mitoTALENs植物线粒体基因编辑载体的构建方法

Construction Method of mitoTALENs Mitochondrial Gene Editing Vector in Plants

【目的】mitoTALENs植物线粒体基因编辑技术能够高效地实现线粒体基因的敲除,进而有效实现线粒体基因功能的研究,但mitoTALENs载体构建过程非常的繁琐复杂且目前仍没有较为系统完整的载体构建方法作为参考。为解决这个问题,结合前人已发表的及本实验室摸索出的方法对mitoTALENs载体构建的完整过程进行了详尽描述,为之后利用mitoTALENs技术进行植物线粒体基因功能研究的研究者们提供重要参考。【方法】以水稻线粒体WA352基因作为目的基因,利用其序列特异性区域设计了靶点TAL,首先采用Platinum gate TALEN assembly的两步组装技术分别构建了mitoTALENs的TALEN-left和TALEN-right载体,然后利用multisite LR反应将TALEN-left、TALEN-right及含有其他功能元件的进入载体和目的载体进行反应,生成最终的表达载体。【结果】第一步组装构建10个载体,第二步组装构建2个载体,最后通过multisite LR反应构建1个终表达载体。【结论】详细介绍了mitoTALENs载体的构建过程,为该技术使用者提供重要参考,以促进植物线粒体基因编辑研究领域的发展。

【Objective】mitoTALENs plant mitochondrial gene editing technology can effectively achieve the knockout of mitochondrial genes,and then effectively conduct the study of mitochondrial gene function.However,the vector construction process of mitoTALENs is very complicated and there is still no relatively systematic and complete vector construction method as a reference.To solve this issue,we described in detail the complete process of mitoTALENs vector construction in combination with the methods published by predecessors and explored by ourselves,providing an important reference for researchers who use mitoTALENs technology to study plant mitochondrial gene function.【Method】With rice mitochondrial WA352 gene as the target gene,a target TAL was designed using its sequence-specific region.Firstly,the TALEN-left and TALEN-right vectors of mitoTALENs were constructed using the two-step assembly technology of Platinum gate TALEN assembly,respectively.Then,multisite LR reaction was used to react TALEN-left,TALEN-right,the entry vector containing other functional elements,and the destination vector to generate the final expression vector.【Result】Ten vectors were assembled in the first step,two vectors were assembled in the second step,and one final expression vector was constructed by multisite LR reaction.【Conclusion】The detailed introduction of mitoTALENs vector construction process provides an important reference for users and promotes the development of plant mitochondrial gene editing research.