长引物PCR法构建白念珠菌SCH9-MYC融合菌

中国真菌学杂志　 2015, Vol. 10 　Issue (3): 129-133.

论著下一篇

长引物PCR法构建白念珠菌SCH9-MYC融合菌

徐秋荣¹, 吕权真², 隋雪^2,3, 王晓娟^2,4, 阎澜², 姜远英^1,2

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

收稿日期:2015-03-08 出版日期:2015-06-28 发布日期:2015-06-28
通讯作者: 阎澜, E-mail:ylan20001228@sina.com;姜远英, E-mail:13761571578@163.com E-mail:ylan20001228@sina.com;13761571578@163.com
作者简介:徐秋荣, 男 (汉族), 硕士研究生在读.E-mail:xuqiurong09@163.com
基金资助:
国家自然科学基金(81470158, 81330083);国家973基金(2013CB531602)

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

XU Qiu-rong¹, LV Quan-zhen², SUI Xue^2,3, WANG Xiao-juan^2,4, YAN Lan², JIANG Yuan-ying^1,2

1. Department of Traditional Chinese Medicine, College of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou 350108, China;
2. Center for New Drug Research, School of Pharmacy, Second Military Medical University, Shanghai 200433, China;
3. School of Life Science and Bio-pharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, China;
4. School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China

Received:2015-03-08 Online:2015-06-28 Published:2015-06-28

摘要/Abstract

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。

关键词: 白念珠菌, MYC, SCH9, 长引物

Abstract:

Objective To construct the MYC-tagged Sch9p 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 pFA-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 Sch9p which had normal phenotypes were selected.Conclusions The MYC-tagged Sch9p 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.

Key words: Candida albicans, MYC, SCH9, long primer

中图分类号:

R379.4

徐秋荣, 吕权真, 隋雪, 王晓娟, 阎澜, 姜远英. 长引物PCR法构建白念珠菌SCH9-MYC融合菌[J]. 中国真菌学杂志, 2015, 10(3): 129-133.

XU Qiu-rong, LV Quan-zhen, SUI Xue, WANG Xiao-juan, YAN Lan, JIANG Yuan-ying. Construction of MYC-tagged Sch9p in Candida albicans by using of long primers PCR amplification[J]. Chinese Journal of Mycology, 2015, 10(3): 129-133.

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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

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