利用CRISPR-Cas9编辑蒺藜苜蓿串联重复基因  

Tandem repeat genes editing using CRISPR-Cas9 system in Medicago truncatula

作　　者：李宇赫 裴悦 沈怡彤 张锐 王宜宜 王劭学 姚莉琦 李力涵 康明明 李登耀 陈玉辉 LI Yuhe;PEI Yue;SHEN Yitong;ZHANG Rui;WANG Yiyi;WANG Shaoxue;YAO Liqi;LI Lihan;KANG Mingming;LI Dengyao;CHEN Yuhui(College of Life Science,Lanzhou University,Lanzhou 730000,Gansu,China)

机构地区：[1]兰州大学生命科学学院,甘肃兰州730000

出　　处：《草业科学》2022年第10期2102-2111,共10页Pratacultural Science

基　　金：细胞活动与逆境适应教育部重点实验室开放基金(lzujbky-2019-kb05)。

摘　　要：DWF7基因编码Δ7甾醇C5去饱和酶,参与植物油菜素甾醇的合成。在豆科模式植物蒺藜苜蓿(Medicago truncatula)中,MtDWF7基因位于6号染色体,具有3个串联重复的拷贝。本研究利用CRISPR-Cas9技术,针对蒺藜苜蓿DWF7基因的3个拷贝,通过序列比对在3个基因外显子的同源区段设计两个编辑靶点,构建基因表达载体,转化野生型蒺藜苜蓿并产生毛根,提取转基因毛根DNA进行检测。经PCR鉴定,35份转基因毛根DNA样品中,靶位点1对3个基因的编辑效率分别为44.43%、5.71%和34.29%,3个基因在靶位点1同时发生编辑的概率为5.71%,而靶位点2未检测到基因编辑。本研究在毛根中实现了对MtDWF7串联重复的3个基因的同时编辑,证明选择合适的靶点可同时编辑3个基因,为串联重复基因功能的研究提供了新的思路。DWF7 gene encodes Δ7-Sterol-C5-desaturase, and it is involved in brassinosteroid biosynthesis. Mt DWF7 gene is located on chromosome 6 and has three tandem repeats in the genome of model legume plant Medicago truncatula. Here, we selected two editing targets for the three copies of Mt DWF7 gene, by aligning the homologous segments of the three genes’ exon region. A CRISPR-Cas9 vector harboring two editing targets was constructed, and transformed to wild-type M. truncatula through Agrobacterium rhizogenes mediated transformation to generate transgenic hairy roots. The DNA of transgenic hairy roots was isolated individually. DNA samples from 35 transgenic hairy roots were obtained, and confirmed by PCR. After PCR confirmation, among these 35 transgenic hairy roots’ DNA samples, the editing efficiency of target site 1 for three genes was found to be 44.43%, 5.71%, and 34.29%, respectively. The probability of three genes being edited simultaneously at target site1 was 5.71%, and no gene editing was observed at target site 2. This study elucidated the simultaneous editing of tandem repeat genes using hairy root transformation, and proved that selecting suitable editing sites can help edit three genes at the same time.This suggests novel way for the functional study of tandem repeat genes in the future.

关 键 词：CRISPR-Cas9 蒺藜苜蓿 串联重复基因 DWF7 基因编辑 毛根转化 油菜素甾醇 

分 类 号：Q943.2[生物学—植物学]