Cloning New Rice Tillering Genes Based On Protein Interaction

Rice is one of the most important cereal crops in the world and provides the staple food for more than half of the world’s population. Previous studies revealed that the rice D3 gene encoding an F-box-LRR protein, which could suppress the outgrowh of tiller buds, was involved in the signal transduction of a new hormone Strigolactone.The purposes of this research are to screen protein partners interacting with the D3 protein using yeast two-hybridization technique and to verify their interaction. This study will improve our understanding of strigolactone signal transduction pathways and shoot branching mechanism. Yeast Two-Hybridization System, based on eukaryotic transcription factor GAL4 and three reporter genes, can sensitively detect weak protein interaction. As a result, we constructed three baits QD3, FBLRR14 and LRR2-14 and transformed them into yeast Y187 cells. Y187-QD3、Y187-FBLRR14 and Y187-LRR2-14 were not able to grow on SD/-Trp/-His/X-a-Gal and SD/-Trp/-Ade/X-a-Gal plates, showing that these three baits did not have self-activation activity. The pGBKT7 expression vector has a c-Myc tag fused in-frame with bait protein. Western blot assay with specific c-Myc antibody proved that QD3、FBLRR14, LRR2-14 fusion proteins could be expressed normally in the yeast.As the D3 gene is involved in signal transduction of Strigolactone, we infer that Strigolactone might enhanc the interaction between D3 and potential target protein. GR24, a synthetic analog of Strigolactone, has the same function with Strigolactone. To observe the effect of strigolactone on protein interaction, we screened the cDNA library of rice tiller buds using QD3、FBLRR14 and LRR2-14 baits without and with 1μL GR24. Screening using QD3 bait identified six and three positive clones without or with GR24, respectively. Nine positive clones were identified using LRR2-14 Bait with GR24. However, no positive clones were identified using FBLRR14 Bait.These important clones were transformed into yeast AH 109 cells and mated separately with FB, LRR, QD3 and LRR2-14 bait on a small scale. Diploids in yeast were retested on QDO/X-a-Gal+GR24 plates. Twelve important clones were confirmed to interact with bait proteins, including A62, A113, A128, B13, B27, GL91, GL96, GL110, GL122, GL136, GL140 and GL158. We speculate that they were likely to interact with the D3 protein, participate in Strigolactone signal transduction pathway and regulate rice tillering.The MBP-D3 fusion protein expression vector was constructed and transfomed into E. coli. The purified MBP-D3 fusion protein would be used to verify the protein interaction via MBP Pull-down assay. To verify protein interactions in vivo, two plant expression vectors was constructed in which the D3 gene was fused in-frame with a 3×FLAG tag at N-terminal or C-terminal. We also constructed four fusion protein expression vectors with TAPa-tag and they have been transformed into rice Nipponbare. These vectors containing a 6×His and a 9×myc would facilitate the separation of D3 protein complex subsequently by tandem affinity purification and the identification of interacting proteins by mass spectrometry.In this study, the important clones screened and verified in the later work provide a basis for further understanding of Strigolactone signal transduction and rice shoot branching mechanism.