Abstract:

Argonaute2 (AGO2) plays an important role in plant resistance and development. In order to create Arabidopsis ago2 nucleotides insertion/deletion mutant materials, structure of AtAGO2 gene was analysed firstly, and then the CRISPR_Cas9 based gene editing expression vector which contains three target sites of AtAGO2 was constructed, and transferred into Arabidopsis by Agrobacterium mediated floral-dip. 62 anti-hygromycin resistant seedlings were obtained by screening the seeds of T₀ generation plants on the medium containing hygromycin antibiotic. And 53 definitively transgenic positive seedlings were further confirmed by PCR amplification using hygromycin B gene specific primers. The fragments of AtAGO2 containing the target sites of 10 random selected transgenic progenies were sequenced, and the results showed that six transgenic lines were successfully edited around the T₁ site, all the 10 lines were edited around the T₂ site, and the third target site was not edited. Varied forms of mutation happened in the nearby area of the target sites, and the most frequent ones were deleting or adding one base before PAM. There were also more than 10 bases deletion forms, and the longest one was 106 bases deletion. This study provided abundant useful genetic materials for further study of the function of AGO2 in Arabidopsis.

Key words: Argonaute2, Arabidopsis, mutant, floral dip, gene editing, CRISPR_Cas9