Isolation Of Genes And Dynamic Change Of Phosphorylated And Ubiquitinated Protein Level In The Self-incompatibility Response Of Brassica Oleracea Var. Acephala

Cell-cell recognition and signal transduction between the pollen and stigma is required to achieve fertilization. Self-incompatibility (SI) allows the pistil to distinguish between self and non-self pollens of the same species and reject self or self-related pollens. Many flowering plants have evolved this physiological strategy to prevent inbreeding and maintain genetic diversity in a population. Studies on signal transduction of SI are not only useful for plant reproduction, but also has practical application in plant breeding. In this study, the molecular mechanism of SI in Brassica oleracea var. acephala was investigated.1. Identification of S_13b haplotypeNine strong self-incompatible strains and 14 self-compatible strains were identified through 3 self-generations from 2 ornamental kales, 'Red Rabbit' and 'White Wave'. The SRK DNA and cDNA sequences were obtained by PCR using a pair of SRK specific primers. The Blastn search showed that SRK sequences obtained was identical to SRK_13b from Brassica oleracea. We carried out the PCR-RFLP and Southern hybridization analysis of SRK, results of which showed that all of these SI strains were a S_13bS_13b homozygous.2. Isolation of SCR_13b from Brassica oleracea var. acephalaBy using SCR specific primers based on the NH₂-terminal region of SCR proteins and anchored oligo(dT)₁₈ primer, SCR cDNA fragment was isolated from Brassica oleracea var. acephala. The SCR cDNA fragment showed a high similarity with SCR₁₃ with 3 nucleic acid (AAG) insertions and 3 nucleic acid (TT and C) deletions. Thus, the SCR was names as SCR_13b. The full length sequence of SCR_13b cDNA was amplified by PCR using a set of SCR_13b specific primers designed from both ends of SCR₁₃ encoding region. The SCR_13b cDNA sequence contained had an ORF of 234 bp, encoding a 78-aa protein. The amino acid sequence of SCR_13b showed 98.3% identity with that of SCR₁₃. The genomic SCR_13b sequence was also obtained by PCR using SCR_13b specific primers. The sequence contained a 758 bp intron with boundary sequences of GT at 5' site and AG at 3' site, which complied with the typical 'GTAG rule' in plants.The coding region of SCR_13b without signal peptide was inserted into the pGEX-KG, pET32a vector. And then, Escherichia. coli DH5αwas transformed with pGEX-KG-SCR_13b and BL21 (DE3) pLysS cell was transformed pET-32a-SCR_13b, the recombinant SCR_13b fusion protein was obtained by using expression and purification in Escherichia. coli. The resultant recombinant GST-SCR_13b and His-SCR_13b proteins were used in a pollination bioassay to show that SCR_13b could induce the SI response in the stigma and disturbe compatible pollination. These results suggested that SCR_13b was an active SCR protein in the self-incompatible strain used.3. Isolation of BoARC1 from Brassica oleracea vat. acephalaBy using a pair of primers based on BnARC1 sequence, BoARC1 was isolated using RT-PCR from stigma. BoARC1 encoded a 663 amino acids protein. The amino acid sequence of BoARC1 showed 94 % identity with that of BnARC1. Southern blot analysis of genomic DNA showed that BoARC1 was a single copy gene found in S_13bS_13b lines. Nothern blot analysis indicated that high levels of BoARC1 mRNA were only detected in the stigma during the flowering period. Yeast two hybrid analysis showed that Arm repeats domain of BoARC1 interacted with the kinase domains of SRK₃, SRK_13b and SRK₉₁₀. These results implied that BoARC1 was a positive component of the self-incompatibility signaling cascade in Brassica.BoARC1 contained U-box domain and ARM repeats domain, which is unique to plant proteins. The phylogenetic analysis showed that BoARCI and BnARC1 clustered with AtPUB17, NtACRE276, StACRE276 and OsACRE. It was suggested that BoARC1 might play a significant role in other signal transduction in plant.4. Dynamic change in protein phosphorylation and ubiqtuitination in SI responseProtein phosphorylation levels were assayed by Western blot analysis using phosphoprotein antibody. The phosphyrylation of membrane fraction proteins of papilla cell reached a peak at 5 min after SI response, cytosolic proteins of papilla cell showd a peak 15 rain after SI response, and then decreased to its basal level. Protein phosphyrylation level was stable in compatible response. Protein ubiquitination levels were assayed by Western blot analysis using ubiquitin-antibody. At 0.5 h after SI response, protein ubiquitination level increased for 30 min, and then the level remained at least for one hour. In compatible combinations, protein ubiquitination level did not increase. These results showed that protein phosphorylation and ubiqtuitination are involved in the events of SI response, which provided an important clue for further study.