关键词: 甘蓝, BoSPx, 酵母双杂交, 生长素, 自花授粉, 自交不亲和

Abstract:

【Objective】Self-incompatibility (SI) is a genetic barrier to inhibit self-pollination and promote hybridization in flowering plants. Here, cloning of a novel gene co-responsive to SI and calcium production during pollination, and spatio-temporal specific expression analysis of the novel gene BoSPx under self-pollination conditions, screening its interaction proteins were conducted to explore. The responding mechanism of BoSPx to self-pollination stimulated stigma in order to provide some further insights into SI process in Brassica oleracea.var.Capitata.【Method】BoSPx was cloned by using transcriptome sequencing, self-pollination and cross-pollination differential screening, and PCR cloning. Amino acid sequence alignment and conserved domain analysis were performed by DNAMAN and Smart software. Expasy online software was used to predict BoSPx protein molecular weight, isoelectric point, secondary structure and transmembrane domain. Phylogenetic tree that constructed by the neighboring method in MEGA6.0 software was used to speculate on the function of BoSPx protein after self-pollination. RT-PCR and qRT-PCR were used to detect BoSPx tissue-specific expression and the relative expression of BoSPx after self-pollination and cross-pollination. The BoSPx-GFP expression vector was constructed and the subcellular localization of BoSPx was observed under confocal microscopy; The interaction proteins were searched by using yeast two-hybrid system. 【Result】A novel gene, which contains a single exon without any introns, named BoSPx was cloned. The open reading frame of BoSPx is 396 bp, encodes a protein with 131 amino acid residues. BoSPx is a hydrophilic protein, no signal peptide and transmembrane, and the theoretical isoelectric point is 4.54. Conserved domains analysis found BoSPx contains three conserved EF-hand motifs (48-60, 64-80, and 81-96). About 500 bp up-stream of BoSPx translation start code contains an auxin response element. RT-PCR analysis found that BoSPx was expressed highest in stigma, BoSPx was also expressed in sepals, leaves, anthers and petals in flowering stage. The BoSPx expression levels in both self-pollinated and cross-pollinated stigma showed “up-down-up” expression pattern. Moreover, The expression of BoSPx in the stigma of self-pollination and cross-pollination increased at first and then decreased down to the highest expression level in stigma in flowering stage. The expression of BoSPx increased rapidly after self-pollination at 15 min and then decreased sharply so that result in SI progress. The decreased value of BoSPx was 1-2 days before flowering. Subcellular location analysis found that BoSPx expression in both the nucleus and cytoplasm. Yeast two-hybrid system did not detect interaction between BoSPx and SRK and ARC1, but BoSPx interacted with auxin family proteins BoSAUR71 and BoPID. 【Conclusion】BoSPx is highly expressed by self-pollination, which may be an auxin-regulated calcium-binding protein, which has a common response to SI and calcium. The protein has multi-tissue expression and nuclear and cytoplasmic properties, indicating that BoSPx may be involved in unknown signaling pathways other than the SRK-ARC1-ExO70A1 pathway.

Key words: Brassica oleracea, BoSPx, yeast two-hybrid, auxin, self-pollination, self-incompatibility