Transposon display for active DNA transposons in rice.
Transposon display for active DNA transposons in rice.
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DOI：
10.1266/ggs.82.109
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发表时间：
2007-04
影响因子：
1.1
通讯作者：
K. Takagi;N. Ishikawa;M. Maekawa;Kazuo Tsugane;S. Iida
中科院分区：
文献类型：
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作者：
K. Takagi;N. Ishikawa;M. Maekawa;Kazuo Tsugane;S. Iida
转座子展示技术是基因标签技术中鉴定转座子整合位点的一种有效方法，是研究基因功能的重要工具。虽然活性内源DNA转座子已被广泛用于玉米的基因标签，只有两个活性内源DNA转座子在水稻中已被确定，0.43-kb的MITE家族的元件mPing和0.6-kb的hAT家族的nDart元件。的nDart转座被证明是诱导杂交与含有其自主元件aDart和稳定的分离aDart在自然生长条件下，而MPING相关的元素被证明转座培养细胞，从花药培养再生的植物，和γ-射线照射的植物。在nDart促进的基因标记中不应发生体细胞克隆变异，因为没有组织培养参与nDart激活。作为在水稻中开发使用nDart的有效标记系统的第一步，我们试图通过TD可视化日本晴中富含GC的nDart相关元件，包括18个0.6 kb的nDart相关序列和63个长于2 kb的nDart相关元件。将TD中观察到的条带与基于可用水稻基因组序列的nDart相关元件的预期虚拟条带进行比较，我们通过优化PCR扩增条件改进了我们的TD方案，并且能够可视化从nDart相关元件产生的约87%的预期条带。为了比较这些nDart相关元素与50个mPing元素和日本晴独特的Ping序列的可视化效率，我们还尝试可视化mPing相关元素;所有mPing相关元素都很容易可视化。在此基础上，我们讨论了影响水稻转座子可视化效率的参数。本文还讨论了nDart元件在水稻功能基因组学基因标签中的应用。
Transposon display (TD) is a powerful technique to identify the integration site of transposons in gene tagging as a functional genomic tool for elucidating gene function. Although active endogenous DNA transposons have been used extensively for gene tagging in maize, only two active endogenous DNA transposons in rice have been identified, the 0.43-kb element mPing of the MITE family and the 0.6-kb nDart element of the hAT family. The nDart transposition was shown to be induced by crossing with a line containing its autonomous element aDart and stabilized by segregating aDart under natural growth conditions, while mPing-related elements were shown to transpose in cultured cells, plants regenerated from an anther culture, and gamma-ray-irradiated plants. No somaclonal variation should occur in nDart-promoted gene tagging because no tissue culture was involved in nDart activation. As an initial step to develop an effective tagging system using nDart in rice, we tried to visualize GC-rich nDart-related elements comprising 18 nDart-related sequences of 0.6-kb and 63 nDart-related elements longer than 2 kb in Nipponbare by TD. Comparing the observed bands in TD with the anticipated virtual bands of the nDart-related elements based upon the available rice genome sequence, we have improved our TD protocol by optimizing the PCR amplification conditions and are able to visualize approximately 87% of the anticipated bands produced from the nDart-related elements. To compare the visualization efficiency of these nDart-related elements with that of 50 mPing elements and a unique Ping sequence in Nipponbare, we also tried to visualize the mPing-related elements; all mPing-related elements are easily visualized. Based on these results, we discuss the parameters affecting the visualization efficiencies of these rice DNA transposons. We also discuss the utilization of nDart elements in gene tagging for functional genomics in rice.