Fonsecaea monophora聚酮合酶基因的CRISPR-Cas9敲除载体的构建  

Construction of CRISPR-Cas9 knockout vector of Fonsecaea monophora polyketide synthase gene

作　　者：李敏英 黄欢 李倩 骆明芬 王晓悦 刘红芳 曾维英 席丽艳 LI Minying;HUANG Huan;LI Qian;LUO Mingfen;WANG Xiaoyue;LIU Hongfang;ZENG Weiying;XI Liyan(Dermatology Hospital, Southern Medical University,Experimental Research Center, Guangzhou 510091, China;Department of Dermatology, SunYat-Sen Hospital, SunYat-Sen University, Guangzhou 510120, China)

机构地区：[1]南方医科大学皮肤病医院,实验研究中心,广州510091 [2]中山大学孙逸仙纪念医院皮肤科,广州510120

出　　处：《中国真菌学杂志》2021年第5期303-307,313,共6页Chinese Journal of Mycology

基　　金：国家自然科学基金(81873960);国自然青年科学基金(81601746);广东省医学科学技术研究基金(A2019345)。

摘　　要：目的改造pFC332质粒,简化实验流程,构建Fonsecaea monophora PKS1基因的CRISPR-Cas9敲除载体。方法设计引物PCR扩增pFC334质粒上的gRNA骨架,获得已经插入AarⅠ酶切位点的gRNA骨架片段后,使用PstI和MerI酶线性化pFC334载体,构建pFC334-AarⅠ载体,使用MreⅠ和BglⅡ酶对其和pFC332质粒进行双酶切,连接后获得pFC332-gRNA-AarⅠ载体,设计gRNA,最后将各片段插入到载体中。结果测序得到16533bp大小的改造后的pFC332-gRNA-AarⅠ质粒,构建了PKS1基因的CRISPR-Cas9敲除载体,通过PCR,酶切以及测序的方法确定敲除载体构建成功。结论成功构建Fonsecaea monophora聚酮合酶基因的CRISPR-Cas9敲除载体,为研究PKS1基因及DHN-黑素在Fonsecaea monophora的生物学功能奠定了良好的基础。Objective To construct a Fonsecaea monophora polyketide synthase 1(PKS1)gene knock-out vector by CRISPR/Cas9 system with pFC332 plasmid.Methods Primers of the gRNA skeleton fragment from pFC334 plasmid were designed,and then the gRNA skeleton fragment inserted with the AarⅠrestriction site was amplified by PCR.After that,the pFC334 plasmid was linearized with PstⅠand MerI enzymes and a pFC334-AarⅠvector was constructed.Subsequently,both pFC334-AarⅠvector and pFC332 plasmid were digested with MreI and BglII.Digested products were ligated together in order to produce a novel recombinant vector called pFC332-gRNA-AarⅠ,with gRNA skeleton fragment.Finally,multiple gRNAs targeting PKS1 were designed and integrated into the pFC332-gRNA-AarⅠvector,respectively.Results The obtained pFC332-gRNA-AarⅠplasmid was 16533bp long,identified by sequencing.PCR,enzyme digestion and sequencing were applied to indicate that the PKS1 gene knock-out vector by CRISPR/Cas9 system was successfully constructed.Conclusion The Fonsecaea monophora polyketide synthase 1(PKS1)gene knock-out vector by CRISPR/Cas9 system was successfully constructed,which laid a good foundation for the research of PKS1 gene and the biological functions of DHN-melanin in Fonsecaea monophora.

关 键 词：Fonsecaea monophora 聚酮合酶基因 CRISPR-Cas9 载体构建 

分 类 号：R379.9[医药卫生—病原生物学]