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Wolfberry lycopene epsilon-cyclase gene and recombinant vector comprising gene

A technology of lycopene and recombinant vector, which is applied in the fields of genetic engineering, plant gene improvement, recombinant DNA technology, etc.

Inactive Publication Date: 2016-01-20
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] With the continuous discovery of the medicinal value and health care 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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  • Wolfberry lycopene epsilon-cyclase gene and recombinant vector comprising gene
  • Wolfberry lycopene epsilon-cyclase gene and recombinant vector comprising gene
  • Wolfberry lycopene epsilon-cyclase gene and recombinant vector comprising gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Lycium barbarum lycopene ε-cyclase gene Lm Clone of LcyE:

[0033] Using RNeasyPlantMiniKit (QIAGEN, German) kit, extract TotalRNA from 100mg fresh wolfberry leaves (Ningxia wolfberry), design the upstream primers according to the Unigene sequence of the transcriptome, Lm The upstream primers of LcyE gene are: Lm LcyEF: 5 ’ -ATGGAATGTGTTGGAGTTCAAAATT-3 ’ . The complete gene sequence was amplified using 3'-FULLRACECoreSetVer.2.0 (TaKaRa, Japan) kit. Specific steps: ① Use TotalRNA as a template and use 3'RACEAdaptor primers for reverse transcription reaction to synthesize 1stStrandcDNA. The reaction system is as follows:

[0034] RNA: 2μl

[0035] 3'RACEAdaptor: 1μl

[0036] 5×M-MLVBuffer: 2μl

[0037] dNTPMixture: 1μl

[0038] RNaseInhibitor: 0.25μl

[0039] ReverseTranscriptaseM-MLV: 0.25μl

[0040] RNaseFreeddH 2 O: 3.5μl

[0041] Reaction conditions: 42°C, 60min; 70°C, 15min.

[0042] ②According to the upstream primer of the gene and the downstream primer 3'RACEoutprimer prov...

Embodiment 2

[0053] Cloning vector pMD-18- Lm The construction process of LcyE

[0054] As shown in the sequence table LmLm The LcyE gene is connected to the pMD-18 vector, and the reaction system is as follows:

[0055] Target PCR fragment: 4μl

[0056] pMD-18 vector: 1μl

[0057] SolutionI: 5μl

[0058] Reaction conditions: 16°C, 30 min. Conversion of ligation products E-Coli. TOP10, spread on the LB plate containing ampicillin. Perform PCR with the target gene as primer (reaction conditions: 94°C, 3min; 94°C, 30sec; 54°C, 30sec; 72°C, 2min30sec; 72°C, 10min, 30 cycles.) The PCR product was obtained, and the electrophoresis band was correct. Then it was sent to Huada Gene Sequencing Company for sequencing, and the sequencing result was Blasted in NCBI, indicating that it was the gene.

Embodiment 3

[0060] Escherichia coli expression vector pET28a -Lm The construction process of LcyE

[0061] First, take pMD-18- Lm LcyE is the template, using the upstream primer F2 shown in the sequence SEQIDNO.5 and the downstream primer R2 shown in the sequence SEQIDNO.6 as primers for amplification Lm The reaction system of LcyE fragment is:

[0062] ddH 2 O18.25μl

[0063] Taq-plusreactionBuffer2.5μl

[0064] dNTP2.0μl

[0065] PrimerF20.5μl

[0066] PrimerR20.5μl

[0067] Template1μl

[0068] Enzyme 0.25μl

[0069] The reaction conditions were 94°C, 4min; 94°C, 30sec; 55°C, 30sec; 72°C, 2min; 72°C, 10min, 30 cycles. The PCR product and pET28a plasmid were Noc I and Xho I double digestion, connect the two digestion products, the reaction system is:

[0070] 5×T4buffer2μl

[0071] pET28a (after digestion and purification) 5μl

[0072] Lm LcyE (after digestion and purification) 1μl

[0073] T4Enzyme 1μl

[0074] ddH 2 O1μl

[0075] The reaction conditions are 16°C, 16hrs.

[0076] The ligation produc...

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Abstract

The invention relates to a wolfberry lycopene epsilon-cyclase gene and a recombinant vector comprising the gene. The gene is selected from one of the following nucleotide sequences: 1) a nucleotide sequence shown in a sequence SEQ ID NO.1; and 2) a homologous sequence or an allelic gene thereof and a derivative nucleotide sequence thereof in which 70% of one or more nucleotides are added, replaced, inserted or deleted in the nucleotide sequence shown in the sequence SEQ ID NO. 1. By extracting the total RNA of fresh wolfberry leaves, a wolfberry lycopene epsilon-cyclase gene Lm LcyE is cloned by using a 3'RACE technology, and an obtained complete genome sequence is 1569. An escherichia coli expression vector pET28a-LmLcyE is constructed, the enzymatic activity of cloned wolfberry isopentenyl pyrophosphate isomerase gene Lm LcyE encoding is identified by using an escherichia coli heterologous expression system, and the wolfberry lycopene epsilon-cyclase gene Lm LcyE can catalyze lycopene for cyclizing the lycopene into delta carotene, so that metabolism flows downstream, thereby improving the content of alpha-carotenoid.

Description

Technical field [0001] The invention relates to a Lycium barbarum lycopene ε-cyclase gene and a recombinant vector containing the gene, [0002] Specifically, wolfberry ( LyciumchinenseMiller ) Lycopene ε-cyclase gene Lm Clone of LcyE. Background technique [0003] The cyclization reaction of lycopene is an important branch point of carotene biosynthesis pathway in plants. Lycopeneε-cyclase (LcyE) can catalyze the formation of a δ-ring at one end of lycopene to generate δ-carotene. [0004] Carotenoids are C 40 Hydrocarbons, fat-soluble pigments widely found in plants, some photosynthetic bacteria and algae, mainly include carotenes (carotenes) and their oxidized derivatives xanthophylls (xanthophylls) two categories. Carotenoids play a vital role in plant photosynthesis. They are an indispensable structural component of the photosynthetic antenna and photoreaction center complex. They can also protect chlorophyll from photooxidation caused by strong light. Carotenoids are the pr...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N9/90C12N15/70C12N1/21
Inventor 季静王罡吴疆刁进进
Owner TIANJIN UNIV
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