| When light that is absorbed by the plant in excess of what can not be utilized through the photosynthetic electron transport pathway, the excess energy, which is not promptly quenched, can reduce the photosynthetic efficiency and result in photooxidative damage and photoinhibition sequentially. Plants have formed many kinds of photoprotective mechanismes, which could minimize the damage caused by excess light energy. The irradiance-dependent xanthophyll cycle plays an important role in the protection of plants under enviromental stress. 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). It is significant to make clear the function and regulation mechanism of xanthophyll cycle.In this reseach, the cDNA of VDE and ZE gene was cloned from wheat using RT-PCR and the expression character was analysised. The antisense expression vector was designed and the change of transgenic tobacco under irradiance stress was explored. The main results are as follows:1. Two specific primers were designed to amplify specific DNA fragment of VDE and ZE gene using cDNA prepared from wheat leaves by RT-PCR.2. Northern blot analysis indicated that the transcript levels of WVDE and WZE gradually increased with the time of irradiance stress. Gene transcript levels were similar for extracts from plants under the high light and chilling stress in the low irradiance, exhibiting a diurnal rhythm.3. The WVDE and WZE genes were subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form antisense constructs, respectively. The constructs were first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method, and antisense transgenic tobacco plants were obtained.WT and transgenic plants with depletion of WVDE were exposed to chilling stress under low irradiance and high light. The results showed that thermal dissipation capacity in atisense transgenic plants of WVDE decreased under irradiance stress. Fv/Fm decreased in both WT and transgenic plants under high light stress, with transgenic plants showing the greater decrease. Fv/Fm also decreased obviously in transgenic plants during chilling stress (4℃) relative to that in wild type plants. Though the degree of decrease was less than it in high light stress, the trend is similar. It showed that the photoinhibition of PSII was aggravated. Under high light stress for 6 and 12 h, the O2 evolution rates of WT and transgenic tomato plants significantly decreased. This decrease was more obvious in transgenic plants than that in the wild type. On the other hand, under chilling stress with low irradiance for 6 and 12 h, the O2 evolution rates of WT and transgenic tobacco plants also decreased, but not significantly compared with that under high light stress. It indicated that the inhibited expression of VDE reduced the stability of O2 evolution system and aggravated the damage of photosynthetic apparatus. The same stress was applied in trangenic tobacco with depletion of WZE gene, but no obvious difference was detected.4. Northern blot analysis of different age leaves such as the young leaves (YL) and mature leaves (ML) in tobacco showed that VDE mainly expressed in the young leaves. The photosynthetic and chlorophyll fluorescence parameters showed that YL seems were not affected by high light stress compared with ML. YL have higher unsaturation of fatty acid in thylakoid membrane than ML, and it is perhaps one of the reasons that the antioxidation ability of YL is more better than ML.
|
PDF Full Text Request