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Phytoene dehydrogenase gene and application thereof

A technology of phytoene and lycopene, which is applied in the field of genetic engineering and can solve problems such as limited sources of phytoene dehydrogenase

Active Publication Date: 2012-08-15
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the sources of phytoene dehydrogenase for lycopene production research in the world are limited, and it is necessary to find new enzyme sources for efficient lycopene synthesis

Method used

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  • Phytoene dehydrogenase gene and application thereof
  • Phytoene dehydrogenase gene and application thereof
  • Phytoene dehydrogenase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: PCR amplification of phytoene dehydrogenase gene of Rhodobacter azotoformans KA25

[0024] Take 5 mL of Rhodobacter azotoformans KA25 culture solution purchased from Japan's NITE Biological Resource Center (NBRC) with a preservation number of 16436, and use the bacterial genome extraction kit to extract the genome. The extraction steps refer to the instructions of the bacterial genome extraction kit from Qiagen. .

[0025] The nucleotide sequences of phytoene dehydrogenase genes of different strains of the same genus were retrieved from GenBank, and a pair of primers F-I and R-I were designed. The primer sequences are as follows:

[0026] F-I: 5'-ATGCCCGCGACCAAGCATGT-3'SEQ ID NO.3

[0027] R-I: 5'-TCATTCCgCggCCAgCCTTT-3'SEQ ID NO.4

[0028] Genomic DNA is used as a template for amplification, and the above primers are used for amplification. The total volume is 50 μL, 18 μL of ultrapure water, 2×GC buffer I (containing MgCl 2 ) 25 μL, dNTP (2.5 mmol / L ea...

Embodiment 2

[0030] Example 2: Expression and purification of Rhodobacter azotoformans KA25 phytoene dehydrogenase gene in Escherichia coli BL21 (DE3)

[0031] Design primers F-I-22b and R-I-22b according to the sequence shown in SEQ ID No.1, respectively introduce the NdeI and SalI restriction sites (shown underlined) that can be inserted into the pET-22b plasmid, and the sequences are as follows

[0032] F-I-22b: 5'-GCG CATATG CCCGCGACCAAGCATGT-3' SEQ ID NO.5

[0033] R-I-22b: 5'-GCG GTC GAC TTCCgCggCCAgCCTTTCA-3' SEQ ID NO.6

[0034] Using the extracted Rhodobacter nitrogen-fixing genomic DNA as a template, PCR amplification was carried out using the above primers. PCR amplification conditions and PCR product recovery method were the same as in Example 1. After recovery of the PCR product, NdeI and SalI (NEB, USA) were added to digest at 37°C for 1 hour for DNA purification, and the pET-22b plasmid vector was treated with the same digestion method. The prepared enzyme gene fragme...

Embodiment 3

[0042] Example 3: Co-expression of Rhodobacter azotoformans KA25 phytoene dehydrogenase gene and plasmid pACCRT-EB in Escherichia coli BL21

[0043] The nitrogen-fixing Rhodobacter phytoene dehydrogenase recombinant plasmid obtained in Example 2 and the plasmid pACCRT-EB were co-transformed into the host strain Escherichia coli BL21 (DE3), and the transformation and induction methods were the same as in Example 2. After centrifugation of the induced recombinant Escherichia coli, the carotenoids were extracted with acetone and detected by HPLC. The chromatographic column was TC-C18 (Agilent, USA), and the mobile phase was methanol / acetonitrile (4 / 6). The flow rate was 1 mL / min. The temperature is 30°C, and the detection wavelength is 474nm.

[0044] figure 2 It shows that the above-mentioned double-plasmid co-expression E. coli strain products mainly contain two kinds of carotenoids. By comparing the mass spectrometry data and the retention time of the standard, the two kinds...

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Abstract

The invention relates to a phytoene dehydrogenase gene, in particular to a phytoene dehydrogenase gene for efficiently synthesizing lycopene and application thereof, belonging to the field of gene engineering. The nucleotide sequence of the phytoene dehydrogenase gene is shown by SEQ ID No.1. The amino acid sequence of the phytoene dehydrogenase coded with the gene is shown by SEQ ID No.2. The phytoene dehydrogenase gene provided by the invention can be used for efficiently producing lycopene. The examination results in that the yield of lycopene in a fermentation liquor is 0.269 mg / L and thecontent of lycopene in the dry weight of bacteria is 0.256 mg / g and accounts for 73.5% the total content of carotenoid; the phytoene dehydrogenase recombinase catalysate of rhodobacter azotoformans uses lycopene as the main pigment component and provides a new enzyme source for producing lycopene.

Description

field of invention [0001] The invention relates to a phytoene dehydrogenase gene, in particular to a phytoene dehydrogenase gene for efficiently synthesizing lycopene and its application, belonging to the technical field of genetic engineering. technical background [0002] Lycopene is currently the strongest antioxidant found in nature. It can efficiently quench singlet oxygen and scavenge free radicals. Its singlet oxygen quenching rate constant is 100 times that of vitamin E. Therefore, lycopene has important physiological functions , such as anti-cancer, anti-aging, improving immunity, etc. [0003] At present, commercial lycopene is mainly obtained from tomatoes. This method requires a large amount of tomatoes to ensure the continuity and low cost of industrial production. Moreover, tomatoes are inconvenient to transport, and their acquisition is greatly affected by climatic conditions. Due to the deterioration of the environment and the improvement of people's health ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/70A61K31/01A23L1/30C12P5/02C12R1/19C12R1/01
Inventor 肖敏张金华卢丽丽
Owner SHANDONG UNIV
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