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。

Expression and molecular analysis of phbB gene in Chlamydomonas reinhardtii

The expression vector containing phbB and ble genes was constructed and transformed into cell-wall-deficient strain Chlamydomonas reinhardtii CC-849 by the glass-head method. The transgenic alga was selected and maintained in the TAP agar plates containing 10 mug/mL Zeomycin. Transgenic alga, which could express phbB at the transcriptional level, was obtained and further confirmed with PCR, Southern blot and RT-PCR-DNA hybridization analysis.

Comparative study of PHBV/TBP and PHBV/BPA blends

In order to clarify the effects of phenols on properties of polyesters, the blends of poly[(3-hydroxybutyrate)-co-(3-hydroxyvalerate)] (PHBV) with 4,4'-dihydroxydiphenylpropane (BPA) and p-tert-butylphenol (TBP) were studied. The FTIR spectra revealed that there was strong hydrogen-bond (H-bond) interaction between PHBV and both phenols. By evaluating the fraction of H-bonded C = O in the blend, it was concluded that BPA showed a stronger tendency than TBP to form H-bonds with PHBV. Accordingly, BPA formed a stronger suppression than TBP on the crystallization of PHBV. When 30 wt% BPA or 50 wt% TBP were added into PHBV, the crystallization of PHBV was completely suppressed in the DSC cooling scan. As the phenol content was increased, the T-g of PHBV/TBP blend decreased while the T-g of PHBV/BPA blend increased. This difference indicated that TBP and BPA acted as plasticizer and physical crosslinking agent, respectively.

Quantitative FTIR study of PHBV/bisphenol A blends

FTIR spectroscopy was used to verify the presence of intermolecular hydrogen bond (inter-H-bond) between poly-(3-hydroxybutyrate co-3-hydroxyvalerate) (PHBV) and bisphenol A (BPA). By monitoring the spectral changes during PHBV crystallization and blends dissociation, the absorptivity ratio of C=O bonds in crystalline and amorphous regions in PHBV and that of H-bonded and free C=O in PHBV/BPA blends were experimentally determined as 1.40 and 1.68, respectively. Using curve-fitting program, the C=O absorptions in spectra of blends were ascribed to three types of bonds: amorphous, crystalline and H-bonded C=O. The crystallinity of PHBV and the fraction of H-bonded C=O were calculated. These results indicated that the H-bond clearly suppressed the PHBV crystallization. Furthermore, the fraction of BPA molecules that simultaneously formed two hydrogen bonds (H-bonds) with C=O was estimated. It revealed that there existed a H-bond network in PHBV/BPA blends. This network was compared with the covalent network by estimating the number of atoms between every two adjacent crosslink points in chain. Up to the high density of H-bond discussed in this paper, there was always a certain part in PHBV that crystallized due to the dynamic character of hydrogen bonds; however, the hydrogen bonds significantly reduced the crystallization rate of PHBV.

The morphology and thermal properties of multi-walled carbon nanotube and poly(hydroxybutyrate-co-hydroxyvalerate) composite

Nanocomposites based on poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and multi-walled carbon nanotubes (MWNTs) were prepared by solution processing. Ultrasonic energy was used to uniformly disperse MWNTs in solutions and to incorporate them into composites. Microscopic observation reveals that polymer-coated MWNTs dispersed homogenously in the PHBV matrix. The thermal properties and the crystallization behavior of the composites were characterized by thermogravimetric analysis, differential scanning calorimetry and wide-angle X-ray diffraction, the nucleant effect of MWNTs on the crystallization of PHBV was confirmed, and carbon nanotubes were found to enhanced the thermal stability of PHBV in nitrogen.

聚（3-羟基烷酸酯）的改性研究

该论文目的是改性细菌合成的聚(3-羟基丁酸酯)(PHB)及其共聚物(PHBV),采用交联或共混的方法,改变其聚集态结构或超分子结构,从而改善其力学性能.加深对高聚物结构与性能之间关系,高聚物结晶规律、及特殊相互作用在高聚物中作用的认识.1.采用反应性加工,用过氧化二异丙苯(DCP)引发PHBV的自由基链转移反应,产生了支化和交联的化学结构.2.用交联助剂二苯甲撑双马来酰亚胺(BMI)实现了PHBV的γ-辐射交联.交联的PHBV熔点和结晶度下降.3.双酚A(BPA)在PHBV/BPA共混物中起到了物理交联剂的作用.4.氢键交联结构使PHBV的链段运动受限,结晶速率下降.5.对叔丁基苯酚(TBP)在PHBV中形成了氢键接枝的超分子结构.6.经过溶液共混,BPA在PHB中起到了物理交联剂的作用,使PHB的断裂伸长率从3%提高到45%.8.二醋酸纤维素(CDA)与PHBV经溶液共混(混合溶剂氯仿/丙酮),PHBV的力学性能没有改善,原因可能是CDA-PHBV分子间的氢键作用较弱,组分间发生严重相分离,不利于性能提高.

聚3-羟基烷酸酯异相成核结晶的研究

本论文以具有巨大应用前景的PHBV作为研究对象，针对PHBV结晶速率低，易产生二次结晶，从而严重影响其加工性能和力学性能的稳定性等方面的缺点，根据PHBV的分子结构和脆性的机理分析，试图采用添加成核剂的方法，提高PHBV的结晶速率和结晶度，从而缩短PHBV的加工成型周期，控制其聚集态结构，提高其制品的稳定性，改善材料的物理力学性能。并在此基础上，探讨PHBV异相成核结晶的机理，加深成核剂对聚合物有效成核机理的认识，更好的理解聚合物结晶过程。1．添加成核剂的量达到0.5wt％时，对苯二甲酸（TPA）对PHBV起到了显著的结晶成核作用。结晶起始温度提高了约20℃，结晶速率达到最大值所对应的温度T_p提高了30℃，结晶烙增加了15J/g，结晶速率提高了4.4倍。这些数据表明，TPA是一种对PHBV极为有效的成核剂。2．加入成核剂TPA的PHBV表现出典型的双熔融行为，主要原因是TPA对PHBV的结晶成核作用和PHBV的熔融再结晶。低温侧的熔融峰对应着PHBV自熔体降温过程形成的结晶，高温侧的对应着PHBV升温过程中形成的结晶。3．TPA的成核作用大大的改变了PHBV的形态结构，使PHBV的球晶尺寸明显减小，球晶数量增大。4．TPA使PHBV晶体在（110）和（020）方向上微晶尺寸变大，晶区和非晶区的电子密度差增大。5．添加0.5wt％的TPA后，PHBV的断裂伸长率从4％提高到10％。6．TPA、IPA、淀粉和山梨醇对PHBV的结晶都具有很明显的成核作用，其原因可能是化学结构上都具有极性基团。7．红外光谱研究没有能够有效的给出PHBV与TPA是否存在特殊相互作用，从而导致TPA对PHBV的结晶成核作用的证据，但是，PHBV拨基伸缩振动谱带随温度的变化却给出了TPA对PHBv的结晶成核作用始于160℃高温。

Thermal and mechanical properties of poly(butylene terephthalate)/epoxy blends

In this study, melt blends of poly(butylene terephthalate) (PBT) with epoxy resin were characterized by dynamic mechanical analysis, differential scanning calorimetry, tensile testing, Fourier transform infrared spectroscopy, and wide-angle X-ray diffraction. The results indicate that the presence of epoxy resin influenced either the mechanical properties of the PBT/epoxy blends or the crystallization of PBT. The epoxy resin was completely miscible with the PBT matrix. This was beneficial to the improvement of the impact performance of the PBT/epoxy blends.

White electroluminescence from a single-polymer system with simultaneous two-color emission: Polyfluorene as blue host and 2,1,3-benzothiadiazole derivatives as orange dopants on the side chain

Four single polymers with two kinds of attachment of orange chromophore to blue polymer host for white electroluminescence (EL) were designed. The effect of the side-chain attachment and main-chain attachment on the EL efficiencies of the resulting polymers was compared. The side-chain-type single polymers are found to exhibit more efficient white EL than that of the main-chain-type single polymers. Based on the side-chain-type white single polymer with 4-(4-alkyloxy-phenyl)-7-(4-diphenylamino-phenyl)-2,1,3-benzothiadiazoles as the orange-dopant unit and polyfluorene as the blue polymer host, white EL with simultaneous orange (lambda(max) = 545 nm) and blue emission (lambda(max) = 432 nm/460 nm) is realised. A single-layer device (indium tin oxide/poly(3,4-ethylenedioxythiophene)/polymer/Ca/Al) made of these polymers emits white light with the Commission Internationale de l'Eclairage coordinates of (0.30,0.40), possesses a turn-on voltage of 3.5 V, luminous efficiency of 10.66 cd A(-1), power efficiency of 6.68 lm W-1, and a maximum brightness of 21240 cd m(-2).

White electrolumineseence from a single-polymer system with simultaneous two-color emission: Polyfluorene as the blue host and a 2,1,3-benzothiadiazole derivative as the orange dopant on the main chain

New single-polymer electroluminescent systems containing two individual emission species - polyfluorenes as a blue host and 2,1,3-benzothiadiazole derivative units as an orange dopant on the main chain - have been designed and synthesized. The resulting single polymers are found to have highly efficient white electroluminescence with simultaneous blue(lambda(max) = 421 nm/445 nm) and orange emission (lambda(max) = 564 nm)from the corresponding emitting species. The influence of the photoluminescence (PL) efficiencies of both the blue and orange species on the electroluminescence (EL) efficiencies of white polymer light-emitting diodes (PLEDs) based on the single-polymer systems has been investigated. The introduction of the highly efficient 4,7-bis(4-(N-phenyl-N-(4-methylphenyl)amino)phenyl)-2,1,3-benzothiadiazole unit to the main chain of polyfluorene provides significant improvement in EL efficiency. For a single-layer device fabricated in air (indium tin oxide/poly(3,4-ethylenedioxythiophene): poly(styrene sulfonic acid/polymer/Ca/Al), pure-white electroluminescence with Commission Internationale de l'Eclairage (CIE) coordinates of (0.35,0.32), maximum brightness of 12 300 cd m(-2), luminance efficiency of 7.30 cd A(-1), and power efficiency of 3.34 lm W-1 can be obtained.

The Heterogeneous Growth of P(3MeTh)--An Ellipsometric Study

Successive thicker P(3MeTh) layers are analysed by ex situ conventional and imaging ellipsometry. Thin films display a smooth surface, are compact and homogeneous while for a growth charge above 20 mC cm(-2) the polymer structure modifies to a still uniform but less dense layer. A two-layer model is used and a mathematical procedure is developed to obtain, simultaneously, from the experimental ellipsometric parameters, Delta and Psi, the thickness and the complex refractive index of P(3MeTh) films grown up to 80 mC cm(-2). Thicker polymer layers are disordered and present a high degree of surface roughness. 

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。

Fabrication of ZnO and its enhancement of charge injection and transport in hybrid organic/inorganic light emitting devices

ZnO nanocrystals were synthesized by hydrolysis in methanol. X-ray diffraction and photoluminescence spectra confirm that good crystallized ZnO nanoparticles were formed. Utilizing those ZnO nanoparticles and poly [2- methoxy-5 - (3',7'-dimethyloctyloxy)- 1,4-phenylenevinylene] (MDMO-PPV), light emitting devices with indium tin oxide (ITO)/poly(3,4-oxyethyleneoxy-thiophene):poly(styrene sulfonate) (PEDOT:PSS)/ ZnO:MDMO-PPV/Al and ITO/PEDOT:PSS/MDMO-PPV/Al structures were fabricated. Electrolummescence (EL) spectra reveal that EL yield of hybrid MDMO-PPV and ZnO nanocrystals devices increased greatly as compared with pristine MDMO-PPV devices. The current-voltage characteristics indicate that addition of ZnO nanocrystals can facilitate electrical injection and charge transport. The decreased energy barrier to electron injection is responsible for the increased efficiency of electron injection. (c) 2007 Elsevier B.V. All rights reserved.

Enhanced photoresponse from the ordered microstructure of naphthalocyanine-carbon nanotube composite film

Naphthalocyanine-sensitized multi-walled carbon nanotube (NaPc-MWNT) composites have been synthesized through the pi-stacking between naphthalocyanine (NaPc) and carbon nanotubes. The resultant nanocomposites were characterized with a scanning electron microscope (SEM), a transmission electron microscope (TEM), and by UV - vis absorption and photocurrent spectra. The long-range ordering was observed in the NaPc - MWNT composites by using a TEM. The enhancement in the absorption intensity and the broadening of the absorption wavelength observed in the composite films, which were due to the attachment of NaPc on the MWNT surface, is discussed based on the measured UV - vis absorption spectra. Furthermore, the photoconductivity of the poly( 3-hexylthiophene)(PAT6) - NaPc - MWNT composite film was found to increase remarkably in the visible region and broaden towards the red regions. These new phenomena were ascribed to the larger donor/acceptor (D/A) interface and the formation of a biconsecutive D/A network structure, as discussed in consideration of the photoinduced charge transfer between PAT6 and NaPc - MWNT.