摘要： 建立一种高效安全的基因定点编辑体系，为研究植物的光合作用途径以及生理代谢过程提供理想材料。针对拟南芥靶基因ALB3设计2个sgRNA，引导Cas9蛋白识别PAM位点，从而实现ALB3基因的大片段敲除；将荧光筛选标记基因mCherry组装到CRISPR/Cas9载体中，构建一种带有荧光筛选标记的CRISPR/Cas9载体；利用农杆菌转化法转化拟南芥，对其后代进行筛选验证，在倒置荧光显微镜下挑选便可获得Cas9-Free的纯合突变种子。测序结果表明，含有可视化筛选标记基因的拟南芥ALB3基因编辑载体序列与预期一致，由此证明载体构建成功；通过倒置荧光显微镜观察，发现阳性转化拟南芥T₀种子含有红色荧光标记，表观观测以及分子鉴定表明T₁植株得到纯合突变后代，条带大小小于野生型，白化突变性状明显。挑选T₁不含荧光的种子进行培育得到的T₂植株，经分子鉴定发现已成功实现Cas9-Free且植株白化。本研究成功构建了一种带有荧光筛选标记且可实现拟南芥ALB3基因敲除的CRISPR/Cas9载体，并获得Cas9-Free的纯合突变植株，为拟南芥基因高效编辑载体的构建以及Cas9-Free基因编辑后代的获得提供了借鉴与参考。

关键词: CRISPR/Cas9, ALB3基因, 拟南芥, 基因编辑, 载体

Abstract: A highly efficient and safe gene site-editing system was established to obtain Cas9-Free Arabidopsis ALB3 homozygous mutant seedlings,providing ideal materials for studying photosynthesis pathways and physiological metabolic processes of plants. Designed two sgRNAs for the Arabidopsis thaliana target gene ALB3, the Cas9 protein was induced to recognize the PAM site, thereby realized large fragment knockdown of the ALB3 gene;Assembled fluorescent screening marker gene mCherry into CRISPR/Cas9 vector to construct a CRISPR/Cas9 vector with fluorescent screening marker;The Agrobacterium transformation method was used to transform Arabidopsis thaliana, and its offspring were screened and verified. The homozygous mutant plants of Cas9-Free were obtained by selecting under an inverted fluorescence microscope.The sequencing results showed that the sequence of A.thaliana ALB3 editing vector containing the visual screening marker gene was consistent with the expectation,thus demonstrated the vector was successfully constructed; observed by inverted fluorescence microscope,positively transformed Arabidopsis T₀ seeds contain fluorescent markers. Apparent observation and molecular identification indicated that the T₁ generation plants obtained homozygous mutant progeny,strip size was smaller than wild type,and the whitening mutation traits were obvious. The T₂ generation plants grown from T₁ generation non-fluorescent seeds had been successfully implemented Cas9-Free and the plants were bleached. This study successfully constructed a CRISPR/Cas9 vector with a fluorescent selection marker and realized large fragment knockdown of the Arabidopsis ALB3,obtained a homozygous mutant of Cas9-Free,provided reference for the construction of A.thaliana gene efficient editing vector and the acquisition of Cas9-Free gene editing progeny.

Key words: CRISPR/Cas9, ALB3 gene, Arabidopsis thaliana, Gene editing, Vector

中图分类号: 

• Q782

• Q786

• Q789