Lonicera japonica chalcone isomerase (LjCHI) gene, LjCHI gene coded protein and their application

A technology of chalcone isomerase and honeysuckle, applied in genetic engineering of medicinal plants, biological field

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

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Problems solved by technology

Before the present invention is published, there is no disclosure or report of the honeysuckle flavonoid enzyme gene and its amino acid sequence mentioned in this patent application

Method used

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  • Lonicera japonica chalcone isomerase (LjCHI) gene, LjCHI gene coded protein and their application
  • Lonicera japonica chalcone isomerase (LjCHI) gene, LjCHI gene coded protein and their application
  • Lonicera japonica chalcone isomerase (LjCHI) gene, LjCHI gene coded protein and their application

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Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1, the construction of honeysuckle full-length cDNA library

[0040] 1. Isolation and detection of honeysuckle total RNA

[0041] Take 2 g of honeysuckle (Lonicera japonica Thunb) flower buds, 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 , 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 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 to the supernata...

Embodiment 2

[0044] Embodiment 2: Cloning of honeysuckle-related genes

[0045] 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 a pictur...

Embodiment 3

[0047] Example 3, bioinformatics analysis of LjCHI gene

[0048] The length of the full-length cDNA of the honeysuckle chalcone isomerase gene involved in the present invention is 1102bp, and the detailed sequence is shown in sequence 1 in the sequence list, wherein the open reading frame is located at 208-930bp. The full-length cDNA sequence of Lonicerae japonica 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 CHI in other species at the amino acid level ( figure 2 and 3 ).

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Abstract

The invention discloses a lonicera japonica chalcone isomerase (LjCHI) gene, a LjCHI gene coded protein and their application. The LjCHI gene is obtained from a lonicera japonica cDNA library for the first time and thus in China, separation and cloning of a chalcone isomerase gene from a traditional Chinese medicine lonicera japonica are realized. The LjCHI gene provided by the invention has a nucleotide sequence of SEG ID NO.1, a homologous nucleotide sequence prepared from the nucleotide sequence of SEG ID NO.1 through addition, replacement, insertion or deletion of one or more nucleotides,or a nucleotide sequence of an allele or an allele derivative. The LjCHI gene coded protein has an amino acid sequence of SEQ ID NO.2, or a homologous amino acid sequence prepared from the amino acidsequence of SEQ ID NO.2 through addition, replacement, insertion or deletion of one or more amino acids. The LjCHI gene can improve lonicera japonica flavonoid content of lonicera japonica flavonoid active components through the biotechnology, is conducive to lonicera japonica medicinal material quality improvement, realizes selective breeding, and has a good application prospect.

Description

technical field [0001] The invention belongs to the field of biological technology, mainly relates to obtaining chalcone isomerase gene and its coded product by constructing honeysuckle cDNA library technology, and belongs 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 mechanism and explains the formation of the quality of medicinal materials, and at the same time brings a broad application space for the use of biotechnology to increase the con...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/61C12N9/90C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A01H5/00
Inventor 黄璐琦秦双双袁媛
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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