茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建

doi:10.13305/j.cnki.jts.2016.04.010

摘要/Abstract

摘要： CRISPR/Cas9技术是一门新兴的基因组定点编辑技术,具有操作简单、高效的优点,可轻松实现对目标基因的敲除、替换和定点突变等操作。该技术刚诞生,就受到了全球生命科学领域研究者的关注,不到3年的时间就已经成功应用于多种动、植物当中。然而CRISPR/Cas9技术在茶树中的应用面临载体构建问题,本文以茶树咖啡碱合成酶为例,联合采用常规PCR、Overlapping PCR和Golden Gate Cloning技术,构建了包含茶树咖啡碱合成酶双靶点的CRISPR/Cas9基因编辑载体,为CRISPR/Cas9介导的基因组编辑技术在茶树中的应用奠定了坚实基础。

关键词: 茶树, 基因组编辑技术, 咖啡碱合成酶, CRISPR/Cas9技术

Abstract: CRISPR/Cas9 technology (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) is a novel and powerful approach for targeted genome editing, such as targeted gene knock out or site-directed mutagenesis in a simple and easy way. Since its establishment, the CRISPR/Cas9 technique has been successfully applied in many eukaryotic organisms, including more than 10 plant species. However, it has not been available for genome editing of tea plant [Camellia sinensis (L.) O. Kuntze] due to the difficulty in constructing CRISPR/Cas9 expression vector. The present work developed an efficient method to construct a CRISPR/Cas9 expression vector for genome editing a tea caffeine synthase (TCS) by using general PCR, overlapping PCR and golden gate cloning technology. The present work would promote the application of CRISPR/Cas9 technology in genomic modification in tea plants.

Key words: Camellia sinensis (L.), genome editing technology, tea caffeine synthase, CRISPR/Cas9 technique

中图分类号:

TS272
Q52

唐雨薇, 刘丽萍, 王若娴, 陈宇宏, 刘仲华, 刘硕谦. 茶树咖啡碱合成酶CRISPR/Cas9基因组编辑载体的构建[J]. 茶叶科学, 2016, 36(4): 414-426. doi: 10.13305/j.cnki.jts.2016.04.010.

TANG Yuwei, LIU Liping, WANG Ruoxian, CHEN Yuhong, LIU Zhonghua, LIU Shuoqian. Development of a CRISPR/Cas9 Constructed for Genome Editing of Caffeine Synthase in Camellia sinensis[J]. Journal of Tea Science, 2016, 36(4): 414-426. doi: 10.13305/j.cnki.jts.2016.04.010.

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