The Differential Protein ITRAQ Analysis And USP Domain Cloning And Expression Of Receptor-kinase STK2 In Maize

Using Ac/Ds transposon tagging technology,we cloned a pollen specific gene STK2 from maize,and its function was studied.We found that STK2 only expressed in tassel,belongs to the class of receptor cytoplasmic protein kinase(RLCK),mainly contains the kinase domain and USP domain.Kinase domain has catalysis,occurs phosphorylation reaction;the USP domain has been shown to be closely associated with stress resistance and growth in other plants.In order to undrerstand the biological function of STK2,we conductged the following research:1.In order to understand the biological significance of STKs,we analyzed STKs genes and proteins by bioinformatics.There are eight members of STKs in maize were obtained from NCBI having more than 80%similarity,and their domains and conserved motifs have similar positions on the sequence and perform similar functions.STKs in maize exhibits expansion and duplication of genes by tandem repeat mode during the evolution.The results of phylogenetic tree revealed that STKs in maize existed before the species differentiation.STKs in maize belonged to paralog relationship,which showed that they might have common ancestral genes,representing that USP domain in STKs was already present before species differentiation.The results of the study would be helpful in better understanding the genetic evolutionary relationship of homological sequences of STK1 and STK2 with its other members,and the similarities as well as differences between the structure and function of each member of the STK gene family.2.Maize pollen was collected and cDNA was obtained by RT-PCR method.PCR amplification techniques was used to obtain STK2-USP domain fragment.Then pET30a-STK2-USP expression vector was successfully constructed,transformed into E.coli BL21 and expressed in vitro.His-STK2-USP fusion protein has a molecular weight of approximately 25 kDa(containing 17 kDa STK2-USP and about 8 kDa His tag)by SDS-PAGE.The vector was detected by SDS-PAGE,and molecular weight of fusion protein was about 25 kDa(containing about 8 kDa His tag).The Higher purity of the fusion protein can be obtained under optimum conditions:37℃,180 r/min induced 4 h by 1.0 mM IPTG.3.Based on the characteristics of ZmSTK2 protein kinase with USP domain and the analysis of GO,KO and protein interaction,we suggested that ZmSTK2 protein kinase affects the main pathway for pollen development as follows:The temperature sensitive protein by receiving extracellular correlation signal,transmits signals to intracellular,and it is also accepted by the cytoplasmic receptor protein kinase ZmSTK2,which ultimately affects the metabolism of carbohydrate,protein synthesis,lipids,photosynthesis,and the unclassified proteins.The differential expression of these proteins in the process of expression or up or down lead to abnormal development of pollen.However,the upstream and downstream substrates of ZmSTK2 protein kinase need further validation for determing the main pathway of ZmSTK2 regulation of pollen development.