Expression And Interaction Analysis Of M Locus Protein Kinase From Ornamental Kale (Brassica Oleracea Var.acephla)

Plant self-incompatibility（SI）is a mechanism that promotes cross-pollination and prevents self-pollination in many flowering plants.M locus protein kinase（MLPK）is a positive regulator of SI signaling in the stigma.Ornamental kale（Brassica oleracea var.acephala）is a cruciferous plant with ornamental value and good cold tolerance,and it is one of the important horticultural plants in the north of China.The ornamental kale self-incompatibility line(S_13-bS_13-b)was used as the material,and the full-length genes of BoMLPKf1 and BoMLPKf2 were cloned and sequenced.We obtained the fusion protein of BoMLPKf1 and BoMLPKf2 shared kinase domain BoMLPK-KD by prokaryotic expression system,and prepared polyclonal antibodies of BoMLPKf1,BoMLPKf2 and BoMLPK-KD.In addition,we examined the expression of BoMLPKf1 and BoMLPKf2 in various tissues of kale.Furthermore,we analyzed the protein expression and localization of the proteoglycan-like protein（PGLP）.Finally,the interaction between BoPGLP and BoMLPK was analyzed using dual-luciferase complementation experiments.The main findings are as follows:1.The full-length sequences of BoMLPKf1 and BoMLPKf2 were obtained by RT-PCR.The open reading frame of BoMLPKf1 is 1215 bp,encoding 404 amino acids,and the open reading frame of BoMLPKf2 is 1233 bp,encoding 410 amino acids.Sequence alignment showed that BoMLPKf1 and BoMLPKf2 were highly similar to homologous proteins in Brassica rapa,Brassica napus and Arabidopsis.2.We constructed the fusion expression vector pCold-SUMO-MLPK-KD,and induced it in E.coli to obtain the BoMLPK-KD fusion protein with a size of about 58 k Da.We specifically designed synthetic peptides based on the N-terminal sequences of BoMLPKf1 and BoMLPKf2,and immunized mice with the synthetic peptides,and the obtained BoMLPK-KD fusion protein to obtain polyclonal antibodies.3.The gene expression levels of BoMLPKf1 and BoMLPKf2 were detected by real-time quantitative PCR,and showed that BoMLPKf1 is highly expressed in the stigma,style and ovary of kale,but lower in the sepal,anther and petal;BoMLPKf2 is abundantly expressed in the stigma,and in other tissues expression is low.The protein expressions of BoMLPKf1 and BoMLPKf2 were detected by immunoblotting,and we found that BoMLPKf1 was expressed at a low level in sepals,and was expressed in petals,anthers,stigma,style and ovary;BoMLPKf2was expressed in stigma specifically.4.The full-length BoPGLP gene was cloned by 5’RACE,and it was found that the length of the BoPGLP open reading frame was 390 bp,encoding 129 amino acids;we constructed the fusion expression vector p ET-14b-PGLP,and purified it in E.coli to obtain the BoPGLP recombinant protein.Then we immunized mice with the recombinant protein to obtain polyclonal antibodies.5.We detected the spatial and temporal expression of BoPGLP protein by western blot.It was found that the expression of BoPGLP was lower in sepals,anthers and ovaries,and higher in petals,stigma and style;the expression of BoPGLP was higher at S2 and S3 stages of stigma development,and lower at S1,S4 and S5 stages.6.We constructed the fusion expression vector p A7-GFP-PGLP and transferred it into onion epidermal cells by vacuum infiltration.We found that the fluorescent signal was concentrated on the onion cell wall,indicating that BoPGLP was localized on the cell membrane.7.The interaction between BoMLPK and BoPGLP was analyzed by dual luciferase complementation assay in tobacco.We constructed fusion expression vectors pCAMIA1300-c LUC-PGLP,pCAMIA1300-n LUC-MLPKf1,pCAMIA1300-n LUC-MLPKf2and pCAMIA1300-n LUC-MLPK-KD.These vectors were separately introduced into tobacco leaves by Agrobacterium transformation.The interaction fluorescence signals between BoPGLP and BoMLPKf1,BoMLPKf2 and BoMLPK-KD were detected,these showed the interaction between BoPGLP and BoMLPKf1,BoMLPKf2 and BoMLPK-KD.