摘要
海洋红酵母作为一种产油微生物富含油脂和类胡萝卜素,但由于没有合适的遗传操作方法,无法对细胞反应器进行理性改进,阻碍了它的进一步发展。首先利用农杆菌介导转化成功构建了海洋红酵母的遗传操作平台。参考完成基因注释的圆红冬孢酵母基因组信息,分离其甘油醛-3-磷酸脱氢酶启动子,以基于圆红冬孢酵母密码子优化的潮霉素作为筛选标记,构建载体转化至农杆菌AGL1中,利用农杆菌介导转化成功得到潮霉素抗性表型正确的转化子,通过表型验证,基因型验证以及Western blot在蛋白水平验证,鉴定了hyg基因的有效表达,证明了外源抗性基因成功导入海洋红酵母菌株中,实现了海洋红酵母新的转化方法的建立。
Rhodotorula mucilaginosa is an oleaginous ocean yeast that of great interest for both lipid and carotenoids production.However,rationally engineering of this cell factory is impeded due to the absence of efficient and reliable genetic tools.Agrobacterium-mediated transformation（ ATMT） was successfully developed for R.mucilaginosa.First,the promoter of glyceraldehyde-3-phosphate dehydrogenase（ GPD） was identified by referring to the completely annotated genome information of Rhodosporidium toruloides.Then,the codon optimized hygromycin（ hyg） gene under GPD was used as both selection marker and functional gene through ATMT transformation in R.mucilaginosa,the integration was confirmed by phenotype,genotype and Western blot analysis in protein level.The results provided a practical method for functional integration and expression of hyg genes in R.mucilaginosa,which would facilitate the development of genetic tools.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2016年第6期81-86,共6页
China Biotechnology
基金
国家自然科学基金面上项目(31370128)资助项目
关键词
海洋红酵母
农杆菌介导转化
潮霉素
Rhodotorula mucilaginosa
Agrobacterium-mediated transformation
Hygromycin
