生物
农杆菌
遗传学
Cas9
突变
清脆的
转化(遗传学)
突变体
转基因
索引
基因
单核苷酸多态性
基因型
作者
Shuang Liu,Xiuyuan Wang,Qianqian Li,Wentao Peng,Zunmian Zhang,Pengfei Chu,Shangjing Guo,Yinglun Fan,Shanhua Lyu
标识
DOI:10.3389/fpls.2022.952428
摘要
Agrobacterium rhizogenes –mediated (ARM) transformation is an efficient and powerful tool to generate transgenic roots to study root-related biology. For loss-of-function studies, transgenic-root-induced indel mutations by CRISPR/Cas9 only with homozygous/biallelic mutagenesis can exhibit mutant phenotype(s) (excluding recessive traits). However, a low frequency of homozygous mutants was produced by a constitutive promoter to drive Cas9 expression. Here, we identified a highly efficient Arabidopsis thaliana gamma- g lutamyl c ysteine s ynthetase promoter, termed AtGCSpro , with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species. AtGCSpro achieved higher homozygous/biallelic mutation efficiency than the most widely used CaMV 35S promoter in driving Cas9 expression in soybean, Lotus japonicus , and tomato roots. Using the p AtGCSpro -Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p 2 × 35Spro -Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the p AtGCSpro -Cas9 system used in ARM transformation, especially its great potential in diploids with multiple-copy genes targeted mutations and polyploid plants with multiplex genome editing. AtGCSpro is conservatively active in various eudicots species, suggesting that AtGCSpro might be applied in a wide range of dicots species.
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