Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice
Generation of a flanking sequence-tag database for activation-tagging lines in japonica rice
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DOI：
10.1111/j.1365-313x.2005.02610.x
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发表时间：
2006-01-01
期刊：
影响因子：
7.2
通讯作者：
An, G
中科院分区：
文献类型：
--
作者：
Jeong, DH;An, S;An, G
我们利用含有四聚化35S增强子序列的激活标签载体，在水稻中构建了47 932个T-DNA标签系。为了便于使用这些品系，我们通过反向聚合酶链式反应分离了插入的T-DNA两侧的基因组序列。对于大多数品系，我们使用两种不同的限制性内切酶对两个方向进行了四组扩增。在对41 234个品系的分析中，共获得27 621个侧翼序列标签，其中12 505个整合到基因区，15 116个整合到基因间区。染色体上的FSTs定位表明，T-DNA整合频率一般与染色体大小成正比。然而，T-DNA插入并不均匀地分布在每条染色体上：在远端较高，在靠近着丝粒的区域较低。此外，几个区域的插入频率出现了极端的峰值和谷值，这表明T-DNA整合的热点和冷点。插入事件的密度在某种程度上与每条染色体上表达的、而不是预测的基因密度相关。对插入的增强子附近的表达模式的分析表明，至少有一半的测试品系表现出更高的标记基因表达。而在大多数增加的品系中，激活后的表达模式与野生型相似，从而保持了内源模式，其余的品系在激活标记的品系中表现出表达的变化。在这种情况下，异位表达最常在成熟叶片中观察到。目前，可以在http://www.postech.ac.kr/life/pfg/risd.上用染色体上的基因座编号或位置来搜索数据库应要求，将向科学界提供T-1或T-2植物的种子。
We have generated 47 932 T-DNA tag lines in japonica rice using activation-tagging vectors that contain tetramerized 35S enhancer sequences. To facilitate use of those lines, we isolated the genomic sequences flanking the inserted T-DNA via inverse polymerase chain reaction. For most of the lines, we performed four sets of amplifications using two different restriction enzymes toward both directions. In analyzing 41 234 lines, we obtained 27 621 flanking sequence tags (FSTs), among which 12 505 were integrated into genic regions and 15 116 into intergenic regions. Mapping of the FSTs on chromosomes revealed that T-DNA integration frequency was generally proportional to chromosome size. However, T-DNA insertions were non-uniformly distributed on each chromosome: higher at the distal ends and lower in regions close to the centromeres. In addition, several regions showed extreme peaks and valleys of insertion frequency, suggesting hot and cold spots for T-DNA integration. The density of insertion events was somewhat correlated with expressed, rather than predicted, gene density along each chromosome. Analyses of expression patterns near the inserted enhancer showed that at least half the test lines displayed greater expression of the tagged genes. Whereas in most of the increased lines expression patterns after activation were similar to those in the wild type, thereby maintaining the endogenous patterns, the remaining lines showed changes in expression in the activation tagged lines. In this case, ectopic expression was most frequently observed in mature leaves. Currently, the database can be searched with the gene locus number or location on the chromosome at http://www.postech.ac.kr/life/pfg/risd. On request, seeds of the T-1 or T-2 plants will be provided to the scientific community.