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首页> 外文期刊>Applied Microbiology >Agrobacterium tumefaciens-Mediated Transformation for Investigation of Somatic Recombination in the Fungal Pathogen Armillaria mellea
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Agrobacterium tumefaciens-Mediated Transformation for Investigation of Somatic Recombination in the Fungal Pathogen Armillaria mellea

机译：根癌农杆菌介导的转化研究真菌病原菌蜜环菌中的体细胞重组。

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Armillaria root disease is one of the most damaging timber and fruit tree diseases in the world. Despite its economic importance, many basic questions about the biology of the causal fungi, Armillaria spp., are unanswered. For example, Armillaria undergoes matings between diploid and haploid mycelia, which can result in a recombinant diploid without meiosis. Evidence of such somatic recombination in natural populations suggests that this reproductive mode may affect the pathogen's ecology. Investigations of the mechanisms and adaptive consequences of somatic recombination are, however, hampered by the lack of a method to reliably synthesize somatic recombinants. Here we report the first genetic transformation system for the genus Armillaria . We transformed A. mellea with selective markers for use in diploid-haploid matings to reliably synthesize somatic recombinants. This was accomplished with Agrobacterium tumefaciens carrying pBGgHg, which carries the hygromycin phosphotransferase gene ( hph ). hph was integrated into transformants, as evidenced by serial transfer to selective media, PCR, reverse transcription-PCR (RT-PCR), and Southern hybridization. Nuclear and mitochondrial markers were developed to genotype synthesized mycelia. In matings between a wild-type diploid and hygromycin-resistant haploids (transgenic), we identified recombinant, hygromycin-resistant diploids and, additionally, hygromycin-resistant triploids, all with the mitochondrial haplotype of the haploid partner. Our approach created no mycelium in which the haploid nucleus was replaced by the diploid nucleus, the typical outcome of diploid-haploid matings in Armillaria . This genetic transformation system, in combination with new markers to track chromosomal and cytoplasmic inheritance in A. mellea , will advance research aimed at characterizing the significance of somatic recombination in the ecology of this important fungus.
机译：蜜环菌根病是世界上最具破坏性的木材和果树病之一。尽管具有重要的经济意义，但仍未回答有关因果真菌Armillaria spp。的生物学的许多基本问题。例如，蜜环菌属在二倍体和单倍体菌丝体之间发生交配，这可能导致重组二倍体无减数分裂。在自然种群中这种体细胞重组的证据表明，这种繁殖方式可能会影响病原体的生态。然而，由于缺乏可靠地合成体细胞重组体的方法，阻碍了体细胞重组的机制和适应性后果的研究。在这里，我们报告了蜜环菌属的第一个遗传转化系统。我们用选择性标记转化了A. mellea，用于二倍体-单倍体交配，以可靠地合成体细胞重组体。这是通过携带带有潮霉素磷酸转移酶基因（hph）的携带pBGgHg的根癌农杆菌来完成的。 hph已整合到转化体中，这可以通过向选择性培养基进行串行转移，PCR，逆转录PCR（RT-PCR）和Southern杂交来证明。核和线粒体标记被开发为基因型合成菌丝体。在野生型二倍体和抗潮霉素的单倍体（转基因）之间的交配中，我们鉴定了重组，抗潮霉素的二倍体，另外还有抗潮霉素的三倍体，均具有单倍体伴侣的线粒体单倍型。我们的方法没有产生菌丝体，其中单倍体核被二倍体核取代，这是蜜环菌属中二倍体-单倍体交配的典型结果。这种遗传转化系统，结合新的标记来追踪A. mellea的染色体和细胞质遗传，将推进旨在表征体细胞重组在这种重要真菌生态学中的重要性的研究。

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