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鱼腥藻脂肪酸代谢途径的遗传改造
Alternative TitleGenetic Modifications of Fatty Acids Metabolism Pathway in Anabaena sp.
盛春霞
Subtype硕士
2008-08-28
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword蜡酯 生物柴油 基因工程 鱼腥藻
Abstract蓝藻是地球上最早出现的能进行放氧型光合作用的原核生物,能够利用太阳光、CO2和无机营养物合成自身生长所需的糖类、蛋白质和脂类等有机物。蜡酯是由长链脂肪酸和长链脂肪醇酯化形成的一类氧酯,在一些动物、植物体内和微生物细胞中有积累。蜡酯可用于精密仪器润滑油原料及药物、食品和化妆品的添加剂等,有很高的商业价值,但资源有限,价格较昂贵。本研究第一部分将细菌和哺乳动物蜡酯合成相关的基因导入蓝藻中,培育能够合成蜡酯的转基因藻株,为能够大量生产低成本蜡酯提供了新的途径。柴油作为一种重要的石油炼制产品,在各国燃料结构中占有较高的比例,而石油资源的日益枯竭促使世界各国开发柴油替代燃料。生物柴油以其优越的环保性能越来越受到重视。本研究的第二部分是将两个细菌的两个乙醇合成基因和一个蜡酯合成基因导入大肠杆菌中,构建能够产生乙酯生物柴油的菌株。 研究内容和结果如下: 1. 从小白鼠KM株中通过RT-PCR克隆了编码脂肪酰辅酶A还原酶1的cDNA序列(FAR1 cDNA),并将该基因和克隆自不动杆菌ADP1株的蜡酯合成酶/二酰甘油酰基转移酶基因(wax/dgat)共同置于蓝藻启动子PrbcL的控制下,构建穿梭表达质粒。通过接合转移将穿梭表达质粒导入鱼腥藻PCC 7120(本文称WT)及其衍生藻株EF113(本文称EF113)中,筛选得到了能够合成蜡酯的转基因藻株。EF113的转基因株产蜡量高于WT的转基因株,可达细胞干重的1%。 2. 从运动发酵单胞菌中克隆了丙酮酸脱羧酶基因(pdc)和乙醇脱氢酶基因(adhB)。构建表达质粒将pdc、adhB和不动杆菌中的wax/dgat三个基因共同以lacZ启动子在E.coli DH5α表达。重组菌株在培养基中外加葡萄糖和油酸钠的条件下能够积累脂肪酸乙脂。
Other AbstractCyanobacteria, the earliest prokaryotes that perform oxygenic photosynthesis on the earth, can utilize sunlight, water, carbon dioxide and inorganic nutrients to synthesize saccharides, proteins, lipids and other organics. Wax esters found in plants, animals as well as microorganisms are oxoesters formed from long-chain fatty acids and long-chain fatty alcohols. Wax esters can be used as raw materials to manufacture lubricants used in accurate instruments, additives to medicine, foods or cosmetics, therefore, have high commercial values. However, natural wax esters are of limited resources and relatively expensive. The first part of my thesis describes the introduction of a bacterial and a mammalian gene, both involved in wax ester synthesis, into Anabaena sp. and a derivative to obtain transgenic strains capable of wax ester synthesis, proposing a new and more cost-saving route for large scale production of wax esters. Diesel, as an important product of oil refining, takes a high position in the world’s fuels markets. Due to the increasingly rapid exhaustion of the earth’s fossil energy resources, many contries are exploring alternative energy sources. For its extrodinary features in environment protection, biodiesel is attracting more and more interst. The second part of my thesis describes the introduction of two bacterial acohol synthesis genes and a wax ester synthesis gene into Escherichia coli to construct a recombinant strain that produces ethyl biodiesel. Experimentation and results are summarized as follows: 1. The mouse FAR1 cDNA encoding a bifunctional acyl-coenzymeA reductase (FAR1) was cloned from Mus musculus strain KM by RT-PCR. The FAR1 gene and wax/dgat encoding wax ester synthase (WS/DGAT), cloned from Acinetobacter baylyi strain ADP1, were both positioned under the control of PrbcL, resulting in an expressive shuttle vector. The shulte vector was introduced into Anabaena sp. PCC 7120 (referred to as WT in this thesis) and a derivative EF113 by conjugation, resulting in transgenic strains showing wax ester synthesis. The strain EF113 was more productive in wax esters than the WT, amounting to ca.1% cell dry weight. 2. pdc encoding pyruvate decarboxylase (PDC) and adhB encoding alcohol dehydrogenase (ADHB) were cloned from Zymomonas mobilis. An expressive plasmid was constructed and used to coexpress the two genes and wax/dgat althogether from the promoter PlacZ in E.coli DH5α. In the presence of exogenous glucose and sodium oleate, the recombinant strain was able to accumulate fatty acid ethyl esters.
Pages68
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12352
Collection学位论文
Recommended Citation
GB/T 7714
盛春霞. 鱼腥藻脂肪酸代谢途径的遗传改造[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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