Development of a high-frequency in vivo transposon mutagenesis system for Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942
Development of a high-frequency in vivo transposon mutagenesis system for Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942
复制标题
集胞藻高频体内转座子诱变系统的开发。
DOI：
10.1007/s11120-015-0082-4
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
T.
中科院分区：
文献类型：
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作者：
Watabe;K.;Mimuro;M.;Tsuchiya;T.
Acaryocholis marinaMBIC 11017 拥有叶绿素 (Chl)da 作为主要叶绿素，使该生物体能够利用远红光进行光合作用。因此，远红光利用的适应机制，包括叶绿素生物合成，受到了广泛的关注，尽管关于这一主题的报道数量有限。为了鉴定负责叶绿素生物合成和适应远红光的基因，对A进行了分子遗传学分析。需要码头。我们为A开发了一个转换系统。 marina并将表达载体引入A中。码头。本研究将我们新近建立的高频体内转座子诱变系统应用于A．码头。结果，我们获得了基因组DNA中不同位置带有转座子的突变体。通过筛选转座子标记的突变体，我们分离出一个在琼脂培养基上形成黄色菌落的突变体（Y1突变体）。在 Y1 突变体中，转座子被插入编码钼辅因子生物合成蛋白 A (MoaA) 的基因中。通过引入themoaAgene或增加培养基中的铵离子来补充Y1突变体的功能。这些结果表明，themoaAgene 的突变降低了 Y1 突变体中需要钼辅因子的硝酸还原酶活性。这是A的首次成功的正向遗传分析。码头，这将导致负责适应远红光的基因的鉴定。
Acaryochloris marinaMBIC 11017 possesses chlorophyll (Chl)das a major Chl, which enables this organism to utilize far-red light for photosynthesis. Thus, the adaptation mechanism of far-red light utilization, including Chldbiosynthesis, has received much attention, though a limited number of reports on this subject have been published. To identify genes responsible for Chldbiosynthesis and adaptation to far-red light, molecular genetic analysis ofA. marinawas required. We developed a transformation system forA. marinaand introduced expression vectors intoA. marina. In this study, the high-frequency in vivo transposon mutagenesis system recently established by us was applied toA. marina. As a result, we obtained mutants with the transposon in their genomic DNA at various positions. By screening transposon-tagged mutants, we isolated a mutant (Y1 mutant) that formed a yellow colony on agar medium. In the Y1 mutant, the transposon was inserted into the gene encoding molybdenum cofactor biosynthesis protein A (MoaA). The Y1 mutant was functionally complemented by introducing themoaAgene or increasing the ammonium ion in the medium. These results indicate that the mutation of themoaAgene reduced nitrate reductase activity, which requires molybdenum cofactor, in the Y1 mutant. This is the first successful forward genetic analysis ofA. marina, which will lead to the identification of genes responsible for adaptation to far-red light.