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Phyllostachys edulis violaxanthin deepoxidase (PeVDE) protein, and coding gene and applications thereof

A technology of cyclooxygenase and violaxanthin, which is applied in the field of genetic engineering, can solve the problems that there is no bamboo violaxanthin decycloxygenase gene, and the molecular basis of bamboo xanthophyll cycle metabolism pathway is unclear, so as to reduce the Cost, effect of high enzyme activity

Inactive Publication Date: 2012-07-04
INT CENT FOR BAMBOO & RATTAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the research on the metabolic pathway of bamboo xanthophyll cycle is still blank, there is no report on the gene and in vitro expression of bamboo violaxanthin decycloxygenase, let alone the artificial synthesis of zeaxanthin by using bamboo violaxanthin decycloxygenase reported that the molecular basis of bamboo lutein cycle metabolic pathway is still unclear

Method used

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  • Phyllostachys edulis violaxanthin deepoxidase (PeVDE) protein, and coding gene and applications thereof
  • Phyllostachys edulis violaxanthin deepoxidase (PeVDE) protein, and coding gene and applications thereof
  • Phyllostachys edulis violaxanthin deepoxidase (PeVDE) protein, and coding gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Acquisition of Phylloxanthin Decycloxygenase Gene PeVDE Sequence in Phyllostachys pubescens

[0028] RNA was extracted from young fresh leaves of Phyllostachys edulis, and reverse-transcribed into cDNA using Promega’s reverse transcription kit (Cat#A3500) as a template. Degenerate primers were designed based on the conserved sequence of the VDE family of enzyme genes, and the coding sequence was amplified by PCR.

[0029] Upstream primer: 5′-CC(A / T)GA(T / C)GA(A / G)AC(T / C / G)GA(A / G)TG(C / T)CA-3′(SEQ ID No.5),

[0030] Downstream primer: 5′-C(C / A)(A / G)CC(A / T)CCATA(T / G)CCATCCCA-3′(SEQ ID No.6)

[0031] PCR amplification products were detected by agarose gel electrophoresis, such as figure 1 shown. Cut out the target band for purification and recovery, connect the recovered DNA fragments to the pGEM-T easy vector, transform Escherichia coli (Esherichia coli) DH5α competent cells, and screen the positive clones with blue and white spots, extract positive clone plas...

Embodiment 2

[0041] Example 2 Construction of a recombinant expression vector carrying the violaxanthin decycloxygenase gene of Phyllostachys pubescens

[0042] Using the cDNA of Phyllostachys pubescens as a template, primers were designed according to SEQ ID No.1, and the deoxyribonucleotide sequence of the mature protein encoded by the violaxanthin decycloxygenase gene of Phyllostachys pubescens was amplified by PCR. Nco I and Xho I restriction sites were introduced at both ends of the primers respectively, and the primer sequences are as follows:

[0043] Upstream: 5′-CATG CCATGG CCACCGATGCTCTCCAAGAC-3', introducing Nco I restriction site (SEQ ID No.11);

[0044] Downstream: 5′-CCG CTCGAG CCTTAGCTTCCTTATTGGC-3', introducing the Xho I restriction site (SEQ ID No. 12).

[0045] Perform agarose gel electrophoresis detection on the PCR amplification product, cut out the target band, purify and recover, connect the recovered DNA fragment to the pGEM-T easy vector, transform Escherichia ...

Embodiment 3

[0047] Prokaryotic expression and protein purification of embodiment 3PeVDE gene

[0048] The recombinant expression vector pET-32b-PeVDE constructed in Example 2 was transformed into Escherichia coli Rosetta-gami B(DE3) competent cells, and a single colony grown on the Amp-resistant plate was picked for enzyme digestion identification and sequencing. Sequencing results showed that the monoclonal colony contained the target gene fragment, which could be used for the induced expression experiment of the protein.

[0049] Correctly identified clones were cultured overnight, and the overnight cultured bacterial solution was added to ampicillin-containing (100 mg L) at a ratio of 1:100. -1 ) cultured in LB medium until OD 600 When the temperature is 0.6-0.8, add IPTG (final concentration 0.4mM), and culture at 20°C and 30°C respectively. Take 50ml of the induced bacterial solution, centrifuge at 5000rpm for 10min, and collect the cells; add 3ml of Tris-EDTA solution and 10μL of ...

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Abstract

The invention provides a Phyllostachys edulis violaxanthin deepoxidase (PeVDE) protein, which refers to proteins as follows: (1) a protein formed by amino acid sequences shown as in SEQ ID NO. 2, or (2) a protein which is obtained by performing substitute, deletion or addition on the amino acid sequences shown as in SEQ ID NO. 2, has equal activity and is derived from the protein formed by the amino acid sequences shown as in SEQ ID NO. 2. The invention also provides a gene for coding the protein. PeVDE provided by the invention is capable of catalyzing violaxanthin to be singly deoxidized to form Antheraxanthin, and the violaxanthin is deoxidized again to form zeaxanthin; the PeVDE has higher enzymatic activity, and a novel selectable is provided for producing the Antheraxanthin and the zeaxanthin through artificially catalyzing the violaxanthin. Moreover, recombinant PeVDE is prepared through prokaryotic expression by the invention, thereby, the difficulty of directly separating the protein from bamboo laminas is overcome, and the cost is reduced.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a violaxanthin decycloxygenase protein, a gene encoding the protein and an application thereof. The invention also relates to an in vitro expression method of the protein. Background technique [0002] Zeaxanthin is a kind of natural pigment widely present in many plants, and it is also a kind of compound necessary for human body. Mainly composed of lutein (C 21 h 30 o 2 , lutein), zeaxanthin (C 40 h 56 o 2 , zeaxanthin) and cryptoxanthin (C 40 h 50 O, cryp toxanthin) and other carotenoids. Research on the function of zeaxanthin has attracted increasing interest, mainly focusing on the relationship between zeaxanthin and eye disease, heart disease and cancer, and their antioxidant properties are also of interest. Studies have shown that lutein and zeaxanthin in food have a direct impact on the metabolic function of the eye, and have a protective effect on th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/63C12N1/21C12P23/00C12R1/19
Inventor 高志民郑波李彩丽
Owner INT CENT FOR BAMBOO & RATTAN
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