摘要
目的在万古霉素产生菌东方拟无枝酸菌HCCB10007内构建attB整合位点,将噬菌体ΦC31重组酶介导的位点特异性整合系统引入该菌,从而提高该工业菌株的遗传操作效率。方法在不破坏结构基因的情况下经同源重组将来源于变铅青链霉菌的attB编码序列插入到东方拟无枝酸菌HCCB10007基因组中,然后将带有绿色荧光蛋白基因(eGFP)的特异性位点重组质粒pLYGYQ2导入该工程菌中。用于验证整合系统的有效性。结果获得基因组中整合有attB位点的基因工程菌LYGYQA。eGFP基因顺利整合入所构建的attB位点,并且获得了表达。同时发现该系统的整合效率较同源重组效率提高了约3倍。结论本研究不但表明来源于链霉菌的位点特异性整合系统可以成功应用于东方拟无枝酸菌中,也证明绿色荧光蛋白基因可以作为报告基因用于该菌的研究。
Objective To construct an artificial attB integration site in Amycolatopsis orientalis HCCB10007 and introduce the site-specific recombination system coming from phage ФC31 into it to improve the genetic manipulation efficiency. Methods Homologous recombination system was applied to construct the attB encoded site（derived from Streptomyces lividans） without damaging the gene structure in A.orientalis HCCB10007.Then the plasmid pLYGYQ2 containing site-specific recombinase gene and eGFP gene was transformed in A.orientalis to test the efficiency of the Specific Recombination System. Results The engineering bacteria LYGYQA containing attB site was constructed and gene eGFP was integrated into the chromosome and expressed successfully. We also discovered that the site-specific recombination system has an efficiency as 4 times as the homologous recombination system. Conclusion The artificial attB integration site derived from S. lividans can be used in A.orientalis and eGFP can be used as a reporter gene in the bacteria as well.
出处
《中国抗生素杂志》
CAS
CSCD
北大核心
2014年第3期193-197,共5页
Chinese Journal of Antibiotics
基金
国家自然科学基金资助(编号:81202440)
