| Self-incompatibility is an important genetic mechanism to avoid self-pollination and promote outcrossing in flowering plants.Due to parental heterozygosity,it is difficult for self-incompatibility species to take advantage of maximum heterosis,thus preventing breeding from achieving maximum gain.The conversion between self-incompatibility and self-incompatibility is very common in plants,which opens up a new way for breeding of self-incompatibility species.Sinomanglietia glauca,a rare plant of Magnoliaceae,is located in the connection between Magnolia and Manglietia,and has important scientific value in evolution.Like other Magnoliaceae,S.glauca is of great ornamental value and has good application prospects.S.glauca shows self-incompatibility.If we can convert S.glauca from self-incompatibility to self-incompatibility and make full use of maximum heterosis,it will open up a new way for breeding S.glauca.Therefore,understanding the self-incompatibility mechanism of S.glauca is of great significance for the breeding of S.glauca.M-locus protein kinase gene(MLPK)is the key gene in sporophytic self-incompatibility(SSI).In SSI plant self-pollination,pollen determinant S-locus cysteine rich protein(SCR)interacts with pistil determinant S-locus receptor kinase(SRK)to activate SRK and trigger self-incompatibility reaction with the participation of MLPK.S.glauca presents late-acting self-incompatibility(LSI)characteristics.Our previous third-generation transcriptome sequencing study also found that S.glauca not only has SRK highly homologous genes,but also MLPK highly homologous genes,but its role in late-acting self-incompatibility was not clear.In this study,SgMLPK gene was cloned from the carpels of self-pollination and cross-pollination of S.glauca;The physicochemical properties of SgMLPK encoding protein were analyzed by online website;The SgMLPK gene family was studied based on bioinformatics technology.Agrobacterium tumefaciens was directly injected into tobacco leaves to identify SgMLPK gene encoding proteins for subcellular localization.Finally,the function of SgMLPK gene was studied by Arabidopsis thaliana heterologous expression technique.The main research results are as follows:(1)Cloning of SgMLPK gene.Based on third generations of transcriptome analysis,SgMLPK gene(Gen Bank Accession No.: MW 139902)was cloned with a total length of1254 bp,encoding 417 amino acids,molecular weight 45.836 k D,isoelectric point 9.15;The protein encoded by SgMLPK gene contains Pkinase＿Tyr functional domain and hydrophobic region,but no transmembrane region;The coding protein had a signal peptide with a tangent point of 29 ～30 AA.SgMLPK gene has typical MLPK gene characteristics.(2)SgMLPK subcellular localization.The recombinant expression vector SgMLPK and the expression vector pCAMBIA1302 with GFP were constructed.The recombinant expression vector was transformed into Agrobacterium tumefaciens and directly injected into tobacco K326 leaves.Thirty six h after the injection,the subcellular location of SgMLPK was observed in the leaf cell of tobacco under fluorescence microscope.The results showed that there was fluorescence in cytoplasm and cell membrane,but the signal in cell membrane was stronger.(3)Evolutionary analysis.OrthoFinder2 was used to analyze gene families of 22 sequenced species with self-incompatibility,and to construct species tree.The SgMLPK homologous genes was detected in all species including self-incompatibility and self-incompatibility,as well as angiosperms,green algae and bryophytes,suggesting that SgMLPK has other functions besides self-incompatibility.The homologous genes of SgMLPK were also found in Chlamydomonas reinhardtii and Physcomitrium patens,indicating that MLPK gene had an early origin and its function in Chlamydomonas Reinhardtii might be the original function.(4)SgMLPK function verification.The recombinant vector containing SgMLPK gene was transformed into Agrobacterium tumefaciens and infected Arabidopsis thaliana by inflorescence infiltration.PCR and fluorescence microscopy showed that SgMLPK gene was integrated into Arabidopsis thaliana genome and expressed.Compared with non-transgenic Arabidopsis thaliana,the normal growth of transgenic Arabidopsis thaliana is affected,the growth is slow,the development is inhibited,and the stem can’t grow.In conclusion,we cloned SgMLPK gene,analyzed its structure,physicochemical properties and evolutionary relationship of the encoding protein,and verified its function through transgenic overexpression.The results of this study provide a basis for further understanding the mechanism of self-incompatibility and provide data for the breeding of S.glauca. |
