食品级重组产朊假丝酵母菌的构建及其遗传稳定性测定  

Construction of a Food-grade Recombinant Candida utilis Strain and Determination of Its Genetic Stability

作　　者：其布日 萨初拉 苏少锋 高娃 王蕴华 刘红葵 吴青海 呼和 Qiburi;Sachula;SU Shao-feng;Gaowa;WANG Yun-hua;LIU Hong-kui;WU Qing-hai;Huhe(Biotechnology Research Center,Inner Mongolia Academy of Agricultural and Animal Husbandry Sciences,Hohhot 010031,China)

机构地区：[1]内蒙古自治区农牧业科学院生物技术研究中心,内蒙古呼和浩特010031

出　　处：《畜牧与饲料科学》2018年第12期6-12,共7页Animal Husbandry and Feed Science

基　　金：内蒙古农牧业创新基金项目(2016CXJJM09)

摘　　要：旨在构建食品级酵母工程菌株,并对其遗传稳定性进行评价。采用重叠延伸PCR(gene splicing by overlap extension PCR,SOE-PCR)方法,将实验室已构建完成的原核表达载体PGZM18中的原核基因序列删除,设计2组引物,以PGZM18原核载体序列为模板进行引物设计,将片段序列分为2个片段P1-1和P2-1,通过重叠延伸PCR技术删除来自原核的DNA序列(包括抗药性标记Amp在内的细菌质粒序列DNA片段及原核复制区),将最终获得的PCR产物GZM18转化至产朊假丝酵母中,通过菌落计数和PCR方法测定外源基因遗传稳定性。结果表明,食品级产朊假丝酵母工程菌株构建成功,外源基因具有较高的遗传稳定性。研究结果为产朊假丝酵母工程菌在微生物饲料开发方面的应用提供了科学依据。The aim of this study was to construct a food-grade yeast engineering strain and to evaluate its genetic stability.The prokaryotic gene sequences in the PGZM18,a previously constructed prokaryotic expression vector,was deleted by SOE-PCR;two sets of primers were designed based on the prokaryotic vector sequence of PGZM18,and the fragment sequence was divided into two fragments,P1-1 and P2-1;the prokaryotic DNA sequences(bacterial plasmid sequence,including antibiotic resistance marker Amp,and prokaryotic replication region)were deleted by SOE-PCR;the finally obtained PCR product GZM18 was transformed into Candida utilis,and the genetic stability of the exogenous gene was determined by colony counting and PCR.The results showed that a food-grade genetically engineered strains of Candida utilis was successfully constructed,and the exogenous gene had high genetic stability.Our results provide a scientific basis for application of the engineering strain of Candida utilis in development of microbial feed.

关 键 词：食品级菌株 产朊假丝酵母 SOE-PCR 遗传稳定性 

分 类 号：Q786[生物学—分子生物学]