T-DNA insertional mutagenesis for functional genomics in rice
T-DNA insertional mutagenesis for functional genomics in rice
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DOI：
10.1046/j.1365-313x.2000.00767.x
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发表时间：
2000-06-01
期刊：
影响因子：
7.2
通讯作者：
An, G
中科院分区：
文献类型：
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作者：
Jeon, JS;Lee, S;An, G
我们已经生产了22 090个携带T-DNA插入的初级转基因水稻植株，这导致了18 358个可育株系。基因组DNA凝胶印迹和PCR分析表明，大约65%的群体含有一个以上的插入T-DNA拷贝。潮霉素抗性试验表明，转基因植株平均含有1.4个T-DNA插入位点。因此，可以估计已经生成了大约25700个标记。在插入中使用的二元载体含有无启动子的β-葡萄糖醛酸酶（GUS）报告基因，其具有内含子和紧邻右边界的多个剪接供体和受体。因此，该基因捕获载体能够检测GUS与内源基因之间的基因融合，该内源基因被T-DNA标记。在5353个株系的叶和根、7026个株系的成熟花和1948个株系的发育中的种子中进行组织化学GUS检测。数据显示，1.6%-2.1%的测试器官是GUS阳性的，并且它们的GUS表达模式是器官或组织特异性的或在植物的所有部分中普遍存在。大量的T-DNA标记株系将有助于鉴定各种基因的插入突变体和发现水稻中的新基因。
We have produced 22 090 primary transgenic rice plants that carry a T-DNA insertion, which has resulted in 18 358 fertile lines. Genomic DNA gel-blot and PCR analyses have shown that approximately 65% of the population contains more than one copy of the inserted T-DNA. Hygromycin resistance tests revealed that transgenic plants contain an average of 1.4 loci of T-DNA inserts. Therefore, it can be estimated that approximately 25 700 taggings have been generated. The binary vector used in the insertion contained the promoterless beta-glucuronidase (GUS) reporter gene with an intron and multiple splicing donors and acceptors immediately next to the right border. Therefore, this gene trap vector is able to detect a gene fusion between GUS and an endogenous gene, which is tagged by T-DNA. Histochemical GUS assays were carried out in the leaves and roots from 5353 lines, mature flowers from 7026 lines, and developing seeds from 1948 lines. The data revealed that 1.6-2.1% of tested organs were GUS-positive in the tested organs, and that their GUS expression patterns were organ- or tissue-specific or ubiquitous in all parts of the plant. The large population of T-DNA-tagged lines will be useful for identifying insertional mutants in various genes and for discovering new genes in rice.