AP3 gene cloning and gene-editing vector construction of Hydrangea macrophylla ‘Dooley’

Abstract

Hydrangea macrophylla is a garden plant widely cultivated in Asia, America, and Europe with its inflorescence as main ornamental feature. It is commonly used in interior decoration and landscape creation. To investigate the role of AP3 gene in hydrangea during calyx formation, H. macrophylla ‘Dooley’ was used as the material. The MADS-box Class B gene HmAP3 was cloned, and its gene function was predicted by bioinformatics analysis. To explore methods for quicker breeding new varieties, highly-specific editing targets were screened and CRISPR/Cas9 gene-editing vectors were constructed. The vector sequence was integrated into the H. macrophylla genome by agrobacterium-mediated transformation. The results were as follows: (1) The cDNA sequence full length of HmAP3 was 546 bp, encoding 181 amino acids. Its amino acid sequence was 100% similar to the reference sequence and 58.8% similar to Arabidopsis thaliana. (2) AP3 differed greatly in different genera. Within the same genus, the main structure of AP3 protein was conserved and differed only in a few motifs. (3) There were two highly specific targets in HmAP3. Sequencing results indicated that two single-target CRISPR/Cas9 gene-editing vectors were constructed successfully. (4) There were five resistant buds with Cas9 sequences in their genomes. However, their target sequences did not change due to the absence of Cas9 expression. In this study, the potential of AP3 gene in the breeding work of double flower phenotype was investigated, and a preliminary exploration of CRISPR/Cas9 gene-editing technology for Hydrangea macrophylla was conducted. These results provide a basis for the breeding of H. macrophylla.

Keywords

• hydrangea macrophylla
• mads-box family
• ap3
• crispr/cas9
• vector construction