Agrobacterium tumefaciens-mediated transformation: an efficient tool for targeted gene disruption in Talaromyces marneffei |
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Authors: | Xing Xiao Jiao Feng Yu Li Zhiwen Chen Minglan Shi Liyan Xi Eleftherios Mylonakis Junmin Zhang |
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Affiliation: | 1.Department of Dermatology and Venerology, Sun Yat-sen Memorial Hospital,Sun Yat-sen University,Guangzhou,China;2.Department of Dermatology and Venerology,The Third Affiliated Hospital of Guangzhou Medical University,Guangzhou,China;3.Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital,Sun Yat-sen University,Guangzhou,China;4.Medical Research Center, Sun Yat-sen Memorial Hospital,Sun Yat-sen University,Guangzhou,China;5.Division of Infectious Diseases, Rhode Island Hospital,Warren Alpert Medical School of Brown University,Providence,USA |
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Abstract: | Talaromyces marneffei causes life-threatening infections in immunocompromised hosts. An efficient tool for genetic manipulation of T. marneffei will allow for increased understanding of this thermally dimorphic fungus. Agrobacterium tumefaciens-mediated transformation (ATMT) was optimized for targeted gene disruption in T. marneffei using the plasmid pDHt/acuD::pyrG. Molecular analyses of transformants were performed by PCR, Southern blot and semi-quantitative RT-PCR. A. tumefaciens strain EHA105 was more efficient at transformation than strain AGL-1 in ATMT via solid co-cultivation. An A. tumefaciens:T. marneffei ratio of 1000:1 in an ATMT liquid co-cultivation led to a relatively high transformation efficiency of 90 transformants per 106 yeast cells. Using ATMT-mediated knockout mutagenesis, we successfully deleted the acuD gene in T. marneffei. PCR and Southern blot analysis confirmed that acuD was disrupted and that the foreign pyrG gene was integrated into T. marneffei. Semi-quantitative RT-PCR analysis further confirmed that pyrG was expressed normally. These results suggest that ATMT can be a potential platform for targeted gene disruption in T. marneffei and that liquid co-cultivation may provide new opportunities to develop clinical treatments. |
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