| Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae, Kluyveromyces lactis, Candida, Bacillus subtilis and so on. In this study, we discuss a new potentially useful strain for industries-Rhodotorula glutinis, which is able to grow in various cheap agricultural raw materials. More importance is that Rhodotorula glutinis has huge biomass and mevalonic acid (MVA), which is the precursor of ubiquinone,β-carotene and ergosterol. In order to elucidate the metabolic regulation in R.glutinis in molecular and genetic level, it is increasingly necessary to develop an efficient and conventional genetic transformation system of R. glutinis.Using homologous recombination integration of foreign genes into the yeast chromosome to achieve stable expression has been widely applied. The target sequences are ARS, URA3 gene,5 and 3'non-coding region of alcohol dehydrogenase gene, rDNA sequence; Transposon. And Yeast conversion technologies become more sophisticated. Protoplast method, LiAC method, electroporation method are very efficient methods.rDNA in the yeast genome having 100-200 repeating units, therefore the vector not only to overcome the usual integration vector only a few copies of limitations, while retaining stability,it is a kind of ideal for the expression of foreign gene delivery systems. Scientists in Netherlands in 1989 and 1991 first reported in constructing high-copy integration vector using S.cerevisiae rDNA fragment and high expression of the PGK and bhaumtin gene. The strategy of rDNA locus for the integration to improve copy number is used in Saccharomyces cerevisiae, Candida utilis yeast and Kluyveromyces a study and other microorganisms.In this thesis, R.glutinis strain As.2.102 having poor molecular biological information is our research object, the novel integrating expression vector, expression system and transformation system constructed for the further theoretical and Practical application studies of Rhodotorula glutinis.In the previous work, morphology, growth and antibiotic susceptibility characteristics, physiological and biochemical characteristics of Rhodotorula glutinis, have been studied.From the yeast genome amplified 621bp of 26SrDNA and 555bp fragment of the 5.8S-ITSrDNA sequence, upload Gene Bank, numbered bankit1350518 and bankit1350528.With the promoter gap from pGAPZA, the bleomycin gene (shble) for the resistance selection marker, green fluorescent protein gene (GFP) as reporter gene,and the two rDNA amplified as the same source integration sites. We have successfully a novel integrated vector pGAPZ/rD/GFP. Improved PEG /lithium acetate method and the optimization of a complete cell transformation, regeneration methods were used. Results showed that the establishment of the transformation method suitable for As.2.102 strains, the GAP and GFP have both been expressed in R.glutinis, also proved pGAPZ/rD/GFP can be integrated into homologous chromosomes. Secondary selection to recombinant cells is fast, simple and intuitive.In this article, We also tried to use the RACE amplification and pMD18T-based promoter probe vector screening for partially digested fragments of the genome with promoter activity, these two strategies to clone R.glutinis'own promoter.We prepared to improve and optimize the expression system with more copy numbers in the next, explain the mechanism of functional components and integrations, more efficient promoter, optimize the composition of the various components to build a secreted, more efficient expression vector.
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