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机构地区:[1]福州大学生物科学与工程学院,福建福州350116
出 处:《宁夏大学学报(自然科学版)》2017年第4期377-382,共6页Journal of Ningxia University(Natural Science Edition)
基 金:国家自然科学基金资助项目(31070093)
摘 要:来自绛红色小单孢菌G1008的genp与来自橄榄星小单孢菌FOM808的forp,均参与3’,4’-双脱羟基,2个基因属于同工异源酶基因.应用同源重组原理,实现forp替换genp,探索同工异源酶基因异源表达的可能性.以温敏型穿梭质粒pKC1139为载体,构建同源重组质粒pFP4.经接合转移将载体导入到G1008基因组中,利用影印筛选和PCR验证的方法,获得基因重组工程菌FTP2.提取工程菌次级代谢产物,经TLC和MS分析.结果表明,工程菌仍然可以产生庆大霉素C组分.forp基因的功能得到了体现,外源基因forp实现了异源表达,进一步证实forp是负责参与3’,4’-双脱羟基的功能基因.Both the genp from Micromonospora purpurea G1008 and the forp from Micromonospora olivoasterospora FOM808 are involved in 3',4'-double dehydroxylation.Both genes belong to Isozyme Heterologous Enzyme and apply to homologous recombination theory to actualize the replacement of forp into genpto explore the probability of Isozyme Heterologous Enzyme expression.Thermo-sensitive type of shuttle palsmid pKC1139 was taken as the carrier to build recombinant plasmid pFP4,and the carrier was introduced into the G1008 genome after conjugational transfer,ultimately,genetic recombinant bacterium FTP2 was obtained with the method of copy screening and PCR identification.The secondary metabolite of engineering bacteria was obtained and under TLC and MS analysis.The result shows that,engineering bacteria still produces gentamicin c components.The function of forpgene was embodied,and exogenous gene was heterologous expressed,which further proves that forpis functioned one involved in the 3',4'-double dehydroxylation.
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