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Paeonia lactiflora 2-C-methyl-D-erythritol-4-cyclodiphaophate synthase (PLIspF) gene, and coded product and application thereof

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

Inactive Publication Date: 2014-02-12
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Before the present invention was published, there was no disclosure or report of the Paeoniae 2-methyl-D-erythritol-2,4-cyclic diphosphate synthase gene and its amino acid sequence mentioned in this patent application

Method used

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  • Paeonia lactiflora 2-C-methyl-D-erythritol-4-cyclodiphaophate synthase (PLIspF) gene, and coded product and application thereof
  • Paeonia lactiflora 2-C-methyl-D-erythritol-4-cyclodiphaophate synthase (PLIspF) gene, and coded product and application thereof
  • Paeonia lactiflora 2-C-methyl-D-erythritol-4-cyclodiphaophate synthase (PLIspF) gene, and coded product and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1. Construction of peony cDNA library

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

[0020] Take 2g of the root of Paeonia lactiflora, quickly grind it into powder with liquid nitrogen in a mortar, and quickly transfer it to 65℃ preheated 10mL extraction buffer (CTAB(W / V)2%, Tris-HCl(pH8) .0)100mmol·L -1 , EDTA 25mmol·L -1 , NaCl 2.0mol·L -1 , PVP402%, spermidine 0.5g / L, mercaptoethanol 2%), shake and mix thoroughly; extract twice with equal volume of chloroform, centrifuge at 7500g for 15 minutes. Add 1 / 4 volume of 10M LiCl to the supernatant, mix well and place it for precipitation overnight at 4°C; centrifuge at 7500g for 20 minutes, and use 500μL SSTE (SDS 0.5%, NaCl 1mol·L) for precipitation -1 , Tris-HCl (pH8.0) 10mmol·L -1 , EDTA 1mmol·L -1 , Dissolve at 65°C for 5 minutes. Extract with an equal volume of chloroform, centrifuge at 13000g for 5 minutes; add 2 volumes of absolute ethanol to the supernatant, and place at -70°C for 2 hours; centrifug...

Embodiment 2

[0023] Example 2: Cloning of peony 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 pipette tip to pick up the single leukoplakia into sterilized water, shake and mix. Add sequentially: Taq buffer 2.5μL, MgCl 2 (25mM) 1.8μL, dNTP (2.5mM) 1μL, M13+ primer (10pmol) 1μL, M13-primer (10pmol) 1μL, Taq enzyme 0.4μL. PCR reaction conditions were 94°C pre-denaturation for 5 minutes, 94°C for 40 seconds, 54°C for 40 seconds, 72°C for 4 minutes, after 35 cycles, 72°C extension for 10 minutes, and 4°C storage. After the PCR reaction enters 4°C, remove the PCR thin-walled tube, take 7ul PCR product and add 3ul bromofin for agarose gel electrophoresis, take a picture after half an hour, observe the gel image, and roughly identify the size and size of the insert based on the gel image Fragment rate. The si...

Embodiment 3

[0025] Example 3 Bioinformatics analysis of PLIspF gene

[0026] The length of the full-length cDNA of the Paeonia lactiflora 2-methyl-D-erythritol-2,4-cyclodiphosphate synthase gene involved in the present invention is 696 bp, and the detailed sequence is shown in sequence 1 in the sequence table, where the open reading frame is located 1-696bp. The full-length cDNA sequence of Paeonia lactiflora was searched for nucleotide homology in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translation+PDB+Swissprot+Superdate+PIR database 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-cyclodiphosphate synthase, IspF) domain. Such as figure 1 .

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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.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and mainly relates to the cloning of 2-methyl-D-erythritol-2,4-cyclodiphosphate synthase gene and its coded product and application by using peony cDNA library, in particular to biosynthesis of pharmacologically active ingredients The organic acid and terpenoid enzyme genes and their encoded products and applications belong to the field of medicinal plant genetic engineering. Background technique [0002] The formation of active ingredients of medicinal plants is the product of a unique gene group in the secondary metabolic pathway of plants. With the extensive and in-depth study of plant functional genomes, the study of functional genes related to the secondary metabolism and synthesis of medicinal plants with unique characteristics and broad application prospects has gradually become a research hotspot. The cloning of these genes will analyze the effective ingredients of medicinal plants The bi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10
Inventor 黄璐琦袁媛汪周勇
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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