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

被引：0
|
作者：
李敏英 [1 ]
黄欢 [1 ]
李倩 [1 ]
骆明芬 [1 ]
王晓悦 [1 ]
刘红芳 [1 ]
曾维英 [1 ]
席丽艳 [1 ,2 ]
机构：
[1] 南方医科大学皮肤病医院实验研究中心
[2] 中山大学孙逸仙纪念医院皮肤科
来源：
中国真菌学杂志 | 2021年 / 16卷 / 05期
关键词：
Fonsecaea monophora; 聚酮合酶基因; CRISPR-Cas9; 载体构建;
D O I：
暂无
中图分类号：
Q78 [基因工程（遗传工程）];
学科分类号：
071007 ; 0836 ; 090102 ;
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。
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页码：303 / 307+313 +313
页数：6
相关论文
共 24 条
  • [1] Fonsecaea monophora对巨噬细胞TLR2、TLR4、Dectin-1和TNF-α表达的影响
    蒋丽
    张军民
    孙九峰
    席丽艳
    蔡文莹
    肖星
    [J]. 中国真菌学杂志, 2014, 9 (03) : 134 - 138
  • [2] Single and Multiplexed Gene Editing in Ustilago maydis Using CRISPR-Cas9
    Schuster, Mariana
    Trippel, Christine
    Happel, Petra
    Lanver, Daniel
    Reissmann, Stefanie
    Kahmann, Regine
    [J]. BIO-PROTOCOL, 2018, 8 (14):
  • [3] Establishment of CRISPR/Cas9 in Alternaria alternata[J] . Maximilian Wenderoth,Christoph Pinecker,Benjamin Vo?,Reinhard Fischer.Fungal Genetics and Biology . 2017
  • [4] Aspergillus Cell Wall Melanin Blocks LC3-Associated Phagocytosis to Promote Pathogenicity
    Akoumianaki, Tonia
    Kyrmizi, Irene
    Valsecchi, Isabel
    Gresnigt, Mark S.
    Samonis, George
    Drakos, Elias
    Boumpas, Dimitrios
    Muszkieta, Laetitia
    Prevost, Marie-Christine
    Kontoyiannis, Dimitrios P.
    Chavakis, Triantafyllos
    Netea, Mihai G.
    van de Veerdonk, Frank L.
    Brakhage, Axel A.
    El-Benna, Jamel
    Beauvais, Anne
    Latge, Jean-Paul
    Chamilos, Georgios
    [J]. CELL HOST & MICROBE, 2016, 19 (01) : 79 - 90
  • [5] Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus
    Fuller, Kevin K.
    Chen, Shan
    Loros, Jennifer J.
    Dunlap, Jay C.
    [J]. EUKARYOTIC CELL, 2015, 14 (11) : 1073 - 1080
  • [6] Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system
    Liu, Rui
    Chen, Ling
    Jiang, Yanping
    Zhou, Zhihua
    Zou, Gen
    [J]. CELL DISCOVERY, 2015, 1
  • [7] Tailor-made TALEN system for highly efficient targeted gene replacement in the rice blast fungus
    Arazoe, Takayuki
    Ogawa, Tetsuo
    Miyoshi, Kennosuke
    Yamato, Tohru
    Ohsato, Shuichi
    Sakuma, Tetsushi
    Yamamoto, Takashi
    Arie, Tsutomu
    Kuwata, Shigeru
    [J]. BIOTECHNOLOGY AND BIOENGINEERING, 2015, 112 (07) : 1335 - 1342
  • [8] Alternaria alternata transcription factor CmrA controls melanization and spore development[J] . Fetzner Ramona,Seither Kristin,Wenderoth Maximilian,Herr Andreas,Fischer Reinhard.Microbiology (Reading, England) . 2014 (Pt 9)
  • [9] Development and Applications of CRISPR-Cas9 for Genome Engineering
    Hsu, Patrick D.
    Lander, Eric S.
    Zhang, Feng
    [J]. CELL, 2014, 157 (06) : 1262 - 1278
  • [10] Melanin in a Meristematic Mutant of Fonsecaea monophora Inhibits the Production of Nitric Oxide and Th1 Cytokines of Murine Macrophages
    Zhang, Junmin
    Wang, Li
    Xi, Liyan
    Huang, Huaiqiu
    Hu, Yongxuan
    Li, Xiqing
    Huang, Xiao
    Lu, Sha
    Sun, Jiufeng
    [J]. MYCOPATHOLOGIA, 2013, 175 (5-6) : 515 - 522
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