A tool for functional plant genomics:: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations
A tool for functional plant genomics:: Chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations
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DOI：
10.1073/pnas.96.15.8774
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发表时间：
1999-07-20
影响因子：
11.1
通讯作者：
May, GD
中科院分区：
文献类型：
--
作者：
Beetham, PR;Kipp, PB;May, GD
由 DNA 和修饰的 RNA 残基组成的自互补嵌合寡核苷酸 (CO) 被评估为一种手段，以 (i) 在核基因中创建稳定的位点特异性碱基取代，以及 (ii) 在植物细胞的核转基因中引入移码。为了证明在基因家族成员中产生等位基因特异性突变，CO 被设计为靶向烟草乙酰乳酸合酶 (ALS) 基因 SuRA 的 Pro-196 密码子。ALS 的 Pro-196 处的 Sn 氨基酸取代赋予了除草剂抗性表型，可用作植物细胞中的选择标记。 CO 被设计为包含一个 25 nt 同源结构域，该结构域由 5 个脱氧核糖核苷酸区域（与天然 ALS 序列存在单碱基错配）组成，两侧各由 10 个核糖核苷酸组成。在选择性培养基上回收除草剂抗性烟草细胞后，DNA 序列分析鉴定了 ALS 基因中 Pro-196 密码子处的碱基转换。为了证明单个碱基在目标基因中的位点特异性插入，CO 被用来恢复失活的绿色荧光蛋白转基因的表达，该转基因被设计为含有单碱基缺失。荧光细胞的回收证实了缺失校正。我们的结果证明了通过催化特定核基因的碱基替换或碱基添加来修改个体遗传位点的技术的应用；这种方法在植物功能基因组学领域应该有很大的用途。
Self-complementary chimeric oligonucleotides (COs) composed of DNA and modified RNA residues were evaluated as a means to (i) create stable, site-specific base substitutions in a nuclear gene and (ii) introduce a frameshift in a nuclear transgene in plant cells. To demonstrate the creation of allele-specific mutations in a member of a gene family, COs were designed to target the codon for Pro-196 of SuRA, a tobacco acetolactate synthase (ALS) gene.,Sn amino acid substitution at Pro-196 of ALS confers a herbicide-resistance phenotype that can be used as a selectable marker in plant cells. COs were designed to contain a 25-nt homology domain comprised of a five-deoxyribonucleotide region (harboring a single base mismatch to the native ALS sequence) flanked by regions each composed of 10 ribonucleotides, After recovery of herbicide-resistant tobacco cells on selective medium, DNA sequence analyses identified base conversions in the ALS gene at the codon for Pro-196, To demonstrate a site-specific insertion of a single base into a targeted gene, COs were used to restore expression of an inactive green fluorescent protein transgene that had been designed to contain a single base deletion. Recovery of fluorescent cells confirmed the deletion correction. Our results demonstrate the application of a technology to modify individual genetic loci by catalyzing either a base substitution or a base addition to specific nuclear genes; this approach should have great utility in the area of plant functional genomics.