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

将编码胚胎发育晚期丰度蛋白（LEA）同源基因的hiC6基因导入酿酒酵母。在酵母GALl启动子的转录控制下，将其插入多拷贝质粒上。免疫化学方法证实HIC6蛋白表达。随着半乳糖诱导时间的延长，该蛋白的表达量逐渐升高。在最长的诱导时间内，经hiC6基因转化的酵母的抗冻能力比对照酵母提高了约3.3倍（存活率从20%提高到66%）。通过分离编码葡萄糖6-磷酸脱氢酶和omega-3去饱和酶的小球藻基因，进一步提高酵母的抗冻能力。根据其他生物中对应基因的序列合成寡核苷酸引物进行PCR扩增。用Poly(A)^+ rna从硬化6 h的小球藻细胞中合成单链cdna。以单链cDNA为模板，PCR扩增葡萄糖6-磷酸脱氢酶和omega-3去饱和酶基因的部分编码区。扩增的片段大小分别为270 bp和900 bp，确认为目标基因的一部分。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.