Preparation of Yarrowia lipolytica P12 haploid

Abstract:

[Background] Currently, with the in-depth study of the yeast and the completion of the whole genome and mitochondrial gene sequence determination, Yarrowia lipolytica is popular for using in experimental research and industrial production. However, compared with conventional yeasts, the lack of effective genetic transformation system is also difficulties in gene regulation for Y. lipolytica. At the same time, the chromosome ploidy of yeast will also affect the transformation. When the haploid cells are as receptors for functional gene modification, it can avoid the effect of interaction between alleles. Solve the problem of incomplete gene knockout in polyploid cells. [Objective] The mutant strain P12 of Y. lipolytica was used as the research object. The haploid protease was isolated by different methods, with the aim of establishing a method of preparing haploid strains of Y. lipolytica. [Methods] Y. lipolytica strain was induced to produce ascospores by solid McClary sporulation medium with the culture condition at 30 °C for 7?14 d and liquid McClary sporulation medium with the culture condition at 200 r/min for 2?4 d, respectively. Then the ascospore cell wall was lysed with 2% snail enzyme at 33 °C for 3 h. Finally, the haploid cells were screened and identified by staining microscopy and PCR. [Results] The growth rate of Y. lipolytica was faster in liquid sporulation medium, and the quality of sporulation was better in solid sporulation medium. In the same field of view, the number of spores in the liquid sporulation medium was about 3.7 times higher than in the solid sporulation medium. Moreover, six strains of Y. lipolytica P12 type B haploid were obtained through preliminary exploration and screening. [Conclusion] This research laid the foundation for the subsequent continued operations of genetic engineering.

Key words:Yarrowia lipolytica P12, ascospores, haploid, MATA/MATB