圆红冬孢酵母诱导性遗传操作平台的构建

发布时间：2018-08-09 09:16

【摘要】：圆红冬孢酵母(Rhodosporidium toruloides)是一株优良的微生物油脂生产菌,在营养限制条件下过量积累胞内油脂;并可用于生产胞外多糖、β-类胡萝卜素、工业用酶等,在生物制药产业和环境治理方面中有广泛的应用前景。同时,圆红冬孢酵母具有生长周期短、利用碳源广泛、油脂积累量多等优点,为生物柴油的能源发展战略提供廉价原料。【目的】利用分子生物学手段和基因工程技术,构建圆红冬孢酵母的诱导型遗传操作平台。【方法】通过生物信息学分析,以R.toruloides NP11基因组DNA为模板,利用聚合酶链式反应(polymerase chain reaction,PCR)方法进行扩增,分离葡萄糖阻遏型启动子Padh2(promoter of alcohol dehydrogenase gene)、半乳糖诱导型启动子Pgal1(promoter of galactokinase)、磷酸盐饥饿诱导型启动子Ppho89(promoter of sodium:inorganic phosphate symporter gene)和终止子Thsp、Tgpd基因片段,然后采用RF(restriction free)克隆方法替换原载体pZPK-pPGK-hyg-tNOS上的组成型启动子Ppgk和终止子Tnos,以潮霉素磷酸转移酶(hygromycin phosphotransferase)基因为报告基因,构建了6种内源性诱导型载体,通过农杆菌介导转化的方法转入单倍体R.toruloides NP11和二倍体R.toruloides Y4基因组中,获得有潮霉素抗性的重组菌株,利用潮霉素抗性表型验证启动子和终止子的功能。在此基础上引入多克隆位点MCS(multiple cloning site),以pZPK-pPGK-hyg-tNOS质粒为基础,构建3种双表达盒诱导型MCS载体,并利用其表达苹果酸酶(malic enzyme)基因。【结果】成功构建了3种圆红冬孢酵母诱导性表达载体,该载体在圆红冬孢酵母中都可表现出启动子和终止子活性,初步了建立圆红冬孢酵母诱导型操作平台,并成功表达了苹果酸酶基因,为进一步优化或改变圆红冬孢酵母的代谢网络和表达调控网络的研究提供便利的平台。【结论】所分离的Ppho89启动子受磷酸盐浓度的严谨调节,Padh2和Pgal1启动子受葡萄糖浓度的严谨调节,诱导响应度高,操作简单经济便捷,为后续圆红冬孢酵母代谢工程研究提供了基本材料。本研究仍在继续完善和优化圆红冬孢酵母表达系统,希望构建出更高效的表达载体,进一步提高目的蛋白的表达量。
[Abstract]:The yeast (Rhodosporidium toruloides) is an excellent microorganism oil producing strain, which can accumulate intracellular oil excessively under the condition of nutrition restriction, and can be used to produce extracellular polysaccharides, 尾 -carotenoids, industrial enzymes and so on. It has a wide application prospect in biopharmaceutical industry and environmental governance. At the same time, Pichia cerevisiae has the advantages of short growth cycle, wide carbon source and abundant oil accumulation, which provides cheap raw materials for the energy development strategy of biodiesel. [objective] to utilize molecular biological methods and genetic engineering techniques. To construct the inducible genetic manipulation platform of Saccharomyces cerevisiae. [methods] by bioinformatics analysis, the genomic DNA of R.toruloides NP11 was amplified by polymerase chain reaction (PCR) (polymerase chain reactions-PCR. The glucose repressor promoter Padh2 (promoter of alcohol dehydrogenase gene), galactose inducible promoter Pgal1 (promoter of galactokinase), phosphate hunger inducible promoter Ppho89 (promoter of sodium:inorganic phosphate symporter gene) and terminator TGPD gene were isolated. Then RF (restriction free) cloning method was used to replace the constitutive promoter Ppgk and Terminator on the original vector pZPK-pPGK-hyg-tNOS. Six endogenous inducible vectors were constructed using hygromycin phosphotransferase (hygromycin phosphotransferase) as the reporter gene. The recombinant strains with hygromycin resistance were obtained by Agrobacterium tumefaciens mediated transformation into haploid R.toruloides NP11 and diploid R.toruloides Y4 genomes. The function of promoter and Terminator was verified by hygromycin resistance phenotype. On the basis of pZPK-pPGK-hyg-tNOS plasmid, three kinds of double expression box inducible MCS vectors were constructed by introducing polyclonal MCS (multiple cloning site),. The expression of malonase (malic enzyme) gene was used to construct three kinds of inducible expression vectors of Rhodozyme rotundii. All of the vectors showed promoter and Terminator activity in Rhodococcus cerevisiae. The inducible operating platform was established and the malonase gene was successfully expressed. It provides a convenient platform for further optimizing or changing the metabolic network and expression regulation network of Rhodococcus cerevisiae. [conclusion] the isolated Ppho89 promoters are rigorously regulated by phosphate concentration of Padh2 and Pgal1 promoters. The strict regulation of sugar concentration, The induction response is high, and the operation is simple, economical and convenient, which provides the basic materials for the further study on the metabolism of Rhodostasia cerevisiae. In order to construct a more efficient expression vector and to further improve the expression of the target protein, this study continues to perfect and optimize the yeast expression system of Aspergillus cerevisiae and hope to construct a more efficient expression vector.
【学位授予单位】：大连工业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q93;TE667

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