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。

Two New Phenolic Glycosides from Viscum articulatum

Two new phenolic glycosides, 1-O-benzyl-[5-O-benzoyl-beta-D-apiofuranosyl (1. 2)]-beta-D-glucopyranoside (1), and 4'-hydroxy-7,3'-dimethoxyflavan-5-O-beta-D-glucopyranoside (2), together with nine known flavanones 3 -11, have been isolated from the dried

Tetranorclerodanes and clerodane-type diterpene glycosides from Dicranopteris dichotoma

The acetone extract of Dicranopteris dichotoma afforded two new tetranorclerodanes, 18-hydroxyaylthonic acid (1) and 18-oxo-aylthonic acid (2), and four new clerodane-type diterpene glycosides, (6S,13S)-6-O-[6-O-acetyl-beta-D-glucopyranosyl-(1 -> 4)-alpha

Quantum Confinement and Electronic Properties of Rutile TiO2 Nanowires

The quantum confinement effect, electronic properties, and optical properties of TiO2 nanowires in rutile structure are investigated via first-principles calculations. We calculate the size- and shape-dependent band gap of the nanowires and fit the results with the function E-g = E-g(bulk) + beta/d(alpha). We find that the quantum confinement effect becomes significant for d < 25 angstrom, and a notable anisotropy exists that arises from the anisotropy of the effective masses. We also evaluate the imaginary part of the frequency-dependent dielectric function [epsilon(2)(omega)] within the electric-dipole approximation, for both the polarization parallel [epsilon(parallel to)(2)(omega)] and the perpendicular [epsilon 1/2(omega)] to the axial (c) direction. The band structure of the nanowires is calculated, with which the fine structure of epsilon(parallel to)(2)(omega) has been analyzed.

Determination and identification of isoflavonoids in Radix astragali by matrix solid-phase dispersion extraction and high-performance liquid chromatography with photodiode array and mass spectrometric detection

The isoflavonoids in Radix astragali were determined and identified by HPLC-photodiode array detection-MS after extraction employing matrix solid-phase dispersion (MSPD). As a new sample preparation method for R. astragali, the MSPD procedure was optimized, validated and compared with conventional methods including ultrasonic and Soxhlet extraction. The amounts of two major components in this herb, formononetin (6) and ononin (2), were determined based on their authentic standards. Four major isoflavonoids, formononetin (6), ononin (2), calycosin (5) and its glycoside (1), and three minor isoflavonoids, (6aR,11aR)-3-hydroxy-9, 10-dimethoxypterocarpan (7), its glycoside (3), and (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavone-7-O-beta-D-glycoside (4), were identified based on their characteristic two-band UV spectra and [M + H](+), [aglycone + H](+) and [A1 + H](+) ions, etc. The combined MSPD and HPLC-DAD-MS method was suitable for quantitative and qualitative determination of the isoflavonoids in R. astragali. (C) 2003 Elsevier B.V. All rights reserved.

Microchip Micellar Electrokinetic Chromatography Based on One Functionalized Ionic Liquid and Its Excellent Performance on Proteins Separation

Herein, one water-soluble functionalized ionic liquid (IL), 1-butyl-3-methylimidazolium dodecanesulfonate (BAS), was designed, investigated and successfully applied to microchip micellar electrokinetic chromatography (MEKC) construction. It possessed the properties of both IL and surfactant. A fairly stable pH value similar to 7.4, which was fit to pH values of general biological buffers, was nicely placed at the optimum concentration of 20 mM BAS solution. While applying BAS solution as running buffer in poly(dimethylsiloxane) (PDMS) microfluidic systems, significantly enhanced electroosmotic flow (8-fold) and resolutions between analytes were obtained than that using other supporting electrolytes or surfactants.

Sensitive, Label-Free Protein Assay Using 1-ethyl-3-methylimidazolium Tetrafluoroborate Supported Microchip Electrophoresis with Laser-Induced Fluorescence Detection

Based on the dimer-monomer equilibrium movement of the fluorescent dye Pyronin Y (PY), a rapid, simple, highly sensitive, label-free method for protein detection was developed by microchip electrophoresis with LIF detection. PY formed a nonfluorescent dimer induced by the premicellar aggregation of an anionic surfactant, SDS, however, the fluorescence intensity of the system increased dramatically when proteins such as BSA, bovine hemoglobin, cytochrome c, and trypsin were added to the solution due to the transition of dimer to fluorescent monomer. Furthermore, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF(4)) instead of PBS was applied as running buffers in microchip electrophoresis.

Ionic liquids Supported Growth of Highly Ordered Microdroplets Induced by Fluidic Leakage at Poly(dimethylsiloxane) Interfaces

"Fluidic leakage" caused by vacuum force at the reversible sealing poly(dimethylsiloxane) (PDMS) interfaces was converted to one useable avenue, which led to formation of highly ordered surfactant microdroplets functionalized with ionic liquids (ILs). Vacuum force is the prerequisite to lead constant microsolutions to diffuse to the PDMS interfaces. Imidazolium ions of ILs rendered structural rearrangement of the surfactant aggregates and the ordered droplets formation.

Sugars-grafted aliphatic biodegradable poly(L-lactide-co-carbonate)s by click reaction and their specific interaction with lectin molecules

A novel biodegradable aliphatic poly(L-lactide-co-carbonate) bearing pendant acetylene groups was successfully prepared by ring-opening copolymerization of L-lactide (LA) with 5-methyl-5-propargyloxycarbonyl-1,3-dioxan-2-one (PC) in the presence of benzyl alcohol as initiator with ZnEt2 as catalyst in bulk at 100 degrees C and subsequently used for grafting 2-azidoethyl beta-D-glucopyranoside and 2-azidoethyl beta-lactoside by the typical "click reaction," that is Cu(I)-catalyzed cycloaddition of azide and alkyne. The density of acetylene groups in the copolymer can be tailored by the molar ratio of PC to LA during the copolymerization. The aliphatic copolymers grafted with sugars showed low cytotoxicity to L929 cells, improved hydrophilic properties and specific recognition and binding ability with lectins, that is Concanavalin A (Con A) and Ricinus communis agglutinin (RCA). Therefore, this kind of sugar-grafted copolymer could be a good candidate in variety of biomedical applications.

Structure identification of a prenylflavonol glycoside from Epimedium koreanum by electrospray ionization tandem mass spectrometry

A novel prenylflavonol glycoside, named acetylicariin, has been isolated from the aerial parts of Epimedium koreanum Nakai. The structure has been identified by electrospray ionization multi-stage tandem mass spectrometry (ESI-MSn) and other chemical evidence, which has been elucidated as 8-prenylkaempferol-4'-methoxyl-3-O-alpha-L-rhamnopyranosyl-7-O-beta-D-(2''-O-acetyl)glucopyranoside.

High-sensitivity molecular mass determination of lysozyme prior to MALDI-MS

Sodium dodecyl sulfate(SDS) is a powerful solubilizing detergent which is often used during the separation of highly complex protein mixtures by one- or two-dimensional (2D) gel electrophoresis. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a widely used technique for mass spectrometric analysis of some protein molecules compared to other techniques. But the presence of SDS or some salts usually leads to signal deterioration when using MALDI-MS. A method for using nitrocellulose membrane as the solid-phase carrier combined with n-octyl-beta-D-glucopyranoside in the matrix highly enhances the sensitivity of the molecular mass determination of lysozyme. This technique has the advantage that the signal-to-noise of the molecular weight profile is improved compared with the mass spectrum and the profile is relatively easy to interpret.

Identification of the flavonoid constituents from leaves of Acanthopanax Senticosus Harms

Four flavonoids from leaves of Acanthopanax Senticosus Harms were observed in negative ion mode in the electrospray mass spectra. Two of them were further isolated and identified as quercitrin (quercetin-3-O-alpha-L-rhamnoside) and hyperin (quercetin-3-O-beta-D-galactoside) on the basis of MS' and NMR data. The other two compounds in the mixtures were tentatively established as quercetin and rutin (quercetin-3-O-rutinoside) in terms of their electrospray tandem mass spectrometry (ESI-MSn) data. Three of the four flavonoids (excluding hyperin) haven't been reported in this plant before.

Electrospray ionization multiple-stage tandem mass spectrometric analysis of diglycosyldiacylglycerol glycolipids from the bacteria Bacillus pumilus

Electrospray ionization (ESI) combined with multiple-stage tandem mass spectrometry (MSn) was used to directly analyze the glycolipid mixture from bacteria Bacillus pumilus without preliminary separation. Full scan ESI-MS revealed the composition of picomole quantities of glycerolglycolipid species containing C-14-C-19 fatty acids, some of which were monounsaturated, Two main components were identified from their molecular masses and fragmentation pathways. The fragmentation pathway of the known compound compared with the investigated compound verified the proposed structure as 1(3)-acyl-2-pentadecanoyl-3(1)-O-[beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-sn-glycerols. A comparison of the multiple tandem mass spectra of the different alkali-metal cation adducts indicates that the intensity of fragments and the dissociation pathways are dependent on the alkali-metal type, The basic structures of glycerolglycolipids were reflected clearly from the fragmentation patterns of the sodium cations, The intense fragments of the sugar residue from the precursor ions were obtained from the lithiated adduct ions. ESI-MSn spectra of [M + K](+) ions did not provide as much fragmentation as [M + Na](+) and [M + Li](+) adducts, but their spectra allow the position of glycerol acylation to be determined. On the basis of MS2 spectra of[M + K](+) ions, it was established that all components have a C-15:0 fatty acid at the sn-2 position of the glycerol backbone and C-14-C-19 acids at the sn-1 position of the glycerol backbone. Copyright (C) 1999 John Wiley & Sons, Ltd.

GLASS-TRANSITION TEMPERATURE AND MOLECULAR-PARAMETERS OF POLYMER

A new relationship, which correlates the glass transition temperature (T(g)) with other molecular parameters, is developed by using Flory's lattice statistics of polymer chain and taking the dynamic segment as the basic statistical unit. The dependences of T(g) on the chain stiffness factor (sigma-2), dynamic stiffness factor (beta = -d ln-sigma-2/dT) and molecular weight of polymer are discussed in detail based on the theory. The theory is compared with experimental data for many linear polymers and good agreement is obtained. It is shown that T(g) is essentially governed by the chain stiffness factor at T(g). Moreover, a simple correlation between the parameter K(g) of the Fox-Flory equation (T(g) = T(g)infinity - K(g)/M(n)) and other molecular parameters is deduced. The agreement between theoretical predictions and experimental measurements of K(g) has been found to be satisfactory for many polymers.

Antioxidative principals of Jussiaea repens: an edible medicinal plant

Jussiaea repens L. (JRL) is an edible medicinal plant and is also used as a vegetable by the local people in southwestern China. The crude extract and its four fractions derived from JRL were evaluated for the 1,1-diphenyl-2-picrylhydrazyl radical-scavenging ability, hydroxyl radical-scavenging capacity and the potassium ferricyanide reduction property. The ethyl acetate-soluble fraction (EAF) and EAF6 (a subfraction derived from EAF) were the most valuable fraction and subfraction, respectively. Furthermore, bioactivity-guided chromatographic fractionation revealed that three pure compounds greatly contributed to the antioxidant activities. Qualitative and quantitative analyses of the major antioxidant constituents in the extract were systematically conducted by NMR, mass spectral analyses and RP-HPLC. The result demonstrated that rosmarinic acid (2.00 mg g(-1) JRL dry weight) quercetin 3-O-beta-D-glucopyranoside (9.88 mg g(-1) JRL dry weight), and kaempferol 3-O-beta-D-glucopyranoside (1.85 mg g(-1) JRL dry weight) were the major antioxidative constituents in JRL. These compounds are reported for the first time from this plant.