CRISPR/Cas9介导靶向敲除拟南芥REVOLUTA基因突变体的鉴定

Identification of Knockout of REVOLUTA Mutant Caused by CRISPR/Cas9 in Arabidopsis

CRISPR/Cas9系统可对植物的内源基因进行定点编辑,为获得基因缺失突变体提供了新的工具。HD-ZipⅢ家族成员REVOLUTA(REV)是植物发育过程中的关键转录因子。本研究利用CRISPR/Cas9基因编辑技术对拟南芥REV进行特异性定点编辑,构建REV基因编辑表达载体,并利用农杆菌介导的花序浸染法将其转入拟南芥。经潮霉素抗性平板筛选和PCR扩增及测序鉴定,获得7棵转基因株系。对转基因植株REV基因的测序结果显示,有3株均成功在靶点1处产生编辑,且其中1株在靶点2处也成功编辑。该基因编辑突变体的获得为后续深入研究REV基因在拟南芥形态发育过程中的作用提供了新的遗传材料。

The clustered regulatory interspaced short palindromic repeat/Cas9(CRISPR/Cas9)genome editing technology is capable of fixed-point editing of endogenous genes in plants,providing a new tool for obtaining gene KO(knock out)mutants.REVOLUTA(REV),a member of the HD-zip III family,is a key transcription factor in plant development.CRISPR/Cas9 system was used to specific fixed-point edit the REV gene in this study,and the expression vector of plant gene editing system was constructed,and then the vector was transferred into Arabidopsis thaliana by Floral Dip method.Total 7 positive transgenic plants were obtained which were confirmed by hygromycin resistance selection as well as PCR amplification and sequencing identification.The REV sequencing results of transgenic progeny showed that,3 positive strains were successfully edited at target site 1,one of which was also occurred edit at target site 2 at the same time.This study provides new genetic materials for further research on the function of REV gene during morphological development in Arabidopsis.