Molecular Cloning And Functional Analysis Of The VDE Gene And Its Promoter From Cucumber

Xanthophyll cycle plays a critical role in protecting photosynthesis apparatus from the damage of excessive light. Violaxanthin de-epoxidase (VDE) is the key enzyme in xanthophyll cycle, which can catalyzed the conversion of violaxanthin (V) via antheraxanthin (A) and then to zeaxalnthin (Z) in high light condition. To intensive study the function of VDE, we have cloned a1449bp Cucumber violaxanthin de-epoxidase gene (CsVDE) and its promoter (1983bp). The expression, subcellular localization and response to abiotic stress of CsVDE were anaylsed, and the histochemical staining,5’deletion and response to light of CsVDE promoter were anaylsed. The mainly results are as follows:The full-length cDNA sequences of CsVDE were coloned by RT-PCR and were analysed for the homology and phylogenetic tree with plant CsVDE. The results show that CsVDE has the characteristics of VDE in the other species and were with high homology to the VDE in Arabidopsis.Using realtime-PCR and western blot analysis indicated that the expression of CsVDE was mainly in leaves. In transcriptional level the highest expression of CsVDE were appear in mature leaves and old leaves, but in protein level the highest expression of CsVDE were appear in young leaves. Using GFP fusion protein and immunogold labeling methods, we show that CsVDE is mainly localized in chloroplasts in cucumber cotyledon cells, mature leaf cells and old leaf cells.CsVDE is quickly induced by cold and drought stress. Under normal light conditions, the transcript level of CsVDE gradually increased at the beginning of the day, and then gradually decreased, whereafter it increased to its original level. Under low light conditions, the expression pattern of the CsVDE transcript was similar with that under normal light conditions, but the peaking time of both the highest and the lowest levels were delayed by2h. Under high light conditions, CsVDE responded more rapidly and reached maximum expression in one hour, and then declined gradually. The de-epoxidation ratio (A+Z)/(V+A+Z) is increased rapidly in0.5h under high light and normal light conditions, and the value is always higher in high light condition than normal light condition. The gold particles were also increased and mainly located in thylakoid membranes under high light condition. The non-photochemical quenching (NPQ), Fv/Fm and the ratio of (A+Z)/(V+A+Z) are reduced in transgenic Arabidopsis down-regulated by the antisense fragment of CsVDE, compared to wild type (WT) Arabidopsis under high light stress. This indicates the decreased functionality of the xanthophyll cycle and the increased sensitivity to photoinhibition of photosystem Ⅱ(PSⅡ) in transgenic Arabidopsis.To characterize the CsVDE promoter in detail, sequence Analysis’ showed a number of potential cis-acting elements involved in light responses and other environmental signals. Histochemical analysis of CsVDE promoter driving GUS expression in Arabidopsis showed high promoter activity in green tissues such as cotyledons, true leaves, stems, sepals and the epidermis of mature fruits.Using a series of constructs with5’deletions of CsVDE promoter showed the truncation from pCsVDEP-3(-1111) to pCsVDEP-4(-741) resulted in a nearly50%decrease for the GUS activity, and the activity was resumed to the highest level when the promoter was further truncated from pCsVDEP-4(-741) to PCsVDEp-5(-498). GUS expression levels decreased gradually as the length of promoter was progressively truncated from the pCsVDEP-5(-498) to pCsVDEP-8(-194). A significant GUS activity decrease was observed by the deletion of a48bp fragment from pCsVDEP-B (-789) to pCsVDEP-4(-741).The activity of CsVDE promoter was altered by hormones and abiotic stresses were negatively by abscisic acid, salicylic acid, polyethylene glycol, mannitol and sodium chloride, positively by gibberellin and indole-3-acetic acid. The activities of GUS driven by the CsVDE promoter fragments were increased when plants were exposed to high light for4h, and the corresponding positive regulation were located to pCsVDEP-D (-533) to pCsVDEP-5(-498) and pCsVDEP-B (-789) to pCsVDEP-4(-741). The response of CsVDE promoter to the first24h normal level light was located in pCsVDEP-5(-498) to pCsVDEP-6(-476).