Molecular characterization and expression analysis of ubiquitin-activating enzyme E1 gene in Citrus reticulata

Ubiquitin-activating enzyme E1 (UBE1) catalyzes the first step in the ubiquitination reaction, which targets a protein for degradation via a proteasome pathway. UBE1 plays an important role in metabolic processes. In this study, full-length cDNA and DNA sequences of UBE1 gene, designated CrUBE1, were obtained from 'Wuzishatangju' (self-incompatible, SI) and 'Shatangju' (self-compatible, SC) mandarins. 5 amino acids and 8 bases were different in cDNA and DNA sequences of CrUBE1 between 'Wuzishatangju' and 'Shatangju', respectively. Southern blot analysis showed that there existed only one copy of the CrUBE1 gene in genome of 'Wuzishatangju' and 'Shatangju'. The temporal and spatial expression characteristics of the CrUBE1 gene were investigated using semi-quantitative RT-PCR (SqPCR) and quantitative real-time PCR (qPCR). The expression level of the CrUBE1 gene in anthers of 'Shatangju' was approximately 10-fold higher than in anthers of 'Wuzishatangju'. The highest expression level of CrUBE1 was detected in pistils at 7 days after self-pollination of 'Wuzishatangju', which was approximately 5-fold higher than at 0 h. To obtain CrUBE1 protein, the full-length cDNA of CrUBE1 genes from 'Wuzishatangju' and 'Shatangju' were successfully expressed in Pichia pastoris. Pollen germination frequency of 'Wuzishatangju' was significantly inhibited with increasing of CrUBE1 protein concentrations from 'Wuzishatangju'.

Expression analyses of CrUBE1 genes in different tissues of 'Wuzishatangju' (WZSTJ) and 'Shatangju' (STJ) (C) and different stages after self-pollination of 'Wuzishatangju' × 'Wuzishatangju' (W * W) and cross-pollination of 'Wuzishatangju' × 'Shatangju' (W * S) (E).83Highlights► Full-length sequences of CrUBE1 are isolated from mandarin and share 99% identity. ► One copy of CrUBE1 exists in genome of both 'Wuzishatangju' and 'Shatangju'. ► The temporal and spatial expression characteristics of CrUBE1 are investigated. ► CrUBE1 protein inhibits pollen germination frequency of 'Wuzishatangju'.