Gene targeting by homologous recombination as a biotechnological tool for rice functional genomics
Gene targeting by homologous recombination as a biotechnological tool for rice functional genomics
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DOI：
10.1104/pp.107.095992
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发表时间：
2007-06-01
期刊：
影响因子：
7.4
通讯作者：
Iida, Shigeru
中科院分区：
文献类型：
--
作者：
Terada, Rie;Johzuka-Hisatomi, Yasuyo;Iida, Shigeru
通过同源重组将内源基因修饰成设计的序列，被称为基因打靶(GT)，对于基础和应用研究具有广泛的意义。水稻基因组全长389Mb，是一种重要的粮食作物和模式植物，利用GT对6号染色体上的单拷贝基因waxy进行了改造，使其每存活愈伤组织的频率为1%。由于该策略独立于特定基因的选择或筛选，因此原则上适用于任何基因。然而，由于异位重组的发生，多基因家族中的一个基因或携带重复序列的基因可能会阻止有效的同源重组促进的GT。在这里，我们描述了一种改进的GT程序，通过该程序，我们获得了9个独立的转化愈伤组织，其酒精脱氢酶2(ADH2)基因的修饰频率约为每个存活愈伤组织2%，随后分离到8个可育的转基因植株，没有伴随着不良异位事件的发生，即使水稻基因组携带4个ADH基因，包括一个新鉴定的Adh3基因，并且ADH2基因附近存在一个高度重复的逆转录元件的拷贝。结果表明，采用强正负选择的GT可以广泛应用于水稻功能基因组学，也可能适用于其他高等植物的功能基因组学。
The modification of an endogenous gene into a designed sequence by homologous recombination, termed gene targeting (GT), has broad implications for basic and applied research. Rice (Oryza sativa), with a sequenced genome of 389 Mb, is one of the most important crops and a model plant for cereals, and the single-copy gene Waxy on chromosome 6 has been modified with a frequency of 1% per surviving callus by GT using a strong positive-negative selection. Because the strategy is independent of gene-specific selection or screening, it is in principle applicable to any gene. However, a gene in the multigene family or a gene carrying repetitive sequences may preclude efficient homologous recombination-promoted GT due to the occurrence of ectopic recombination. Here, we describe an improved GT procedure whereby we obtained nine independent transformed calli having the alcohol dehydrogenase2 (Adh2) gene modified with a frequency of approximately 2% per surviving callus and subsequently isolated eight fertile transgenic plants without the concomitant occurrence of undesirable ectopic events, even though the rice genome carries four Adh genes, including a newly characterized Adh3 gene, and a copy of highly repetitive retroelements is present adjacent to the Adh2 gene. The results indicate that GT using a strong positive-negative selection can be widely applicable to functional genomics in rice and presumably in other higher plants.