【作者】 李丽；

【导师】 杜林森； 宋艳茹；

【作者基本信息】 首都师范大学 ， 植物学， 2001， 硕士

【摘要】 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，具有广泛应用价值。该聚酯具有与合成塑料相似的性质并可被微生物完全降解，研究PHA在植物中合成并提高其含量可为解决环境污染提供新途径。 聚-β-羟基丁酸酯（PHB）为分布广泛、研究最早、研究较为清楚的PHAs。前期工作已通过聚合酶链式反应扩增并克隆了罗氏真养菌（Ralstonia eutrophus）中PHB合成三个关键酶基因——phbA（编码3-酮硫裂解酶）、phbB（编码乙酰乙酰CoA还原酶）、phbC（编码PHB合酶），并分离种子特异性启动子（7S promoter）和质体导肽序列（ctp），利用已克隆的三个基因，分别构建含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，并由导肽将基因产物定位质体。经根癌农杆菌介导转化油菜（Brassica napus L.）H165，分子检测表明，已获分别整合三个基因且在转录水平表达的转基因植株。 上述表达框架中三个酶基因均采用相同启动子和导肽序列，较多同源序列会增加基因沉默的可能性，我们拟从油菜品系H165中分离新的启动子（napinBpromoter），优化现有表达框架。NAPIN是由基因家族编码的油菜（Brassica napus）种子贮存蛋白，napinB基因已经分离和鉴定。Radke（1988）等人已证明翻译起始密码子前300bp序列就足以启动下游嵌合基因在转基因油菜中相应发育阶段正确表达。根据引物设计原则和已知DNA序列，设计并合成特异性引物，通过PCR技术从油菜（H165）基因组DNA中扩增到一条约300bp的带。测序结果与napinB启动子比较显示，同源性达97%以上。除TACACAT框内一个碱基由A变为T、B-box中两个碱基突变之外，其余调控序列完全吻合；非保守区有5个碱基不同源，三处因引入了酶切位点，另两个非同源碱基推测可能该启动子在不同油菜品系间差异所致。以上结果分析表明，nap300可启动外源基因的种子特异性表达。我们将扩增到的片段分别与gus和phbA基因相连，构建鉴定其功能的表达框架。经冻融法转化农杆菌 摘 要 LBA4404，并通过叶盘法分别对烟草（Nlcotjma tabac伽 cv．Wisconsin38）进行转化， PCR和 PCR－Southern结果表明我们已获转基因植株，取其种子做 GUS和卜酮硫裂解 酶活性分析，因烟草生长周期较长，目前鉴定工作还在进行中。我们将用其替换原 二价和三价表达框架中相同的7S启动子，以期获得高PHB表达量的转基因植株；同 时，利用己有的三种种子特异性表达载体继续油菜三系转化工作，已获分别整合相 应基因的转基因油菜植株，为田间杂交工作提供更多的试材，为早日实现PHB商品 化生产提供理论依据。更多还原

【Abstract】 As a carbon reserve, Polyhydroxyalkanoates (PHAs) are produced in many species of bacteria when grown in an unbalanced nutrient condition. PHAs, with its water-resistance, good physical and piezoelectric properties, have the properties to rubber like materials, making them suitable polymers for a wide variety of uses. Especially, with the distinct advantage of being biodegradable and biocompatibility, PHAs are ideal alternatives to petrochemically derived plastics, which caused serious environmental pollution. Poly- P -hydroxybutyrate(PHB) is the best understood species of PHAs. The three enzymes involved in PHIB synthesis, phbA (encodes the first key enzyme of PHIB synthesis, 3- ketothiolase), phbB and phbC have been amplified and cloned from chromosomal DNA of Ralstonia eutrophus by PCR amplification. An effective way to reduce the harm to plant caused by accumulated polyester is to confine PHB in plastid of seed by seed-specific promoter and ctp gene (encoding chloroplastid transit peptide, CT?). 7s promoter and ctp gene were isolated and seed-specific expression vectors pSCB containing phbC and phbB and pSCAB containing phbC, phbB and phbA were constructed. cq gene led the enzymes involved in the PHB synthesis into plastids. Transgenic rapeseeds, which were confirmed by PCR and Southern analysis, were obtained. RT-PCR results showed that each gene has been expressed at the transcription level. In order to avoid the possibility of gene silence caused by high homology of promoter, it is necessary to isolate a new promoter to improve the constructed expression vectors. One of the napin genes, napinB, was isolated from Brassica napus (rape) by Mats et al. Radke et al have shown that 300 bp of the 5?flanking sequence in front of the translation initiation codon are sufficient to give a developmentally faithfhl expression of a chimeric gene construct in transgenic Brassica napus plants. Moreover, most of the important elements necessary for the expression of napin gene are contained in this region. A fragment of 286bp (nap3 00) in napinB promter region was isolated by PCR from genomic DNA of Brassica napus H 165. Nap300 and the corresponding sequence of napinB Ill promoter shared 97% homology at the nucleotide level, except for eight different bases. One in the TACACAT conserved motif (allowing one base mismatch) changed from A to T, which is more favorite in Brassica napus, two in B-box and the other five in non- consensus region which may be due to differences among varieties of Brassica napus and designed enzyme site. Two expression vectors harboring the nap300 promoter fused with GUS gene and phbA were constructed for functional analysis, respectively. Tobacco plants were transformed by Agrobacterium tumefaciens-mediated method. PCR and Southern results showed that nap300 had been integrated into genomic DNA of tobacco successfully. The assays of GUS and 3-ketothiokase activity in the seeds of transgenic tobaccos are still being carried out to identify the function of the promoter for the long growth period needed for tobacco plants to bear fruits. In our further work, the promoter would take the place of 7s promoter used repeatedly in the expression vectors pSCB and pCAB. We hopefully think that the transgenic plants with the improved expression vectors could produce high content of P1-lB and also provide scientific basis for the commercialization of PHB in soon early time. Transformatio更多还原