| Xanthophyli cycle has been widely concerned since the finding that it has the function in heat dissipation. With the trans-thylakoid pH gradient,zeaxanthin (Z) together with antheraxanthin (A) can absorb excess energy from chlorophyll and release it as heat,thus protect the photosynthetic apparatus from photodamage by high light. Violaxathin de-epoxidase (VDE) is the key enzyme in xanthophylls cycle. Under low pH conditions,VDE converts violaxanthin (V) to A and Z within minutes. In this study,we not only cloned genes encoding VDE enzyme from rice and spinach,but also transferred Svde gene into tobacco. It will provide us to further study the function of xanthophyll cycle in photoprotection. The major results are as following:Two cDNA sequences encoding violaxanthin de-epoxidase were cloned from japonica rice (jRvde) and indica rice (iRvde) with the full-length of 1887bp and 1647bp,respectively. The homology of the open reading frame is 98% identity between two Rvde genes,and more than 60% identities with those of other species. It is deduced to encode 446 amino acids with the transpeptide of 98 amino acids. The amino acid sequences of their mature proteins are completely homologous. They also share 87.4% identity with wheat.The genomic DNA sequences of Rvde genes were also amplified through PCR. jRvde gene has four introns as 105bp,327bp,81bp and 69bp,respectively,in its encoding region. In contrast,the introns of iRvde gene are the same as that ofjRvde except that the second is 425bp. The amounts of A+T are ranged from 60 to 63 percents. According to the rice genomic database,the homology between their promoters is about 69% identity,suggesting that the difference of photoinhibition between indica rice and japonica rice might be somewhat related to the difference of the promoter sequences.The bacterial expression vector of jRvde gene was constructed as name pET-Rvde. The foreign VDE protein was largely expressed in E. coli strain DL21(DE3) induced by 0.4 mmol/L IPTG. SDS-PAGE and Western blotting indicated that the molecular weight of the expressed protein is 43kDa,similar to that of the native VDE enzyme. The expression quantity increased with the induction time by IPTG,and accounted for 25 percent of the total proteins after 4-hour induction. Absorption spectrum together with xanthophyll pigments quantification by HPLC demonstrated that the expressed VDE has its enzyme activity,which can de-epoxidate V into A and Z in vitro.Svde gene was also amplified from spinach by RT-PCR. The plant expressionvector pCB-antiSvde was constructed,in which Svde gene was controlled by CaMV35S promoter and nos terminator. Antisense transgenic tobacco plants v. ere obtained from leave explants via Agrobacterium tumefaciens-medialed transformation. GUS histochemical assay showed that the regenerated callus became blue when they were stained with X-Gluc. The predicted bands were amplified from both TO and TI generation transgenic plants by PCR,indicating that the hygromycin resistance gene (hpt) and Svde gene were both transferred into tobacco. When the seeds from TO transgenic plants were cultured on medium supplemented with 50mg/L hygromycin,the ratio of germinated to non-germinated seeds is 3:1,which is consisted with Mendel's first law. Southern blotting demonstrated that the Svde gene had been integrated into the genome of tobacco plants. The VDE enzyme activity in A29 transgenic plant is 3.2 units,amounting for 45.7 percent of the wild-type tobacco,indicating that VDE was inhibited. Under high light treatment,the formation of A and Z decreased,and the values of non-photochemical quenching (NPQ) and Fv/Fm in A29 were both lower than those in the wild-type plant. These results demonstrate that the heat dissipation ability in the transgenic plants decreases,suggesting that xanthophyll cycle has the function in photoprotection.Besides,a DNA minipreparation method suitable for screening and identification large amounts of transgenic plants was established. Using this rapid and efficient method,one person can...
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