| Cabbage (Brassica oleracea L. var. capitata) is one of the most important crop plants in Brassica oleracea L.. This plant is widely planted in our country, and now it is one of the most important vegetable crops in our country. Cabbage is the plant which belongs to the male and female in the same flower. It has the SI system which might inhibit self-pollination, it could be applied to the heterosis. The studies showed that Self-incompatibility in Brassica (including cabbage) is controlled by a single multiallelic locus (designated as S locus), and recognition of self-SCR by S-locus receptor kinase is the first and determining step in the SI signal transduction, as the SRK and SCR genes are two key factors in SI reaction. But now we do not know much clearly about the core sites of interaction between SCR and SRK, and the core sites were also a hot spot. The extracellular domains of SRK (named eSRK) and open reading frame of SCR were amplified from Brassica oleracea L.’D3’, acquired the cDNA, using the PCR, at last we detected the interaction between SCR and SRK in Brassica oleracea by using the yeast two-hybrid system. The main research contents and results are as follows:1. Cloning and bioinformatics analysis of SCR.Total RNA was extracted from D3, and their corresponding cDNAs were obtained through reverse transcription. Primers were designed to amplify SCR using5’-end conserved amino acids and3’-ploy (A). The cDNA sequence was319bp in length respectively, containing the ORF,3’UTR and3’-ploy (A). The SCR gene encoded could be translated into a protein consisting with58amino acids in the open reading frame. The structure of this protein contains the α-helix and the β-sheet. SCRs of D3have the same sequences as SCR3. The results for of the phylogenetic tree showed that SCR3and SCR20are revolutionaries closely linked, whereas the SCR of S3, S20and the SCR of S28are more distant relatives.2. The amplification of different truncated fragments SCR and sequence analysis.The different truncated fragment SCRn were amplified from RNA in Brassiea oleracea L.’D3’by PCR. The analysis results shows that the fragment sequences were292bp,256bp, and239bp,66bp,90bp,135bp,141bp,144bp,60bp,108bp,78bp,54bp,63bp and33bp, and could be translated into protein with49,39,31,22,30,45,47,38,20,36,26,18,21and11amino acid in the open reading frame.3. The toxicity and autoaetivation detection of recombinant plasmids.The transformants of Y2HGold[pGBKT7SCRn] and Y2HGold[PGBKT7] were cultured on SD/-Trp plates, after three days, we can find clones on the plates. As the size and colure for clones showed that the recombinant plasmids pGBKT7SCRn were no toxic to the yeast cell. In addition, Y2HGold[pGBKT7SCRn] could grow on SD/-Trp/x-a-Gal and the clones did not turn blue, but could not grow on SD/-Trp/x-a-Gal/AbA plates, The results indicates that the bait were confirmed not activateing the expression of reporter genes by the test for autoactivation.4. Detection of Interactions between SCR and SRK in Brassica oleracea L. by Yeast Two-Hybrid System.The recombinant plasmids were successfully transformed into the diploid yeast mating cell. Two groups of pGADT7eSRK D3×pGBKT7SCR4and pGADT7eSRKD3×pGBKT7SCR5could emerge significantly blue clones, and then removed blue clones on SD/-Trp/-Leu/x-a-Gal/AbA plate. After three days, we can find the blue clones on the plate, which may indicate that activated the reporter gene. The results showed that the yeast two-hybrid system could be used to study the interaction domain between SCR and eSRK, the core region in the SCR was located in97and186bp, and this is the study with initial success had been. Moreover, the result also indicated that the two groups of pGADT7eSRK D3×pGBKT7SCR1and pGADT7eSRKD3×pGBKT7SCR13could not emerge strikingly blue clones. It showed that the splicing site of signal peptides of this haplotype SCR and its several adjacent amino acid residues could affect the interaction. |
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