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Molecular Cloning and Functional Analysis of Violaxanthin Deepoxidase Gene from Phyllostachys Edulis

Author: ZhengBo
Tutor: GaoZhiMin
School: Chinese Academy of Forestry
Course: Tree Genetics and Breeding
Keywords: Bamboo Xanthophyll cycle Non - photochemical quenching Apparent photosynthetic electron transport rate Violaxanthin cyclooxygenase
CLC: S795.7
Type: Master's thesis
Year: 2011
Downloads: 20
Quote: 0
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Abstract


Adapt to a variety of lighting conditions is the the plant survival instinct, xanthophyll cycle in plant adaptation to high-light environments, Photoprotection and adapt to the low light environment, and improve the process of light use efficiency plays an important role. The xanthophyll cycle contains three components, namely violaxanthin, anthers yellow substance and corn yellow substance. When the plant absorption of light energy exceeds its ability to transform the body cavity thylakoid over-acidification low the pH be able to activate Violaxanthin cyclooxygenase, reaction of zeaxanthin catalytic violaxanthin generated. Zeaxanthin excess energy to the form of heat is dissipated, and could remove some of the oxygen free radicals, in order to reduce the excess solar energy plants produce injury. In low light, zeaxanthin zeaxanthin role of cyclooxygenase into violaxanthin, reduce the heat dissipation of the light energy to improve the plant's solar energy utilization. Moso bamboo (Phyllostachys edulis) is distributed in scattered bamboo species in the subtropical region of China, has a high economic value. The paper with bamboo seedling material, research carried out on the basis of the study chlorophyll fluorescence parameters Moso bamboo xanthophyll cycle key enzyme - Violaxanthin the cyclooxygenase period for understanding bamboo xanthophyll cycle in different light environments provide the basis of the molecular biology, the main findings are as follows: first, Bamboo chlorophyll fluorescence kinetics study. Chlorophyll fluorescence techniques to study the Moso bamboo seedling chlorophyll fluorescence parameters of non-photochemical quenching (NPQ) and the variation of the electron transport rate (ETR). The results showed that, after full dark adaptation, along with the gradual increase of photosynthetically active radiation (PAR), NPQ increased, while the ETR increased first and then decreased. Its that the NPQ to play an important role in the process of excess solar energy heat dissipation. Second, gene cloning. Different species violaxanthin de cyclooxygenase (VDE) homologous protein sequences to compare design degenerate primers get of Moso bamboo VDE gene partial cDNA sequence by RACE method to obtain the 5 'and 3' terminal sequence obtained by splicing the gene full-length cDNA (1723 bp) coding region for the 1356 bp encoding 451 amino acids (including the N-terminal 103 amino acids of the transit peptide), the gene named PeVDE. The primers were designed according to the coding region of the PeVDE genomic DNA as a template for amplification and sequencing results show that the sequence of the Moso bamboo PeVDE genes coding region of the genome containing four introns and five exons. Third, prokaryotic expression of the gene. The mature protein encoded PeVDE gene sequence was cloned into the prokaryotic expression vector pET-32b, and the resulting recombinant expression vector containing the desired gene. The expression vector was transformed into E. coli expression strain (Rosetta-gamin B (DE3)) competent cells, prokaryotic inducible expression. Induced by optimizing the conditions, under the conditions of 30 ° C, 0.4 mmol · L-1 IPTG induction 4 h, the soluble protein expression abundance. Fourth, the enzyme activity assay. Total protein extract PeVDE recombinant bacteria, by sonication, the reaction substrate to violaxanthin, dark reaction at 25 ° C and pH 5.1 under the conditions 15 min, HPLC assay of the reaction product. The results show that the reaction products in addition to violaxanthin, anther zeaxanthin and zeaxanthin component, thus proving the recombinant protein with biological activity, violaxanthin sequentially converted to the anthers zeaxanthin and zeaxanthin . Fifth, Western blotting analysis. Prokaryotic expression resulting recombinant protein was used to immunize New Zealand white rabbits, polyclonal antibody. The Moso bamboo seedling root, stem, leaf sheath and leaf tissue total protein were extracted and separated by SDS-PAGE of polyclonal antibody of PeVDE as a probe to detect. Western Blotting The results showed that can be detected in stem, leaf sheaths and blades PeVDE, wherein the highest content of the leaves, the molecular weight of about 50 kDa, and is not detected in the root.

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CLC: > Agricultural Sciences > Forestry > Forest tree species > Bamboo > Phyllostachys
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