The Studies On Expression And Protein Phosphorylationof ZmSTKR In Maize

Protein phosphorylation is a modification of post-translation in prokaryotes and eukaryotes, is a process that protein kinase catalyize the transfering a gamma phosphate group from ATP or GTP to amino acid residues of target protein. In plant species, protein phosphorylation involved in a wide range of biological processes, such as signal transduction, cell division, development, stress response. The researches in our laboratory revealed that Zm STKR which encodes a serine/threonine protein kinase receptor is a candidate of QTL for kernel number per row of maize. In this study, we assayed the spatial and temporal expression pattern of Zm STKR in a set of near isogenic line using reverse-transcription PCR(RT-PCR) and quantitative PCR(QPCR); Subcellular localization of Zm STKR using 35S-Ca M promoter drives the expression of Zm STKR-GFP in onion epidermal cell; Protein expression and Purification by prokaryotic expression system; The autophosphorylation of Zm STKR and the phosphorylation between Zm STKR and its interaction protein LG2 using phosphorylation analysis in vitro to reveal the regulation pathway of Zm STKR. The main results achieved as follows:1. The results of spatial and temporal expression analysis showed that the expression pattern of Zm STKR is constitutive expression, but the expression level is different in different tissues. There are three development stages(SPM, SM, FM). In SPM and SM, the Zm STKR expression level of SL57-6 is higher than Ye478 significantly. In addition, There are no significant difference in Ye478. So there are expression difference between Ye478 and SL57-6 and tissue and temporal specificity expression in the development process of corn. Hence, The expression difference in the inflorescence development process may result in the difference of kernel number per row between Ye478 and SL57-6.2. Subcellular localization of Zm STKR by onion epidermal cell showed that Zm STKR-GFP localized to the cytoplasm and nuclear, this indicated that Zm STKR may play a role in cytoplasm and nuclear.3. we obtained ATP binding site mutation(Zm STKR-74R)、active-site mutation(Zm STKR-172E)、double mutation of the two site(Zm STKR-double) By site-directed mutation and we also obtaind the mutation protein GST/Zm STKR-74 R 、 GST/ Zm STKR-172 E and GST/ Zm STKR-double by Prokaryotic expression system and purification. The analysis of phosphorylation in vitro showed that Zm STKR and Zm STKR-172 E both couldn’t autophosphorylate, but Zm STKR-74 R and Zm STKR-double was found to autophosphorylate, so the 74 th amino acid plays an important role on the autophosphorylation of Zm STKR. Science the proteins on our studies are based on Ye478 allelic gene sequences of Zm STKR, however, there is an amino acid difference between Ye478 and its nearly isogenic line SL57-6 on the ATP binding site of Zm STKR. We speculate that the difference of the amino acid may result in Zm STKR difference in protein function, leading to the differences of kernel number per row. In addition, the results of Zm STKR Phosphorylate its interaction protein LG2 showed that Zm STKR and three kinds of mutation protein all couldn,t phosphorylate LG2. so Zm STKR and LG2 interaction, but LG2 may not be the target protein of Zm STKR phosphorylation.