Cloning And Characterization Of Mitogen-activated Protein Kinase ZmMAPK4 In Maize

Maize(Zea mays L.) is the most important sources of food and feed in the world, so it is an important part to sustain increase the maize production of ensuring world food security. There are many adverse situations during of the maize whole life, such as the biotic stress and abiotic stress. Viruses, bacteria and the fungi are the main biotic stresses, and the abiotic is contain the drought, high or low temperature, salt, the reactive oxygen and so on. They all have a great impact on the maize yield and quantity. As a the result, searching, cloning and analyzing the function of maize resistance genes, and revealing function and mechanism from the molecular levels, to find the resistance of the molecular mechanism and the relate singling pathway in the cell, so enhance the resistance and improve its immunity are important to the theoretical and practical meaning.In this study, we use the maize inbred line B73 for the basic material, because the whole genome sequences of B73 has been sequenced. We cloned the ZmMAPK4 gene by the method of homologous cloning, which has a high similarly with Arabidopsis thaliana Mitogen-activated protein kinase 4 (AtMPK4) and Oryza sativa Mitogen-activated protein kinase 4(OsMAPK4). Under the different stress conditions, we studied the rhythm expression pattern of ZmMAPK4 by fluorescence quantitative RT-PCR; Then we constructed the transient expression vector and the over expression vector of ZmMAPK4 gene, and carried out subcellular localization on the onion epidermis transient expression assay, and transformed the over expression vector of ZmMAPK4 gene into the Arabidopsis with the Agrobacterium disseminated Floral Dip method. Analyzed relationship of the expression pattern of ZmMAPK4 among PR1, ABA1 and ABA2. The main results are as follows:Using the method of orthologus gene cloning, we got the ZmMAPK4 gene from the maize. The total ORF is 1125bp, Contains 6 exons and 5 introns, which has 375 amino acids, and the protein molecular weight is 42.44 KDa, isoelectric point is 6.5. By phylogenetic analysis, we find the ZmMAPK4, AtMAPK4 and OsMAPK4 at the near neighbor evolutionary branch. The structure of protein indicates that they containing the Pkc_like domain, so predicates it may be homologue with AtMAPK4 or OsMAPK4, have a similar function.We used fluorescence quantitative RT-PCR to research the ZmMAPK4 gene expression pattern analysis under different biotic and abiotic conditions. The results show that after one week of inoculated with the Sugarcane mosaic virus on the maize leaves, the ZmMAPK4 gene expression has a significant increase along with the time; but under the abiotic stresses, such as the drought, high temperature, and salt stress, the ZmMAPK4 gene expression pattern analysises have different results. They may provide some advises for the functional identification in the future transgenic.We have constructed GFP Fusion expression vector pCAMBlA-ZmMAPK4 and high efficiency over expression vector pCAMBIA1304-ZmMAPK4-2. A transient expression assay in onion epidermal cells by gene gun bombardment, the result showes that ZmMAPK4 protein localed in the nucleus followed by the GFP in the cells.Using the Floral Dip method, the over expression vector pCAMBIA1304-ZmMAPK4-2 and the pCAMBIA1304 vector were transformed into the Arabidopsis for the fundamental functional research. Under the biotic stress, the PR1 gene in the T3 generation positive plants have a high expression, so it may engender to increase the plants resistance to the biotic in the single pathway; The same as the biotic stress, the abiotic has a good phenomenon when the adverse situation come out, and the ABA1 and ABA2 gene have a significant increase also, because we all know that the ABA1 and ABA2 genes are the main genes in ABA biosynthesis? so it is good for increase the maize resistance.