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' × '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'.

Cloning and expression analysis of S-RNase homologous gene in Citrus reticulata Blanco cv. Wuzishatangju.

S-RNase-based self-incompatibility is the most widespread form of genetically controlled mate selection in plants and that S-RNase controls pollination specificity in the pistils. 'Wuzishatangju' (Citrus reticulata Blanco), a nature bud mutant from a self-compatible (SC) cultivar 'Shatangju', displays gametophytic self-incompatibility (GSI). In this study, full-length sequences of cDNA and DNA of the S-RNase homologous gene were obtained from 'Wuzishatangju' and 'Shatangju'. There was no difference in ORF sequences of the S-RNase cDNA between 'Wuzishatangju' and 'Shatangju'. However, 13, 9 and 6 consecutive bases were missing in 'Wuzishatangju' cDNA 5' UTR, 3' UTR and genomic DNA, respectively. Tissue-specific expression of the S-RNase gene was detected using semi-quantitative RT-PCR and quantitative real-time PCR. The expression level of the S-RNase gene in styles of 'Wuzishatangju' was approximately 10- and 5-fold higher than that in leaves and pollen, respectively. When 'Wuzishatangju' was self-pollinated, the expression of S-RNase in pistils peaked at 3 days, which was approximately 10-fold higher than that at 4h and 7 days, while in cross-pollination of 'Wuzishatangju' x 'Shatangju' the expression was very weak at 3 days. Results from a Southern blot showed that two copies of the S-RNase gene existed in genomic DNA of both 'Wuzishatangju' and 'Shatangju'.