Functional Analyis Of A Mitogen-Activated Protein Kinase Gene ZmSIMK1in Zea Mays

In the process of growth and development, the plants meet a variety of adverseenvironmental conditions (such as, salt, drought, waterlogging, temperature, pathogenivvasion, mechanical damage and so on). Plants have developed mechanism that help in theadaptation or adjustment of an organism to these stresses, including the adaption changes oforganism, gene expression, increased intracellular osmolytes (proline, soluble sugar), theactivities of antioxidant enzymes, desease resistance related protein accumulation. These helpplants to adapt to environmental stresses.MAPK cascades play important roles in signal transduction of biotic and abiotic stresses.The purpose of this study is to analysis of function of MAPK from maize through geneticengineering method. This provides a new basis for further genetic breeding and cultivation ofresistant varieties. The main results are as follows:（1）ZmSIMK1was expressed in E.coli, and anti-ZmSIMK1was obtained. ZmSIMK1was expressed in the strain BL21(DE3), the protein with a His-tag was purified and wasindentified by SDS-PAGE. The anti-ZmISMK1was obtained by immunizing the mices withthe purified protein and specificity of antibody was detected by Weston blot.（2）ZmSIMK1was expressed in yeast and the analysis of osmotic stress tolerance wasperformed. The short cycle of yeast is extremely convenient to be used as a model oforganism to study the functions of genes. For this point, ZmSIMK1was expressed inS.cerevisiae, and the result showed that the overexpression of ZmSIMK1improved yeasttolerance to osmotic stress.（3）The analysis of expression pattern of ZmSIMK1. The results of qRT-PCR showedthat ZmISMK1had a higher expression in leaves relative to roots and stems. ZmSIMK1transcript was induced by salt, drought, ABA, SA, and bacterial pathogen (Pst DC3000).（4）The analysis of ZmSIMK1promoter. We investigated the promoter sequence of ZmSIMK1by biological software (plantcare). Sequence analysis showed that it containedTATA-box, CAAT-box and transcripton start site+1, which are characteristics of eukaryoticpromoter. In addition, it contains some cis-elements, including Box-W1(response to fungiexcitation), TCA element (response to SA), ABRE (response to ABA), and MBS (MYBtranscription factor response element). The result of GUS staining indicated that ZmSIMK1expression could be induced by salt, drouht stesses, and Pst DC3000.（5）Subcelluar localization of ZmSIMK1protein. Usually, the function of a protein isclosely associated with its subcellular localization. Our study presented that ZmSIMK1waslocated in nulear. And CD domain located the C-terminus of ZmSIMK1was required for itsnuclear localization.（6）The generation of ZmSIMK1-overexpressing transgenic tobacco. The ORF sequence(1187bp) of ZmSIMK1was cloned into pBI121vector and the reconstructed plasmid wastransformed into tobacco leaves by agrobacterium LBA4404. T2generation transgenictobacco was obtained by screening based on RT-PCR and kanamycin resistance. And theexpression level of ZmSIMK1in T2generation transgenic tobacco was analyzed by qRT-PCR.（7）The tolerance analysis of transgenic and WT tobacco to drought stress. Thetransgenic and WT tobacco were treated with20%PEG. At the end of treatment, it was foundthat the wilting speed of WT tobacco was significantly faster than in the transgenic tobacco,and the WT accumulated more reactive oxygen species (ROS), malondialdehyde (MDA),ABA, and had a higher relative conductance and lower germination rate in comparison withthe transgenic plants. In addition, the transgenic tobacco had higher antioxidant enzymeactivities, proline and ascorbic acid compared to the WT. In order to elucidate the molecularmechanism of drought stress tolerance, the transcripts of seven drought-response genes wereanalyzed. It is found that the genes encoding the antioxidant enzymes, NtLEA5and ABAsynthase were up-regulated to higher level in transgenic lines than those in the WT afterdrought stress treatment.（8）Overexpression of ZmSIMK1increased transgenic tobacco resistance to thepathogen, Pst DC3000. Pst DC3000was used to infect the transgenic and WT tobacco, theresults indicated that transgenic plants generated hypersensitivity (HR) more fasterly relativeto the WT, and the HR limited the growth rate of Pst DC3000. Moreover, the transgenic tobacco had more transcripts of PR, WRKY and NPR1gene and SA accumulation compared tothe WT. Finally, the systemic acquired resistance (SAR) was activated in the transgenictobacco.