解脂亚罗酵母P12单倍体的制备

Preparation of Yarrowia lipolytica P12 haploid

【背景】目前解脂亚罗酵母在实验研究和工业生产方面的应用越来越广泛,但相较于常规酵母而言,解脂亚罗酵母缺乏简便有效的遗传转化体系,致使其在基因表达调控方面存在较大困难。同时,酵母的染色体倍性也会对基因敲除效果产生影响,选择单倍体细胞作为功能基因改造的受体可以避免等位基因之间相互作用的影响,解决多倍体细胞基因敲除不完全的问题。【目的】以解脂亚罗酵母诱变菌株P12为研究对象,以不同方法分离得到单倍体菌株,建立解脂亚罗酵母单倍体的制备方法。【方法】分别采用固体和液体McClary产孢培养基诱导解脂亚罗酵母菌株产生子囊孢子,培养条件为30℃,固体7-14 d;液体200 r/min,2-4 d。以2%浓度的蜗牛酶33℃水浴裂解子囊孢子细胞壁3 h,通过染色镜检和PCR鉴定筛选单倍体细胞。【结果】镜检结果表明,解脂亚罗酵母在液体产孢培养基中产孢速度较快,相同视野下孢子数约为固体产孢培养基的3.7倍,在固体产孢培养基中产孢质量较好。初步探索并筛选得到6株解脂亚罗酵母P12 B型单倍体菌株。【结论】解脂亚罗酵母P12 B型单倍体菌株的获得可为后续继续开展基因工程操作奠定基础。

[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 ℃ 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 ℃ 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.