Molecular breeding of yeast using mitochondria as vecter

我们尝试开发一种将异种基因导入酵母细胞的新方法。该方法利用整合载体质粒将异质基因导入线粒体DNA (mtDNA)，异质基因与标记基因连接在质粒上。我们选择了细胞色素b （Cob）作为标记，它是酵母呼吸链上的一种酶，缺乏Cob的酵母失去了呼吸能力。该酶分子量相对较低，其基因似乎适合作为基因标记。将Cob基因相关基因导入Cob缺陷mtDNA，恢复呼吸能力，寻找新基因的引入。我们按照以下步骤构建了上述基因导入体系：(1)建立有效的线粒体制备方法并将其导入酵母细胞。(2)建立有效的mtDNA制备方法。(2) PCR法克隆Cob基因。下一步，我们打算在Cob基因中引入Lac Z基因，诱导不完整Cob基因，然后将其引入mtDNA，从而引入Cob缺陷突变体。最后，完成了“Cob缺陷细胞-Cob窝藏载体体系”的构建。

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.