| Many plants have adopted self-incompatibility (SI) systems, which enable the plant to discriminate between "self" and "non-self" pollen, to avoid inbreeding depression and promote out-crossing in the population. The Brassica SI is not only a model system for cell-cell recognition and signal transduction, but also an important event for breeding in plant. In recent years, female and male S1determinants were identified as a receptor-type protein kinase (SRK) and its ligand (SP11/SCR) in Brassica. However, little is known about the molecular mechanism of SI signalling transmitting in papilla cell and inhibition of self-pollen growth thus far. In this study, the molecular mechanism of SI in ornamental kale(Brassica oleracea var. acephala) was investigated as following.1Isolation of BoExo70Al of ornamental kale and interaction analysis with ARC1The BoExo70A1gene was isolated from stigma of ornamental kale S13-b homozygotes by reverse transcriptase PCR and rapid amplification of cDNA ends PCR. BoExo70A1cDNA was2184bp in the full length, which contained a5'-untranslated region (5'-UTR) of56bp, a3'-UTR of211bp, and an opening reading frame of1917bp encoding a638predicted amino acids residue protein. The genomic BoExo70A1sequence was4076bp, contained12exons and11introns ranging in size from74bp to453bp with boundary sequences of GT at5'site and AG at3'site, which complied with the typical'GT-AG rule' in plants. The deduced amino acid sequence of BoExo70Al shared99%and94%identity with Brassica napus Exo70A1and Arabidopsis thaliana Exo70A1, respectively. The C-terminus of this protein contains a typical Exo70domain.Southern hybridization analysis showed that there were multiple copies of BoExo70A1gene in genome. Northern hybridization analysis showed that the BoExo70A1was ubiquitous expressed in stem, petal, anther, stigma, style and ovary, as well as a relative low expression in leaf and relative high expression in stem and petal. The yeast two-hybrid analysis indicated that the N-terminus of BoExo70A1(BoExo70A11-270) interacted with the UND domain (U-box N-terminal domain) of ARC1.2Isolation and characterization of JDP1from ornamental kale and interaction analysis with ARC1A J domain protein (J domain-containing protein1, JDP1) was isolated using a yeast two-hybrid screen against a stigma cDNA library with ARC1as bait. The nucleotide sequence of putative full-length cDNA was1326bp, containing a short5'(73bp)-and a long3'(218bp)-untranslated region and an open reading frame of1035bp which encoded a peptide of344amino acids with the predicted molecular mass of38.4kDa and the calculated pI of7.04. The N-terminus of JDP1contains a J domain, but the remainder of the protein showed no similarity with any domains in the SMART database or Pfam database. RT-PCR analysis showed that JDP1was expressed in the anther, stigma, style and ovary of non-pollinated flowers as well as in the stem, petal, and leaf. Western blot analysis using polyclonal antibody against JDP1, revealed that JDP1expressed in all of the tissues examined. And JDP1accumulation in stigma was at low levels during early developmental stages, but gradually increased before flowering and reached its highest level on the day of anthesis.The MBP-ARC1and His-JDP1fusion protein were obtained by prokaryotic expression and purification system, and BoARC1bound to full-length JDP1in vitro binding assays. In addition, the C-terminal of JDP1(JDP169-344) was required and sufficient for interaction with ARC1in yeast two-hybrid assays and in vitro binding assays. JDP169-344regulated the trafficking of ARC1from the cytoplasm to the plasma membrane by interacting with ARC1in Arabidopsis mesophyll protoplasts. A point mutation of JDP1at amino acid position8substituting Tyr for Phe (Y8F) resulted in a strong interaction of JDP1with BoARC1, indicating that the Tyr8regulates the interaction between JDP1and BoARCl.3Characterization of differentially expressed proteins in stigma during the process of self-pollination and cross-pollination in ornamental kaleTotal proteins of self-pollinated or cross-pollinated stigmas were extracted by TCA-acetone precipitation method. Afer purification and quantification, the proteins were separated by two-dimensional difference gel electrophoresis (2-D DIGE) following the user manual. In self-pollinated stigma, a total of38protein spots showed significant changes in self-pollinated samples compared to control samples. A total of11protein spots were increased and27protein spots were decreased. In cross-pollinated stigma, a total of16protein spots showed significant changes in cross-pollinated samples compared to control samples, with6spots increased and10spots decreased in abundance.The differentially expressed protein spots were excised, in-gel trypsin digested and analyzed using MALDI-TOF-TOF MS. The identified proteins covered a wide range of molecular functions, including metabolism, photosynthesis, energy, protein folding and degradation, signaling, cellular structure, cell wall, stress response and defense. In self-pollination, the differentially expressed proteins were classified into8categories, and the largest groups were metabolism (36%) and photosynthesis (19%). In cross-pollination, the differentially expressed proteins were classified into7categories, and metabolism (26%) and energy (20%) formed the largest groups. Differential expression proteins were isolation and analysis in this study, which provided an important clue for further SI study.
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