Molecular breeding of yeast using mitochondria as vecter

以线粒体为载体的酵母分子育种

• 批准号: 04806017
• 负责人: SAKAI Takuo
• 金额: $ 1.22万
• 依托单位: University of Osaka Prefecfure
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04806017/
• 关键词: yeast; mitochondria; molecular breeding; apocytochrome b; ミトコンドリアDNA; 遺伝子

We attempted to develop new method to introduce heterogeneous gene to yeast cell. In the method, heterogeneous gene is introduced to mitochondria DNA (mtDNA) using integration vecter plasmid on which heerogeneous gene is conected with marker gene. As the marker, we selected apocytochrome b (Cob) , wich is an enzyme on respiration chain of the yeast, and Cob-deficient yeast lose its respiration ability. The enzyme has relatively low molecular weight and its gene seemed to be suitable for the gene marker. If the gne connected with Cob gene is introded on Cob deficient mtDNA,the respiration ability is restore so as to be found introduction of new gene.We constructed the above gene introduction system, according the following steps(1) Development of effective method to prepare mitochondria and its introduction to yeast cells.(2) Development of effective method to prepare mtDNA.(2) Cob gene cloning by PCR method.In the next step, We intend to introduce Lac Z gene in Cob gene to induce incomplete Cob gene, then, introduce it on mtDNA resulting to introduce Cob deficent mutant.Finally, construction of "Cob deficient cell-Cob harboring vecter system" is accomplished.

本研究试图探索一种将外源基因导入酵母细胞的新方法。该方法是利用整合载体质粒将外源基因导入线粒体DNA中，外源基因在载体质粒上连接有标记基因。选择了酵母呼吸链上的脱辅基细胞色素B（Co B）作为标记，发现Co缺陷的酵母丧失了呼吸能力。该酶相对分子质量较低，其基因可能适合作为基因标记。将与Cob基因相连的基因导入Cob缺陷型mtDNA，使其呼吸功能恢复，从而发现新基因的导入，我们按照以下步骤构建了上述基因导入系统：（1）建立有效的线粒体制备方法，并将其导入酵母细胞; (2)线粒体DNA有效制备方法的建立。(2)通过PCR方法克隆Cob基因，将Lac Z基因导入Cob基因中，诱导Cob基因不完全表达，再将其导入mtDNA中，形成Cob缺失突变体，最终构建“Cob缺失细胞-Cob载体系统”。