热带假丝酵母ctfat1p基因在吸收脂肪族化合物中的功能研究  

Function of gene ctfat1p in Candida tropicalis on the absorption of aliphatic compounds

作　　者：石莹[1] 杨晓慧[1] 马春玲[1] 王腾飞[1] 肖静[1] 王瑞明[1] 汪俊卿[1] 

机构地区：[1]齐鲁工业大学生物工程学院山东省微生物工程重点实验室,山东济南250353

出　　处：《中国酿造》2017年第3期132-137,共6页China Brewing

基　　金：山东省自主创新及成果转化专项(No.201422CX02602)

摘　　要：以热带假丝酵母(Candida tropicalis)1798中的ctfat1p基因为研究对象,利用重叠PCR将ctfat1p基因内约500 bp片段与G418抗性基因(kan^r)相连接,经末端单酶切后电转化至C.tropicalis 1798感受态细胞中,通过一次同源单交换,将抗性基因kan^r插入至ctfat1p基因内部,实现目的基因的敲除,并通过发酵实验分析Ctfat1p在热带假丝酵母脂肪酸跨膜转运过程中的功能。结果表明,经过G418抗性筛选和基因组PCR鉴定,成功获得ctfat1p基因缺失菌株C.tropicalis 1798△ctfat1p;分析发现ctfat1p基因敲除对C.tropicalis 1798以油脂为底物培养12h后重组菌OD_(600nm)值仅为野生型菌株的47.9%,表明ctfat1p基因敲除后影响C.tropicalis 1798对油脂吸收利用。通过基因敲除手段构建ctfat1p基因缺失菌株,可以减弱细胞对长链脂肪酸的摄取,验证了ctfat1p基因为热带假丝酵母油脂吸收和利用的关键基因。Using the gene ctfatlp in Candida tropicalis 1798 as research object, the 500 bp gene ctfatlp fragment was connected with G418 resistance gene （karO by using overlapping PCR. After a single enzyme digestion at the end of the fragment, then the recombinant gene fragment was transformed into C. tropicalis 1798 competent cells. Through a homologous single exchange, the resistance gene katr was inserted into the gene ctfatlp, and then the target gene was knocked successfully. The function of Ctfat lp in the process of fatty acid transmembrane-movement of C. tropicalis was analyzed by fermentation experiments. The results showed that a gene ctfatlp deletion strain （named C. tropicalis 1798Actfatlp） was obtained successfully by G418 resistance screening and genome PCR identification. The analysis was founded that the OD^0~ value of C. tropicalis 1798Actfatlp with oil as the substrate cul^ring for 12 h was only 47.9% of the wild type strain, which indicated that deletion of gene ctfatlp affected absorption and utiliza- tion of C. tropicalis 1798 on oil. The establishment of gene ctfatlp deletion strain by the gene knockout method could weaken intake of cell on long chain fatty acid, which verified that the gene ctfatlp was a key gene of the absorption and utilization of C. tropicalis on oil.

关 键 词：ctfat1p基因 热带假丝酵母 基因敲除 同源单交换 

分 类 号：Q815[生物学—生物工程]