Study On Interaction Protein Of DsMAPK From Dunaliella Salina

Dunaliella salina,which is recognized as a salt tolerant eukaryote,is a model organism for studying plant adaptation to high salinity.Mitokine-activated protein kinase(MAPK)belongs to serine/threonine(Ser/Thr)protein kinase and is widely distributed in eukaryotes.A large number of studies have shown that serine/threonine protein kinases are widely involved in plant responses to drought and salt stress.It is of great scientific significance to study the mechanism of MAPK in order to elucidate the molecular mechanism of salt tolerance and the molecular pathway of response to salt stress signals in D.salina.In this study,the interaction proteins of DsMAPK(GenBank: JQ782412)in D.salina were screened by immunoprecipitation and yeast two-hybrid techniques.The experimental methods and results of this study are as follows:1.The E.coli BL21 expression strain containing the prokaryotic expression vector pGS21a-DsMAPK was induced by IPTG to successfully express the MAPK gene.The soluble protein was purified by Ni-NTA(pre-packed Gravity Column)to obtain a high purity fusion protein.The fusion protein was used as antigen to immunize animals.Polyclonal antibodies were prepared.The purified antibody titer of the antiserum was determined by ELISA(> 512K).Using endogenous target protein as bait,target protein antibodies were co-incubated with total cell proteins,and protein A/G agarose beads were used to purify the complex.165 proteins interacting with DsMAPK were detected by mass spectrometry.Through GO and KEGG analysis,it was found that these differential proteins were mainly involved in biological regulation processes such as metabolism,transmission of genetic information and signal transduction.Analysis of protein interaction network revealed that there were four proteins directly interacting with DsMAPK.2.Expression of DsMAPK interacting proteins by real-time fluorescence quantitative polymerase chain reaction.The antisense expression vector pMDCaMGN-Cat plasmid was transformed into D.salina cells by LiAc/PEG mediated method.Genetically engineered D.salina with relative resistance were screened by chloramphenicol.The expression of DsGAPDH,DsRbcS,DsPKC and DsPGK1 in transgenic D.salina under salt stress for 1 hour was analyzed by qRT-PCR.The results showed that the expression of DsMAPK in transgenic D.salina after 1 hour of salt stress was significantly lower than that in wild D.salina(P < 0.05),indicating that the introduction of antisense genes effectively inhibited the expression of DsMAPK.The expression levels of DsPKC and DsGAPDH in transgenic D.salina were significantly lower than those in wild D.salina(P < 0.05).At the same time,the expression of DsRbcS,DsPGK1 and DsGPDH decreased in varying degrees,which indicated that the expression of these genes was correlated with DsMAPK.3.The open reading frames of DsPKC and DsMAPK genes were cloned into yeast expression vectors pGBKT7 and pGADT7 by DNA recombination technology,and the correct reading frame was verified by sequencing.The yeast expression vectors of DsPKC and DsMAPK were constructed.The yeast expression plasmids pGBKT7-DsPKC and pGADT7-DsMAPK were transformed into Y2 H Gold competent cells.The plasmids were successfully transformed into Y2 H Gold by colony PCR.The solid medium SD/-Trp was coated with pGBKT7-DsPKC/Y2 H Gold and SD/-Leu was coated with pGADT7-DsMAPK/Y2 H Gold.The results showed that the yeast strains containing yeast expression vectors and yeasts containing empty vectors had the same colony size and growth status in the same culture time,which proved that the protein had no toxicity.Colonies successfully transferred to yeast Y2 H Gold were transfected on the SD/-Leu/-Trp/-His/-Ade/X-?-gal solid medium for self-activation assay.The results showed that neither of these proteins was self-activating.The pGBKT7-DsPKC and pGADT7-DsMAPK were co-transformed into Y2 H Gold competent cells,and the co-transformed cells were directly coated on the SD/-Leu/-Trp/-His/-Ade,SD/-Leu/-Trp/-His/-Ade/X-?-gal solid medium,the experimental group(pGADT7-DsMAPK+pGBKT7-DsPKC)grew and turned blue,indicating that DsMAPK interacted with DsPKC.The results of this study provide a reference for elucidating the function of DsMAPK in salt algae at the protein level,which provides new information for us to study the molecular mechanism of D.salina in response to salt stress..