| The Self-incompatibility is a common phenomenon in flower plants and it is a effective way to avoid inbreeding depression and promote out-crossing.Self-incompatibility is most important to plant evolution.The Arm Repeat-Containing protein 1(ARC1),a U-box E3 ubiquitin ligase,which is identified the only downstream transfer factor in SI pathway.It is widely believe that ARC1 can specifically mediated SI pathway.The research of SI has significant to flower plant such as reproductive biology and Hybrid production.In our research,we have obtain the following achievements.1.The ARC1-F,ARC1-N and ARC1-C genes was isolated from stigma ornamental kale S13-b,which were instrted in pET-14b express vector.The three vectors,named 14b-ARC1-F,14b-ARC1-N and 14b-ARC1-C,which were transformed DH5a.After PCR analysis,the three plasmids were extracted from this three germs.Through the enzyme digestion analysis,three plasmids were transformed BL21 respectively.Finally,we obtained three express germs,named ARC1-F-BL21,ARC1-F-BL21 and ARC1-F-BL21.Used prokaryotic expression technology,ARC1-F(1-663),ARC1-N(1-279)and ARC1-C(361-663)were purified.2.In vitro ubiquination assays were performed using a yeast E1 enzyme,a E2 enzyme UBC7,ubiquitin and ARC1.Western blot results showed that ARC1 mediated the polyubiquitination of proteins with anti-ubiquitin antibodies.Further,a point mutation of U-box domain at amino acid position 232 substituting Pro for Ala or omission of any of the components resulted in a loss of protein ubiquitination.We speculate that ARC1 is an E3 ubiquitin ligase and U-box domain has very important function.3.Two Balb/C mice were immunized by ARC1-C.After three times immunizations and once enhanch immunization,the spleen cell was fused with myeloma cell SP2/0.Two positive hybridoma cells were screened out by high throughput screening and used subcloning limited dilution we obtained two mAbs,which was named anti-ARC1-C mAb-8 and anti-ARC1-C mAb-10 respectively.The result of ELISA indicated that the anti-ARC1-C mAb-8 is better than anti-ARC1-C mAb-10.Western blot analysis showed that the anti-ARC1-C mAb-8 and anti-ARC1-C mAb-10 could interact with ARC1-F and ARC1-C but could not interact with ARC1-N.4.We extracted stem,petal,anther,style,ovary and stigma(0-4 mm,4-6 mm,6-8 mm,8-10 mm,>10 mm)from ornamental kale S13-b.Western blot analysis showed that ARC1 is only accumulation in stigma and but gradually increased before flowering and reached its highest level on the day of anthesis. |
