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FAD-dependent oxidase Ma-1 in anabolic pathway of Diels-Alder type adduct and application of FAD-dependent oxidase Ma-1

A technology of adducts and compounds, applied in FAD-dependent oxidase Ma-1 and application fields, can solve problems such as unidentified

Active Publication Date: 2020-09-29
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

However, none of the above oxidases and [4+2]cyclases have been identified so far

Method used

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  • FAD-dependent oxidase Ma-1 in anabolic pathway of Diels-Alder type adduct and application of FAD-dependent oxidase Ma-1
  • FAD-dependent oxidase Ma-1 in anabolic pathway of Diels-Alder type adduct and application of FAD-dependent oxidase Ma-1
  • FAD-dependent oxidase Ma-1 in anabolic pathway of Diels-Alder type adduct and application of FAD-dependent oxidase Ma-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1, Screening and cloning of FAD-dependent oxidase gene in D-A adduct synthetic metabolic pathway

[0086] 1. Screening of FAD-dependent oxidase genes in the synthetic metabolic pathway of D-A adducts

[0087] The mulberry (Morus alba L.) transcriptome database constructed in this experiment was searched with Cannabis tetrahydroxycannabinate synthase (THCA), and the FAD-dependent oxidase genes with lower E-Value and higher FPKM were selected for analysis.

[0088] 2. Cloning of FAD-dependent oxidase gene in mulberry

[0089] 1. Take the mulberry suspension culture cells grown to 14 days, use the Trizol method to extract the total RNA of the mulberry, and use the 5' RACE kit of invitrogen to amplify the 5' end of the FAD-dependent oxidase gene to obtain the full-length sequence of the candidate gene.

[0090] 2. Cloning and sequencing of full-length cDNA

[0091] Splice the 5' RACE results and the 3' ends in the transcriptome database, search the ORF region, and...

Embodiment 2

[0099] Example 2, FAD-dependent oxidase gene eukaryotic expression and functional analysis

[0100] 1. Construction of yeast expression vector

[0101] According to the nucleotide sequences of the Ma-1 gene (SEQ ID No.1) and the Ma-2 gene (SEQ ID No.2), two pairs of primers Ma-1 with EcoR I and Not I restriction sites were designed respectively -F2 / Ma-1-R2 and Ma-2-F2 / Ma-2-R2.

[0102] Ma-1-F2: 5'-ATCCTACGTA GAATTC AAAAATGTCT-AAGTACTTTTCATTATCTT CGTCATTTG-3';

[0103] Ma-1-R2: 5'-AATTAATTC GCGGCCGC TTAATGATGATGATGATGATG-CACAAGAAGAGATGGAATGC-3'.

[0104] Ma-2-F2: 5'-ATCCTACGTA GAATTC AAAAATGTCT-CAGTACTTTTTCCTTCCCTTCAT-3';

[0105] Ma-2-R2: 5'-AATTAATTC GCGGCCGC TCAATGATGATGATGATGATG-CATTGCTGAATGTAGAGG-3'.

[0106] Using the recombinant vector pGEM-T-Ma-1 obtained in Example 1 as a template, using Ma-1-F2 / Ma-1-R2 as primers for PCR amplification, and then using restriction enzymes EcoR I and Not I double enzymes The amplified product was cut, recovered from the gel,...

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Abstract

The invention discloses an FAD-dependent oxidase Ma-1 in an anabolic pathway of a Diels-Alder type adduct and an application of the FAD-dependent oxidase Ma-1. The invention provides the following proteins: an Ma-1 protein as shown in SEQ ID No. 3 and / or an Ma-2 protein as shown in SEQ ID No. 4. Ma-1 and Ma-2 have FAD-dependent oxidase characteristic structural domains, the Ma-1 can catalyze isopentenyl side chains of isopentenyl phenolic compounds to be dehydrogenated and rearranged to form dienes, and catalyze the isopentenyl side chains of the isopentenyl phenolic compounds to react with isopentenyl ortho-hydroxyl to form a ring after being dehydrogenated; and the Ma-2 can catalyze [4+2] cyclization reaction between the diene and double bonds of chalcone compounds to form a Diels-Aldertype adduct. The FAD-dependent oxidase Ma-1 disclosed by the invention has important theoretical and practical significance for producing the Diels-Alder type adduct and researching an enzymatic Diels-Alder type [4+2] cyclization reaction mechanism.

Description

technical field [0001] The invention relates to the field of medicinal plant genetic engineering, in particular to FAD-dependent oxidase Ma-1 in the Diels-Alder type adduct synthetic metabolic pathway and its application. Background technique [0002] The formation of active ingredients of medicinal plants is the product of specific 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 and synthesis of medicinal plants with unique characteristics and broad application prospects has gradually become a research hotspot. The biosynthetic pathway and its regulatory mechanism provide a theoretical basis for the formation of the quality of medicinal materials, and at the same time bring a broad application space for the use of biotechnology to increase the content of target components or directly produce active components or intermediates. [0003] Mulberry is a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12P17/06C12P17/04
CPCC12N9/0004C12P17/06C12P17/04
Inventor 王瑞杉戴均贵王升郭娟康传志黄璐琦
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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