Peony 2-methyl-d-erythritol-2,4-cyclic diphosphate synthase gene and its encoded product and application

A technology of cyclic diphosphate synthase and cyclic diphosphate, applied in the field of biological and medicinal plant genetic engineering, to achieve the effect of increasing the content of terpenoids

CN103571860BInactive Publication Date: 2016-03-23INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2016-03-23
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention discloses a PLIspF gene and a coded protein and application thereof. The gene is cloned from Paeonia lactiflora by constructing a cDNA library for the first time, so a gap in separation and cloning of a 2-C-methyl-D-erythritol-4-cyclodiphaophate synthase gene from the traditional Chinese medicine--Paeonia lactiflora is filled in. The PLIspF gene provided by the invention has a nucleotide sequence shown in SEQ ID NO. 1, or a homologous sequence obtained through addition, substitution, insertion or deletion of one or a plurality of nucleotides of the nucleotide sequence shown in SEQ ID NO. 1, or an allele of the gene or a nucleotide sequence derived from the allele. The protein coded by the gene has an amino acid sequence shown in SEQ ID NO. 2 or a homologous sequence obtained through addition, substitution, insertion or deletion of one or a plurality of amino acids of the amino acid sequence shown in SEQ ID NO. 2. The PLIspF gene can increase the content of metabolites of 2-phospho-4-(cytidine 5'-phospho)-2-C-methyl-D-erythritol in Paeonia lactiflora through biotechnology and has good application prospects in aspects like quality improvement of the medicinal material Paeonia lactiflora and synthetic biology of active components of Paeonia lactiflora.
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Description

technical field

[0001] The invention belongs to the field of biotechnology, and mainly relates to cloning 2-methyl-D-erythritol-2,4-cyclic diphosphate synthase gene and its coded product and application by using peony cDNA library, especially relates to biosynthesis of pharmacologically active ingredients The organic acid, terpenoid enzyme genes and their encoded products and applications belong to the field of genetic engineering of medicinal plants. Background technique

[0002] The formation of medicinal plant active ingredients is the product of a unique group of genes in plant secondary metabolic pathways. With the extensive and in-depth study of plant functional genomes, the study of functional genes related to secondary metabolism synthesis of medicinal plants with unique characteristics and broad application prospects has gradually become a research hotspot. It provides a theoretical basis for the biosynthetic pathway and its regulation mechanism and explains the fo...

Examples

Embodiment 1

[0018] Embodiment 1, the construction of peony cDNA library

[0019] 1. Isolation and detection of peony total RNA

[0020] Take 2 g of the root of Paeonia lactiflora, quickly grind it into powder with liquid nitrogen in a mortar, and quickly transfer it to 10 mL of extraction buffer (CTAB (W / V) 2%, Tris-HCl (pH 8. 0) 100mmol·L -1 , EDTA25mmol·L -1 , NaCl2.0mol L -1 , PVP402%, spermidine 0.5g / L, mercaptoethanol 2%), fully shake and mix; extract twice with equal volume of chloroform, and centrifuge at 7500g for 15 minutes. Add 1 / 4 volume of 10MLiCl to the supernatant, mix well, and place it at 4°C to precipitate overnight; centrifuge at 7500g for 20 minutes, and use 500 μL SSTE (SDS0.5%, NaCl1mol L -1 , Tris-HCl (pH8.0) 10mmol L -1 , EDTA1mmol·L -1 , dissolved at 65°C for 5 minutes. Extract with an equal volume of chloroform, centrifuge at 13,000g for 5 minutes; add 2 times the volume of absolute ethanol to the supernatant, and store at -70°C for 2 hours; centrifuge at 1...

Embodiment 2

[0023] Example 2: Cloning of Paeoniae officinalis-related genes

[0024] Randomly pick 5000 single clones for colony PCR identification. Take an appropriate amount of PCR thin-walled tubes, place them on ice, and add 17.3ul of sterilized water to each tube. Use a sterilized 10ul small tip to pick up the monoclonal white spot into sterilized water, shake and mix. Add in sequence: Taqbuffer 2.5μL, MgCl 2 (25mM) 1.8μL, dNTP (2.5mM) 1μL, M13+ primer (10pmol) 1μL, M13-primer (10pmol) 1μL, Taq enzyme 0.4μL. The PCR reaction conditions were 5 minutes of pre-denaturation at 94°C, 40 seconds at 94°C, 40 seconds at 54°C, 4 minutes at 72°C, 35 cycles of extension at 72°C for 10 minutes, and storage at 4°C. After the PCR reaction enters 4°C, remove the PCR thin-walled tube, take 7ul of PCR product and add 3ul of bromofinland for agarose gel electrophoresis, take a picture half an hour later, observe the gel map, and roughly identify the size of the insert fragment and small fragments a...

Embodiment 3

[0025] Embodiment 3, the bioinformatics analysis of PLIspF gene

[0026] The length of the full-length cDNA of the peony 2-methyl-D-erythritol-2,4-cyclic diphosphate synthase gene involved in the present invention is 696bp, and the detailed sequence is shown in sequence 1 in the sequence list, wherein the open reading frame is located at 1-696bp. The full-length cDNA sequence of Peony was searched for nucleotide homology in Non-redundantGenBank+EMBL+DDBJ+PDB and Non-redundantGenBankCDStranslation+PDB+Swissprot+Superdate+PIR databases using BLAST program. The gene has high homology with IspF in other species at the amino acid level, and has a typical MECDP_synthase (2-C-methyl-D-erythritol-2, 4-cyclodiphosphatesynthase, IspF) domain. Such as figure 1 .