Agrobacterium tumefaciens-mediated transformation: an efficient tool for targeted gene disruption in Talaromyces marneffei
Agrobacterium tumefaciens-mediated transformation: an efficient tool for targeted gene disruption in Talaromyces marneffei
复制标题
根癌农杆菌介导的转化：马尔尼菲踝节菌靶向基因破坏的有效工具
DOI：
10.1007/s11274-017-2352-0
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发表时间：
2017-10-01
影响因子：
4.1
通讯作者：
Zhang, Junmin
中科院分区：
文献类型：
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作者：
Xiao, Xing;Feng, Jiao;Zhang, Junmin
马尔尼菲Talaromyces marneffei在免疫功能低下的宿主中引起危及生命的感染。一个有效的工具，遗传操作的T. marneffei将允许增加对这种热二态真菌的了解。利用质粒pDHt/acuD::pyrG对农杆菌介导转化（Agrobacterium tumefacien -mediated transformation， ATMT）进行了基因靶向破坏优化。利用PCR、Southern blot和半定量RT-PCR对转化子进行分子分析。在ATMT共培养条件下，瘤胃拟酵母菌EHA105的转化效率高于AGL-1。a结核菌：T。在ATMT液体共培养条件下，马尼菲比为1000:1，转化效率较高，每106个酵母细胞可转化90个转化子。利用atmt介导的基因敲除诱变技术，我们成功地删除了T. marneffei的acuD基因。PCR和Southern blot分析证实acuD基因被破坏，外源pyrG基因被整合到T. marneffei中。半定量RT-PCR分析进一步证实pyrG表达正常。这些结果表明，ATMT可能是一个潜在的平台，在马氏弓形虫中进行靶向基因破坏，液体共培养可能为开发临床治疗提供新的机会。
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.