Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system
Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system
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DOI：
10.1046/j.1365-313x.1999.00561.x
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发表时间：
1999-09-01
期刊：
影响因子：
7.2
通讯作者：
Han, CD
中科院分区：
文献类型：
--
作者：
Chin, HG;Choe, MS;Han, CD
在水稻中，通过使用插入诱变剂，特别是异源转座子如玉米Ac/Ds来鉴定基因的努力有限。我们构建了Ac和基因陷阱Ds载体，并通过农杆菌介导的转化将它们导入水稻基因组。在这份报告中，含有单一和简单的插入T-DNA的水稻植株进行了分析，以评估基因标签的效率。检查Ds的3'端的推定剪接供体位点。如在玉米中观察到的，在水稻中的Ds的3'端鉴定出三个剪接供体位点。当AccDNA在CaMV 35 S启动子下表达时，近80%的Ds元件从原始T-DNA位点切除。重复再生培养进行诱导新的易位Ds在新的植物来自扦插。约30%的植株携带至少一个Ds，这些Ds在后来的培养中经历了二次转座。转座Ds元件中有8%在穗部各组织中表达GUS。与克隆的DNA相邻的Ds，插入位点的基因组复杂性进行了研究，通过Southern杂交。一半的Ds插入位点表现出简单的杂交模式，可以很容易地利用来定位Ds。我们的数据表明，Ac/Ds介导的基因陷阱系统可以证明是一个很好的工具，在水稻基因功能的分析。我们讨论了遗传策略，可用于大规模诱变水稻异源Ac/Ds家庭。
In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analysed in order to evaluate the gene-tagging efficiency. The 3' end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3' end of the Ds in rice. Nearly 80% of Ds elements were excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds which underwent secondary transposition in the later cultures. Eight per cent of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybridization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds-mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a large scale mutagenesis using a heterologous Ac/Ds family in rice.