Cytochrome P450 gene participated in anabolism of tanshinone compound as well as coding product and application thereof

A technology of biosynthesis and tanshinone, applied in the direction of plant gene improvement, application, plant products, etc.

Active Publication Date: 2014-04-02
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

Before the publication of the present invention, the CYP76AH3 gene and its amino ac

Method used

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  • Cytochrome P450 gene participated in anabolism of tanshinone compound as well as coding product and application thereof
  • Cytochrome P450 gene participated in anabolism of tanshinone compound as well as coding product and application thereof
  • Cytochrome P450 gene participated in anabolism of tanshinone compound as well as coding product and application thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0016] Example 1: Cloning of Cytochrome P450 Genes in Salvia Miltiorrhiza

[0017] 1. The root of Salvia miltiorrhiza in full flowering stage was taken, the total RNA of Salvia miltiorrhiza was extracted by Trizol method, and the 5' and 3' end sequences of CYP76AH3 gene were obtained by using the 5' and 3' RACE kits of invitrogen to amplify respectively.

[0018] 2. Cloning and sequencing of full-length cDNA

[0019] The 5' and 3' RACE results were spliced, the ORF region was searched, full-length gene primers were designed, and cDNA was used as a template for amplification. Agarose gel electrophoresis showed that a specific fragment appeared at about 1500bp, the target fragment was recovered by the agarose gel recovery kit (Takara), cloned into the pGEM-T easy vector (Promega), positive clones were identified and verified by sequencing) (Beijing Huada Gene) for the construction of expression vectors.

Embodiment 2

[0020] Example 2, Bioinformatics of CYP76AH3 Gene Sequence

[0021] The length of the full-length open reading frame (ORF) of the cytochrome P450 gene CYP76AH3 gene of the Danshen diterpene synthetic metabolic pathway involved in the present invention is 1485bp, encoding 494 amino acids, and the detailed sequence is shown in SEQ ID No.1 and SEQ ID in the sequence listing No.2. The full-length open reading frame of CYP76AH3 was searched for homology in the NCBI database by BLAST program. The comparison analysis of the gene at the amino acid level showed that the amino acid sequence of the protein encoded by the Danshen CYP76AH3 gene had low homology with other species.

Embodiment 4

[0022] Example 4, Eukaryotic expression and functional analysis of CYP76AH3 gene

[0023] 1. Construction of yeast expression vector

[0024] By designing primers with EcoR I and Spe I restriction sites, the open reading frame of the cloned Danshen cytochrome P450 gene was inserted into the EcoR I and Spe I of the yeast expression vector pESC-His by using PCR method Between the restriction sites, the recombinant plasmid pESC-CYP76AH3 was obtained, which was verified by sequencing at Beijing Huada Gene Company.

[0025] 2. Induced expression

[0026] The constructed pESC-CYP76AH3 plasmid was transformed into YJ35, an engineered strain of the ferruginous yeast, and the expression was induced by galactose.

[0027] 3. Activity analysis of CYP76AH3 in Danshen

[0028] Introduce the CYP76AH3 gene expression vector into the Saccharomyces cerevisiae engineering strain producing ferruginol, pick 3 single clones and inoculate them in 5ml SD liquid medium, 30°C, 200rmin -1 Cultivate f...

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Abstract

The invention discloses a cytochrome P450 gene which participates in anabolism of tanshinone compound as well as coding product and application thereof, and the invention belongs to the field of medicinal plant gene engineering. The gene is obtained by clone from red sage root for the first time, and is a key enzyme gene in anabolism pathway of tanshinone compound, and can catalyze ferruginol into 11-hydroxy ferruginol. The CYP76AH3 gene provided by the invention has a nucleotide sequence shown in the SEQ ID No.1. The gene coding protein comprises an amino acid residue sequence shown in the SEQ ID No.2 of the sequence table and a protein derived from the SEQ ID No.2 whose activity is identical to that of the amino acid residue sequence shown in the SEQ ID No.2. The CYP76AH3 gene provided by the invention is closely related to anabolism of tanshinone compound, and has important theoretical and real meanings for regulating plant diterpenoids compounds and increasing contents of diterpene active component tanshinone in red sage root by biological technology.

Description

technical field [0001] The invention relates to the cytochrome P450 gene related to the metabolic pathway of tanshinone compounds, and uses the whole cell catalytic strategy to analyze the functional activity of the protein encoded by the gene, and relates to the cytochrome P450 gene and its coding in the biosynthetic pathway of tanshinone compounds, the main active ingredient of Danshen The product and application belong to the field of medicinal plant genetic engineering. 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 t...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/81C12N5/10C12N1/19C12P15/00A01H5/00
Inventor 黄璐琦蔡媛郭娟马晓惠马莹
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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