CYP450 (Cytochrome P450) gene participating in tanshinone biosynthesis and coded product as well as application thereof

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

Active Publication Date: 2012-09-19
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 CYP76Q1 gene and its amino acid sequence involved in this patent application have not been disclosed or reported

Method used

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  • CYP450 (Cytochrome P450) gene participating in tanshinone biosynthesis and coded product as well as application thereof
  • CYP450 (Cytochrome P450) gene participating in tanshinone biosynthesis and coded product as well as application thereof
  • CYP450 (Cytochrome P450) gene participating in tanshinone biosynthesis and coded product as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Screening of CYP450 genes in the synthetic metabolic pathway of tanshinone compounds

[0019] Through the transcriptome database constructed in this experiment, the differentially upregulated CYP450 genes were selected for bioinformatics analysis and showed that these CYP450 genes with induced expression upregulation mainly came from CYP76, CYP716, CYP81 and CYP71 families, among which CYP76 and CYP71 families are important in terpene synthesis 15 candidate CYP450 genes from these two families were analyzed.

Embodiment 2

[0020] Example 2: Cloning of CYP450 genes in Salvia miltiorrhiza

[0021] 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' end of CYP450 gene was amplified by Invitrogen's 5'RACE kit, and a total of 15 full-length sequences of CYP450 genes were obtained.

[0022] 2. Cloning and sequencing of full-length cDNA

[0023] The 5' RACE results and the 3' ends in the transcriptome database 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), and positive clones were identified and verified by sequencing (Beijing Hua large gene) for the construction of expression vectors.

Embodiment 3

[0024] Example 3, Bioinformatics Analysis and Tissue Expression Analysis of CYP76Q1 Gene Sequence

[0025] 1. The length of the full-length open reading frame (ORF) of the CYP450 gene CYP76Q1 gene CYP76Q1 gene of the Danshen diterpene synthetic metabolic pathway involved in the present invention is 1488bp, encoding 495 amino acids, and the detailed sequence is shown in SEQ ID No.1 and SEQ ID in the sequence list No.2. The full-length open reading frame of CYP76Q1 was searched for homology in the NCBI database using BLAST program, and the comparison analysis of the gene at the amino acid level showed that the amino acid sequence of the protein encoded by the Danshen CYP76Q1 gene had low homology with other species, It has a homology of up to about 50% with the Verbenax hybrida cytochrome P450 gene that has not been functionally verified.

[0026]2. Extract the RNA of the root, stem and leaf of Salvia miltiorrhiza in full flowering stage, use the reverse transcription kit (Ferm...

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Abstract

The invention discloses a CYP450 (Cytochrome P450) gene participating in tanshinone biosynthesis and a coded product as well as application thereof, and belongs to the field of medicinal plant genetic engineering. The gene is firstly obtained through cloning from salvia miltiorrhiza bunge, is a key enzyme gene for a biosynthesis pathway of a tanshinone compound and is a key step of catalyzing miltiradiene to ferruginol. The CYP76Q1 gene provided by the invention has a nucleotide sequence shown by SEQ ID NO. 1. Protein coded by the gene has an amino acid residue sequence shown by SEQ ID No. 2 in a sequence table and protein with the same activity as that of the amino acid residue sequence shown by the SEQ ID No. 2 and derived from the SEQ ID No. 2. The CYP76Q1 gene provided by the invention is closely associated with biosynthesis of the tanshinone compound, has great theoretical and actual significance to regulating and producing a plant diterpene compound and improving the content of the diterpene active ingredient tanshinone in the salvia miltiorrhiza bunge through a biotechnology, contributes to quality improvement of the salvia miltiorrhiza bunge medicinal material and variety selection, also has the capacity of producing ferruginol monomer with pharmacological activity by constructing engineering bacteria using the gene, and has excellent application prospect.

Description

technical field [0001] The invention relates to the use of the Salvia miltiorrhiza transcriptome library to screen the CYP450 gene related to the metabolic pathway of tanshinone compounds, and uses the method of in vitro enzymatic reaction to analyze the functional activity of the protein encoded by the gene. The gene and its coded product and application belong to the field of genetic engineering of medicinal plants. Background technique [0002] The formation of active ingredients of medicinal plants is the product of specific gene expression in plant secondary metabolism pathways. With the extensive and in-depth study of plant functional genomes, the unique and broad application prospects of medicinal plant secondary metabolism synthesis related functional genes The research of these genes has gradually become a research hotspot. The cloning of these genes will provide a theoretical basis for the interpretation of the biosynthetic pathway and its regulatory mechanism of t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/63C12N5/10C12N1/19C12P7/22A01H5/00
Inventor 黄璐琦郭娟申业张夏楠高伟
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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