Expression In Vitro And Functional Analysis Of Violaxanthin De-epoxidase Gene In Tomato

The radiant energy absorbed by the photosynthetic apparatus will finally be partially converted into steady chemical energy via photochemical processes. When a plant absorbs more light than it can utilize through the photosynthetic electron transport pathway, the excess energy can reduce the photosynthetic efficiency and result in photoinhibition and photooxidative damage sequentially. Plants have formed many kinds of photoprotective mechanisms, which could minimize the damage caused by excess light energy. The irradiance-dependent the xanthophyll cycle plays an important role in the protection of plants under enviromental stress. The xanthophyll cycle involves the interconversions between the three pigments, violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z). When the energy is excessive, V is convered to Z via A in the presence of ascorbate and an acidic lumen generated by the proton pump, this reaction is thought to occur in the lumen of thylakoids, catalyzed by violaxanthin de-epoxidase, and ZE catalyzes the reverse reaction thought to be located in the stromal side. The cycle is catalyzed by two key enzymes, violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE).In this reseach, a full length cDNA of VDE gene was cloned from tomato using RT-PCR and its function was analysed. The main results are as follows:1. Two specific primers were designed to amplify specific DNA fragment using cDNA prepared from tomato leaves according to the VDE sequences from other plant species. The clone, which named LeVDE (Acession number: FJ648424), contains 1670bp, an open reading frame (ORF) of 1437 bp comprising 478 amino acid residues with the predicted molecular mass of 53 KD.2. A recombinant of prokaryotic expression vector pET::LeVDE was constructed and transformed to E.Coli BL21 (DE3) to express. The strong induced fusion protein bands were collected and used to immunize white mice to obtain antiserum. Western blot analysis revealed the LeVDE protein level remained constant in leaves. Moreover, there was no different among leaves exposed to chilling stress under low irradiance and high light.3. Tobacco (Nicotiana tabacum) transformed with the sense and antisense constructs of LeVDE was obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). RT-PCR and western blot analysis demonstrated that the exogenous gene was integrated into the tobacco genome. Overexpression and suppression of LeVDE in transgenic tobacco plants resulted in the change of the level of de-epoxidation compared with WT under excessive light energy.4. De-epoxidation ratio of the xanthophyll cycle pigments (A+Z)/(V+A+Z) and NPQ significantly increased during high light and chilling stress with low irradiance. The level of (A+Z)/(V+A+Z) and NPQ in sense transgenic plants increased markedly relative to that in WT, while that in antisense lines was lower than that in WT. The higher de-epoxidation ratio of the xanthophyll cycle and higher NPQ in sense transgenic tobacco plants showed that overexpression of LeVDE increased the xanthophyll-cycle-dependent energy dissipation under high light and chilling stress with low irradiance.5. In order to investigate the relationship of expression of LeVDE and the tolerance of photosystem to high light and chilling stress with low irradiance, photosynthetic and chlorophyll fluorescence parameters were measured. The maximal photochemical efficiency of PSâ…¡(Fv/Fm) and the photosynthetic rates (Pn) in the sense line descreased less, while Fv/Fm and Pn in the antisense line decreased obviously. It is demonstrated that the PSII of sense plants are tolerant to damage under high light and chilling stress with low irradiance, while that of antisense transgenic plants are susceptible to these conditions.