Molecular Cloning And Functional Analysis Of Violaxanthin Deepoxidase Gene From Phyllostachys Edulis

It is a potential ability for plants to survive under naturally varying light conditions. The xanthophyll cycle plays an important role both in protecting plants from photo-oxidative damage under strong light and in improving photosynthetic efficiency under inadequate light. The cycle consists of three components, i.e. violaxanthin, antheraxanthin and zeaxanthin. Under strong light the energy input exceeds the photosynthetic capacity and leads to overacidification of the thylakoid lumen. This drop in lumen pH leads to activation of the violaxanthin deepoxidase (VDE), thus shifting the xanthophyll balance from violaxanthin (V) toward zeaxanthin (Z) through antheraxanthin (A). Z is able to enhance energy dissipation as heat and efficiently scavenges reactive oxygen species which are harmful to organisms. Under low light Z is converted back to V through A by zeaxanthin epoxidase (ZE), which can increase light-harvesting efficiency by avoiding unnecessary quenching of excitation energy. Moso bamboo (Phyllostachys edulis), an economic species with great value located in subtropical regions of southern China, was selected as material for experiment. Based on the chlorophyll fluorescence parameters analysis of P. edulis, research on violaxanthin de-epoxidase was carried out, which was expected to understand the role of the xanthophyll cycle in photoprotection. The main results are as follows:First, chlorophyll fluorescence parameter measurement. The chlorophyll fluorescence parameters such as nonphotochemical quenching (NPQ) and apparent electron transport rate (ETR) characteristics of Moso bamboo leaves were measured with a portable pulse amplitude modulation fluorometer (Imaging PAM). The results showed that NPQ enhanced with the increasing of photosynthetically active radiation (PAR) after dark adaptation adequately, while ETR increased firstly, then decreased. It indicated that NPQ played an important role in the dissipation of excess energy.Second, gene isolation. Degenerate primers for the conserved domain of VDE were designed based on the homologous protein sequence of different species and the RACE primers were designed according to the gained conserved domain sequence. A full length cDNA encoding violaxanthin de-epoxidase was obtained by the methods of RT-PCR and RACE and named as PeVDE. The cDNA was 1723 bp which contained an open reading frame (ORF) encoding 451 amino acids. The protein structure analysis showed that the putative protein consisted of transit peptides (103 amino acids at the N-terminal end) and mature protein of VDE. Homology analysis showed that the deduced mature protein was highly homologous to other VDE proteins from different species. Based on the ORF sequence of PeVDE, primers were designed to clone the genomic sequence. Sequence analysis showed that the genomic sequence was 1921 bp containing four introns and five exons.Third, prokaryotic expression of PeVDE. The sequence encoding the mature protein of PeVDE was subcloned into pET-32b and transformed into Rosetta-gamin B (DE3) induced by IPTG. Analysis by SDS-PAGE of cell extracts indicated that the recombinant protein was expressed effectively at 30℃induced by 0.4 mmol·L-1 IPTG for 4 h.Fourth, the enzymatic activity assay. The bacterial pellet was lysed using an ultrasonic cell disruptor, and the crude protein was used for activity assay. The enzymatic reaction was performed with pH 5.1 at 25℃in dark for 15 min. The mixture of reaction was extracted and analyzed by HPLC. The result showed that it contained not only V but also A and Z, which indicated that the recombinant protein exhibited enzymatic activity and could catalyze V into Z through A.Fifth, western blotting. The recombinant protein was purified and used to immunize white rabbit to obtain polyclonal antibody. The total protein from the roots, stems, leaf sheaths and leaves were extracted respectively. The polyclonal antibody of PeVDE was used as probe for western blotting. The result showed that the violaxanthin de-epoxidase was only detected in Moso bamboo stems, leaf sheaths and leaves which was richest, and the molecular weight was about 50 kDa.