The Study On Ubiquitination Of The S-RNase By Pollen RING Type E3Ubiquitin Ligase In Apple

Self-incompatibility is a widespread barrier to reproduction in flowering plants that can prevent inbreeding and promote outcrossing, by rejecting self pollen while leaving non-self pollen for fertilization. The apple and other rosaceae plants belong to the gametophytic self-incompatibility. When pollinated by non-self pollen, the style determinant S-RNase uptake by the pollen tube will be ubiquitinated and degraded by the26S proteasome, leading to normal fertilization; while the self S-RNase would not be marked by ubiquitin and it will keep active and degrade the pollen tube rRNA, which leading to death of the pollen tube. Until now there have been some research on ubiquitination of S-RNase in petunia and antirrhinum, but nothing has been reported in apple and other rosaceae plants. Here we cloned three components of the SCF complex SKP1, Cullinland F-box. Based on their expression patterns, we detected their interaction relationship and simulated the ubiquition system to determine whether the SCF complex could mediate the ubiquition of S-RNase. The following are our results:1. Based on the’Golden Delicious’ genome and EST database, we got19F-box genes and found that10of them were located on the telomere of17chromosome, around the S-RNase; the others on chromosome2,8,10,12,15and16, respectively.2. We cloned these19F-box genes by designing specific and full-length primers. RT-PCR analysis showed that the10genes located on the17chromosome were expressed specially in pollen; the other9genes were expressed in all the organs examined.3. Using16different apple cultivars as PCR templates and specific primers for the10genes, we found that:only4of them has haplotype specificity. So we named them S2-MdSFBB1, S2-MdSFBB2, S2-MdSFBB3and S2-MdSFBB4, respectively; the other6genes S2-MdSFBL1, S2-MdSFBL2, S2-MdSFBL3, S2-MdSFBL4, S2-MdSFBL5and S2-MdSFBL6, respectively.4. We performed a yeast-two-hybrid experiments between these F-box genes and S1-, S2-, S3-, S5-, S7-and S9-RNase, the results showed that:S2-MdSFBB1only can interact with S2-and S3-RNase, S2-MdSFBB2only interact with S7-RNase, while S2-MdSFBB3and S2-MdSFBB4could interact with S3-, S5-and S9-RNase. What is more, S2-MdSFBL2only interacted with S3-RNase, S2-MdSFBL5with S7-RNase, while S2-MdSFBL4could interact with S1, S3, S5and S9-RNase.5. Using the conserved amino acid of C terminal domain of SSK in petunia, antirrhinum, pear and sweet cherry, we performed Blast search of the apple genome and obtained three candidate SKPl-like genes. Two of the SKP1-like genes contained eight sheets and two helices and had a shared sequence of five amino acids following the WAFE motif, whereas another SKP1like gene contained only the C terminal domain and the WAFE motif, and lacked～140amino acids at the N terminal. RT-PCR analysis showed the three genes were all expressed specifically in pollen. Using the same method, we cloned a homologous gene of CUL1, it was expressed specifically in pollen and were named MdCullin1. In yeast two hybrid and Pull down, only one SKP1-like gene could interact with MdSFBB and Cullinl so we think this gene to be the SLF-interacting Skp1-like1in apple and named it as MdSSK1.6. We cloned a homologous gene of PiSBP1,MdSBP1. MdSBP1is a RING-HC protein and it is expressed in pollen and other organs examined. Except interacting with the hypervariable region of S-RNase, it could not bind with the full length of S-RNase, SFB and Cullinl in yeast two-hybrid and Pull-down.7. After incubated by25μg m1-1His-S2-RNase for2h, we extracted the pollen total protein and performed Westernblot using anti-S2-RNase, the results showed that:there existed two bands that higher than S2-RNase, which indicated that the S2-RNase had been tagged ubiquitin; In vitro experiments using MdSSKl, S2-MdSFBBl and MdCUL1proteins incubated with S2-RNase and ubiquitin showed only one band higher than S2-RNase. These results showed that the number of ubiquitin that tagged to S-RNase might be different in vivo and vitro. While the incubation containing MdSBP1and S2-MdSFBBl, MdCUL1or alone with S2-RNase did not show any band except the S-RNase band.Taken together, we think the style determinant S-RNase could be ubiquitinated by the SCF complex, which is connected by MdSSK1.