Lycium chinese Miller isopentenyl pyrophosphate isomerase (LmIpi) gene, recombinant vector comprising gene, host cell comprising gene, and application of gene

A technology of Lycium barbarum isoprene pyrophosphate and Lycium barbarum isoprene pyrophosphate, which is applied in the fields of molecular biology and biology

Inactive Publication Date: 2012-06-27
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 chinese Miller isopentenyl pyrophosphate isomerase (LmIpi) gene, recombinant vector comprising gene, host cell comprising gene, and application of gene
  • Lycium chinese Miller isopentenyl pyrophosphate isomerase (LmIpi) gene, recombinant vector comprising gene, host cell comprising gene, and application of gene
  • Lycium chinese Miller isopentenyl pyrophosphate isomerase (LmIpi) gene, recombinant vector comprising gene, host cell comprising gene, and application of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Cloning of Lycium barbarum isoprene pyrophosphate isomerase gene LmIpi:

[0041] Using the RNeasy Plant Mini Kit (QIAGEN, German) kit, Total RNA was extracted from 100 mg of fresh Lycium barbarum leaves, and the upstream primer was designed according to the Unigene sequence of the transcriptome. The upstream primer of the LmIpi gene was: LmIpiF: 5'-ATGTCGCTGACTACTGCACCTTC-3'. 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′RACEAdaptor primers for reverse transcription reaction to synthesize 1st Strand cDNA. The reaction system is as follows:

[0042] RNA: 2 μl

[0043] 3′RACE Adapter: 1μl

[0044] 5×M-MLV Buffer: 2μl

[0045] dNTP Mixture: 1μl

[0046] RNase Inhibitor: 0.25μl

[0047] Reverse Transcriptase M-MLV: 0.25μl

[0048] RNase Free dH2O: 3.5 μl

[0049] Reaction conditions: 42°C, 60min; 70°C, 15min.

[0050] ②According to the upstream primer of the ...

Embodiment 2

[0061] Construction process of cloning vector pMD18-T-LmIpi

[0062] The LmIpi gene shown in the sequence listing is connected to the pMD18-T vector, and the reaction system is as follows:

[0063] Target PCR fragment: 4 μl

[0064] pMD18-T vector: 1 μl

[0065] Solution I: 5μl

[0066] Reaction conditions: 16°C, 30min. The ligation product was transformed into E-Coli.TOP10 and spread on LB plates containing ampicillin. Use the target gene as a primer to perform PCR (reaction conditions: 94°C, 3min; 94°C, 30sec; 54°C, 30sec; 72°C, 50sec; 72°C, 10min, 30 cycles.) to obtain PCR products with correct electrophoresis bands. Then it was sent to Huada Gene Company for sequencing, and the sequencing results were blasted in NCBI, indicating that it was the gene. The schematic diagram of the carrier is as figure 1 .

Embodiment 3

[0068] Construction process of Escherichia coli expression vector pET28a-LmIpi

[0069] First, use pMD18-T-LmIpi as a template, and P3 and P4 as upstream and downstream primers to amplify the LmIpi fragment respectively. The reaction conditions are 94°C, 3min; 94°C, 30sec; 55°C, 30sec; 72°C, 1min50sec; 72°C , 10min, 30 cycles. A BamHI restriction site (GGATCC) was introduced into P3, and an XhoI restriction site (CTCGAG) was introduced into P4. Then, the PCR product and the pET28a plasmid were digested by BamHI and XhoI respectively, and the digested products were ligated: 16°C, 16hrs, ligation (2μl 10×T4buffer, 0.5μl T4 DNA ligase, 5μl carrier DNA, 7.5μl exogenous DNA, 5 μl ddH2O). The ligation product was transformed into E coli.Top10, and spread on the LB plate containing Amp. Perform PCR with the target gene as a primer to obtain a 882bp product, and identify the target band by enzyme digestion, if 2 is the enzyme digestion electrophoresis pattern. Finally, it was sent...

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Abstract

The invention relates to an LmIpi gene, a recombinant vector comprising the gene, a host cell comprising the gene, and an application of the gene. A nucleotide sequence for forming the gene is selected from one of the following nucleotide sequences: 1, a nucleotide sequence represented by SEQ ID NO.1; and 2, a nucleotide sequence which is obtained by adding, substituting, inserting or deleting one or more nucleotides to the nucleotide sequence represented by the SEQ ID NO.1 and has a 70% homology with the nucleotide sequence represented by the SEQ ID NO.1, or a nucleotide sequence of an allele and a derivative of the gene. Total RNAs are extracted from fresh Lm leaves, and the LmIpi gene is cloned through a 3'RACE technology to obtain a complete gene sequence which has bps with the number of 882. An Escherichia coli expression vector pET28a-LmIpi is constructed, an Escherichia coli heterogenous expression system is applied to identify the activity of an enzyme coding the obtained cloned LmIpi gene, and the LmIpi gene can catalyze the transformation of isopentenyl pyrophosphate into dimethylallyl pyrophosphate, makes the metabolism flow to the downstream, and can improve the content of beta-carotenoid.

Description

technical field [0001] The invention relates to the cloning, recombination and application of isoprene pyrophosphate isomerase gene LmIpi in Lycium chinense Miller, belonging to the field of molecular biology and biotechnology. Background technique [0002] Lycium barbarum is rich in carotenoids. It is reported that the β-carotene content of Ningxia wolfberry fresh fruit is 19.65mg / 100g, and the total carotenoid content is 295.27mg / 100g, which is 3 to 4 times that of carrots. Carotenoids are mushroom compounds formed by the polymerization of isoprenoids, and the precursor for biosynthesis is isoprenyl pyrophosphate (IPP) with 5 carbons. The vast majority of carotenoids are hydrocarbon tetraterpenoids (carotene) and their oxygen-containing derivatives containing 40 carbons, which are polymerized by 8 IPP units. In the carotenoid biosynthetic pathway, Isopentenyl pyrophosphate isomerase (IPI) catalyzes the conversion of isoprene pyrophosphate into dimethylpropylene pyrophosph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N15/70C12N9/90C12N1/21C12R1/19
Inventor 季静王罡李招娣吴疆关春峰金超
Owner TIANJIN UNIV
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