Functional Confirmation Of Receptor-like Cytoplasmic Kinase Gene ZmSTK2_USP In Maize (Zea Mays L.)

Pollen development and pollen tube growth is an important event during sexual reproduction in maize,which directly affects the yield and quality of maize.Therefore,the molecular mechanism of pollen development and pollen tube growth is extremely complex,in addition to including a series of organ differentiation and a series of strictly controlled cells,physiological and biochemical changes,but also involves a large number of gene expression and regulation.This study found that a novel pollen development-related receptor-like cytoplasmic kinase gene ZmSTK2_USP,its biological function can regulate process of pollen development and pollen tube growth.The main contents of the study were as follows:(1)The bioinformatics analysis showed that the encoded protein ZmSTK2_USP had a N-terminal universal stress protein A(Usp A)receptor domain and a predicted C-terminal serine/threonine kinase(S_TK)domain.ZmSTK2_USP gene was divided into the RLCK-IXb subfamily according to the phylogenetic tree constructed by the kinase domain of the maize pollen development-related RLKs.And its promoter(proZmSTK2_USP)was found to be a pollen-specific promoter with typical promoter elements including TATA-box,CAAT-box,G-box,GATA-box,and necessary elements of tissue-specific expressive prom oters including GTGA and AGAAA.(2)Pollen viability studies showed that the pollen viability of the mutant zmstk2_usp decreased by 43.30%and the germination rate decreased by 45.08%,resulting in a decrease of 60.00%in the filled grains per spike rate,and these difference were extremely significant level.(3)In this study,ZmSTK2_USP gene was successfully cloned.Northern blot analysis showed that the ZmSTK2_USP gene was specifically expressed only in mature pollen and was not detected in other tissues(organs).Fluorescence quantitative PCR analysis showed that the ZmSTK2_USP gene began to express in the middle stage of maize pollen development and was specifically expressed in mature pollen.(4)Two expression vectors ZmSTK2_USP-GUS and ZmSTK2_USP-GFP were also successfully constructed.The transgenic plants of maize were obtained by Agrobacterium-mediated transformation of maize germinating embryos.The transgenic plants were tested by GUS histochemical staining for flower organs and three stages of pollen development.GUS activity was not detected in maize transgenic ZmSTK2_USP-GUS with young male flowers,mature male flowers,mature anthers and pollen ESPs,but GUS activity was detected during the pollen MSP period until the pollen tube elongation.Subcellular localization of onion epidermis and tobacco leaves by Agrobacterium-mediated transformation were observed.Two results showed that the transient expression of ZmSTK2_USP-GFP fused protein in onion epidermis and tobacco leaves were localized in the cytoplasm,indicating that the protein encoded by ZmSTK2_USP gene belongs to cytoplasmic protein,which also provides the basis for the investigation of ZmSTK2_USP as a receptor-like cytoplasmic protein kinase in bioinformatics analysis.(5)In this study,the promoter of ZmSTK2_USP gene(named proZmSTK2 JUSP)was successfully cloned,and the expression vector proZmSTK2_CUSP-GUS was successfully constructed.The maize transgenic plants were obtained by Agrobacterium-mediated maize germination embryo transformation.GUS histochemical staining was found that proZmSTK2_USP regulates gene expression during maturation of pollen and pollen germination,and proZmSTK2_USP is a pollen-specific promoter,which also validates the results of bioinformatics analysis.(6)In this study,the kinase domain(ZmSTK2_USP-KD)of ZmSTK2_USP was successfully cloned and the expression vector pET30a-His-ZmSTK2_USP-KD with His tag was constructed and transformed into strain BL21(E.coli)for in vitro prokaryotic expression.The target fused protein is inclusion body protein.The optimum induction condition is IPTG concentation 0.5mM,induction temperature 37℃ and induction time 5hr,and the precipitated bacteria was dissolved in 8M urea solution.Then using,2P radioisotope imaging and its kinase detection of universal antibody,these results showed that the kinase domain of ZmSTK2_USP could autophosphorylate and also could catalyze the phosphorylation of histone H1.At the same time,ZmSTK2_USP has Ser/Thr protein kinase activity and does not have Tyr protein kinase activity,indicating that it is a non-double substrate specific kinase.