Screening Interacting Proteins Of StMAPKK1 And Functional Analysis Of These Protein Genes In Potato

Mitogen-activated protein kinase（MAPK）cascades is one of the important signaling networks in plant growth and development,hormones and stress response signaling pathways.As one of its main members,mitogen-activated protein kinase kinases（MAPKKs）is a kind of kinase located in the middle of the pathway in this cascades and plays a key role in signaling collection and divergence.Five MAPKK genes in potato（Solanum tuberosum L.）were identified in our laboratory.The qRT-PCR of StMAPKK genes in potato under different stress treatments showed that the StMAPKK1 gene of this gene family increased most significantly under drought stress.Therefore,the purpose of this study was to explore and analyze the regulatory functions of StMAPKK1 gene and its interacting proteins in signal pathways which were rarely reported,so as to lay a foundation for further study on the molecular mechanism and metabolic network of MAPK、MAPKK and MAPKKK signaling cascade in potato on anti-stress responsing.For this reason,the sequence and function of StMAPKK1 gene were analyzed by bioinformatics.Vectors were constructed by directional cloning of homologous recombination,including the yeast（Saccharomyces cerevisiae）two-hybrid（Y2H）system bait vector pGBKT7-StMAPKK1,subcellularlocalizationexpressionvector pCEGFP-StMAPKK1,bimolecular fluorescence complementation（BiFC）vectors pSPYCE-StMAPKK1 and pSPYNE-StMAPKK1.By using yeast two-hybrid technique,proteins interacting with StMAPKK1 were obtained from extensive screening in the yeast two-hybrid cDNA library of potato‘Zihuabai’.The gene function annotation of StMAPKK1 interacting protein genes were analyzed by bioinformatics.The results are as follows:1.By using bioinformatics analysis,it was found that the StMAPKK1 were stable and hydrophilic proteins with isoelectric point of 5.47,no signal peptide and transmembrane domain,containing serine and threonine-dominated protein phosphorylation sites,and containing the Protein kinase domain（PF00069）domain.StMAPKK1 gene were localized in cytoplasm and contained cis-acting elements involved in plant hormone ABA,GA₃ and ethylene response,plant stress drought response,anaerobic induction and damage response,and light response.There were significant changes of gene expression under BABA or P.infestans treatment in biotic stresses responsing,and under ABA,BAP or water stress treatment in biotic stresses responsing.From STRING v11.0,a total of five StMAPKK1 interacting proteins were predicted,including two StMAPKKK proteins,one StMAPK protein,one eukaryotic translation initiation factor 3（eIF-3）,and one unidentifiable protein.2.In this experiment,StMAPKK1 gene was cloned,and the yeast two-hybrid bait vector pGBKT7-StMAPKK1 was constructed by homologous recombination method.The bait vector pGBKT7-StMAPKK1 was transformated into Y187 and its nontoxic and no self-activating activity were tested.It can be used for further yeast two-hybrid experiment.3.Using yeast two-hybrid method,the StMAPKK1 bait protein was hybridized with potato cDNA library.Hybrids were coated on 50 QDO mediums.Then there were 406 larger diameter,white or light pink single colonies selected for preliminary X-α-Gal staining screening and identification.Among them,210 colonies grew and showed blue within 48 h,which were regarded as positive clones,and the positive rate was 51.72%.The assumed positive clones were detected by PCR electrophoresis,and sequenced,after combining with repeated and redundant sequencing results,five StMAPKK1 interacting protein positive clones with different sequences were screened in the end,named C1-C5,respectively.All five positive clones verified the authenticity of the interaction by small-scale hybridization.4.From Blast analysis of StMAPKK1 positive clone sequencing results,the C1-C5 of five StMAPKK1 interacting protein genes were identified:hydrolase（hydrolyzing O-glycosyl compound）,RING-H2 subgroup RHE protein,cyanate hydratase,ARF GTPase activator,and a C2 domain-containing protein.By bioinformatics analysis,it was found that all the StMAPKK1 interacting proteins were hydrophilic proteins,and all of them contained protein phosphorylation sites in varying numbers.With the exception of C2 gene that may be located in chloroplast thylakoid membrane or nucleus,all the other StMAPKK1 interacting protein genes may be located in cytoplasm.StMAPKK1 interacting protein genes contained plant growth-related,hormone response-related and stress response-related cis-acting elements.And there were specific expression of these StMAPKK1 interacting protein genes in tissue and in response to biotic and abiotic stresses.5.The subcellular localization expression vector pCEGFP-StMAPKK1 was constructed which could be used for the subcellular localization study of StMAPKK1gene.The bimolecular fluorescence complementary expression vector pSPYCE-StMAPKK1 and pSPYNE-StMAPKK1 were constructed which would be used for the bimolecular fluorescence complementary experiments of StMAPKK1and its 5 interacting proteins to go a step further for verifing the above-mentioned 5pair protein interaction.