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Lycium chinense miller lycopene beta-cyclase gene, recombinant vector containing gene, host cell and application

A technology of lycopene and recombinant carrier, which is applied in the direction of introducing foreign genetic material and modifying cells, application, genetic engineering, etc., to meet nutritional needs and increase production

Inactive Publication Date: 2012-01-18
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] With the continuous discovery of the medicinal value and health care function of carotenoids, the demand for the types and yields of carotenoids will also increase. However, carotenoids are difficult to synthesize by chemical methods

Method used

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  • Lycium chinense miller lycopene beta-cyclase gene, recombinant vector containing gene, host cell and application
  • Lycium chinense miller lycopene beta-cyclase gene, recombinant vector containing gene, host cell and application
  • Lycium chinense miller lycopene beta-cyclase gene, recombinant vector containing gene, host cell and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] For the experimental methods that do not specify specific conditions in the examples, usually follow the conventional conditions and the conditions described in the manual, or follow the conditions suggested by the manufacturer.

[0051] Cloning of lycopene β-cyclase gene LmLycB from Lycium barbarum:

[0052] With RNeasy Plant Mini Kit (QIAGEN, German) kit, Total RNA was extracted from 100 mg of fresh Lycium barbarum leaves. Amino Acid Sequence A degenerate primer P1 was designed, such as the sequence shown in SEQ ID NO.3 in the sequence listing. The complete gene sequence was amplified using the 3′-FULL RACE Core Set Ver.2.0 (TaKaRa, Japan) kit. Specific steps: ① Using Total RNA as a template, use 3′RACE Adapter primers for reverse transcription reaction to synthesize 1st Strand cDNA. The reaction system is as follows:

[0053] RNA: 2 μl

[0054] 3′ RACE Adaptor: 1 μl

[0055] 5×M-MLV Buffer: 2μl

[0056] dNTP Mixture: 1 μl

[0057] RNase Inhibitor: 0.25μl

[00...

Embodiment 2

[0072] Construction process of intermediate vector pBS-T-LmLycB

[0073] The LmLycB gene shown in SEQ ID NO.1 was connected to the pBS-T vector, and the reaction system was as follows:

[0074] Target PCR fragment: 3μl

[0075] pBS-T vector: 1 μl

[0076] 2×T4DNA Rapid Ligation Buffer: 5μl

[0077] T4DNA Ligase: 1μl

[0078] Reaction conditions: 23°C, 10 min. The ligation product was transformed into E-Coli.TOP10 and spread on LB plates containing ampicillin. PCR was carried out with the target gene as a primer (reaction conditions: 94°C, 3min; 94°C, 30sec; 54°C, 30sec; 72°C, 2min30sec; 72°C, 10min, 30 cycles.) to obtain a 1800bp product, and the extracted plasmids were subjected to BamHI Double enzyme digestion with PstI: 37°C, 16hrs, digestion (15μl plasmid DNA, 3μl R buffer, 0.5μl BamHI exonuclease, 0.5μl PstI exonuclease, 13μl ddH2O) to obtain the target band, and finally sent to Huada Gene Sequencing Company Sequencing results showed that the vector was constructed ...

Embodiment 3

[0080] Construction process of Escherichia coli expression vector pMON-LmLycB

[0081] First, use pBS-T-LmLycB as a template, and P3 and P4 as upstream and downstream primers to amplify the LmLycB fragment respectively. The reaction conditions are 94°C, 3min; 94°C, 30sec; 55°C, 30sec; 72°C, 2min30sec; 72°C , 10min, 30 cycles. A BamHI restriction site (GGATCC) and a base A were introduced into P3, and a SalI restriction site (GTCGAC) was introduced into P4. Then, the PCR product and the pMON38201 plasmid were digested with BamHI and SalI respectively, and the digested products were ligated: 16°C, 16hrs, ligation (2μl 10×T4buffer, 0.5μl T4DNA ligase, 5μl carrier DNA, 7.5μl exogenous DNA, 5 μl ddH 2 O). The ligation product was transformed into Ecoli.DH5α, and spread on the LB plate containing Amp, such as figure 1 Shown is the schematic diagram of the constructed pMON-LmLycB vector. PCR was performed with the target gene as a primer to obtain a 1500bp product, and the targe...

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Abstract

The invention discloses a lycium chinense miller lycopene beta-cyclase gene, a recombinant vector containing the gene, a host cell and application. Total RNA (Ribonucleic Acid) of fresh lycium chinense miller leaf is extracted, and lycopene beta-cyclase gene LmLycB is cloned by using 3' RACE (Rapid Amplification of cDNA Ends) technology to obtain a complete gene sequence of 1,506bp. An Escherichia coli expression vector pMON-LmLycB is constructed; an escherichia coli heterogenous expression system is applied; the activity of cloned LmLycB gene-coded enzyme is identified; and two beta-lonone rings can be added at the tail end of lycopene by LmLycB to produce beta-carotene. A maize inbred line immature embryo genetic transformation system is optimized; a plant expression vector pCAMBIA2300-LmLycB-Bar containing a target gene is transformed into maize callus to finally obtain a transgenic positive plant; through HPLC (High Performance Liquid Chromatography) detection, the total carotinoid content of transgenic maize leaf is 165.70mug / g FW and is 12.76 percent greater than that of a wild maize; and beta-carotene content is 80.17mug / g FW and is 54.83 percent greater than that of the wild maize.

Description

technical field [0001] The invention relates to a wolfberry (Lycium chinense Miller) lycopene beta-cyclase gene, a recombinant vector including the gene, a host cell and application. Background technique [0002] In the biosynthetic pathway of plant carotenoids, the cyclization reaction of lycopene is the most important branch point. Symmetrical cyclization forms two β-ionone rings at both ends of linear lycopene, and then produces β-carotenoids white. Lycopene β-cyclase (LycB) is an important rate-limiting enzyme in the carotenoid biosynthesis pathway, which catalyzes the conversion of lycopene to β-carotene [Bartley GE, Scolnik PA, Plant carotenoids: Pigments for photoprotection, visual attraction, and human health, Plant Cell, 1995, 7: 1027-1038]. So far, LycB genes have been isolated from plants such as soybean, tomato, pepper, Arabidopsis, corn, daffodil, and wild tobacco. [0003] Carotenoids are C40 hydrocarbons, fat-soluble pigments widely present in plants, some ...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/63C12N15/70C12N15/82C12N1/21C12N5/10A01H5/00
Inventor 季静王罡马然关春峰
Owner TIANJIN UNIV
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