Plant flavone methyltransferase protein as well as encoding gene and application thereof

A plant and encoding technology, applied in the field of protein IiOMT1 and its encoding gene and application, can solve the problem that the encoding gene has not been identified

Active Publication Date: 2020-04-28
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

Oxymethylation modification is catalyzed by methyltransferases. Oxymethyltransferases (O-methyltransferases) convert methyl groups from activated S-adenosyl-L-methionine (S-adenosyl-L-methionine ,SAM) donors are transferred to hydroxyl gro...

Method used

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  • Plant flavone methyltransferase protein as well as encoding gene and application thereof
  • Plant flavone methyltransferase protein as well as encoding gene and application thereof
  • Plant flavone methyltransferase protein as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1, the preparation of recombinant methyltransferase

[0072] One, the acquisition of the coding gene (IiOMT1 gene) derived from the methyltransferase of Isatis indigo

[0073] The inventors of the present invention have discovered the coding gene (IiOMT1 gene) of the methyltransferase in Isatis sativa through a large number of experiments. The nucleotide sequence of the IiOMT1 gene is shown as sequence 1 in the sequence listing. The IiOMT1 gene encodes the protein IiOMT1, and the amino acid sequence of the protein IiOMT1 is shown in Sequence 2 in the sequence listing.

[0074]The amino acid sequence of protein IiOMT1 was searched for homology in Non-redundantGenBank CDS translation+PDB+Swissprot database using BLAST program in NCBI database.

[0075] OMT phylogenetic tree (neighbor-joining method) see figure 1 . The results showed that the protein IiOMT1 had high homology with OMT in other species at the amino acid level and had a typical active site doma...

Embodiment 2

[0101] Example 2. Recombinant methyltransferase decomposes luteolin to generate golden eriodictin

[0102] The crude enzyme solution (induced by IPTG) of the recombinant bacteria A prepared in Example 1 was used as the solution to be tested.

[0103] 1. Prepare the reaction system. The reaction system consisted of 300 μL of the solution to be tested, 1.5 μL of luteolin solution (solvent in methanol, concentration 40 mM) and 3 μL S-adenosyl-L-methionine (solvent in methanol, concentration 32 mM).

[0104] 2. Take the reaction system prepared in step 1 and put it in a water bath at 30°C for 12 hours.

[0105] 3. Take the reaction system that has completed step 2, add 2 times the volume of pure methanol (for the purpose of terminating the reaction), and shake to extract.

[0106] 4. After completing step 3, centrifuge at 13000rpm for 15min and collect the supernatant.

[0107] 5. After completing step 4, take the supernatant, filter it with a filter membrane with a pore size o...

Embodiment 3

[0114] Example 3. Recombinant methyltransferase decomposes isoorientin to generate auroisogenistein

[0115] The 1.5 μL luteolin solution (solvent is methanol, concentration is 40mM) in embodiment 2 is replaced with 1.5 μL isorientin solution (solvent is methanol, concentration is 40mM); 1-5 are the same, and the filtrate B is obtained.

[0116] The filtrate B and the isogenistein standard were subjected to UPLC (ultra high performance liquid chromatography) analysis.

[0117] UPLC conditions are: chromatographic column: ACQUITY UPLC HSS T3 (2.1mm×50mm, 1.7μm); mobile phase: containing 0.1% formic acid-acetonitrile (A) and containing 0.1% formic acid-water (B); elution gradient: 10 -21%A(0-3min), 35-45%A(5-7min), 55-65%A(9-12min), 95%A(12.5-14min); flow rate: 0.4mL / min; detection wavelength The concentration is 350 nm, the column temperature is 40°C, and the injection volume is 2 μL.

[0118] Experimental results such as Figure 4 As shown, wherein IiOMT1+Isoorientin is th...

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Abstract

The invention relates to a plant flavone methyltransferase protein as well as an encoding gene and application thereof. Enzyme activity experiment of IiOMT1 protein encoded by an IiOMT1 gene shows that transgenosis experiment that the IiOMT1 gene is introduced into isatis indigotica shows that contents of 3'-O-methylflavones such as chrysoeriol and/or isoscoparin in a plant, namely transgenic isatis indigotica in which the IiOMT1 gene is expressed, can be increased when being compared with those in receptor isatis indigotica, it means that the IiOMT1 gene is a gene related to the contents of the 3'-O-methylflavones such as chrysoeriol and/or isoscoparin in the plant, the IiOMT1 gene and an encoding gene thereof can be adopted to increase the contents of the 3'-O-methylflavones such as chrysoeriol and/or isoscoparin in the plant.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to protein IiOMT1 and its coding gene and application. Background technique [0002] Woad (Isatis indigotica Fort.) is a biennial herb of the genus Isatis in the family Cruciferae. Isatis indigo root is Radix Isatidis, and its leaves are one of the sources of Daqingye. They are commonly used traditional Chinese medicines. They have the effects of clearing away heat and detoxification, cooling blood and relieving throat. They are often used clinically for influenza, mumps, and Japanese encephalitis. , acute and chronic hepatitis, herpes zoster and other diseases. Flavonoids mainly exist in the above-ground leaves of Isatis indica, which is one of the important material bases for its pharmacological activity. Modification reactions such as the degree of oxidation, hydroxylation, methylation, and glycosylation of the carbon chain of flavonoids enrich the structural diversity o...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/82C12P17/06A01H5/00A01H6/20
CPCC12N9/1007C12Y201/01C12P17/06C12N15/8243
Inventor 黄璐琦唐金富谭宇萍杨健郭娟曾雯
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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