TALEN-Based Mutagenesis of Lipoxygenase LOX3 Enhances the Storage Tolerance of Rice (Oryza sativa) Seeds.
TALEN-Based Mutagenesis of Lipoxygenase LOX3 Enhances the Storage Tolerance of Rice (Oryza sativa) Seeds.
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DOI：
10.1371/journal.pone.0143877
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Wang T
中科院分区：
文献类型：
--
作者：
Ma L;Zhu F;Li Z;Zhang J;Li X;Dong J;Wang T
稻谷的变质降低了稻米的品质，给农民造成严重的经济损失。脂氧合酶（LOXs）催化多不饱和脂肪酸与至少一种顺式，顺式-1，4-戊二烯的二氧合形成氢过氧化物，这是影响种子寿命和活力的主要因素。最近，基因组编辑作为反向遗传学中的一种重要工具，已被实验性地用于研究基础植物生物学或修改作物植物以改善重要的农业性状。在这项研究中，我们进行了靶向诱变水稻转录激活因子样效应核酸酶（TALENs），以提高种子的耐贮性。采用改良的连接非依赖性克隆方法（LIC）以允许TALEN单体模块快速且有效地定向插入到植物中使用的目的载体中。我们通过开发一组用于基因组编辑的模块化载体来证明该技术的可行性和灵活性。在构建和验证后，TALEN对被用于通过农杆菌介导的转化来创建稳定的转基因水稻品系。从25个转基因抗NPTII品系中回收到1个杂合突变体（4%），该突变体遗传到下一代。进一步的分子水平和蛋白水平的实验证实了LOX 3的缺陷，并证明了种子耐贮性的改善。我们的工作为改善重要的农艺性状提供了灵活的基因组编辑工具，并直接证明Lox 3对种子寿命的影响有限。
The deterioration of rice grain reduces the quality of rice, resulting in serious economic losses for farmers. Lipoxygenases (LOXs) catalyze the dioxygenation of polyunsaturated fatty acids with at least one cis,cis-1,4-pentadiene to form hydroperoxide, which is a major factor influencing seed longevity and viability. Recently, genome editing, an essential tool employed in reverse genetics, has been used experimentally to investigate basic plant biology or to modify crop plants for the improvement of important agricultural traits. In this study, we performed targeted mutagenesis in rice using transcription activator-like effector nucleases (TALENs) to improve seed storability. A modified ligation-independent cloning method (LIC) was employed to allow for the quick and efficient directional insertion of TALEN monomer modules into destination vectors used in plants. We demonstrated the feasibility and flexibility of the technology by developing a set of modular vectors for genome editing. After construction and validation, the TALEN pairs were used to create stable transgenic rice lines via Agrobacterium-mediated transformation. One heterozygous mutant (4%) was recovered from 25 transgenic NPTII-resistant lines, and the mutation was transmitted to the next generation. Further molecular and protein level experiments verified LOX3 deficiency and demonstrated the improvement of seed storability. Our work provides a flexible genome editing tool for improving important agronomic traits, as well as direct evidence that Lox3 has only a limited impact on seed longevity.