长引物PCR法构建白念珠菌SCH9-MYC融合菌  

Construction of MYC-tagged Sch9p in Candida albicans by using of long primers PCR amplification

作　　者：徐秋荣[1] 吕权真 隋雪[2,3] 王晓娟[2,4] 阎澜[2] 姜远英[1,2] 

机构地区：[1]福建中医药大学药学院中药学教研室,福州350108 [2]第二军医大学药学院新药研究中心,上海200433 [3]沈阳药科大学生命科学与生物制药学院,沈阳110016 [4]中国药科大学药学院,南京210009

出　　处：《中国真菌学杂志》2015年第3期129-133,共5页Chinese Journal of Mycology

基　　金：国家自然科学基金(81470158;81330083);国家973基金(2013CB531602)

摘　　要：目的构建白念珠菌SCH9-MYC融合菌。方法运用长引物PCR扩增含有MYC标签和ARG4筛选标记的质粒序列,采用醋酸锂转染法将质粒序列同源重组到白念珠菌SN152的SCH9基因开放阅读框的C末端,在SC-Leu-选择性培养基上筛选阳性克隆,抽取阳性克隆基因组进行PCR验证,将验证为阳性的转染子进行生长曲线测定、spot assay、菌丝诱导实验,进一步筛选出表型正常的融合菌。结果通过PCR验证鉴定出3株融合菌构建正确,通过生长曲线测定、spot assay、菌丝诱导实验筛选出两株表型正常的融合菌菌株。结论运用长引物PCR扩增方法同源重组可以正确构建白念珠菌SCH9-MYC融合菌菌株。Objective To construct the MYC-tagged Sch9 p in Candida albicans.Methods Using a pair of the long primers to amplify sequences containing the MYC tag and the ARG4 selection markers from the plasmid p FA-ARG4-MYC.The amplified plasmid sequences were transformed into the C-terminus of SCH9 open reading frame（ ORF） in C.albicans SN152 by homologous recombination.The positive colonies were selected in the SC-Leu-selective solid culture medium. Afterwards,the positive colonies with correct integration were confirmed by PCR of genomic DNA.Finally,these positive transfectants were examined by time-growth curve testing,drug sensitivity spot assays,and mycelium inducing experiments.Results Strains with MYC-tagged Sch9 p which had normal phenotypes were selected.Conclusions The MYC-tagged Sch9 p of C.albicans strains can be constructed correctly by using of long primers to amplify the plasmid sequences containing the MYC tag and ARG4 selection markers which were integrated into the C-terminus of SCH9 ORF in C.albicans by homologous recombination.

关 键 词：白念珠菌 MYC SCH9 长引物 

分 类 号：R379.4[医药卫生—病原生物学]