Expression Characteristics And Transgenic Analysis Of Mitogen- Activated Protein Kinase From Zea Mays Roots, And Construction Of The CDNA Liabrary

Up to date, a number of MAPKs have been isolated from different plants, and considerable progresses have been made in understanding plant MAPKs. Current research of plant MAPK mostly foucs on dicot plant spe-cies (such as Arabidopsis, tobacco, and alfafa etc.), but relatively less study on Zea mays. In this paper, Zea mays (Zheng 958) seedlings are treated with different environmental stresses and signal substances, and expression pat-tern of MAPKs in Zea mays are analyzed under these conditions. The anti-body is made according to induce-expressed ZmOSMAPK1. Stress tolerance of transgenic Arobidopsis and transgenic tobacco are also investigated with different treatments. Meanwhile, the cDNA library of Zea mays roots treated with PEG is also constructed. The main results are as follows: 1 Expression analysis of MAPKs in Zea mays roots with treatments of drought, salt and low-temperatureExpression analysis of MAPKs in Zea mays with treatments of drought, salt and low-temperature were investigated using RT-PCR. The results indicate that only ZmOSMAPK1 has no expression only in the control, while other MAPKs have different combinational expression level. Sig-nificant differences exist in the expression level of ZmOSMAPK1 with treatments of PEG and NaCl. A detailed time course experiment with treatments of PEG and NaCl was conducted, and the results demon-strated that the expression level of ZmOSMAPK1 in Zea mays roots treated with NaCl is higher than that with PEG in the first 5 hours but reversed in the later time course. These indicate that ZmOSMAPK1 is involved in the signal pathway induced by PEG and NaCl respectively and mainly related with the signal pathway induced by drought. The ex-pression level of MAPKs at 4℃in light is higher than 4℃that in dark-ness. The result indicated that light might be a stimulus in the MAPK cascades and has effect on the regulation of gene expression. 2 Expression analysis of MAPKs in Zea mays roots with treatments of plant hormones and signal substancesThe expression pattern of a MAPK in Zea mays roots was generally similar with treatments of plant hormones GA and signal substances CaCl2 and SA, except ABA. The results indicated that the expression level of MAPK cascades components would be up-regulated with treatments of GA, CaCl2 and SA, while the effect of ABA might be similar to PEG and NaCl.3 Influence of mechanical injury and UV-irradiation on MAPKs expres-sion in Zea mays rootsMAPKs in Zea mays roots could be induced by mechanical injury at different level. Results also showed that there are no rules in the expres-sion pattern of MAPKs after UV-irradiation. These indicated that MAPKs in Zea mays roots were involved in the signal transduction in-duced by mechanical injury and UV-irradiation.4 Expression of ZmOSMAPK1 gene in E.coli, preparation of polyclone antibody and expression analysis of ZmOSMAPK1 protein with differ-ent stressesAfter cloning the encoding region of ZmOSMAPK1 into prokaryotic ex-pression vector pET-30a-c(+), we obtained the soluble fusion proteins in BL21 strain. Special polyclone antibody was prepared by injecting fu-sion proteins into immune mice and the titer reached up to 1:100000. Western blot analysis demonstrated that ZmOSMAPK1 could be de-tected in different levels with different stresses, and the expression pat-tern was accordant to that in transcriptional levels. These indicated that the expression of ZmOSMAPK1 gene was consistent in transcriptional and translational level.5 Tolerance analysis of transgenic Arabidopsis and transgenic tobacco The ZmOSMAPK1 gene was transformed into Arabidopsis and tobacco. The results showed that the transgenic Arabidopsis had ability of bear- ing drought and high salinity. And the transgenic Arabidopsis showed increased ability of bearing low-temperature. These results indicated that ZmOSMAPK1 gene played important roles in response to environ-mental stresses.6 Construction of the cDNA library of Zea mays roots The cDNA library was constructed with Zea mays roots which were treated with different concentrations of PEG for different time. Some of the screening results show that the capacity of the library was rather big for us to screen, and the inserted segments were relative large.