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  

Cloning of Chalcone Isomerases(CHI1A) Gene of Soybean for Transformation with Schizosaccharomyces pombe Expression Vector

作　　者：张卓[1] 王丕武[1] 付永平[1] 刘洪禹[1] 

机构地区：[1]吉林农业大学生物技术中心,吉林长春130118

出　　处：《河南农业科学》2010年第11期23-26,共4页Journal of Henan Agricultural Sciences

基　　金：国家自然科学基金(30971805);吉林农业大学博士点基金(20070193005)

摘　　要：利用RT-PCR克隆Ⅱ型大豆查尔酮异构酶基因(CHI1A),然后将目的片段与克隆载体pMD18-T质粒相连接,检测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测试验结果,表明获得了CHI1A完整开放阅读框(670bp),与已报道序列(NO:AY595413)的同源性达到99%。PCR和酶切鉴定表明,CHI1A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。Flavonoids and isoflavonoids are major secondary metabolites that mediate diverse biological functions.There are a series of protein enzymes in the compound process.Chalcone isomerases（CHIs） is the key enzymes in the phenypropanoid pathway that produces flavonoids and isoflavonoids.Type Ⅱ chalcone isomerase gene（hereafter termed CHI1A） had been cloned in the way of reverse transcriptional polymerase chain reaction and connected with pMD18-T plasmid.After checking the plasmid,adding the CHI1A gene into the MCS fragment that consist in the Schizosaccharomyces pombe expression vector.Then the recombined plasmid had been transformed into the S.pombe yeast cell with electrical transformation device.The recombinant had been identified by polymerase chain reaction and double enzymes digested.The results indicated that the cloned fragment of CHI1A contained 670 base pairs,and shared a sequence homology of 99% with that from GenBank accession number AF595413（CHI1A）.It is feasible to produce flavonoids and isoflavonoids in a primary method depending on the expression of the recombined vector in which CHI1A involved.

关 键 词：大豆 查尔酮异构酶基因 粟酒裂殖酵母 表达载体 

分 类 号：S565.1[农业科学—作物学]