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CYP76B74 protein participating in biological synthesis of alkannin as well as encoding gene and application thereof

A technology of shikonin and coding, which is applied in the fields of plant gene improvement, application, genetic engineering, etc., and can solve problems such as unobtained amino acid sequence of hydroquinone

Active Publication Date: 2018-05-22
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

But the amino acid sequence of hydroquinone 2-geranoquinone-3” hydroxylase has not been obtained so far

Method used

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  • CYP76B74 protein participating in biological synthesis of alkannin as well as encoding gene and application thereof
  • CYP76B74 protein participating in biological synthesis of alkannin as well as encoding gene and application thereof
  • CYP76B74 protein participating in biological synthesis of alkannin as well as encoding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Embodiment 1, the acquisition of CYP76B74 protein and its coding gene

[0100] Through a large number of sequence analysis, expression analysis and functional verification of Zinnia xinjiang, a DNA coding sequence was found, and the encoded protein is shown in sequence 1 of the sequence list.

[0101] The protein shown in Sequence 1 of the Sequence Listing is named as CYP76B74 protein, which consists of 496 amino acid residues. The gene encoding CYP76B74 protein is named as CYP76B74 gene, and its open reading frame is shown as sequence 2 in the sequence listing.

Embodiment 2

[0102] Example 2, CYP76B74 protein function analysis

[0103] 1. Construction of recombinant strains

[0104] 1. Construction of recombinant plasmid pESC-Trp-CYP76B74. According to the sequencing results, the structure of the recombinant plasmid pESC-Trp-CYP76B74 is described as follows: the plasmid pESC-Trp is used as the starting vector, and the DNA molecule shown in sequence 2 of the sequence table is inserted before the FLAG tag.

[0105] 2. Transform the recombinant plasmid pESC-Trp-CYP76B74 prepared in step 1 into Saccharomyces cerevisiae BY-T20 to obtain the recombinant strain BY-T20-CYP76B74; transform the plasmid pESC-Trp into Saccharomyces cerevisiae BY-T20 to obtain the recombinant strain BY-T20-Trp (hereinafter referred to as the empty vector strain).

[0106] 2. In vitro enzymatic experiment

[0107] TEG buffer: TE buffer containing 20% ​​(v / v) glycerol.

[0108] Precipitation buffer: TESB buffer containing 0.225M NaCl and 0.15g / mL PEG4000.

[0109] TESB buff...

Embodiment 3

[0169] Example 3, Analysis of Enzymatic Kinetic Parameters of CYP76B74 Protein

[0170] 1. Prepare the reaction system. The reaction system is 1 mL, consisting of microsomal protein solution (500 μg protein content), Tris-HCl (pH7.5) 50 mM, NADPH 1 mM, FAD 5 μM, FMN 5 μM, 6-phosphate-glucose 50 mM, 6-phosphate-glucose dehydrogenation Enzyme 1U, GHQ and distilled water composition. The concentration of GHQ in the reaction system was 0 μM, 5 μM, 10 μM, 15 μM, 30 μM, 40 μM, 60 μM or 100 μM (each concentration was repeated three times).

[0171] 2. Take the reaction system prepared in step 1, and carry out an in vitro enzymatic reaction to obtain a reaction product.

[0172] Reaction conditions: 30°C, 150rpm for 30min.

[0173] 3. Add an equal volume of ethyl acetate to the reaction product, vortex for 1 min, centrifuge to collect the supernatant organic phase, and keep the lower bacterial liquid.

[0174] 4. Add an equal volume of ethyl acetate to the lower bacterial solution...

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Abstract

The invention discloses CYP76B74 protein participating in biological synthesis of alkannin as well as an encoding gene and application thereof. An amino acid sequence of the CYP76B74 protein is shownas a sequence 1 in a sequence table. An experiment proves that the CYP76B74 protein is closely related to the synthesis of alkannin type compounds and can be used for catalyzing hydroquinone2-geranylquinone into 3''-hydroxyl-hydroquinone2-geranylquinone. The CYP76B74 protein disclosed by the invention has important theoretical and practical significance of adjusting and producing plant naphthoquinones compounds and improving the content of a naphthoquinones active component, i.e., alkannins, in arnebiae through a biological technology.

Description

technical field [0001] The invention belongs to the field of genetic engineering of medicinal plants, and specifically relates to a CYP76B74 protein involved in shikonin biosynthesis, its coding gene and application. Background technique [0002] The active ingredients of medicinal plants are usually a class of small molecular organic compounds produced by secondary metabolism in the process of plant growth and adaptation to the environment. The analysis of biosynthetic pathways of active ingredients is the core content of the research on secondary metabolites of medicinal 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. The biosynthetic pathway and its regulatory mechanism provide a theoretical basis for the formation of the quality of medicinal material...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12P7/66
CPCC12N9/0073C12P7/66C12Y114/13116
Inventor 黄璐琦王升郭兰萍王瑞杉刘谈
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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