Fonsecaea monophora多聚酮合酶基因的扩增及敲除载体的构建

中国真菌学杂志　 2016, Vol. 11 　Issue (2): 70-74.

Fonsecaea monophora多聚酮合酶基因的扩增及敲除载体的构建

肖星¹, 张军民¹, 冯姣¹, 陈志文¹, 陈春梅¹, 何娅¹, 贺丹², 孙九峰³, 席丽艳¹

1. 中山大学孙逸仙纪念医院皮肤科, 广州 510120;
2. 吉林大学基础医学院病原生物学系, 吉林大学真菌研究中心·教育部人兽共患病重点实验室, 长春 130021;
3. 广东省疾病预防控制中心, 广州 510120

收稿日期:2015-12-22 出版日期:2016-04-28 发布日期:2016-04-28
通讯作者: 张军民,E-mail:junminmx@163.com E-mail:junminmx@163.com
作者简介:肖星,女(汉族),硕士研究生在读.E-mail:136591052@qq.com
基金资助:
国家自然科学基金(81571970)

The amplification and targeting vector construction of a polyketide synthases gene of Fonsecaea monophora

XIAO Xing¹, ZHANG Jun-min¹, FENG Jiao¹, CHEN Zhi-wen¹, CHEN Chun-mei¹, HE Ya¹, HE Dan², SUN Jiu-feng³, XI Li-yan¹

1. Department of Dermatology, Sun Yat-Sen Hospital, Sun Yat-Sen University, Guangzhou 510120, China;
2. Department of Pathogenobiology, Jilin University Mycology Research Center, College of Basic Medical Sciences, Jilin University, Changchun 130021, China;
3. Guangdong Provincial Center for Disease and Prevention, Guangzhou 510120, China

Received:2015-12-22 Online:2016-04-28 Published:2016-04-28

摘要/Abstract

摘要：

目的扩增Fonsecaea monophora中控制黑素合成的多聚酮合酶(polyketide synthases,PKS)基因并测序,构建PKS基因敲除载体。方法扩增PKS基因,根据测序结果设计引物,从Fonsecaea monophora基因组DNA扩增PKS基因5'-同源臂和3'-同源臂,从质粒pBHt1中扩增潮霉素B抗性标记基因(hyg),最后将各片段插入载体PDHt/sk中。结果测序得到5389 bp大小的PKS基因序列,构建了PKS基因的敲除载体,用酶切及测序等方法鉴定载体构建成功。结论成功构建Fonsecaea monophora PKS基因敲除载体,为研究PKS基因及黑素的生物学功能奠定了良好的基础。

关键词: Fonsecaea monophora, 多聚酮合酶基因, 根癌农杆菌, 载体构建

Abstract:

Objective To amplify and sequence a polyketide synthases gene of Fonsecaea monophora, and to construct its targeting vector.Methods A polyketide synthases gene of Fonsecaea monophora was amplified and sequenced.Then we designed primers to amplify the 5' and 3' flanking region of PKS gene from Fonsecaea monophora genomic DNA, and the Hygromycin B resistance gene (hyg) from plasmid pBHt1.At last, all fragments were inserted into the vector PDHt/sk.Result The sequence of PKS gene was acquired, and its length was 5 389 bp.The targeting vector of PKS gene was constructed, and the vector was identified by restriction enzyme digestion and nucleotide sequencing.Conclusion This study acquired the targeting vector of PKS gene of Fonsecaea monophora successfully, and laid a good foundation for biological functions research of PKS gene and melanin.

Key words: Fonsecaea monophora, polyketide synthases gene, Agrobacterium tumefaciens, vector construction

中图分类号:

R379.9

肖星, 张军民, 冯姣, 陈志文, 陈春梅, 何娅, 贺丹, 孙九峰, 席丽艳. Fonsecaea monophora多聚酮合酶基因的扩增及敲除载体的构建[J]. 中国真菌学杂志, 2016, 11(2): 70-74.

XIAO Xing, ZHANG Jun-min, FENG Jiao, CHEN Zhi-wen, CHEN Chun-mei, HE Ya, HE Dan, SUN Jiu-feng, XI Li-yan. The amplification and targeting vector construction of a polyketide synthases gene of Fonsecaea monophora[J]. Chinese Journal of Mycology, 2016, 11(2): 70-74.

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