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首页> 外文期刊>Applied and Environmental Microbiology >Transkingdom Genetic Transfer from Escherichia coli to Saccharomyces cerevisiae as a Simple Gene Introduction Tool
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Transkingdom Genetic Transfer from Escherichia coli to Saccharomyces cerevisiae as a Simple Gene Introduction Tool

机译：作为一种简单的基因导入工具，从大肠埃希氏菌到啤酒酵母的Transkingdom遗传转移

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Transkingdom conjugation (TKC) permits transfer of DNA from bacteria to eukaryotic cells using a bacterial conjugal transfer system. However, it is not clear whether the process of DNA acceptance in a recipient eukaryote is homologous to the process of conjugation between bacteria. TKC transfer requires mobilizable shuttle vectors that are capable of conjugal transfer and replication in the donor and recipient strains. Here, we developed TKC vectors derived from plasmids belonging to the IncP and IncQ groups. We also investigated forms of transfer of these vectors from Escherichia coli into Saccharomyces cerevisiae to develop TKC as a simple gene introduction method. Both types of vectors were transferred precisely, conserving the origin of transfer (oriT) sequences, but IncP-based vectors appeared to be more efficient than an IncQ-based vector. Interestingly, unlike in agrobacterial T-DNA (transfer DNA) transfer, the efficiency of TKC transfer was similar between a wild-type yeast strain and DNA repair mutants defective in homologous recombination (rad51Δ and rad52Δ) or nonhomologous end joining (rad50Δ, yku70Δ, and lig4Δ). Lastly, a shuttle vector with two repeats of IncP-type oriT (oriTP) sequences flanking a marker gene was constructed. TKC transfer of this vector resulted in precise excision of both the oriTP loci as well as the marker gene, albeit at a low frequency of 17% of all transconjugants. This feature would be attractive in biotechnological applications of TKC. Taken together, these results strongly suggest that in contrast to agrobacterial T-DNA transfer, the circularization of vector single-stranded DNA occurs either before or after transfer but requires a factor(s) from the donor. TKC is a simple method of gene transfer with possible applications in yeast genetics and biotechnology.
机译：Transkingdom共轭（TKC）允许使用细菌共轭转移系统将DNA从细菌转移到真核细胞。但是，尚不清楚接受者真核生物中DNA的接受过程是否与细菌之间的缀合过程同源。 TKC转移需要可动的穿梭载体，该载体能够在供体和受体菌株中进行结合转移和复制。在这里，我们开发了来自属于IncP和IncQ组的质粒的TKC载体。我们还研究了这些载体从大肠杆菌转移到啤酒酵母中的形式，以开发TKC作为一种简单的基因导入方法。两种类型的载体均被精确地转移，从而保留了转移起点（ oriT ）的序列，但是基于IncP的载体似乎比基于IncQ的载体更有效。有趣的是，与农杆菌T-DNA（转移DNA）转移不同，野生型酵母菌株与同源重组缺陷的DNA修复突变体（rad51Δ和rad52Δ ）或非同源末端连接（rad50Δ，yku70Δ和lig4Δ）。最后，构建了带有两个重复的IncP型 oriT （ oriT P ）序列的穿梭载体。该载体的TKC转移可精确切除 oriT P 基因座和标记基因，尽管在所有转导结合体中的发生率较低，仅为17％。此功能在TKC的生物技术应用中将很有吸引力。综上所述，这些结果强烈暗示与农杆菌T-DNA转移相反，载体单链DNA的环化发生在转移之前或之后，但是需要来自供体的因子。 TKC是一种简单的基因转移方法，可能在酵母遗传学和生物技术中得到应用。

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