大豆查尔酮异构酶基因的克隆及粟酒裂殖酵母表达载体的构建.PDF

DOI :10.15933/j.cnki .1004 -3268.2010.11.001 河南农业科学 ＊ 张　卓, 王丕武 ,付永平, 刘洪禹 ( , 130118) :利用 RT-PCR 克隆Ⅱ型大豆查尔酮异构酶基因(CH I1 A ), 然后将目的片段与克隆载体 pM D18-T质粒相连接,检 后将目的基因重组于粟酒裂殖酵母表达载体 pESP-2 的MCS 序列之 中,使用电击法将重组质粒转化进入粟酒裂殖酵母中, 用PCR 和双酶切的方法来检 试验结果,表 明获得了CH I1A 完整开放阅读框(670 bp), 与已报道序列(NO:A Y595413)的同源性达到99%。 PCR 和酶切鉴定表明,CH I1A 已导入到酵母表达载体中。查尔酮异构酶基因的克隆、粟酒裂殖酵 母表达载体的构建, 为该基因的应用提供了依据。有望利用基因工程技术将该基因重组于酵母基 因组中并表达目的蛋白, 以此来催化合成黄酮和异黄酮类化合物。 :大豆;查尔酮异构酶基因;粟酒裂殖酵母;表达载体 :S565.1 　　:A 　　:1004-3268(20 10)11-0023-04 Cloning of Ch lcone Isomer ses (CH I1A )Gene of Soybe n for T r nsform tion w ith S ch iz osaccharomy ces p ombe Expression Vector ＊ ZHANG Zhuo,WANG Pi-w u ,FU Yong-ping, LIU Hong-yu (Biotechnologic l Center of Jilin Agricultur l University, Ch ngchun 130118, Chin ) Abstract:Fl vonoids nd isofl vonoids re m jor second ry met bolites th t medi te diverse bio - logic l functions .There re series of protein enzymes in the compound process .Ch lcone isomer ses (CHIs)is the key enzymes in the phenyprop noid p thw y th t produces fl vonoids nd isofl vonoids.Type Ⅱch lcone isomer se gene (here fter termed CH I1A )h d been cloned in the w y of reverse tr nscription l polymer se ch in re ction nd connected with pM D18-T pl s- mid.After checking the pl smid, dding the CH I1A gene into the M CS fr gment th t consist in the S ch iz osaccharomy ces p ombe expression vector.Then the recombined pl smid h d been tr ns- formed into the S .p ombe ye st cell with electric l tr nsform tion device.The recombin nt h d been identified by polymer se ch in re ction nd double enzymes digested .The results indic ted th t the cloned fr gment of CH