Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology
Creation of fragrant rice by targeted knockout of the OsBADH2 gene using TALEN technology
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利用TALEN技术定向敲除OsBADH2基因培育香米
DOI：
10.1111/pbi.12312
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发表时间：
2015-08-01
影响因子：
13.8
通讯作者：
Gao, Caixia
中科院分区：
文献类型：
--
作者：
Shan, Qiwei;Zhang, Yi;Gao, Caixia
香米因其香味宜人而受到全世界的喜爱。编码甜菜碱醛脱氢酶（BADH 2）的缺陷型badh 2等位基因的存在导致合成2-乙酰基-1-吡咯啉（2AP），其是主要的香料化合物。在这里，转录激活因子样效应物核酸酶（TALEN）被工程化以靶向并破坏OsBADH 2基因。从20个转基因潮霉素抗性株系中回收了6个杂合突变体（30%）。桑格测序证实，这些细胞系在TALEN靶位点处具有各种indel突变。所有六个将BADH 2突变传递到T1代;并且测试的四个T1突变系也有效地将突变传递到T2代。通过在T1和T2代中分离获得仅携带所需DNA序列变化但不携带TALEN转基因的突变体植物。突变纯合的T_1代水稻籽粒中2AP含量从0增加到0.35-0.75mg/kg，与突变阳性对照品种的2AP含量相近。我们还通过轰击将靶向三个单独的水稻基因的三对不同的TALEN同时引入水稻细胞中，并获得了在一个、两个和所有三个基因中具有突变的品系。这些结果表明，使用TALEN的靶向诱变是产生重要农艺性状的有用方法。
Fragrant rice is favoured worldwide because of its agreeable scent. The presence of a defective badh2 allele encoding betaine aldehyde dehydrogenase (BADH2) results in the synthesis of 2-acetyl-1-pyrroline (2AP), which is a major fragrance compound. Here, transcription activator-like effector nucleases (TALENs) were engineered to target and disrupt the OsBADH2 gene. Six heterozygous mutants (30%) were recovered from 20 transgenic hygromycin-resistant lines. Sanger sequencing confirmed that these lines had various indel mutations at the TALEN target site. All six transmitted the BADH2 mutations to the T1 generation; and four T1 mutant lines tested also efficiently transmitted the mutations to the T2 generation. Mutant plants carrying only the desired DNA sequence change but not the TALEN transgene were obtained by segregation in the T1 and T2 generations. The 2AP content of rice grains of the T1 lines with homozygous mutations increased from 0 to 0.35-0.75mg/kg, which was similar to the content of a positive control variety harbouring the badh2-E7 mutation. We also simultaneously introduced three different pairs of TALENs targeting three separate rice genes into rice cells by bombardment and obtained lines with mutations in one, two and all three genes. These results indicate that targeted mutagenesis using TALENs is a useful approach to creating important agronomic traits.