无指盘臭蛙皮肤抗菌肽Odorgrin A和Odorgrin C基因表达载体的构建及其在毕赤酵母中的表达

研究背景与目的：近二十年来，抗生素的广泛使用以及一些不当应用导致临床上出现大量的耐药性病原菌，所以不易产生耐药性的抗菌肽就成为目前研究的热点。本课题组此前的研究表明无指盘臭蛙（Odorrana grahami）皮肤抗菌肽具有广谱抗菌活性，但对真核细胞没有毒性，因此有成为新型药物的潜力。本研究采用毕赤酵母真核表达系统来生物合成抗菌肽Odorgrin A和Odorgrin C，为大量获取抗菌肽资源提供技术支撑。 方法：依照Odorgrin A和C的氨基酸序列、采用酵母偏爱密码子分别设计并化学合成了相应的目的基因序列。目的片段从合成质粒上用Xho Ι和EcoR Ι双酶切下后，与经同样限制酶完全酶切pPIC9K载体所获得的两个大片段直接连接，并转化至大肠杆菌DH5α。用PCR扩增、酶切及测序检测，鉴定正确的重组质粒。提取大量表达载体pPIC9K - Odo A和C并使之线性化后经电击法分别转化毕赤酵母（Pichia pastoris）GS115宿主菌，用营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序检测，鉴定并筛选出对G418具高抗性的Odorgrin A和C重组酵母菌。用甲醇对之进行诱导表达，SDS - PAGE电泳及反相层析检测表达产物，并做抑菌活性检测。 成果：PCR扩增、酶切及测序等结果表明表达载体pPIC9K - Odo A和C构建成功。营养缺陷型筛选、遗传霉素抗性筛选、PCR扩增和测序等证实pPIC9K - Odo A和C已整合入酵母基因组中。SDS - PAGE电泳及反相层析结果表明抗菌肽Odorgrin A和C成功地获得了分泌表达。而抑菌活性实验则检测到部分阳性克隆菌诱导分泌表达的抗菌肽Odorgrin A和C都对测试菌的生长具有较高（>94%）的抑制率。 结论：无指盘臭蛙皮肤抗菌肽Odorgrin A和Odorgrin C基因的表达载体都构建成功，并且都在毕赤酵母系统中获得了成功表达。 Background ＆ Objective: In the recent twenty years, a lot of pathogenic bacteria have come forth in clinic with durable trait derived from making use of and abusing the traditional antibiotics. Therefore, studying antimicrobial peptides, not be easy to be invalidated by durable bacteria, are becomimg popular and important. The skin antimicrobial peptides of Odorrana grahami with broad spectrum antibacterial activity and no toxicity to eukaryotic cell, discovered by previous research work of our workgroup, are looked forward to being potential medication. Pichia pastoris expressional system was used for biosynthesis antimicrobial peptides Odorgrin A and Odorgrin C in this study, for producing abundant antimicrobial peptides. Methods: The foreign fragments which included Odorgrin A or Odorgrin C gene according to their amino acid sequence respectively were synthesized based on the biased codon usage of yeast. The DNA fragments, obtained from the plasmids containing them by digested with Xho Ι and EcoR Ι, were directly ligated with the two bigger fragments obtained from the vector pPIC9K by digested with the same restriction enzymes. And then they were transformed into Escherichia coli DH5α to be selected and amplified positive colonies. The recombinants were testified by using PCR amplification, enzymes digestion and sequencing of the foreign fragment. After the expressional vector pPIC9K - Odo A and pPIC9K - Odo C were linearized, they were transformed into Pichia pastoris GS115 strain by the electroporation. Then the positive colonies which were of the highest geneticin resistant were selected through auxotrophic screening, genetic resistant screening, PCR amplification and sequencing of the inserted fragment. Methanol was used to induce the recombinant yeasts to express the foreign gene. SDS-PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment were used to testify the expressional products. Results: The evidences of PCR, enzymes digestion and sequence analysis confirmed that the expressional vector pPIC9K - Odo A and pPIC9K - Odo C have been constructed correctly. The results of auxotrophic screening, of genetic resistant screening, of PCR and sequencing of the foreign fragment showed that Odorgrin A and Odorgrin C gene have been homologous integrated with the Pichia pastoris genome. And it was also testified that antimicrobial peptides Odorgrin A and Odorgrin C have been expressed successfully by using SDS - PAGE electrophoresis, reversed phase chromatography and antibacterial activity experiment. Conclusion: The expressional vector of the skin antimicrobial peptides Odorgrin A and Odorgrin C gene of Odorrana grahami have been constructed correctly and both of the genes have been expressed successfully in Pichia pastoris system in this study.

簇毛麦（ Dasypyrum villosum）矮秆突变体的研究

株高是农作物的重要农艺性状之一，适度矮化有利于农作物的耐肥、抗倒、高产等。20世纪50年代，以日本的赤小麦为矮源的半矮秆小麦的培育和推广，使得世界粮食产量显著增长，被誉为“绿色革命”。迄今为止，已报到的麦类矮秆、半矮秆基因已达70多个，但由于某些矮源极度矮化或者矮化的同时伴随不利的农艺性状，使得真正运用于育种实践的矮源较少。因此，发掘和鉴定新的控制麦类作物株高的基因，开展株高基因定位、克隆及作用机理等方面的研究，对实现麦类作物株高的定向改良，具有重要的理论意义和应用价值。簇毛麦(Dasypyrum villosum，2n＝14，VV)是禾本科簇毛麦属一年生二倍体异花授粉植物，为栽培小麦的近缘属。本课题组在不同来源的簇毛麦杂交后代中发现了一株自然突变产生的矮秆突变体。观察分析了该突变体的生物学特性，对矮秆性状进行了遗传分析，对茎节细胞长度、花粉的活力进行了细胞学观察，考察了该突变体内源赤霉素含量及不同浓度外施赤霉素对突变体的作用，分析了赤霉素生物合成途径中的内根贝壳杉烯氧化酶（KO）和赤霉素20氧化酶(GA20ox)的转录水平，对赤霉素20氧化酶和赤霉素3-β羟化酶(GA3ox)进行了克隆和序列分析，并对GA20ox进行了原核表达和表达的组织特异性研究。主要研究结果如下：1. 该突变体与对照植株在苗期无差异，在拔节后期才表现出植株矮小，相对对照植株，节间伸长明显受到抑制，叶鞘长度基本不变。在成熟期，对照植株的平均株高为110cm，而突变株的平均株高为32cm，仅为对照植株的1/3 左右。除了株高变矮以外，在成熟后期，突变株还表现一定程度的早衰和雄性不育。I2-KI染色法观察花粉活力结果表明，对照植株花粉90%以上都是有活力的，而突变植株的花粉仅20%左右有活力。2. 突变株与对照植株的杂交F1代均表现正常株高，表明该突变性状为隐性突变。F1代植株相互授粉得到的168株F2代植株中，株高出现分离，正常株高（株高高于80cm）与矮秆植株（株高矮于40cm）的株数比为130：38，经卡方检验，其分离比符合3：1的分离比，因此推测该突变体属于单基因的隐性突变。3. 用ELISA方法检测突变株和对照植株的幼嫩种子中内源性生物活性赤霉素（GA1＋3）含量，结果表明突变株的赤霉素含量为36 ng/ml，而对照植株的赤霉素含量为900 ng/ml。对突变株外施赤霉素，发现矮秆突变株的株高和花粉育性均可得到恢复。这些结果表明该突变株为赤霉素缺陷型突变。4. 用荧光定量PCR方法比较突变株与对照植株中内根贝壳杉烯氧化酶和赤霉素20氧化酶的转录水平，结果表明突变株的KO转录水平比对照植株分别提高了6倍（苗期）和16倍（成熟期），突变株的GA20ox转录水平与对照植株在苗期无明显差异，在成熟期突变株较对照植株则提高了10倍左右。这些结果表明该矮秆突变体与赤霉素的生物合成途径密切相关，而且极有可能在赤霉素的生物合成途径早期就发生了改变。5. 以簇毛麦总基因组为模板，同源克隆了GenBank登录号为EU142950，RT-PCR分离克隆了簇毛麦的GA3ox基因cDNA全长序列，分析结果表明该cDNA全长1206bp，含完整编码区1104bp，推测该序列编码蛋白含368个氨基酸残基，分子量为40.063KD，等电点为6.27。预测的氨基酸序列含有双加氧酶的活性结构，在酶活性中心2个Fe离子结合的氨基酸残基非常保守。该序列与小麦、大麦和水稻的GA3ox基因一致性分别为98%、96%、86%。基因组序列与cDNA序列在外显子部分一致，在478-715bp和879-1019bp处分别含238bp和140bp的内含子。6. 通过RT-PCR技术克隆了簇毛麦的GA20ox基因全长，命名为DvGA20ox，GenBank登录号为EU142949。该基因全长1080个碱基，编码359个氨基酸，具有典型的植物GA20ox基因结构。该基因编码的蛋白质与小麦、大麦、黑麦草等GA20ox蛋白的同源性分别为98%，97% 和91%。该序列重组到原核表达载体pET-32a(+)上，将获得的重组子pET-32a(+)-DvGA20ox转化大肠杆菌BL21pLysS后用IPTG进行诱导表达。SDS-PAGE分析表明，DvGA20ox基因在大肠杆菌中获得了高效表达，融合蛋白分子量为55kDa。定量PCR分析表明，该基因在簇毛麦不同器官中的表达差异明显：叶片中表达水平最高，根部表达水平次之，茎部和穗中表达较弱。在外施赤霉素后，该基因的表达水平在两小时以后急剧下降，表明该基因的表达受自身的反馈调节。本研究结果认为，(1)该簇毛麦矮秆突变体为单基因的隐性突变；(2)该矮秆突变体为赤霉素敏感突变，内源赤霉素含量检测表明突变体的内源性赤霉素含量仅为对照植株的1/30；(3)荧光定量PCR结果表明突变株的赤霉素生物合成途径的关键酶基因表达水平比对照植株高，而且突变植株的赤霉素生物合成改变很可能发生在赤霉素生物合成途径的早期；(4)GA20ox有表达的组织特异性，且受到自身产物的反馈调节。 Plant height is an impotrant agronomic trait of triticeae crops.Semi-dwarf cropcultivars, including those of wheat, maize and rice, have significantly increased grainproduction that has been known as “green revolution”. The new dwarf varieties couldraise the harvest Index at the expense of straw biomass, and, at the sametime, improvelodging resistance and responsiveness to nitrogen fertilizer. Moreover, dwarf traits ofplant are crucial for elucidating mechanisms for plant growth and development aswell. In many plant species, various dwarf mutants have been isolated and theirmodles of inheritance and physiology also have been widely investigated.The causesfor their dwarf phenotypes were found to be associated with plant hormones,especially, gibberellins GAs.Dasypyrum villosum Candargy (syn.Haynaldia villosa) is a cross-pollinating,diploid (2n = 2x = 14) annual species that belongs to the tribe Triticeae. It is native toSouthern Europe and West Asia, especially the Caucasuses, and grows underconditions unfavorable to most cultivated crops. The genome of D. villosum,designated V by Sears, is considered an important donor of genes to wheat for improving powdery mildew resistance, take-all, eyespot, and plant and seed storageprotein content. A spontaneous dwarf mutant was found in D. villosum populations.The biological character and modles of inheritance of this dwarf mutant are studied.The cell length of stem cell is observed. The influence of extraneous gibberellin tothe dwarf mutant is also examined; the transcript level of key enzyme of gibberellinbiosynthesis pathway in mutant and control plants is compared. GA3ox and GA20oxare cloned and its expression pattern is researched.1. The dwarf mutant showed no difference with control plants at seedlingstage.At mature stage, the average height of control plants were 110cm and the dwarfplants were 33cm. The height of the mutant plant was only one third of the normalplants due to the shortened internodes. Cytology observation showed that theelongation of stem epidermal and the parenchyma cells were reduced. The dwarfmutant also shows partly male sterile. Pollen viability test indicates that more than80% of the pollen of the mutant is not viable.2. The inheritance modle of this dwarf mutant is studied. All The F1 plantsshowed normal phenotype indicating that the dwarfism is controlled by recessivealleles. Among the 168 F2 plants, there are 130 normal plants and 30 dwarf plants, thesegregation proportion accord with Mendel’s 3:1 segregation. We therefore proposethat this dwarf phenotype is controlled by a single recessive gene.3. Quantitative analyses of endogenous GA1+3 in the young seeds indicated thatthe content of GA1+3 was 36ng/ml in mutant plants and 900ng/ml in normal plants.The endogenous bioactive GA1+3 in mutant plants are only about 1/30 of that innormal plants. In addition, exogenously supplied GA3 could considerably restore themutant plant to normal phenotype. These results showed that this mutant wasdefective in the GA biosynthesis.4. More than ten enzymes are involved in GA biosynthesis. KO catalyzes thefirst cytochrome P450-mediated step in the gibberellin biosynthetic pathway and themutant of KO lead to a gibberellin-responsive dwarf mutant. GA20ox catalyze therate-limited steps so that their transcript level will influence the endogenous GAbiosynthesis and modifies plant architecture. The relative expression levels of genesencoding KO and GA20ox were quantified by real time PCR to assess whether thechanges in GA content correlated with the expression of GA metabolism genes andwhere the mutant occurred during the GA biosynthesis pathway. In mutant plants,the transcript levels of KO increased about 6-fold and 16-fold at the seedling stage and elongating stage respectively comparing with the normal plants. For theseedlings, there was no notable difference in the expression of GA20ox betweenmutant and normal plants. At the elongating stage, GA20ox transcript increased 10times in mutant plants, suggesting that the GA biosynthesis pathway in mutant plantshad changed from the early steps rather than the late steps.5. A full length cDNA of D. villosum gibberellin 3β-hydroxylase homology(designated as DvGA3ox) was isolated and consisted of 1206bp containing an openreading frame of 1104bp encoding 368 predicted amino acid residues. Identityanalysis showed that the gibberellin 3β-hydroxylase nucleotide sequence shared 98%,96% and 86% homology with that of wheat, barley and rice. The predicted peptidecontained the active-site Fe of known gibberellin 3β-hydroxylase and the regionhomologous to wheat, barley and Arabidopsis. The genomic clone of gibberellin3β-hydroxylase has two introns.6. The full-length cDNA of D. villosum gibberellin 20 oxidase (designated asDvGA20ox) was isolated and consisted of 1080-bp and encoded 359 amino acidresidues with a calculated mol wt of 42.46 KD. Comparative and bio-informaticsanalyses revealed that DvGA20ox had close similarity with GA20ox from otherspecies and contained a conserved LPWKET and NYYPXCQKP regions. Tissueexpression pattern analysis revealed DvGA20ox expressed in all the tissues that wereexamined and the highest expression of DvGA20ox in expanding leaves followed byroots. Heterologous expression of this cDNA clone in Escherichia coli gave a fusionprotein that about 55KD. Transcript levels of DvGA20ox dramatically reduced twohours after application of biologically active GA3, suggesting that the biosynthesis ofthis enzymes might be under feedback control.

RNAi沉默烟草GA 20-氧化酶基因研究

赤霉素是一种高效能的广谱植物生长调节剂，为五大植物激素之一，具有重要的生物学功能。目前利用赤霉素突变体研究生物合成途径和信号转导已经成为热点。 GA 20-氧化酶是GA生物合成中的一类关键酶，它位于GA合成途径的中心位置。本研究根据烟草（Nicotiana tabacum）GA 20-氧化酶基因序列，设计2对分别含有特定酶切位点的特异引物，以烟草基因组DNA为模板，扩增目的基因(约250 bp)片段。将正、反向目的片段分别插入中间载体的内含子两侧，再经BamH I和Sac I双酶切回收约700 bp的目的片段，插入到双元载体质粒p2355中，成功构建了含GA 20-氧化酶基因片段反向重复序列的植物表达载体p23700。分别将p2355质粒和p23700质粒导入根癌农杆菌（Agrobacterium tumefaciens）EHA105中并转化烟草叶片细胞，经卡那霉素选择培养，PCR及GUS组织染色鉴定，获得转基因烟草植株。以EHA105-p2355转化的烟草，获得41株转基因植株，均没有矮化表型；而以EHA105-p23700转化的烟草，获得转基因植株14株，其中具有矮化表型的烟草10株，表明反向重复序列转录产物能形成发夹RNA(hpRNA)，产生小分子干扰RNA（small interferring RNA，简称siRNA），干扰目的基因的表达。 赤霉素含量测定表明矮化植株中赤霉素合成途径的最终产物GA3总含量明显低于野生型烟草植株。荧光定量PCR结果表明，矮化转基因烟草的GA 20-氧化酶基因表达量受到明显抑制，表达量明显低于野生型对照。同时对上游内根-贝壳杉合成酶（Ent-kaurene synthase，KS）基因，下游的GA-3β羟化酶基因进行了RT-PCR分析，结果显示上游基因的表达没有规律性变化，而下游基因表达量亦降低。上述结果表明，GA 20-氧化酶基因的表达被有效地干扰了，表达受到抑制，从而影响植株体内GA3的合成，影响植株的生长发育，导致植株矮化。并推测，GA 20-氧化酶基因受到抑制，可能影响下游基因的表达。并且通过干旱胁迫测试，发现矮化植株相对于野生型植株及不含干扰片段的转基因植株，对干旱的耐受力有了很大的提高，具有更强的耐受力。 研究结果为进一步进行相关研究奠定基础。 Gibberellin(GA) is an efficient plant growth regulator. As one of five major plant hormones, it plays an important biological function. Using GA mutant for investigating biosynthetic pathways and signal transduction has become high lights. GA 20-oxidase is a crucial enzyme involved in gibberellin biosynthesis. According to tobacco (Nicotiana tabacum) GA 20-oxidase enzyme gene sequence and based on binary vector p2355, we constructed a plant expression vector p23700, which habors an inverted repeat DNA fragment of GA 20-oxidase gene drivered by Cauliflower mosaic virus promtor (CaMV 35Sp). Binary plasmid p2355 had no inverted repeat DNA fragment of GA 20-oxidase gene. The vector p2355 and p23700 were introduced into Agrobacterium tumefaciens EHA105 and tobacco leaf transformation was conducted. After selected by kanamycin and characterized by PCR and GUS hischemical reaction, transsgenic plants were obtained. Fourtheen transgenic plants, which were transformed by EHA105-p23700, were obtained. Among them, 10 were dwarf mutants. However, 41 transgenic plants with the same normal phenotype as wild type,which were transformed by EHA105-p2355, were obtained. Analysis of Gibberellin contents showed that it was lower in dwarf mutants than in normal phenotype plants. Moreover, comparing to normal phenotype plants including wild type and transgenic plants with no interference fragment, the drought tolerance of dwarf plants have greatly increased. And their proline content increased obviously after drought test. Fluorescence quantitative real time PCR (RT-PCR) showed that GA 20-oxidase gene expression was significantly inhibited in dwarf transgenic tobacco. Meanwhile, the expression of the upstream gene ent-kaurene synthase (KS) gene and downstream gene GA-3β hydroxylase gene was also detected by RT-PCR. The results presented that KS gene expression had no regular change while GA-3β hydroxylase gene expression reduced. It implied that inhibiting GA 20-oxidase gene probably reduce the expression of downstream genes. The results showed that the transcriptional products of the foreign inverted repeat fragment can form hairpin RNA (hpRNA) to induce RNAi. It presented that GA 20-oxidase gene expression was effectively interfered, resulting in reducing GA3 synthesis and inhibiting plant growth and development, then dwarf plants were produced. However, the dwarf plants had higher tolerance of drought.

微生物法生成辅酶Q10的研究

本文根据我们实验室建立的发酵产物中辅酶Q10定性定量检测方法，筛选得到一株可以代谢产生较多辅酶Q10的野生菌株放射形土壤杆菌(Agrobacterium radiobacter No.50)。 为了提高放射形土壤杆菌的辅酶Q10的产量，本实验利用液体培养研究了单因素对菌株辅酶Q10产量的影响，并用正交法确定了最佳液态发酵条件。最佳发酵培养基是：葡萄糖20g,蔗糖40g, 硫酸铵10g,玉米浆30g, 酵母膏3g，K2HPO4 3g,MgSO4.7H2O 1g，蒸馏水1000mL,pH 7.0-7.2。最佳发酵条件是：转接斜面菌种到种子培养基, 转速220r/min、温度28。C培养24h后，转入发酵培养基(250mL三角拼装液量为50mL，pH 7.0), 接种量为10％，转速220r/min、温度28。C，培养120h。在此条件下，菌体湿重约为50g/L，辅酶Q10含量约为20mg/L。 本文以放射形土壤杆菌为出发菌株进行诱变育种，以期获得辅酶Q10高产菌。根据微生物育种原理、参照辅酶Q10的代谢调控机制，以野生型放射形土壤杆菌(Agrobacterium radiobacter No.50)为出发菌株，采用紫外线和亚硝基胍复合诱变技术，依次筛选得到菌体提取物M抗性菌ARM－7、烟草提取物T抗性菌株ARMT-26、Vk3抗性菌株ARMTV-25、链霉素抗性菌株ARMTVS-32，菌株ARMTVS-32产量达到了36.8mg/L，与原始出发菌株相比，产量提高了77％。 研究了茄尼醇、对羟基苯甲酸、橘子皮提取物D、胡萝卜提取物E、烟草提取物对ARMTVS-32合成辅酶Q10的影响，结果表明这些物质对菌体合成辅酶Q10有一定促进作用，添加0.2g/L茄尼醇时，辅酶Q10含量提高了17%，达到了40.7mg/L；添加1.2g/L橘子皮提取物D时，辅酶Q10含量提高了13.8%，达到了39.6mg/L；添加0.5g/L胡萝卜提取物E时，辅酶Q10含量提高了25.3% ，达到了43.6mg/L；添加8g/L烟草提取物时，辅酶Q10含量提高了12.6%，达到了39.2mg/L。 Production of Coenzyme- Q10 (CoQ10) by fermentation is considered as a process with broad prospects.Quantitative Analysis of CoQ10 in the culture of microbe by TLC—UV spectrophotometry was developed, by using this method we got the strain Agrobacterium radiobacter,which was isolated from forest soil of southwest of China. The effect of the single factor on CoQ10-production ability of the strain was examined by liquid cultured, and its best optimum cultivation conditions were established by orthogonal method. The results showed that the optimum fermentation conditions were as following: carbon sources glucose 20g/L,sucrose 40g/L; nitrongen sources (NH4)2SO4 10g/L,maize liquid 30g/L;yeast extract 3g; K2HPO4 3g/L,MgSO4.7H2O 1g/L; initial pH was 7 and volume of medium(medium volume vs flask volume) was 50mL/500mL, incubating for 120h on a rotary shaker at 220 rpm and 28℃.Under these conditions, the biomass and CoQ10 concentration reached 50g/L and 20mg/L respectively. According to the biosynthesis mechanism of CoQ10 and breeding theory, CoQ10 over-production strains were screened by UV--NTG. mutation using Agrobacterium radiobacter No.50 as parent strain. A microbe-juice resistant mutant ARMTVS-32, which also could resist tobacco-juice, VK3 and streptomycin, was screened out from an agar plate. The CoQ10 content of ARMTVS-32 reached 36.8mg/L, which was 77% higher than the initial strain. In addition, We discussed the effects of some organic substrates on the synthesis of CoQ10 in ARMTVS-32. The results showed that solanesol, orange juice D, carrot juice E and tobacco juice could promote the CoQ10 accumulation in the cells. The CoQ10 content of ARMTVS-32 reached 40.7mg/L when added 0.2g/L solanesol,it reached 39.6mg/L when added 1.2g/L orange juice D，it reached 43.6mg/L when added 0.5g/L carrot juice E. it reached 39.2mg/L when added 8g/L tobacco juice.

Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures

Plant cell cultures have been suggested as a feasible technology for the production of a myriad of plant-derived metabolites. However, commercial application of plant cell culture has met limited success with only a handful of metabolites produced at the pilot- and commercial-scales. To improve the production of secondary metabolites in plant cell cultures, efforts have been devoted predominantly to the optimization of biosynthetic pathways by both process and genetic engineering approaches. Given that secondary metabolism includes-the synthesis. metabolism and catabolism of endogenous compounds by the specialized proteins, this review intends to draw attention to the manipulation and optimization of post-biosynthetic events that follow the formation of core metabolite structures in biosynthetic pathways. These post-biosynthetic events-the chemical and enzymatic modifications, transport, storage/secretion and catabolism/degradation have been largely unexplored in the past. Potential areas are identified where further research is needed to answer fundamental questions that have implications for advanced bioprocess design. Anthocyanin production by plant cell cultures is used as a case study for this discussion, as it presents a good example of compounds for which there are extensive research publications but still no commercial bioprocess. It is perceived that research on post-biosynthetic processes may lead to future opportunities for significant advances in commercial plant cell cultures. (C) 2002 Elsevier Science Inc. All rights reserved.

The Early Diagenetic Formation of Organic Sulfur in the Sediments of Mangrove Lake, Bermuda

Due to a low mineral content, the sapropelic sediments depositing in Mangrove Lake, Bermuda, provide an excellent opportunity to explore for possible additions of sulfur to organic matter during the early stages of diagenesis. We evaluated early diagenetic organic sulfur transformations by monitoring the concentrations and stable isotopic compositions of a number of inorganic and organic sulfur pools, thereby accounting for all of the sulfur in the sediments. We have identified and quantified the following sulfur pools: porewater sulfate, porewater sulfide, elemental sulfur, pyrite sulfur, hydrolyzable organic sulfur (HYOS), chromium-reducible organic sulfur (CROS), and nonchromium-reducible organic sulfur (Non-CROS). Of the organic sulfur pools, the Non-CROS pool is by far the largest, followed by CROS, and finally HYOS. By 60 cm depth these pools contribute, respectively, to 85, 7.9, and 3.6% of the total solid phase sulfur. The HYOS pool is probably of biological origin and shows no interaction with the sulfur compounds produced during diagenesis. By contrast, CROS is produced, most likely, from the diagenetic addition of polysulfides to functionalized lipids in the upper, H2S-poor, elemental sulfur-rich, region of the sediment. A portion of this sulfur pool is unstable and decomposes on contact with the H2S-rich porewaters. The portion of CROS that remains in the sulfidic waters appears to readily exchange sulfur isotopes with H2S. While some of the Non-CROS pool is of biological origin, some is also formed by the diagenetic addition of sulfur to organic compounds in the upper H2S-poor region of the sediment. By contrast with CROS, Non-CROS is not diagenetically active in the H2S-rich porewaters. Overall, somewhere between 27 and 53 % of the organic sulfur buried in Mangrove Lake sediments is of diagenetic origin, with the remaining organic sulfur derived from biosynthesis. We extrapolate our Mangrove Lake results and calculate that in typical coastal marine sediments between 11 and 29 μmol g−1 of organic sulfur will form during early diagenesis, of which 2–5 μmol g−1 will be chromium reducible.

Synthesis of polysaccharide-stabilized gold and silver nanoparticles: a green method

A simple, green method was developed for the synthesis of gold and silver nanoparticles by using polysaccharides as reducing/stabilizing agents. The obtained positively charged chitosan-stabilized gold nanoparticles and negatively charged heparin-stabilized silver nanoparticles were characterized with UV-vis spectroscopy and transmission electron microscopy. The results illustrated the formation of gold and silver nanoparticles inside the nanoscopic polysaccharide templates. Moreover, the morphology and size distribution of prepared gold and silver nanoparticles varied with the concentration of both the polysaccharides and the precursor metal salts.

Kinetic study of paracetamol on prolidase activity in erythrocytes by capillary electrophoresis with Ru(bpy)(3)(2+) electrochemiluminescence detection

We explored the CE with Ru(bpy)(3)(2+) electrochemiluminescence detection for the kinetic study of drug-enzyme interaction. Effects of four nonsteroidal anti - inflammatory drugs including aspirin, paracetamol, sodium salicylate and phenacetin on prolidase (PLD) activity in erythrocytes were investigated. Aspirin enhanced PLD activity whereas the other three had inhibiting effects. This may reveal their different effects on the collagen biosynthesis and catabolism that influence tumor invasiveness. Kinetic study of paracetamol on PLD showed that the value of Michaelis constant Km for PLD was 1.23 mM. The mechanism of PLD inhibition by paracetamol is noncompetitive inhibition, and the inhibitor constant K-i value obtained in our research was 9.73 x 10(3) mu g/L.

Synthesis and characterization of poly(beta-hydroxybutyrate) and poly(epsilon-caprolactone) copolyester by transesterification

To synthesize the copolyester of poly(beta-hydroxybutyrate) (PHB) and poly(epsilon-caprolactone) (PCL), the transesterification of PHB and PCL was carried out in the liquid phase with stannous octoate as the catalyzer. The effects of reaction conditions on the transesterification, including catalyzer concentration, reaction temperature, and reaction time, were investigated. The results showed that both rising reaction temperature and increasing reaction time were advantageous to the transesterification. The sequence distribution, thermal behavior, and thermal stability of the copolyesters were investigated by C-13 NMR, Fourier transform infrared spectroscopy, differential scanning calorimetry, wide-angle X-ray diffraction, optical microscopy, and thermogravimetric analysis. The transesterification of PHB and PCL was confirmed to produce the block copolymers. With an increasing PCL content in the copolyesters, the thermal behavior of the copolyesters changed evidently. However, the introduction of PCL segments into PHB chains did not affect its crystalline structure. Moreover, thermal stability of the copolyesters was little improved in air as compared with that of pure PHB.

Miscibility, crystallization kinetics, and morphology of poly(beta-hydroxybutyrate) and poly(methyl acrylate) blends

The miscibility, spherulite growth kinetics, and morphology of binary blends of poly(beta-hydroxybutyrate) (PHB) and poly(methyl acrylate) (PMA) were studied with differential scanning calorimetry, optical microscopy, and small-angle X-ray scattering (SAXS). As the PMA content increases in the blends, the glass-transition temperature and cold-crystallization temperature increase, but the melting point decreases. The interaction parameter between PHB and PMA, obtained from an analysis of the equilibrium-melting-point depression, is -0.074. The presence of an amorphous PMA component results in a reduction in the rate of spherulite growth of PRE. The radial growth rates of spherulites were analyzed with the Lauritzen-Hoffman model. The spherulites of PHB were volume-filled, indicating the inclusion of PMA within the spherulites. The long period obtained from SAXS increases with increased PMA content, implying that the amorphous PMA is entrapped in the interlamellar region of PHB during the crystallization process of PHB. All the results presented show that PHB and PMA are miscible in the melt. (C) 2000 John Wiley & Sons, Inc.

A potent anti-oxidant property: fluorescent recombinant alpha-phycocyanin of Spirulina

To express and product a fluorescent antioxidant holo-alpha-phycocyanin (PC) of Spirulina platensis (Sp) with His-tag (rHHPC; recombinant holo-alpha-phycocyaninof Spirulina platensis with His-tag) in 5-l bench scale. A vector harbouring two cassettes was constructed: cpcA along with cpcE-cpcF in one cassette; ho1-pcyA in the other cassette. Lyases CpcE/F of Synechocystis sp. PCC6803 (S6) could catalyse the 82 site Cys in apo-alpha-PC of Sp linking with bilin chromophores, and rHHPC was biosynthesized in Escherichia coli BL21. The constant feeding mode was adopted, and transformant reached the biomass of rHHPC up to 0.55 g l(-1) broth in 5-litre bench scale. rHHPC was purified by Ni2+ affinity column conveniently. The absorbance and the fluorescence emission spectra of rHHPC had lambda(max) at 621 and 650 nm, respectively. The IC50 values of rHHPC were 277.5 +/- 25.8 mu g ml(-1) against hydroxyl radicals and 20.8 +/- 2.2 mu g ml(-1) against peroxyl radicals. Combinational biosynthesis of rHHPC was feasible, and the constant feeding mode was adopted to produce good yields of rHHPC. Fluorescent rHHPC with several unique qualitative and quantitative features was effective on scavenging hydroxyl and peroxyl radicals. A potent antioxidant rHHPC was co-expressed, produced and characterized for nutritional and pharmacological values, which would help to develop phycobiliproteins' applications in their fluorescent and biological activities.

Cloning and characterization of beta-carotene ketolase gene promoter in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis accumulates a highly valuable ketocarotenoid, astaxanthin, under various environmental stresses. beta-carotene ketolase (BKT) plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, an approximate 700 bp 5'-flanking region of the bkt gene containing a putative promoter was cloned through walking upstream. The results of the sequence analysis showed that this bkt 5'-flanking region might have cis-acting elements such as sterol regulatory element (SRE-1)-like motifs, the C-repeat/dehydration responsive element (DRE) and al-3 proximal element (APE)-like motifs, except for typical TATA and CCAAT boxes. The results of the P-galactosidase assay and the transient expression of lacZ driven by a series of sequential deletions revealed that a minimal promoter-like region might exist from -630 to -408 bp, and the highest promoter activity was observed to span the positions from -630 to -308 bp. The results of the site-directed mutagenesis of a C-repeat/DRE and two APE-like motifs in a promoter-like region (-630 to -308 bp) suggested that two APE-like motifs might be essential for transcriptional control of the bkt gene.

Hymeniacidon perleve associated bioactive bacterium Pseudomonas sp NJ6-3-1

Among marine bacteria isolated from the cytotoxic sponge Hymeniacidon perleve, one strain NJ6-3-1 classified as Pseudomonas sp. showed both cytotoxic and antimicrobial activities. Fatty acid analysis indicated that the bacterial strain consists mainly of C16:1, C16:0, C18:1, C18:0, C15:0, C14:0. One unusual 9,10-cyclopropane-C17:0 fatty acid and C26:0 also constitute major components, as well as the existence of squalene, the precursor of triterpenoids. The major metabolites in the culture broth were identified as alkaloids, including diketopiperazines and indole compounds, namely 3,6-diisopropylpiperazine-2,5-dione, 3-benzyl-3-isopropylpiperazine-2,5-dione, 3,6-bis-(2-methylpropyl)-piperazine-2,5-dione, indole-3-carboxaldehyde, indole-3-carboxylic acid methyl ester, indole-3-ethanol, and quinazoline-2,4-dione.

Diet-induced changes in lipid and fatty acid composition of Artemia salina

The influence of diet on lipid and fatty acid composition of the brine shrimp Artemia salina nauplii was investigated. Various diets with different lipid composition and fatty acid profiles were fed to nauplii for 2 weeks. The lipid composition of microalgal diets, Isochrysis galbana, Phaeodactylum tricornutum and Nannochloropsis oculata and baker's yeast was analyzed. Newly hatched nauplii were examined before the feeding experiment. It was shown that Artemia was able to incorporate and selectively concentrate some dietary lipids. Depot lipids were more sensitive to changes in the dietary lipid composition than the main structural lipids, polar lipids and sterols. Variations in the content of the lipid classes correlated with stage of development of the animal. The fatty acid composition of the animal varied with that of diet. The concentrations of saturated fatty acids were apparently supported in the nauplii by biosynthesis de novo. The acid 16:1(n-7) originated from the food. The concentration range of n-6 polyunsaturated fatty acids (PUFAs) remained constant through the accumulation from the diet. The proportion of n-3 PUFAs varied with their level in the diet. The dynamics of alteration of 20:5(n-3) content in Artemia fed on Isochrysis, which is poor in this acid, suggested a limited capacity for elongation and desaturation of 18:3(n-3) to 20:5(n-3). None of the diets provided dietary input of 22:6(n-3). (C) 1998 Elsevier Science Inc. All rights reserved.