Screening And Preliminary Verification Of USP Domain Interaction Proteins Of Maize Cytoplasmic Receptor Protein Kinase ZmSTK1

Corn is an important food crop with important application merit in feed processing.Whether the fertility of corn pollen is normal,it is an important prerequisite for ensuring fertilization and the yield formation.The research team found that the maize pollen receptor cytoplasmic protein kinase ZmSTK1 is expressed in mature tassels.The germination rate of corn pollen was significantly reduced after the mutation.The ZmSTK1 protein includes the universal stress protein USP domain and kinase domain.Among them,the kinase domain can interact with enolase1 to regulate the synthesis of enolpyruvate in the glycolytic pathway.The USP domain is involved in stress responses in plant,so it is speculated that ZmSTK1 may be involved in stress responses during pollen development.In this study,the co-immunoprecipitation and yeast two-hybrid technique were used to screen and validate the interaction protein of USP domain in ZmSTK1,which provided a theoretical basis for exploring the cell signal transduction pathway and its molecular regulation mechanism during pollen development.The experimental methods and results are as follows:1.According to the prokaryotic expression vector p ET30a-ZmSTK1-USP successfully constructed in the previous stage of the research group,the recombinant protein of ZmSTK1-USP was induced and the recombinant protein was affinity purified.The experimental results show that the molecular weight of the expressed protein is consistent with the theoretical molecular weight and the purification effect is good,which can be used for antibody preparation.The Co-IP method was used to screen interacting proteins from pollen proteins.The eluted proteins were detected by SDS-PAGE after co-immunoprecipitation.And two more obvious bands were selected for mass spectrometry.Ten candidate proteins which may interact with ZmSTKl-USP were obtained,respectively: 5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase,two Actin1,Cell division cycle protein 48,Elongation factor 1-alpha,Translation elongation factor Tu,Heat shock 90 k Da protein,Heat shock 70 k Da protein and Histone H4.2.Prediction of proteins that may interact with ZmSTK1.The results showed that five of the ten candidate proteins belonged to eukaryotic ribosomal proteins,suggesting that ZmSTK1 protein may interact with ribosome-associated proteins and participate in ribosome translation and processing.According to the mass spectrometry screening results of Co-IP,the ZmSTK1-USP and six candidate genes were amplified by PCR,and the target fragments of ZmSTK1-USP,Actin1,EF1 A,HSP90,MTR5,H4 and EF-Tu genes were obtained.Seven yeast recombinant expression vectors constructed successfully.Proteins interacting with ZmSTK1-USP were verified by the yeast two-hybrid system.The recombinant yeast recombinant expression vector p GBKT7-ZmSTK1-USP+p GADT7 and the empty vector p GBKT7 were transformed into yeast AH109,respectively.The results showed that the exogenous protein ZmSTK1-USP had no toxic effect yeast growth,and the false negative in the experiment was excluded.The bait recombinant expression fusion protein has no self-activation effect,and the downstream reporter gene can not be activated separately,and the false positive in the experiment is excluded.The bait plasmid was co-transformed into the yeast AH109 with the capture plasmid.The results showed that ZmSTK1-USP interacted with EF-Tu and had no interaction with other candidate proteins.This result lays a foundation for further exploration of the mechanism of action of ZmSTK1.