植物生产生物可降解塑料PHB的研究：真养产碱杆菌H16的phbB和phbC的基因克隆、植物表达载体构建和转基因马铃薯植株的获得

利用聚合酶链式反应(PCR)技术从Alcaligenes eutrophus H16染色体DNA中扩增并克隆了调控聚-3-羟基丁酸(poly-3-hydroxy-butyrate，PHB)生物合成的两个关键酶基因：依赖NADPH的乙酰乙酰CoA还原酶基因(phbB)和PHB合成酶基因(phbC)。限制性内切酶图谱和核苷酸序列分析证实了克隆结果，并表明克隆的基因与国外所报道的有很高的同源性。经过基因拼接，构建了块茎特异性表达的高等植物表达载体pPSAGB（嵌合phbB）、pBIBGC(嵌合phbC)和pPSAGCB（嵌合phbB和phbC）。并以试管薯(microtuber)为外植体经Agrobacterium介导转化了虎头、京丰、Bintje、Favorita、高原4号和88-5共6个马铃薯品种，获得49个株系。经PCR检测导入phbB的株系共有44个，对其中30个株系进行DNA dot blot分析，结果表明phbC导入呈阳性的株系有20个。深入的鉴定工作还在进行中。

Synthesis and characterization of maleated poly(3-hydroxybutyrate)

Graft copolymerization of maleic anhydride (MA) onto poly(3-hydroxybutyrate) (PHB) was carried out by use of benzoyl peroxide as initiator. The effects of various polymerization conditions on graft degree were investigated, including solvents, monomer and initiator concentrations, reaction temperature, and time. The monomer and initiator concentrations played an important role in graft copolymerization, and graft degree could be controlled in the range from 0.2 to 0.85% by changing the reaction conditions. The crystallization behavior and the thermal stability of PHB and maleated PHB were studied by DSC, WAXD, optical microscopy, and TGA. The results showed that, after grafting MA, the crystallization behavior of PHB was obviously changed. The cold crystallization temperature from the glass state increased, the crystallization temperature from the melted state decreased, and the growth rate of spherulite decreased. With the increase in graft degree, the banding texture of spherulites became more distinct and orderly. Moreover, the thermal stability of maleated PHB was obviously improved, compared with that of pure PHB.

Isothermal crystallization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Isothermal crystallization behavior of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was investigated by means of differential scanning calorimetry and polarized optical microscopy (POM). The Avrami analysis can be used successfully to describe the isothermal crystallization kinetics of PHBV, which indicates that the Avrami exponent n = 3 is good for all the temperatures investigated. The spherulitic growth rate, G, was determined by POM. The result shows that the G has a maximum value at about 353 K. Using the equilibrium melting temperature (448 K) determined by the Flory equation for melting point depression together with U-* = 1500 cal mol(-1), T-infinity = 30 K and T-g = 278 K, the nucleation parameter K-g was determined, which was found to be 3.14+/-0.07 x 10(5) (K-2), lower than that for pure PHB. The surface-free energy sigma = 2.55 x 10(-2) J m(-2) and sigma(e) = 2.70+/-0.06 x 10-2 J m(-2) were estimated and the work of chain-folding (q = 12.5+/-0.2 kJ mol(-1)) was derived from sigma(e), and found to be lower than that for PHB. This implies that the chains of PHBV are more flexible than that of PHB.

Effect of nucleating agents on the crystallization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

The effect of nucleating agents on the crystallization behavior of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was studied. A differential scanning calorimeter was used to monitor the energy of the crystallization process from the melt and melting behavior. During the crystallization process from the melt, nucleating agent led to an increase in crystallization temperature (T-c) of PHBV compared with that for plain PHBV (without nucleating agent). The melting temperature of PHBV changed little with addition of nucleating agent. However, the areas of two melting peaks changed considerably with added nucleating agent. During isothermal crystallization, dependence of the relative degree of crystallization on time was described by the Avrami equation. The addition of nucleating agent caused an increase in the overall crystallization rate of PHBV, but did not influence the mechanism of nucleation and growth of the PHB crystals. The equilibrium melting temperature of PHBV was determined as 187degreesC. Analysis of kinetic data according to nucleation theories showed that the increase in crystallization rate of PHBV in the composite is due to the decrease in surface energy of the extremity surface.

Nonisothermal crystallization and melting behavior of poly(3-hydroxybutyrate) and maleated poly(3-hydroxybutyrate)

Nonisothermal crystallization and melting behavior of poly(3-hydroxybutyrate) (PHB) and maleated PHB were investigated by differential scanning calorimetry using various cooling rates. The results show that the crystallization behavior of maleated PHB from the melt greatly depends on cooling rates and its degree of grafting. With the increase in cooling rate, the crystallization process for PHB and maleated PHB begins at lower temperature. For maleated PHB, the introduction of maleic anhydride group hinders its crystallization, causing crystallization and nucleation rates to decrease, and crystallite size distribution becomes wider. The Avrami analysis, modified by Jeziorny, was used to describe the nonisothermal crystallization of PHB and maleated PHB. Double melting peaks for maleated PHB were observed, which was caused by recrystallization during the heating process.

The kinetics of the thermal decomposition of poly(3-hydroxybutyrate) and maleated poly(3-hydroxybutyrate)

The thermal decomposition mechanism of maleated poly(3-hydroxybutyrate) (PHB) was investigated by FTIR and H-1 NMR. The results of experiments showed that the random chain scission of maleated PHB obeyed the six-membered ring ester decomposition process. The thermal decomposition behavior of PHB and maleated PHB with different graft degree were studied by thermogravimetry (TGA) using various heating-up rates. The thermal stability of maleated PHB was evidently better than that of PHB. With increase in graft degree, the thermal decomposition temperature of maleated PHB gradually increased and then declined. Activation energy E. as a kinetic parameter of thermal decomposition was estimated by the Flynn-Wall-Ozawa and Kissinger methods, respectively. It could be seen that approximately equal values of activation energy were obtained by both methods.

Study on the biodegradation process of poly (3-hydroxybutyrate)

Poly (3-hydroxybutyrate) (PHB) films were biodegraded by DS9701. The degradation process was monitored by using SEM. It was shown that the PHB degradation occurred firstly in the amorphous part of PHB and then in the crystalline part, especially from the center of PHB spherulites. PHB deplymerase produced by DS9701 mainly attacked the second ester bond of PHB and the degraded product was dimmer, determined by using mass spectrometer.

转基因植物生产生物可降解塑料PHB的研究-phbA功能鉴定、多价表达载体的构建及转基因植物的获得

聚-β-羟基链烷酸（PHA）是许多微生物作为碳源、能源的一类贮藏性聚酯，具有广泛的应用价值。该聚酯可被微生物完全降解且有与塑料相似的性质，因而研究并提高PHA在植物中的合成为解决环境污染提供了新的解决途径。 聚-β-羟基于酸酯（PHB）是研究的最早、研究的最清楚的一种PHA。用聚合酶链式反应扩增并克隆了真养产碱杆菌（Alcaligenes eutrophus）中合成PHB的一个关键酶——3-酮硫裂解酶基因phbA。DNA序列分析表明所克隆的基因与国外报道序列同源性很高，只有一个碱基对的区别。为了检测该基因的功能及导肽的定位效率，构建了带有导肽基因的组成型表达载体，由根癌农杆菌介导转化烟草（Nicotiana tabacum cv. Wisconsin 38）得到转基因植株。蛋白质电泳结果表明导肽可以将外源蛋白定位于质体，phbA基因能翻译成相应大小的蛋白。酶活性分析证实了转基因烟草中phbA编码的3-酮硫裂解酶可以催化乙酰-CoA合成乙酰乙酰-CoA。 将携有导肽序列的phbC（编码PHB合酶）和phbB（编码乙酰乙酰-CoA还原酶）连入pBIB-HYG得到组成型表达载体pZCB，用冻融法转入根癌农杆菌，介导转化烟草。烟草为已获得的具有卡那霉素抗性整合并表达phbA的转基因烟草。通过二次转化将携有潮霉素抗性的phbB基因和phbC基因导入已整合phbA的烟草，各基因均由质体导肽控制，最后得到整合PHB合成的三个酶基因的转基因烟草。转基因烟草经PCR、PCR-Southern检测，初步确定整合phbB和phbC烟草植株。以气相色谱初步分析，转基因烟草中PHB的含量可达鲜重的0.233%。 结果表明phbB和phbC基因可以在真核表达系统中编码相应的蛋白。通过色素分析、荧光动力学等手段分析了PHB在叶绿体中的累积对其功能的影响。 为了提高底物乙酰-CoA的供应能力及减少惰性聚酯对植物体的伤害，分离了种子特异性启动子和质体导肽序列，利用忆经克隆的合成PHB的三个关键酶基因，通过一系列DNA重组，分别构建了含有种子特异性启动子的嵌合phbC、phbB的二价表达载体pSCB及嵌合phbC、phbA、phbB的三价表达载体pSCAB，并由导肽将基因表达产物定位于质体。经根癌农杆菌介导转化油菜（Brassica napus L.) H165，获得转基因油菜植株，并进行了PCR、Southern blot及RT-PCR-DNA杂交等分检测。结果表明，三基因已经分别整合到相应的转基因油菜中，并已在转录水平表达。同时转化了油菜不育系、恢复系和保持系，获得批量转化株，并移入温室栽培。

利用转基因植物生产生物可降解塑料(PHB)的研究

聚 3 一控基丁酸酯 (Poly – 3 - hydroxybutyrate,PHB) 及其它类型的聚 3-泾基链烷酸醋同属于聚酯类物质 , 是自然界中多种细菌的碳源及能源储备物。这种聚酯的物理化学特性与传统塑料相似 , 并具有生物可降解性 , 如能取代化学合成塑料将减少环境中的塑料废弃物 , 从源头治理 " 白色污染 " 问题。微生物发酵法生产的 PHB 价格过高 , 无法在市场上与化学合成塑料竞争。随着分子生物学的发展 , 人们逐渐将视线转向植物生物反应器。转基因植物能够利用二氧化碳为碳源、太阳能为能源合成目的产物 , 大大降低生产成本 , 为生产具有市场 竞争力的新型生物可降解塑料提供可行途径。在此领域虽然己取得一定进展 , 但远未达到商业化生产水平。大规模商业化生产要求转基因植物能够在确保环 境安全性的前提下高效、稳定地生产 PHB 。本文尝试改善植物中 PHB 的生产体系 ,为环保型塑料早日进入市场作出努力。 1. 由于表达框架中多次使用同一启动子会导致基因沉默 , 本文克隆了另一 种子特异性启动子 nap300, 以替换重复使用的7S启动子,减轻“共抑制”。将 nap300 与 GUS 基因相连进行功能鉴定。荧光检测和组织化学染色的结果都证明此仅 30Obp 的 DNA 序列足以调控基因进行种子特异性表达。尽管 B 盒作为 高度保守区在种子特异性表达中起重要作用 , 位于此处的两个碱基替代型突变 并未使 nap300 的活性明显降低 , 对启动子的时空表达模式也无明显影响。将 nap300 、 7S 分别与 phbA 基因 ( 编码 3-酮硫裂解酶) 相连 , 在相似表达环境中 对二者功能进行比较 , 发现两个启动子表达模式基本相同并在同一时期达到活 性高峰 , 因此 nap300 可用于改善 PHB 合成基因在植物体内的表达调控。通过 对种子特异性启动子的比较可加深对其表达模式的了解 , 为植物基因工程中的 精细调控提供依据。 2. 叶绿体基因工程是随着植物遗传转化技术发展刚刚兴起的生物技术 , 具 有超量表达外源基因 , 为原核基因提供适宜表达环境 , 消除 “位置效应”和基因沉默 , 环境安全性好等优点 , 较更适合用于植物生物反应器方面的研究。本研究在国内率先探讨将叶绿体转化技术引入植物生产生物可降解塑料这一领域 的可行性 ( 国外仅有日本一例 ), 构建了叶绿体转化及表达载体 pTRV-PHB, 通过基因枪法将 PHB 合成相关基因导入烟草叶绿体基因组。转基因烟草顺利达到同质化，其形态和生长发育均无异常。 Northern 点杂交检测表明与 PHB 合成相关的三个基因均能在转录水平表达 , 未出现核转化中经常发生的“基因沉默”现象。通过 RT-PCR 进一步检测表明叶绿体型转基因烟草中目的基因的表达水平明显比核转化植株中相应基因的表达水平高。气相色谱分析确证转基因植株具有合成 PHB 的能力。这些都表明叶绿体转化适合用于转基因植物生产 PHB的研究。虽然叶绿体型转基因烟草中产物含量偏低 , 并未达到预期结果 , 但经进一步改进与完善 , 终将会成功地用于生产高附加值产品的植物基因工程中。 3. 为初步探讨叶绿体转化中在同源重组反应介导下整合外源基因的机理 , 从油菜叶绿体基因组中分离两段序列作为同源片段 , 基因枪法转化烟草 , 结果显示即使供体所含同源片段与受体叶绿体基因组相应区域差异高达 10%, 转化效率也无降低。这一现象的发现有助于促进“通用载体”　的改进 , 扩展叶绿体转化受体范围乃至达到商业化应用水平。 4. 成功地通过二次转化获得整合并表达多基因的转基因烟草 , 缩短了研究周期 , 对相关转基因植物的研究有一定参考价值。本文还优化了油菜转化体系 , 使转基因油菜同时整合三个 PHB 合成相关基因的效率由 7.69% 增加至 16.0% 。 田间试验与产物分析正在进行中。

Hybrid bulk heterojunction solar cells from a blend of poly(3-hexylthiophene) and TiO2 nanotubes

Hybrid bulk heterojunction solar cells based on blend of poly(3-hexylthiophene) (P3HT) and TiO2 nanotubes or dye(N719) modified TiO2 nanotubes were processed from solution and characterized to research the nature of organic/inorganic hybrid materials. Compared with the pristine polymer P3HT and TiO2 nanoparticles/P3HT solar cells, the TiO2 nanotubes/P3HT hybrid solar cells show obvious performance improvement, due to the formation of the bulk heterojunction and charge transport improvement. A further improvement in the device performance can be achieved by modifying TiO2 nanotube surface with a standard dye N719 which can play a role in the improvement of both the light absorption and charge dissociation. Compared with the non-modified TiO2 nanotubes solar cells, the modified ones have better power conversion efficiency under 100 mW/cm(2) illumination with 500W Xenon lamp. (C) 2008 Elsevier B. V. All rights reserved.

Electropolymerized poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) film on ITO glass and its application in photovoltaic device

Poly(3,4-ethylenedioxythiopliene):poly(styrene sulfonate) (PEDOT:PSS) films have been electrochemically polymerized in situ on ITO glass substrate in boron trifluoride diethyl etherate electrolyte (BFEE). Cyclic voltammograms show good redox activity and stability of the PEDOT films. These films had been directly used to fabricate organic-inorganic hybrid solar cells with the structure of ITO/PEDOT/ZnO:MDMC-PPV/Al. The solar cells made of electrochemically polymerized films exhibit higher energy conversion efficiencies compared with that prepared by the spin-coating method, and the highest value is 0.33%. This in-situ electropolymerized method effectively simplifies fabricating procedures and may blaze a facile and economical route for producing high-efficiency solar cells.

Morphology and crystalline transition of poly (3-butylthiophene) associated with its polymorphic modifications

Poly (3-butylthiophene) (P3BT) is a much less studied conjugated polymer despite its high crystallizability and thus excellent electrical property. In this work, morphology of P3BT at different crystalline polymorphs and solvent/thermal induced phase transition between form I and U modifications have been intensively investigated by using optical microscopy, electron microscopy, differential scanning calorimetry, and X-ray diffraction. It is shown that a direct deposition from carbon disulfide (CS2) at fast evaporation results in P3BT crystals in form I modification, giving typical whiskerlike morphology. In contrast, low evaporation rate from CS, leads to formation of form II crystals with spherulitic morphology, which is so far scarcely observed in polythiophene.

Effects of fullerene solubility on the crystallization of poly(3-hexylthiophene) and performance of photovoltaic devices

The substantial crystallization suppression of poly(3-hexylthiophene) (P3HT) in the untreated P3HT:C60 composite film prepared from o-dichlorobenzene (ODCB) solution has been revealed. Besides, the effective conjugation length of P3HT in this composite has been nearly maintained to that in the solution. The different crystallization behaviors of P3HT in its composites with C60 and [6,6]-phenyl C-61 butyric acid methyl ester (PCBM) are mainly attributed to the relative solubility of C60 and PCBM with respect to P3HT in ODCB. The solution to overcome this disadvantage of chain conformation and crystallinity of P3HT in the composite with C60 is thus proposed and finalized by resorting to the addition of low volatile solvent with much higher solubility of C60 than P3HT into the main solvent used, so as P3HT can crystallize before C60 forms crystallites in the solution. The feasibility of this approach has been proven by the improved efficiency of devices based on composites of P3HT and the low cost C60 without resorting to post-treatments.

Solvent Vapor-Induced Self Assembly and its Influence on Optoelectronic Conversion of Poly(3-hexylthiophene): Methanofullerene Bulk Heterojunction Photovoltaic Cells

Nanoscale-phase separation of electron donor/acceptor blends is crucial for efficient charge generation and collection in Polymer bulk heterojunction photovoltaic cells. We investigated solvent vapor annealing effect of poly(3-hexylthiophene) (P3HT)/methanofullerene (PCBM) blend oil its morphology and optoelectronic properties. The organic solvents of choice for the treatment have a major effect oil the morphology of P3HT/PCBM blend and the device performance. Ultraviolet-visible absorption spectro,;copy shows that specific solvent vapor annealing can induce P3HT self-assembling to form well-ordered structure; and hence, file absorption in the red region and the hole transport are enhanced. The solvent that has a poor Solubility to PCBM Would cause large PCBM Clusters and result in a rough blend film. By combining an appropriate solvent vapor treatment and post-thermal annealing of the devices, the power conversion efficiency is enhanced.