Effects Of Caffeic Acid On DNA Methylation In Drought-stressed Cucumber Leaves And Functional Analysis Of One Differently Methylated Cucumber Gene

Plant always suffers from abiotic stresses such as drought and chilling. Cucumber which has shallow root systems and large leaves is one of main cultivated vegetables in our country. So it needs more water and particularly sensitive to moisture response. Drought is the main problem of cucumber production in summber and autumn. Hence, it is of great significant to investigate the impaction of dehydration stress on cucumber and to explore the way alleviating it. The paper explored the mechanism of dehydration stress mitigated by exogenous caffeic acid(CA). We investigated the effects of exogenous CA on the indexes of physiological and biochemical and the changes of genomic DNA methylation. Then the DNA methylation differently fragments were separated and further cloned and sequenced. This paper reveals the mechanism of exogenous CA alleviating dehydration stress on the macro, micro, physiological and molecular levels, which provides more theoretical and practical basis for the application of exogenous CA and the drought-resistant production of cucumber. Furthermore, the paper applies the quantitative real time PCR to determine the transcript expressions of two differential methylation genes which were obtained from exogenous hydrogen peroxide(H2O2) alleviating the cucumber dehydration stress in different tissues of cucumber seedings. The present article tried the transformation of one differential methylation gene into cucumber ovaries by microinjection, the antisense transgenic cucumber plants was obtained by PCR method. And then makes functional analysis of the gene. This lays the foundation for elucidating the mechanism of cucumber in response to stress. The main findings are as follows:(1) Effects of exogenous CA alleviate dehydration stress on cucumber seedling growth parameters, antioxidant enzymes and related indexesIn this study, dehydration stress led to the growth indexes of(Cucumis sativus L) cv. Jinchun no.4 decreased significantly, while exogenous CA treatment before dehydration stress significantly increased the growth indexes in cucumber seedlings. Therefore, dehydration stress inhibits the growth of cucumber seedlings, while exogenous CA treatment alleviates the growth inhibition.Compared to control, dehydration increased the levels of malonaldehyde(MDA) and ROS(endogenous H2O2 and O2.-) in cucumber leaves(the second true leaves) in this paper. Meanwhile, after pretreatment with CA, the activities of antioxidant enzyme including superoxide dismutase(SOD), catalase(CAT), guaiacol peroxidase(GPX), glutathione peroxidase(GSH-Px), ascorbate peroxidase(APX), dehydroascorbate reductase(DHAR), monodehydroascorbate reductase(MDHAR) and glutathione reductase(GR) and the activities of antioxidants including ascorbate(AsA) and glutathione(GSH) were significantly increased. Meanwhile we observed the elevated levels of proline and soluble sugars. Hence, the levels of MDA and ROS in cucumber leaves were decreased. We concluded that exogenous CA pretreatment increases antioxidant enzymes and non-enzymatic antioxidants activities in cucumber leaves and decreases the ROS levels.(2) Effects of exogenous CA on DNA methylation of cucumber genome under dehydration stressDNA of cucumber leaves were used for MSAP analysis. According to the results of MSAP, dehydration stress effects the level of genomic DNA methylation of the cucumber leaves, and CA pretreatment further changes the DNA methylation.(3) The expression of differently methylated cucumber genes HG-1 and HG-2 induced by exogenous H2O2 under drought treatment and the functional analysis of HG-2In comparison to PEG treatment, the transcript levels of gene HG-1 were significantly decreased in the root, the second true leaves and the first true leaves of H2O2+PEG treatment, but they were significantly increased in stem and cotyledon. Meanwhile, the expression levels of gene HG-2 in different tissues were significantly decreased in H2O2+PEG treatment when compared to the PEG treatment, thus leading to “downgrade” expression tendency.The full-length cDNA sequences HG-2 which was obtained by PCR was subcloned into the expression vector pBI121. Then the construct was introduced into cucumber by ovary-injection method. The transgenic cucumber plants were verified by PCR, and make functional analysis of them.