Cloning and functional analysis of bog bilberry E3 ligase VuARI2 gene in cold resistance

Abstract [Objective] This study aims to explore the function of VuARI2 gene in response to low temperature stress, by cloning and expressing VuARI2 gene in Arabidopsis. [Methods] VuARI2 gene was cloned from the stem of bog bilberry by RACE and RT-PCR and conducted a comprehensive bioinformatics analysis. Expression of VuARI2 in different tissues after cold acclimation was analyzed by RT-qPCR. Furthermore, the transgenic Arabidopsis was analyzed for the expression of VuARI2, physiological indexes, and freezing resistance. [Results] VuARI2 was successfully cloned,with an open reading frame of 1 770 bp, encoding 589 amino acids. The multi-sequence alignment and phylogenetic tree indicated that ubiquitin ligase VuARI2 was closely related to the amino acid sequence of ARI2 proteins in wild camellia, coffee tree, grape, and riverside grape. RT-qPCR showed that the expression of VuARI2 in leaves and flower buds of bog bilberry was higher than that in roots and stems, and the expression of VuARI2 in stems and flower buds was induced under low temperature. After cold acclimation, expression levels of VuARI2 gene in transgenic plants were higher in wild type, and chlorophyll content was lower than the wild type. Proline content and soluble sugar content were higher than the wild type. Superoxide dismutase activity, peroxidase activity, and catalase activity were higher than the wild type, while relative conductivity and malondialdehyde content were lower than the wild type. After cold acclimation, the survival rate of transgenic plants was higher than the wild type,and the relative conductivity was lower than the wild type at freezing temperature,enhancing freezing resistance of plants. [Conclusions] VuARI2 gene is cloned from bog bilberry and expressed in Arabidopsis. Overexpress of VuARI2 gene enhances the cold resistance of plants.