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

通过引入小球藻的发光诱导基因增强面包酵母的冷冻耐受性

• 批准号: 08660164
• 负责人: HONJOH Ken-ichi
• 金额: $ 1.47万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08660164/
• 关键词: Chlorella; Yeast; Freezing to tolerance; LEA protein

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.

将编码晚期胚胎发生丰富蛋白(LEA)同源基因的hiC6基因导入酿酒酵母。它被插入到酵母胆汁启动子转录控制下的多拷贝质粒上。免疫组织化学方法检测HIC6蛋白的表达。随着半乳糖诱导时间的延长，该蛋白的表达水平逐渐升高。在最大诱导时间下，转化hiC6基因酵母的耐冻性约为对照酵母的3.3倍(存活率在20%~66%之间)。为了进一步提高酵母的耐冻性，我们尝试分离编码葡萄糖6-磷酸脱氢酶和omega-3脱饱和酶的小球藻基因。为了扩增这两个基因，根据其他生物中相应基因的序列，合成了寡核苷酸引物。用Poly(A)^+RNA从6h硬化的小球藻细胞中合成单链cDNA。以单链cDNA为模板，通过聚合酶链式反应扩增了葡萄糖6-磷酸脱氢酶和omega-3去饱和酶基因的部分编码区。扩增片段大小分别为270bp和900bp，证实为目的基因的一部分。现在，我们正试图克隆编码它们的全长基因，使用扩增的Fhages作为探针。