| Self-incompatibility is fascinating molecular "lock-and-key" mechanisms preventing self-fertilization in flowering plants, cross-incompatibility occurs between different individuals that have the same incompatibility type when Self-incompatibility occurs between the same haploid pollen and pistil. Members of the Brassicaceae family have a SSI (sporophytic SI) system, the SSI response of Brassica oleracea results from Recognition of SRK and self-SCR which are encoded by two genes at S locus, then the response of recognition between stigma and self pollen is transmitted to internal cells. THL is separated from SRK and is released into cytoplasm. At the same time, ARC1 is activated by the kinase domain of SRK. Then the response of ARC1 and SRK is passed to EXO70A1, then the subsequent cascade of reactions ultimately lead to self-incompatibility.In this study, a yeast two-hybrid system was used to investigate the interaction between ARC1 and the Kinase Domain of SRK. The sequences of SRK Kinase Domain (SRKJ) and different-length ARCla, ARClb, ARClc and ARC1d of Brassica oleracea were amplified by PCR, then SRKJ was subcloned into pGADT7 and different longth ARC1 were subcloned into pGBKT7 vectors. After sequencing, the plasmids were transformed into the yeast cells, and the interaction between the truncated fragments of ARC1 from Brassica oleracea var. acephala and kinase domain of SRK from Brassica oleracea var. capitata L were tested by Yeast Two-Hybrid System provides some insights into the molecular mechanism of self-incompatibility in Brassica.The results showed:1 Cloning and analysis of sequenceThe results indicate that the gDNA, consisting of 1992 bp without introns, encodes a 663 amino acid ORF which has 99.7%similarity to Brassica oleracea var acephala (sequence accession:EU344909), containing a total of four amino acid polymorphisms. It has 45 amino acid differences when compared to B. napus (AF024025), with which it shares a 92.9%similarity. The ARC1 has no signal peptide, but has six phosphorylation sites; Sequence analysis also demonstrated that the ARC1 protein coding region contains U-box and ARM areas. Vector NTITM analysis indicated that there were 5 amino acid differences contained in the U-box region and 14 amino acid differences in the ARM region between the cloned Brassica oleracea var. acephala and Brassica napus. The sequences of Kinase Domain of SRK (SRKJ) was amplified by PCR from Brassica oleracea var. apitata L. the length of SRKJ is 1131bp and encodes 376 amino acid ORF which has 91.0%similarity to Kinase Domain of SRK from Brassica oleracea var acephala, and has 12 similarityamino acid in46 differences amino acid; there is a hypervariable region in Kinase Domain of SRK and located between the sequence of 250 amino acids and 270 amino acids.2 The toxicity and Autoactivation detection of recombinant bait plasmidsThe yeast which were transformed by Y2HGold [pGBKT7-ARC1a], Y2HGold [pGBKT7-ARClb]、Y2HGold [pGBKT7-ARClc]、Y2HGold [pGBKT7-ARCld]、Y2HGold [pGBKT7-ARCla] Y2HGold [pGBKT7] were cultured on SD/-Trp plates at 30℃. After three days, there were white clones in the plates. The result showed that the recombinant bait plasmids pGBKT7-ARC1 were confirmed not toxic to yeast. In addition, four experimental groups could grow on SD/-Trp, SD/-Trp/x-a-gal plates but not grow on SD/-Trp/x-a-gal/AbA plates, the colonies did not turn blue on SD/-Trp/x-a-gal plates. The results indicates that the baits were confirmed not activate the expression of reporter genes by the test for autoactivation.3 The toxicity detection of recombinant prey plasmid pGADT7-SRKJThe empty vector pGADT7 and recombinant prey plasmid pGADT7-SRKJ were transformed into Y187, the yeast of Y187 [pGADT7] and Y187 [pGADT7-SRKJ] grew well on the SD/-Leu plates. The results suggested that recombinant plasmid pGADT7-SRKJwas successfully transformed into Y187 yeast cells and was not toxic to yeast cell Y187.4 The interaction detection between different truncated fragments of ARCl and SRKJThree experimental groups Y2HGold [pGBKT7-ARCla]×Y187 [pGADT7-SRKJ], Y2HGold [pGBKT7-ARClc]×Y187 [pGADT7-SRKJ] and Y2HGold [pGBKT7-ARCld]×Y187 [pGADT7-SRKJ] could grow on QDO/x/A nutritional media with transcription activation of the reporter gene AUR1-C, MEL1, HIS3, ADE2, it indicated that there exists interaction between ARC1 from Brassica oleracea var. acephala L and SRK from Brassica oleracea var. capitata L and the interaction domain was located on ARM repeats, and the fifference at amino acid level with the ARC1 of Brassica oleracea var. capitata L is not enough to change the conformation in the interaction region. All above mentioned provides some insights into the molecular mechanism of self-incompatibility in Brassica. |
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