利用可重复使用的URA3标记基因建立热带假丝酵母基因敲除系统  

Development of a genetic transformation system for Candida tropicalis based on a reusable selection marker of URA3 gene

作　　者：项峥[1] 陈献忠[1] 张利华[1] 沈微[1] 樊游[1] 陆茂林[2] 

机构地区：[1]江南大学生物工程学院、工业生物技术教育部重点实验室,无锡214122 [2]江苏省微生物研究所有限责任公司,无锡214063

出　　处：《遗传》2014年第10期1053-1061,共9页Hereditas（Beijing)

基　　金：江苏省科技支撑计划项目(编号:BE2012618)资助

摘　　要：热带假丝酵母(Candida tropicalis)在发酵工业中具有重要的应用潜力,但二倍体遗传结构和较低的遗传转化效率限制了其代谢工程育种技术的应用。建立可靠的遗传转化技术并高效的删除目的基因是代谢工程改造热带假丝酵母的重要前提。文章以C.tropicalis ATCC 20336为出发菌株,通过化学诱变筛选获得了尿嘧啶缺陷型突变株C.tropicalis XZX(ura3/ura3)。以丙酮酸脱羧酶(Pyruvate decarboxylase,PDC)基因作为靶基因构建了两端包含同源臂并在选择性标记C.tropicalis URA3(Orotidine-5′-phosphate decarboxylase,乳清酸核苷-5-磷酸脱羧酶)基因两侧同向插入源于沙门氏菌(Salmonella typhimurium)的hisG序列的基因敲除盒PDC1-hisG-URA3-hisGPDC1(PHUHP),并转化宿主菌株C.tropicalis XZX,筛选获得PHUHP片段正确整合到染色体的PDC基因位点的转化子XZX02。在此基础上,将转化子XZX02涂布于5-FOA(5-氟乳清酸)选择培养基上,筛选得到URA3基因从PHUHP片段中丢失的营养缺陷型菌株XZX03。进一步构建了第2个PDC等位基因的删除表达盒PDCmURA3-PDCm,并转化C.tropicalis XZX03菌株,获得转化子C.tropicalis XZX04。经PCR和DNA测序确认转化子C.tropicalis XZX04细胞染色体上的两个PDC等位基因被成功敲除。文章建立了一种营养缺陷型标记可重复使用的热带假丝酵母遗传转化技术,利用该技术成功敲除了细胞的PDC基因,为进一步利用代谢工程改造热带假丝酵母奠定了基础。Candida tropicalis, a diploid asporogenic yeast, is frequently utilized in industrial applications and＆amp;nbsp;research studies. However, the low efficiency of genetic transformation limits the strain improvement by metabolic engineering. A reliable transformation and efficient deletion of target gene are prerequisite for molecular improve-ment ofC. tropicalis. In this study, an efficient approach for genetic transformation ofC. tropicalis was devel-oped based on the URA3 gene as a reusable selection marker and both ofPDC allele genes encoding pyruvate decar-boxylase were successfully deleted by this approach. Firstly, an auxotrophic mutant strain ofC. tropicalis XZX which is defective in orotidine-5′-phosphate decarboxylase （URA3） was isolated by chemical mutagenesis combined with nystatin enrichment selection and 5-fluoro-orotic acid （5-FOA） resistance selection using C. tropicalisATCC 20336 as the parent strain. Then, the firstPDC deletion cassettePDC1-hisG-URA3-hisG-PDC1（PHUHP） which contains a 1.6 kb URA3 marker gene, two copies of 1.1 kbSalmonellahisG fragments and homologous arms of target gene was con-structed and transformed intoC. tropicalis XZX cells. Transformants with a single copy ofPDC deleted were isolated and identified by PCR and DNA sequencing, which was designated asC.tropicalis XZX02. TheC.tropicalis XZX02 cells were spread on the minimal medium containing 5-FOA to generate mutant C. tropicalis XZX03 in whichURA3 marker gene was excised from PHUHP fragment integrated into thePDC gene site. The secondPDC gene dele-tion cassettePDCm-URA3-PDCm（MUM） was constructed and transformed intoC. tropicalis XZX03 to generate C.tropicalis XZX04 in which both ofPDC allele genes were deleted. All strains were confirmed by PCR and DNA sequencing. This efficient genetic transformation approach laid a foundation for further metabolic engineering ofC. tropicalis.

关 键 词：热带假丝酵母 遗传转化 同源重组 URA3基因 基因敲除 

分 类 号：Q78[生物学—分子生物学]