Molecular Characterization and Expression Analysis of S1 self-incompatibility Locus-linked Pollen 3.15 Gene in Citrus reticulata

Gametophytic self-incompatibility (GSI) is controlled by a highly polymorphic locus called the S-locus, which is an important factor that can result in seedless fruit in Citrus. The S1 self-incompatibility locus-linked pollen 3.15 gene (S1-3.15) belongs to a type of S locus gene. The role of S1-3.15 in the SI reaction of Citrus has not yet been reported. In this study, full-length sequences of cDNA and DNA encoding the S1-3.15 gene, referred to as CrS1-3.15, were isolated from Wuzishatangju' (Self-incompatibility, SI) and Shatangju' (Self-compatibility, SC). The predicted amino acid sequences of CrS1-3.15 between Wuzishatangju' and Shatangju' differ by only three amino acids. Compared to Wuzishatangju', three bases were substituted in the genomic DNA of CrS1-3.15 from Shatangju'. Southern blot results showed that one copy of CrS1-3.15 existed in the genomic DNA of both Wuzishatangju' and Shatangju'. The expression level of the CrS1-3.15 gene in the ovaries of Shatangju' was approximately 60-fold higher than that in the ovaries of Wuzishatangju'. When Wuzishatangju' was cross-pollinated, the expression of CrS1-3.15 was upregulated in the ovaries at 3 d, and the highest expression levels were detected in the ovaries at 6 d after cross-pollination of Wuzishatangju' x Shatangju'. To obtain the CrS1-3.15 protein, the full-length cDNA of CrS1-3.15 genes from Wuzishatangju' and Shatangju' was successfully expressed in Pichia pastoris. Pollen germination frequency of Wuzishatangju' was inhibited significantly with increasing CrS1-3.15 protein concentrations from SI Wuzishatangju'.