Two new diterpenes from Euphorbia kansuensis

Two new lathyrane diterpenes, 3 beta,5 alpha-dihydroxy-15 beta-cinnamonyloxy-14-oxoladiyra-6Z, 12E- diene (1) and 3 beta,5 alpha,20-trihydroxy-15 beta-cinnamoyloxy-14-oxolathyra-6Z, 12E-diene (2), were isolated from the roots of Euphorbia kansuensis. Their structures were determined by spectroscopic methods. (C) 2008 Elsevier B.V. All rights reserved.

Brittle-ductile transition of polypropylene/ethyllene-propylene-diene monomer blends induced by size, temperature, and time

To study the brittle-ductile transition (BDT) of polypropylene (PP)/ethylene-propylene-diene monomer (EPDM) blends induced by size, temperature, and time, the toughness of the PP/EPDM blends was investigated over wide ranges of EPDM content, temperature, and strain rate. The toughness of the blends was determined from the tensile fracture energy of the side-edge notched samples. The concept of interparticle distance (ID) was introduced into this study to probe the size effect on the BDT of PP/EPDM blends, whereas the effect of time corresponded to that of strain rate. The BDT induced by size, temperature, and time was observed in the fracture energy versus ID, temperature, and strain rate. The critical BDT temperatures for various EPDM contents at different initial strain rates were obtained from these transitions. The critical interparticle distance (IDc) increased nonlinearly with increasing temperature, and when the initial strain rate was lower, the IDc was larger. Moreover, the variation of the reciprocal of the initial strain rate with the reciprocal of temperature followed different straight lines for various EPDM contents. These straight lines were with the same slope.

Compatibilization of PP/EPDM blends by grafting acrylic acid to polypropylene and epoxidizing the diene in EPDM

In this article, ethylene-propylene-diene-rubber (EPDM) was epoxidized with an in situ formed performic acid to prepare epoxided EPDM (eEPDM). The eEPDM together with the introduction of PP-g-AA was used to compatibilize PP/EPDM blends in a Haake mixer. FTIR results showed that the EPDM had been epoxidized. The reaction between epoxy groups in the eEPDM and carboxylic acid groups in PP-g-AA had taken place, and PP-g-EPDM copolymers were formed in situ. Torque test results showed that the actual temperature and torque values for the compatibilized blends were higher than that of the uncompatibilized blends. Scanning electron microscopy (SEM) observation showed that the dispersed phase domain size of compatibilized blends and the uncompatibilized blends were 0.5 and 1.5 mu m, respectively. The eEPDM together with the introduction of PP-g-AA could compatibilize PP/EPDM blends effectively. Notched Izod impact tests showed that the formation of PP-g-EPDM copolymer improved the impact strength and yielded a tougher PP blend.

Toughening of nylon with epoxidised ethylene propylene diene rubber

Blends of nylon-6 and epoxidised ethylene propylene diene (eEPDM) rubber were prepared through reactive mixing. It is found that the toughness of nylon-6 can be much improved by this method, and that the particle size of eEPDM is much smaller than that of unexpoxidised EPDM (uEPDM) rubber in a nylon-6 matrix. This indicates that the epoxy group in eEPDM could react with a nylon-6 end group to form a graft copolymer which could act as an interfacial compatibiliser between the nylon-6 and the eEPDM rubber dispersed phase. (C) 1998 Elsevier Science Ltd. All rights reserved.

(+/-)-7,7,9,9-Tetramethyl-2,3 : 4,5-dinaphthol,6,8-trioxa-7,9-disilacyclonona-2,4-diene

The title compound, C24H24O3Si2, is a twofold symmetric silicocrown ether with the two dimethylsilyl groups attached to the O atoms of 1,1'-bi-2-naphthol, and bridged by another O atom.

Toughened blend of polycarbonate and epoxidized ethylene propylene diene rubber

A super-tough polycarbonate (PC) blend was obtained by using epoxidized ethylene propylene diene (eEPDM) rubber as modifier. The notched Izod impact strength of PC/eEPDM (96/4) blend shows a great improvement, with a value about 25 times of that of pure PC. Finely and homogeneously dispersed rubber particles (0.2-0.8 mu m) in the PC matrix indicated good adhesion between the eEPDM rubber phase and the PC matrix. (C) 1997 Elsevier Science Ltd.

In situ epoxidation of ethylene propylene diene rubber by performic acid

In this paper, epoxidation of ethylene propylene diene rubber by in situ generated performic acid is discussed. The samples have been characterized by infra-red and H-1-nuclear magnetic resonance analyses. Quantitative analysis of the reaction products is made possible by using the methyl deformation band at 1377 cm(-1) as internal standard. The conversion of double bonds increases rapidly within the first 1 h, then gradually, over 2 h, has only a slight increase. The maximum conversion ratio of double bonds is about 70%. The relative content of epoxy groups has a top value at about 7 h. The side reactions are also discussed. (C) 1997 Elsevier Science Ltd.

Toughening of poly(butylene terephthalate) with epoxidized ethylene propylene diene rubber

In this paper, unepoxidized ethylene propylene diene rubber (uEPDM) was first epoxidized with formic acid and H2O2, and then the epoxidized ethylene propylene diene rubber (eEPDM) was melt-mixed with PET resin in a Brabender-like apparatus. Toughening of PET matrix was achieved by this method. The dispersion of rubber particles and phase structure of the blends were also observed by SEM. It has been suggested that the epoxy groups in the eEPDM could react with PET end groups to form a graft copolymer which could act as an interfacial compatibilizer between the PBT matrix and eEPDM rubber dispersed phase. This is beneficial to the improvement of the impact performance of PBT. (C) 1997 Elsevier Science Ltd.

Toughened poly(butylene terephthalate) by epoxided ethylene-propylene-diene-rubber

Blends of poly (butylene terephthalate) (PBT) and epoxided ethylene-propylene-diene terpolymer (EEPDM) were prepared. Their mechanical properties and morphology were studied by Izod impact test machine and scanning electronic microscope respectively, It was found that the notched Izod impact strength of blend PBT/EEPDM was as about 23 times as that of pure PET and about 10 times as that of blend PBT/EPDM at room temperature, The dispersed rubber particles were much smaller and the phase boundary was more blurred in blend PBT/EEPDM than in blend PBT/EPDM. The toughness of blend PBT/EEPDM was much more better than that of blend PET and PBT/EPDM, which was in good agreement with the difference between their morphologies.

青蒿素生物合成相关基因的克隆、大肠杆菌表达与分子分析

利用现代分子生物学和基因工程技术手段，克隆青蒿素生成途径的关键酶基因，研究关键酶基因对青蒿素生物合成的调控规律，是打破青蒿素生物合成的限速步骤，大幅度提高青蒿素含量，最终达到利用植物生物技术工业化生产青蒿素的目的必须解决的关键问题。本论文基于此目的，开展了青蒿素生物合成相关基因的分子克隆工作。 用RACE方法从青蒿高产株系001中克隆了一个新的1886bp的全长倍半萜合酶cDNA。克隆的倍半萜合酶氨基酸序列与烟草马兜铃烯合酶、莨菪岩兰螺旋二烯合酶、棉花杜松烯合酶的一致性分别为39%，38%和41%;与青蒿柏木脑合酶、紫穗槐二烯合酶和一个推测的倍半萜合酶克隆cASC125的一致性为50%，48%和59%。cDNA编码区序列被克隆进原核表达载体pET-30a，并在大肠杆菌BL21（DE3）中诱导表达，但过量表达的蛋白主要是以不溶性蛋白形式存在。RT-PCR分析表明此基因在茎、叶和花中表达，在根中没有表达。 用RT/PCR方法从青蒿高产株系001中克隆了amorpha-4, 11-diene合酶cDNA。将该cDNA插入原核表达载体pET3d并在大肠杆菌BL21（DE3）中过量表达。Southern blot分析表明AMS基因在青蒿基因组中至少有3个拷贝。AMS基因组DNA有一个复杂的结构，包含有7个外显子和6个内含子。RT/PCR分析表明AMS基因在叶片、茎和花中表达，而在根中没有表达。 用RACE方法首次从青蒿中克隆了一个1539 bp全长鲨烯合酶cDNA。青蒿鲨烯合酶氨基酸序列与拟南芥、烟草、人类、酵母鲨烯合酶的一致性分别为70%、77%、44%、39%。青蒿鲨烯合酶基因组DNA有一个复杂的结构，包括14个外显子和13个内含子。全长的或C末端截短的鲨烯合酶cDNA被克隆进原核表达载体pET30a并在大肠杆菌BL21（DE3）中诱导表达。但在含有全长的鲨烯合酶cDNA的大肠杆菌中并没有观察到预期大小的鲨烯合酶表达，而C末端截短30个疏水氨基酸的鲨烯合酶可在大肠杆菌中过量表达。

青蒿素生物合成相关基因的功能分析及遗传转化研究

从中国传统药用植物青蒿（Artemisia annua L.）中提取的青蒿素及其半合成衍生物如蒿甲醚等是一类新型的抗疟特效药，特别是对抗氯喹的恶性疟疾和脑型疟疾有很好的疗效。由于青蒿素在植物中的含量极低，使得其价格很高，特别是对于亚非拉等第三世界国家来说。因此如何提高青蒿素的产量成为近年来研究的热点。各种传统的育种、生理生化手段和细胞培养技术均未取得较好的结果，因此，利用植物基因工程技术提高青蒿素产量已成为研究的重点之一。 本论文围绕青蒿素的生物合成途径开展了以下的工作： 一、中药青蒿紫穗槐二烯合酶的大肠杆菌表达、纯化与功能鉴定 利用RT-PCR方法，从中药青蒿高产株系001中克隆到的中药青蒿紫穗槐二烯合酶(ADS) cDNA, 其推测编码蛋白与前人报道的有两个位点的突变。将其开放阅读框插入到原核表达载体pET30a(+)的BamHⅠ和XhoⅠ酶切位点之间，构建N端携带有HIS6表达标签的紫穗槐二烯合酶重组表达载体pETADS。将pETADS转入大肠杆菌BL21(DE3)， IPTG (Isopropyl-beta -D-thiogalactoside)诱导重组紫穗槐二烯合酶的表达。表达产物经镍琼脂糖柱纯化。纯化蛋白加入酶促反应体系（含FPP），GC-MS分析酶促反应体系的正己烷萃取物，结果显示重组紫穗槐二烯合酶可以催化FPP向紫穗槐二烯的转化。体外酶促动力学分析表明，两个位点的氨基酸突变，并没有影响到青蒿紫穗槐二烯合酶的催化活性。基因组DNA杂交表明，紫穗槐二烯合酶基因在001株系基因组中至少有4个拷贝。 二、中药青蒿鲨烯合酶的大肠杆菌表达、纯化与功能鉴定 将经RACE方法克隆到的中药青蒿鲨烯合酶cDNA(AF302464) 开放阅读框的3'末端截短99 bp，插入到原核表达载体pET30a(+)的NcoⅠ和BamHⅠ酶切位点之间，构建N端和C端均携带有HIS6表达标签的鲨烯合酶重组表达载体pETSSA。将pETSSA转入大肠杆菌BL21(DE3)， IPTG (Isopropyl-beta-D-thio galactoside)诱导重组鲨烯合酶的表达。表达产物经镍琼脂糖柱纯化。纯化蛋白加入酶促反应体系（含FPP和NADPH），GC-MS分析酶促反应体系的正己烷萃取物，结果显示重组鲨烯合酶可以催化FPP向鲨烯的转化。青蒿鲨烯合酶的功能鉴定，为进一步利用反义或RNAi技术限制甾类生物合成，从而提高青蒿中的青蒿素含量提供了基础。 三、中药青蒿法呢醇合酶原核表达、纯化与功能鉴定 将经RACE方法克隆到的中药青蒿倍半萜合酶cDNA ( AF304444) 开放阅读框插入到原核表达载体pET30a(+)的NcoⅠ和BamHⅠ酶切位点之间，构建N端和C端均携带有HIS6表达标签的重组表达载体pET30SESQ。将pET30SESQ转入大肠杆菌BL21(DE3)， IPTG (Isopropyl-beta-D-thioga lactoside)诱导蛋白表达，表达产物经镍琼脂糖柱纯化。纯化蛋白加入酶促反应体系（FPP），GC-MS分析酶促反应体系的正己烷萃取物，结果显示此重组酶可以催化FPP向法呢醇的转化。 四、中药青蒿FPS、ADS双功能酶基因的构建、表达与功能鉴定 将青蒿素生物合成途径中催化两步连续反应的酶：法呢基焦磷酸合酶和紫穗槐二烯合酶的基因进行融合，经大肠杆菌表达后鉴定融合蛋白的功能，结果表明融合蛋白具有了双功能酶活性。进一步将融合酶基因转入酿酒酵母中，发酵后检测紫穗槐二烯的含量，并与同时转入法呢基焦磷酸合酶和紫穗槐二烯合酶单个基因的酵母、单独转入紫穗槐二烯合酶基因的酵母进行了比较，结果表明，转入双功能酶的酵母发酵获得的紫穗槐二烯含量要比两个对照酵母高，这表明，获得的双功能酶的催化效率要比两个单独酶的催化效率高。 五、过量表达青蒿紫穗槐二烯合酶对青蒿中青蒿素及其前体物含量的影响 利用根癌农杆菌介导，将青蒿紫穗槐二烯合酶转入青蒿株系001，分子检测证明，紫穗槐二烯合酶整合到了青蒿基因组中并在mRNA水平得到了高效表达。部分转基因青蒿的青蒿素含量有明显增加，最多的比001株系提高了41%。青蒿酸和二氢青蒿酸含量测定表明，转基因青蒿株系的青蒿酸和二氢青蒿酸含量最多的比对照分别提高了47%和79%。这些结果表明，紫穗槐二烯合成在青蒿素生物合成途径中是一个限速步骤，同时，也显示青蒿酸或二氢青蒿酸的进一步转化也可能是青蒿素生物合成中下游的限速步骤。

青蒿素生物合成相关基因对烟草遗传转化的研究

　　青蒿素是存在于中药青蒿(Artemisia annua L.)中的一种含有过氧桥的倍半萜内酯化合物，是中国科学家研发出的当今最有潜力的抗疟药剂，较传统抗疟药很少或无毒副作用，因此青蒿素的生产备受人们关注。目前，青蒿素的生产主要以植物提取为主，但由于青蒿植株中青蒿素的含量很低（约占干重的0.01%～0.8%），从而导致青蒿素价格昂贵，使许多贫困地区的疟疾患者无法得到医治，故提高青蒿植株中青蒿素的含量或扩大青蒿素的来源，降低生产青蒿素的成本具有重要的意义。　　 　　本论文基于扩大青蒿素的来源和提高青蒿植株中青蒿素含量的目的，开展了以下两方面的工作： 一、紫穗槐二烯在烟草中组合生物合成的研究 　　紫穗槐二烯合酶（amorpha-4,11-diene synthase，ADS）是青蒿素生物合成的关键酶之一，为了能在烟草中合成青蒿素的前体，本研究将青蒿的紫穗槐二烯合酶基因置于CaMV 35S启动子控制下，通过根癌农杆菌介导转入烟草（Nicotiana tobacum L.），并获得了转ADS基因烟草植株。经PCR及Southern杂交分析表明，ADS基因已经整合到转基因烟草基因组中；RT-PCR及对转基因烟草中ADS酶活性和产物中紫穗槐二烯和植物甾醇的测定分析，进一步证明整合的ADS基因在转录、翻译水平上均已经表达。上述结果表明，利用基因工程将青蒿素生物合成途径的关键酶基因导入植物，转基因植物中能够合成青蒿素的前体，这一研究结果为利用转基因植物生产青蒿素或其前体奠定了基础。 二、青蒿鲨烯合酶双链干涉基因对烟草的遗传转化研究 　　鲨烯合酶（squalene synthase, SQS）是甾醇类生物合成分支途径的关键酶之一，利用RNA干扰技术(RNA interference，RNAi)抑制目标基因表达的技术已日趋成熟。本文根据植物中hpRNA(hairpin RNA)的原理，在与烟草SQS同源性高达80％的青蒿ASQS序列的5/端保守区选择622 bp作为构建RNAi的序列，借助中间克隆载体，经过三次亚克隆，最后形成含ASQS－RNAi表达盒的双元表达载体pART27－ASQS，并转入农杆菌EHA105。采用农杆菌介导的烟草叶盘转化法，共获得了12棵转基因植株。转基因植株经过PCR和PCR-Southern blotting 检测，证实外源ASQS基因已经导入烟草中，并已经成功整合到烟草基因组中；通过RT-PCR分析说明，转基因烟草中SQS基因的表达已被成功抑制，部分转基因植株中内源SQS的干扰效果高达90％以上。对SQS的直接产物鲨烯和最终产物植物甾醇的检测显示，转基因烟草的植物甾醇和鲨烯的含量明显低于对照。本实验的结果为下一步将此RNA干扰载体导入青蒿，抑制青蒿中ASQS基因的表达，从而提高青蒿素的含量提供了可能。