Molecular cloning and expression analysis of <italic>BoPUB9</italic> in self-incompatibility <italic>Brassica oleracea</italic>

Self-incompatibility (SI) refers to the characteristic that the pistil of a plant can recognize its own pollen and allogeneic pollen so as to inhibit the germination of its own pollen. PUB (Plant U-Box) proteins play an important role in plant stress resistance and signal transduction. In this study, through the analysis of stigma transcriptome data of Brassica oleracea self-pollination at 0-60 minute(s), a PUB protein coding gene BoPUB9 was screened and up-regulated by self-pollination. The BoPUB9 cDNA sequence was 1368 bp, gDNA of 1720 bp in length, containing one arm repeat domain and one U-box domain. The relative expression levels showed that BoPUB9 gene was expressed in different tissues of Brassica oleracea, and the relative expression level was the highest in sepals, followed by petals, stamens and stigmas, and relatively lower in pollen buds, which was consistent with the results of GUS staining. The relative expression level of BoPUB9 gene was up-regulated rapidly in self-pollination 0-30 minute(s), the expression level after 15 min was more than 20 times that after cross-pollination, and the difference was the biggest after 30 min, the expression level after self-pollination was more than 40 times that after cross-pollination. Subcellular localization revealed that BoPUB9 protein was expressed in both nucleus and cytoplasm, and BoPUB9 protein was successfully induced in E. coli, and the relative molecular weight of 51 kD was consistent with the predicted results. The results of yeast two-hybrid and poll-down indicated that the intracellular domain of SRK interacted with PUB9 protein. It was speculated that BoPUB9 might be a novel gene involved in the process of self-incompatibility in Brassica oleracea, which providing a new content for further research and utilization of self-incompatibility in Brassica oleracea.