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Flavanone-6-hydroxylase (SbF6H) gene in scutellaria baicalensis georgi as well as product coded by same and application of gene

A technology of Scutellaria flavanone and baicalenone, applied in the field of biological and medicinal plant genetic engineering

Inactive Publication Date: 2013-10-30
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Before the present invention was published, there was no disclosure or report of the Scutellaria baicalensis gene and its amino acid sequence mentioned in this patent application

Method used

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  • Flavanone-6-hydroxylase (SbF6H) gene in scutellaria baicalensis georgi as well as product coded by same and application of gene
  • Flavanone-6-hydroxylase (SbF6H) gene in scutellaria baicalensis georgi as well as product coded by same and application of gene
  • Flavanone-6-hydroxylase (SbF6H) gene in scutellaria baicalensis georgi as well as product coded by same and application of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1, the construction of Scutellaria baicalensis full-length cDNA library

[0038] 1. Isolation and detection of Scutellaria baicalensis total RNA

[0039] Take 2 g of roots of Scutellaria baicalensis Georgi, quickly grind them into powder with liquid nitrogen in a mortar, and quickly transfer them to 10 mL of extraction buffer (CTAB (W / V) 2%, Tris-HCl (pH 8 .0)100mmol·L -1 , EDTA 25mmol·L -1 , NaCl 2.0mol·L -1 , PVP40 2%, 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 10M LiCl 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 (SDS 0.5%, NaCl 1mol L -1 , Tris-HCl (pH8.0) 10mmol L -1 , EDTA 1mmol·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 t...

Embodiment 2

[0042] Example 2: Cloning of Scutellaria baicalensis-related genes

[0043] 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: 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. After each reagent is added, shake it on the centrifuge to make it sink to the bottom, and place it on the PCR instrument. 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 thin-walled PCR tube, take 7ul of the PCR product and add 3ul of bromine Finland to run electrophoresis, take...

Embodiment 3

[0045] Embodiment 3, the bioinformatics analysis of SbF6H gene

[0046] The length of the full-length cDNA of the Scutellaria baicalensis flavanone-6-hydroxylase (SbF6H) gene involved in the present invention is 1002bp, and the detailed sequence is shown in sequence 1 in the sequence list, wherein the open reading frame is located at 109-921bp. 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 databases using BLAST program. The gene has high homology with F3H in other species at the amino acid level, and has a typical 2OG-Fe(II) oxygenase superfamily and flavanone-3-hydroxylase domain. Such as figure 2 and 3 .

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Abstract

The invention discloses a flavanone-6-hydroxylase (SbF6H) gene in scutellaria baicalensis georgi as well as a protease coded by the same and application of the gene. The gene is cloned from scutellaria baicalensis georgi by constructing a full-length cDNA (complementary deoxyribonucleic acid) library for the first time and fills in the gap in separating and cloning the flavanone-6-hydroxylase (SbF6H) gene from the traditional Chinese herbal medicine scutellaria baicalensis georgi in China. The flavanone-6-hydroxylase (SbF6H) gene has nucleotide sequences shown in SEQ ID NO.1 or homologous sequences obtained through addition, substitution, insertion or deletion of one or a plurality of nucleotides or nucleotide sequences derived from alleles of the gene and the gene. A protein coded by the gene has amino acid sequences shown in SEQ ID NO.2 or homologous sequences obtained through addition, substitution, insertion or deletion of one or a plurality of amino acids. The flavanone-6-hydroxylase (SbF6H) gene provided by the invention can increase the content of baicalein in scutellaria baicalensis georgi through biotechnology, is conductive to improvement of the quality of the medicinal scutellaria baicalensis georgi, simultaneously can carry out variety breeding and has good application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and mainly relates to the use of a full-length cDNA library to clone the baicalin flavanone-6-hydroxylase gene and its encoded product and its application, in particular to the biosynthesis of the flavonoid enzyme gene and its encoded product with pharmacologically active ingredients The invention and application belong to the field of genetic engineering of medicinal plants. Background technique [0002] The formation of active ingredients (secondary metabolites) of medicinal plants 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 me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/63C12N5/10A01H5/00
Inventor 黄璐琦袁媛胡国强伍翀汪周勇
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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