性碳同位素测定结果却表明木岩黄芪为C3植物。因此，我们认为C3豆科植物木岩黄芪体内可能存在着C4光合途径，这种机制使得其对于流动沙丘的胁迫环境有着很强的适应性和很高的资源利用效率。

菠菜甜菜碱醛脱氢酶的研究

本实验以菠菜叶片为材料，分离并纯化了甜菜碱醛脱氢酶（BADH，EC 1.2.1.8），并对其某些性质进行了研究。此外，还提取并纯化了poly (A)+RNA，并对其完整性进行了分析，主要结果如下： 1.菠菜甜菜碱醛脱氢酶存在于60％硫酸铵沉淀部分，70％硫酸铵沉淀部分未检出其活性。用层析法纯化该酶，使纯化倍数达到405.3倍。菠菜甜菜碱醛脱氢酶有两个同工酶。 2.菠菜甜菜碱醛脱氢酶主要定位于胞液中。其中，在过氧化物酶体及微粒体中有一定量活性存在，但在叶绿体中未检出其活性。 3.菠菜甜菜碱醛脱氢酶活性有较广的pH值范围，其最适pH范围为9.5左右。该酶以NAD作为特异性辅酶，其Km值为8.0×10-6M, Vmax为0.143nmol/min。该酶以甜菜碱醛作为特异性底物，其Km值为1.82×10-4M，Vmax为0.182nmol/min。该酶活性为0.125～1M的NaCl、KCl和脯氨酸所抑制，但0.125～1M的蔗糖及甜菜碱对其活性没有影响。 4.PCMB和Mersalyl抑制该酶的活性，DTT可逐步恢复被抑制的活性。稀土元素LaCl3对该酶活性没有影响，但CeCl则使其完全失活。另外，Mn2+和Mo6+离子对其活性没有影响，Mg2+离子可增加其活性。 5.菠菜叶片中有菠菜甜菜碱醛脱氢酶的抑制因子存在，该因子可能是一种小分子化合物。 6.应用酚-氯仿方法分离了菠菜叶片的poly (A)+RNA，并在-80℃低温下长期贮存。

不同葡萄糖/果糖类型桃果实发育期间可溶性糖的变化规律和代谢

以成熟果实中不同葡萄糖/果糖（G/F）类型的6个桃品种（G/F≈1品种：‘冈山白’、‘山一白桃’和‘燕红’;高G/F品种：‘张黄7号’、‘龙246’和‘临白7号’）为试材，采用高效液相色谱法测定果实发育期果实和叶片中可溶性糖含量，并在盛花后74 d或101 d测定了‘冈山白’、‘山一白桃’、‘张黄 7号’和‘龙 246’新梢韧皮部中可溶性糖的含量;测定了果实发育过程中‘山一白桃’和‘临白7号’果实中的可溶性糖和淀粉代谢相关酶的活性。研究成熟果实中不同G/F类型桃果实内G/F差异的部位和时期;分析桃果实内G/F差异的可溶性糖代谢调控机理。 成熟果实中不同G/F类型桃果实中均以蔗糖作为主要碳水化合物积累形式，花后43–85 d蔗糖含量很低，随后持续快速积累直至果实成熟;花后43–85 d山梨醇有升高趋势，在果实成熟前40 d左右迅速降低;葡萄糖和果糖含量在果实发育早期较高，之后逐渐降低;但两类不同G/F桃在整个果实发育过程中G/F值与果实成熟时相似。叶片中贮藏的可溶性糖主要是蔗糖和山梨醇，在果实整个发育期间，G/F≈1品种叶片中G/F约1-3，而高G/F品种叶片中G/F约为2-7。G/F≈1品种‘冈山白’和‘山一白桃’与高G/F品种‘张黄 7号’和‘龙 246’韧皮部中山梨醇占总可溶性糖47-63％，显著高于蔗糖、葡萄糖和果糖的含量，G/F为0.8-0.91，且两类不同G/F桃品种间G/F值不存在显著差异。 成熟果实中G/F≈1类型的‘山一白桃’和高G/F值类型的‘临白7号’整个果实发育过程中，葡萄糖、山梨醇和淀粉的含量在这两个品种间一般没有明显差异;‘山一白桃’果实中的果糖含量显著高于‘临白7号’果实中的果糖;果实最后迅速生长期，‘山一白桃’果实中的蔗糖明显高于‘临白7号’。‘山一白桃’和‘临白7号’果实中的NAD+依赖型山梨醇脱氢酶（NAD+-SDH）活性低，两者有相似的变化趋势，一般无显著差异。‘临白7号’果实中的NADP+依赖型山梨醇脱氢酶（NADP+-SDH）和山梨醇氧化酶（SOX）活性一直高于‘山一白桃’，两者NADP+-SDH和SOX的活性分别在花后93-123 d和花后43-93 d有显著差异。‘临白7号’果实中的果糖激酶（FK）活性一般高于‘山一白桃’。花后43-93 d，‘临白7号’果实中的磷酸蔗糖合成酶（SPS）和蔗糖合成酶（SS）活性一般显著‘山一白桃’。果实最后迅速生长期，蔗糖快速积累，葡萄糖、果糖、山梨醇和淀粉含量迅速降低，同时伴随有SPS和SS活性的迅速升高。在整个果实发育过程中，两个品种果实中的淀粉酶活性较高，其果实中的淀粉含量和淀粉酶活性都有明显的下降趋势。 研究结果表明，整个果实发育过程中桃果实中均存在G/F≈1和高G/F现象，光合产物在韧皮部的运输对桃果实的G/F没有显著的影响，果实中G/F的差异主要由于果实内糖代谢差异所导致。‘临白7号’果实中山梨醇向果糖方向的转化能力与‘山一白桃’一般没有显著差异，由于不同时期较高的NADP+-SDH和SOX活性，使得山梨醇向葡萄糖方向的转化能力明显高于‘山一白桃’，同时，‘临白7号’果实中的FK活性一般高于‘山一白桃’，因此导致‘临白7号’果实中G/F高于‘山一白桃’。

桃不同组织中的葡萄糖/果糖比值特点及己糖代谢

以不同葡萄糖/果糖（G/F）类型的桃品种（正常G/F 品种：‘燕红’、‘冈山白’和‘山一白桃’；高G/F 品种：‘龙124’、‘龙246’、‘张黄7 号’和‘临白7 号’）为试材，测定果实发育期果实、叶片、韧皮部和木质部中糖和淀粉含量，并分别在果实第一迅速生长期、硬核期和成熟期测定了‘燕红’、‘山一白桃’、‘龙124’、‘龙 246’和‘临白7号’果实和叶片中己糖相关酶。研究不同G/F类型桃品种产生G/F差异的组织器官和时期，并且分析相关代谢酶调控机理。 两类不同G/F 桃果实中均以蔗糖作为主要碳水化合物积累形式，花后70 d前蔗糖含量很低，随后快速积累直至果实成熟；山梨醇含量较为稳定，高G/F品种‘龙124’两年间在未成熟果实中山梨醇含量高于正常 G/F品种；葡萄糖和果糖含量在果实第一迅速生长期积累，之后逐渐降低。高 G/F 品种‘龙124’和‘临白7号’成熟果实中葡萄糖含量高于‘龙246’和正常 G/F 品种。正常G/F品种果实、叶片、韧皮部和木质部中葡萄糖和果糖含量基本相等，G/F基本保持在0.7-1.5。高G/F品种果实、叶片中葡萄糖显著高于果糖，果实中G/F在1.6-8.8，叶片中G/F在果实未成熟时为2.5-9.3，在果实成熟期为14.5-21.3。然而韧皮部和木质部中葡萄糖略高于果糖或基本相等，但较正常G/F品种高。因此，光合产物在韧皮部的运输对桃果实的G/F 没有显著影响。 在第一迅速生长期和成熟期时，所有供试桃品种果实和叶片中合成己糖的NAD+-SDH 和 SOX较为活跃，而分解己糖的FRK、GLK和PGI则保持在较低水平；在果核硬化期则相反，果实和叶片中合成己糖的NAD+-SDH 和 SOX活性较低，而分解己糖的FRK、GLK和PGI则较为活跃。高G/F品种‘龙124’和‘龙246’在果核硬化期果实中的FRK、NADP+-SDH 和GLK活性显著高于正常G/F品种，而高G/F品种‘临白7号’则与正常G/F品种没有明显差异。可见，高G/F品种间己糖代谢调控机制也有所差异。此外，叶片中两种G/F类型间的己糖代谢相关酶差异并无明显规律，由此我们认为叶片存在与果实类似但相对独立的调控机制。

衣藻糖酵解途径的定位及其与卡尔文循环之间的协调

糖酵解作为细胞的基本能量代谢途径广泛存在于各类生物中。真核生物的糖 酵解一般都在细胞质中进行。然而，有报道发现少数物种如原生生物的动基体类、 少数藻类（如硅藻）中糖酵解途径却并非发生在胞质中，而是分别发生在糖酵解 体和线粒体中。在处于关键进化地位的单细胞绿藻—衣藻中，其糖酵解途径的亚 细胞定位一直存在争议。本文针对衣藻糖酵解途径的亚细胞定位问题开展了如下 两方面的研究工作，获得了一些重要结果： 1 衣藻糖酵解途径亚细胞定位的实验研究 本实验室曾利用生物信息学方法 对衣藻糖酵解途径的酶进行了鉴定和定位预测的理论分析，结果发现该途径的前 7 步主要发生在叶绿体，后3 步则主要发生在胞质。据此，本文选取了糖酵解途 径中具有代表性的几个酶(PFKa、PFKb、NAD+-GAPDH 和TIM)进行了GFP 定 位实验，以期通过实验验证该结论。结果表明，参与糖酵解途径第3、5、6 步反 应的酶（分别为PFK、TIM 和NAD+-GAPDH）确实定位到叶绿体。本文的研究 结果在一定程度上证实了以往的生物信息学理论分析结果--衣藻糖酵解途径的 前七步发生在叶绿体中。 2 衣藻叶绿体内糖酵解与卡尔文循环二者之间协调机制的研究 既然衣藻糖 酵解途径发生在叶绿体中，这势必与同样发生在叶绿体中的卡尔文循环途径发生 冲突。因为这两个途径有五步反应是逆向重叠的，它们之间是如何协调的呢？为 了探讨这一问题，本文开展了如下研究：首先，我们利用生物信息学方法对衣藻 全基因组中参与上述五步逆向重叠反应的酶进行了鉴定，并对它们的定位进行了 预测。结果发现，第一步有两个定位到叶绿体的PFK 催化糖酵解途径的反应， 其逆向反应则是由FBP 来催化；第四步在叶绿体中有两个利用不同辅酶（NAD+ 和NADP+）的GAPDH；第二步有两个拷贝的FBA（FBAa 和FBAb）定位到叶 绿体；而参与第三步与第五步的TIM 和PGK 仅有一个拷贝且是叶绿体定位。其 次，我们对这些参与五步逆向重叠反应且定位到叶绿体的酶进行了real time RT-PCR 实验，得到了它们的转录表达谱。PFK、FBAb 和NAD+-GAPDH 在整个 光照和黑暗中均表达恒定波动不大，但相对于PFK 和NAD+-GAPDH，FBAb 的 表达量却极低；而FBP、FBAa 和NADP+-GAPDH 在黑暗条件下表达量低且恒定， 而进入光照后表达量急剧增长，1 小时之后即能达到最高，表明这些酶的表达是 受光调节的；PGK 的转录表达情况则与FBP、FBAa 和NADP+-GAPDH 类似,也 是光照条件下表达量剧增，说明它也是受光调节的；TIM 在光照条件下也是有上 调趋势的，只是幅度较小，推测可能与其作为异构酶催化效率高有关。因此我们 认为PFK、FBAb 和NAD+-GAPDH 是专职参与糖酵解途径的，而FBP、FBAa 和 NADP+-GAPDH 是专职催化卡尔文循环反应的，通过光对它们表达的调节而 来协调它们各自参与的反应；而TIM 和PGK 则是两个代谢途径所共有的，它们 是通过光照、底物浓度等综合因素来调节它们所参与的反应方向进而达到两个途 径之间的协调。 该研究工作不仅对衣藻糖酵解途径的亚细胞定位进行了实验验证，还首次揭 示了衣藻同处叶绿体中的糖酵解与卡尔文循环两个途径之间的协调机制。

镇海棘螈繁殖群体生态和遗传结构研究

genetics, such as: population size, reproduction, mating system, growth, development,genetic structure and systematics status; The main results are presented below: The seasonal variation of the operational sexual ratio of this animal was found in the field and the ration always bias the female in the breeding season. Aiming at this character and considering the distance of time and space of both sexual habitat in breeding season, we census female population first by toe-clipping mark-recapture method, then estimated the population size with the definitive sexual ratio. Up to now, this species was found only at the Beilun district of the Ningbo City. The population size of the Ruiyan Temple Forest Park approximates to 369. The status of this population is extremely endangered, so besides protecting this population at the original locality, we also suggested to breed the salamander in fenced locality and to hatch embryos artificially, and send metamorphosed juveniles back to nature. We can transfer some individuals to other similar habitats or breed them under artificial conditions for saving this species from extinction. The early developmental stage of the Chinhai salamander is the same as its relative species, E. andersoni. Their balanceres are poorly developed and disappear very early. Temperature and moisture significantly influence the embryonic development of the Chinhai salamander. The embryonic stage is approx. 29 days under room temperature. The hatchling grows in a logarithmic curve. The larvae stage in water is approx. 58- 88 days. Many factors influence the nomal development, including two aspects of internal and external. Due to these factors, the effective protected measures were presented in detail. The breeding migration of E. chinhaiensis takes place at late March～late April every year. This salamander's hatching rate is high, but the rate of hatchling migrating into water is low. The average effectiveness of all the nest sites is 36.7%. The maternal self-conservation was contrary to the reproductive success of the egg-laying strategy. In the strategy of egg-laying behavior, the first factor selected by the female was its self-conservation, the second is embryonic survival rate, and the last is rate of hatchling survival rate. The oviposition selection is significant for the survival of the larvae. Based on the analysis of the evolutionary process of reproductive behaviors nad egg-laying site selections of all genera of the family Salamandridae, we deduced that perhaps Echinotriton is a transitional type in the evolutionary process from water to land. Due to its location in the adaptive stage in the terrestrial evolution, Echinotriton chinhaiensis's terrestrial nest may be one of important reason that causes this species to be endangered. The genetic deversity analysis shows that although the population size of the Chinhai salamander is quite small compared to other Chinese salamandrid species, the genetic diversity of this population is not reduce remarkably. We explain this phenomena with the polygamy mating system of this species. The result of 4 families' parenthood determinations shows that the parenhood determination can be taken without any paternal information. The "children" of every female include rich genetic information from at least two "fathers". It implies that female Chinhai salamander mates more than once with different males in a breeding season. The molecular evidence, the behavioral observation evidences and the sperm evidence in the female cloaca proved that this species has a polygamy mating system. The kin recognition in the mating of adult salamander was first discussed. The taxonomic status and phylogenetic relationships of 12 species representing 6 genera in the family Salamandridae were studied using DNA fingerprinting. The results showed that the DNA fingerprinting. The results showed that the DNA fingerprinting patterns demonstrated rich genetic diversity and species diversity, and also revealed the taxonomic status and phylogenetic relationshipes of higher taxa to a certain extent. The results are highly consistent with those obtained from the studies based on the morphology, ecology, cytology and molecular biology. The compreshensive analysis indicate that Tylototrition hainanensis and T. wenxianensis should be valid species; Echinotriton should be a valid genus;Tylotortriton is a natural cluster; Tylotortriton asperrimus should be put in Tylototrition rather than in Echinotriton, Hypselotriton and Allomestriton are synonyms of Cynops and Paramesotriton, respectively. There are three main groups in Chinese salamandride: Cynops, Paramesotriton and Pachytrition from the first group, the species of the Tylototriton from the second, and E. chinhaiensis composes the third.