Pichia pastoris is an ascomycetous budding yeast, which can exist in a haploid or a diploid state, and has asexual and sexual reproductive cycles. Haploid Pichia pastoris is a perfect material for breeding and genetic manipulation. The sporulation conditions and preparation methods of haploid cells were studied with diploid P. pastoris X33. The results showed that P. pastoris X33 sporulated on nitrogen-limiting agar. Then, the haploid cells could be prepared by 2 ways: (1) treat ascospore with 3% snailase at 30℃ for 2 h followed by incubation at 58℃ for 12 min, by which the yield of haploid cells was 60%; (2) vortex in 50% diethyl ether at room temperature for 5min, by which the yield of haploid cells was 37%.%毕赤酵母(Pichia pastoris)是子囊孢子型的出芽酵母,以单倍体或二倍体形式存在,能够进行无性繁殖和有性繁殖,在酵母育种或基因功能研究方面,单倍体细胞起着重要的作用.本研究以P.pastoris X33二倍体细胞为研究材料,探讨了子囊孢子诱导、单倍体制备方法.结果表明,P pastoris X33二倍体细胞在氮源限制培养基上能够诱导形成子囊.随后可经2种方法获得单倍体:(1)3％蜗牛酶30℃处理2h,58℃处理12min,单倍体获得率为60％；(2)50％乙醚涡旋处理5min,单倍体获得率为37％.
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