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Scutellaria baicalensis Georgi caffeoyl-CoA-3-O-methyltransferase (SbCCOMT2) gene, and coding product and application thereof

A technology of methyltransferase and acyl coenzyme, which is applied in the genetic engineering of medicinal plants and the field of biology

Inactive Publication Date: 2012-05-23
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 publication or report of the baicalein acid enzyme gene and its amino acid sequence mentioned in this patent application

Method used

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  • Scutellaria baicalensis Georgi caffeoyl-CoA-3-O-methyltransferase (SbCCOMT2) gene, and coding product and application thereof
  • Scutellaria baicalensis Georgi caffeoyl-CoA-3-O-methyltransferase (SbCCOMT2) gene, and coding product and application thereof
  • Scutellaria baicalensis Georgi caffeoyl-CoA-3-O-methyltransferase (SbCCOMT2) gene, and coding product and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Construction of Scutellaria baicalensis full-length cDNA library

[0043] 1. Isolation and detection of total RNA from Scutellaria baicalensis

[0044] Take 2 g of Scutellaria baicalensis Georgi root, 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) preheated at 65°C. .0)100mmol·L -1 , EDTA 25m mol·L -1 , NaCl 2.0mol·L -1 , PVP40 2%, spermidine 0.5g / L, mercaptoethanol 2%), fully shaken and mixed; extracted twice with an equal volume of chloroform, and centrifuged at 7500g for 15 minutes. Add 1 / 4 volume of 10M LiCl to the supernatant, mix well and place it at 4°C to precipitate overnight; centrifuge at 7500g for 20 minutes, use 500μL SSTE (SDS 0.5%, NaCl 1mol·L for the precipitation) -1 , Tris-HCl (pH8.0) 10mmol·L -1 , EDTA 1mmol·L -1 , dissolve at 65°C for 5 minutes. Extracted with an equal volume of chloroform, centrifuged at 13,000g for 5 minutes; adde...

Embodiment 2

[0050] Example 2: Cloning of Scutellaria baicalensis related genes

[0051]5000 single clones were randomly picked for colony PCR identification. Take an appropriate amount of PCR thin-walled tubes, put them on ice, and add 17.3 ul of sterilized water to each tube. Pick the monoclonal vitiligo with a sterilized 10ul small pipette tip into sterile water, shake and mix well. Add in order: Taq buffer 2.5μL, MgCl 2 (25mM) 1.8 μL, dNTP (2.5 mM) 1 μL, M13+ primer (10 pmol) 1 μL, M13- primer (10 pmol) 1 μL, Taq enzyme 0.4 uL. After all the reagents have been added, shake them on the centrifuge to make them sink to the bottom and place them on the PCR machine. PCR reaction conditions were pre-denaturation at 94°C for 5 minutes, 94°C for 40 seconds, 54°C for 40 seconds, 72°C for 4 minutes, and after 35 cycles, 72°C extension for 10 minutes, and storage at 4°C. After the PCR reaction entered 4°C, remove the PCR thin-walled tube, take 7ul of PCR product and add 3ul of bromine to run ...

Embodiment 3

[0053] Example 3. Bioinformatics analysis of SbCCOMT2 gene

[0054] The length of the full-length cDNA of the Scutellaria baicalensis oxymethyltransferase gene gene involved in the present invention is 873 bp, and the detailed sequence is shown in Sequence 1 in the sequence table, wherein the open reading frame is located at 59-818 bp. The full-length cDNA sequence of Scutellaria baicalensis 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 CCOMT in other species at the amino acid level, and has a typical caffeoyl-CoA O-methyltransferase domain. like figure 2 and 3 .

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Abstract

The invention discloses a Scutellaria baicalensis Georgi caffeoyl-CoA-3-O-methyltransferase (CCOMT) gene, and a protease coded by the gene and application thereof. The gene is for the first time cloned from Scutellaria baicalensis Georgi by constructing overall length cDNA library and fills the blank of separating and cloning caffeoyl-CoA-3-O-methyltransferase from traditional Chinese medicine Scutellaria baicalensis Georgi. The caffeoyl-CoA-3-O-methyltransferase provided by the invention has a nucleotide sequence shown as a SEQ ID NO.1 or a homologous sequence by adding, substituting, inserting or deleting one or more nucleotides, or an allele thereof, and a nucleotide sequence derived therefrom. The protein coded by the gene has an amino acid sequence shown as a SEQ ID NO.2 or a homologous sequence by adding, substituting, inserting or deleting one or more nucleotides. The caffeoyl-CoA-3-O-methyltransferase provided by the invention can increase a content of phenylalanine metabolite in Scutellaria baicalensis Georgi through biotechnology, so as to facilitate quality improvement of the medicinal material Scutellaria baicalensis Georgi; besides the caffeoyl-CoA-3-O-methyltransferase can be used for variety breeding and has good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, mainly relates to the use of a full-length cDNA library to clone an oxymethyltransferase gene and its coded product and application, in particular to the biosynthesis of a phenolic acid enzyme gene with pharmacologically active components, its coded product and application. The field of medicinal plant genetic engineering. Background technique [0002] The formation of active ingredients (secondary metabolites) 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 secondary metabolism and synthesis of medicinal plants with unique characteristics and broad application prospects has gradually become a research hotspot. It provides a theoretical basis for the biosynthesis pathway and its regulation mechanism, and explains the formation of medicina...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A01H5/00
Inventor 袁媛黄璐琦杨兆春胡国强秦双双
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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