Isolation and preliminary functional analysis of FvICE1, involved in cold and drought tolerance in Fragaria vesca through overexpression and CRISPR/Cas9 technologies

Abstract

Cold and drought stresses are serious problems of strawberry cultivation in temperate and subtropical regions. In the molecular regulation system of cold and drought stresses, ICE transcription factors (TFs) are crucial. In this research, the FvICE1 was isolated from Fragaria vesca ‘Hawaii 4’, a bioinformatics analysis was conducted, overexpression vector and CRISPR/cas9 vector were constructed. The results showed that FvICE1 was a member of the bHLH TF family, with a length of 1608 bp, encoding 535 amino acids, and its molecular formula was C2504H3987N745O811S22. By observing the fusion protein 35S-FvICE1-GFP, it was found that FvICE1 was a nuclear protein. The qRT-PCR results demonstrated that FvICE1 was significantly upregulated in different tissues of Fragaria vesca after cold, drought, salt and heat treatments. The wild type (WT) strawberry was selected as the control group, FvICE1-overexpression strawberries showed high tolerance to cold and drought treatments at the phenotypic and physiological levels. On the contrary, fvice1 mutant strawberries obtained by CRISPR/cas9 editing technology had lower tolerance to cold and drought treatments. Moreover, the expression of FvCBF1, FvCBF2, FvCBF3, FvCOR413, FvRD22 and FvKIN1 was positively regulated in the FvICE1-overexpression strawberries and inhibited in fvice1 mutant strawberries. Overall, the current results suggested that FvICE1 functioned as a positively regulator of cold and drought resistances.