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Molecular Cloning And Gene Expression Of Wheat Violaxanthin De-epoxidase

Posted on:2004-12-11Degree:DoctorType:Dissertation
Country:ChinaCandidate:B LiFull Text:PDF
GTID:1100360092990259Subject:Botany
Abstract/Summary:PDF Full Text Request
The radiant energy absorbed by the photosynthetic apparatus will finally be partially converted into steady chemical energy via photochemical processes. When plants absorb more light than that can be used for photosynthesis, the excess energy, which is not promptly quenched, can reduce the photosynthetic efficiency and result in photoinhibition, even photooxidative damage to the photosynthetic reaction center. The heat dissipation depended on xanthophyll cycle has been wildly concerned recently. It is regarded as an important photoprotective way because it can deal with the excess energy and protect the photosynthetic apparatus from photodamage. The xanthophyll cycle involves the interconversions between the three pigments, violaxanthin(V), antheraxanthin(A) and zeaxanthin(Z). The cycle is catalyzed by two enzymes, violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). When the energy is excessive, VDE converts V to Z via A in the presence of ascorbate and an acidic lumen generated by the proton pump, and ZE catalyzes the reverse reaction in the dark. The study on the function of xanthophyll cycle in reducing photoinhibition and photodamage has been a hot field in the research of photosynthesis. In this experiment, the gene, which was named WVDE, encoding VDE enzyme from wheat was cloned by RT-PCR. The GenBank accession number is AF265294. The WVDE cDNA is 1,746bp long. The length of the open reading frame is 1,365bp and it encodes 464 amino acids. The length of the transpeptide is 464 amino acids, and mature protein is 348 amino acids. Its molecular weight is 39.8 Kda. The amino acid sequence of WVDE showed 78.7%, 62.6%, 58.3%, 60.8%, 60.6% and 61.2% identity with those of the rice, Arabidopsis thaliana, tobacco, lettuce, spinach and tea, respectively.The 5' end sequence of WVDE was amplified by RT-PCR, and its reverse sequence was inserted into the plant expression vector pBI121. About 80 antisense transgenic tobacco plants were obtained from leaves' explants via Agrobacterium tumefaciens-mediated transformation in 60 days. It has been confirmed that the antisense WVDE gene was transferred into thegenome of the transgenic tobacco plants by analysis of Southern blot and RT-PCR amplification. In natural condition, the PSII maximal photochemical efficiency (Fv/Fm) of wheat leaves declined quickly in according with the increasing of the light intensity in the morning, and touched bottom at 12 o'clock, which indicated that the photoinhibition occurred. At the same time, non-photochemical quenching (NPQ) increased and reached maximum at 12 o'clock, and the diurnal changes of NPQ was reverse to those of Fv/Fm. The results of RT-PCR southern blot indicated there was a linear relationship between NPQ and WVDE expression in wheat leaves. There was no great difference between Fv/Fm and NPQ of the transgenic and wild-type tobacco plants at the beginning of the strong light treatment or under the low light intensity in the morning, but the obvious difference appeared when Fv/Fm was declined and NPQ was increased following the increase of the light treatment time and light intensity. Similar changes of Fv/Fm and NPQ between the transgenic and wild-type tobacco plants were noted under the salt stress. The decline of their Fv/Fm was almost accordant at the beginning of the treatment, but along with the extending of treating time, Fv/Fm of the transgenic plants dropped rapidly. Under the 4℃ temperature stress, the decrease of Fv/Fm in the transgenic and wild-type tobacco leaves was similar for the first 3 h. After 3 hour's chilling stress, Fv/Fm in the transgenic plants decreased more sharply than that of the wild-type plants, but it could recover to the initial value of wild-type tobacco plants. These results demonstrated that transferring antisense WVDE gene inhibited VDE activity and led to the decrease of heat dissipation in the transgenic tobacco plants. Under the stress condition, the carbon assimilation was inhibited, and the light energy was more excessive. If VDE was inhibited a...
Keywords/Search Tags:Wheat, Xanthophyll Cycle, Violaxanthin De-Epoxidase, Photoprotection
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