Enbancement of freezing tolerance of baker, s yeast by introduction of haedening-inducible genes from Chlorella

hiC 6基因，编码一个同源的晚期胚胎发育丰富（莱亚）蛋白，被引入到酿酒酵母。将其插入在酵母GAL 1启动子的转录控制下的多拷贝质粒上。免疫组化法证实了BMP 6蛋白的表达。随着半乳糖诱导时间的延长，该蛋白的表达量逐渐增加。在最大诱导时间下，hiC 6基因转化酵母的抗冻性比对照酵母提高了3.3倍（存活率从20%提高到66%），本研究尝试分离编码葡萄糖-6-磷酸脱氢酶和ω-3去饱和酶的小球藻基因，以期进一步提高酵母的抗冻性。为了PCR扩增这两个基因，基于来自其他生物体的相应基因的序列合成寡核苷酸引物。用来自6小时硬化的小球藻细胞的Poly（A）^+ RNA合成单链cDNA。以单链cDNA为模板，通过PCR扩增编码葡萄糖6-磷酸脱氢酶和ω-3去饱和酶的基因的部分编码区。扩增片段大小分别为270 bp和900 bp，证实为目的基因的部分片段。现在，我们正试图克隆编码它们的全长基因，使用扩增的片段作为探针。

The hiC6 gene, encoding a homologue of a late embryogenesis abundant (LEA) protein, was introduced into Saccharomyces cerevisiae. It was inserted on a multicopy plasmid under the transcriptional control of the yeast GALl promoter. Expression of HIC6 protein was confirmed by immunochemical methods. Expression level of the protein was increased gradually with the induction-time by galactose. With maximum induction time, the freeze-tolerance of yeast transformed with hiC6 gene was approximately 3.3 times (from 20% to 66% survival rate) higher than that of the control yeast.The isolation of Chlorella genes, which encode glucose 6-phosphate dehydrogenase and omega-3 desaturase, were tried for further enhancement of freezing tolerance of yeast by introduction of them. For PCR amplification of the two genes, oligonucleotide primers were synthesized based on the sequences of the corresponding genes from other organisms. Single strand cDNAs were synthesized using Poly(A)^+RNAs from 6-h hardened Chlorella cells. Parts of coding regions of the genes encoding glucose 6-phosphate dehydrogenase and omega-3 desaturase were amplified by PCR using the single stranded cDNA as templates. The sizes of the amplified fragments were 270 bp and 900 bp, respectively, and confirmed to be parts of the objective genes. Now, we are trying to clone the full-length genes encoding them, using the amplified fhagments as probes.