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Salviamiltiorrhizabge-cytochrome P450 (SmP450) gene as well as coded protein and application thereof

A cytochrome, Salvia miltiorrhiza technology, applied in application, genetic engineering, plant genetic improvement and other directions

Inactive Publication Date: 2010-12-29
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

Before the present invention is published, there is no publication or report of the cytochrome P450 gene and its amino acid sequence mentioned in this patent application

Method used

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  • Salviamiltiorrhizabge-cytochrome P450 (SmP450) gene as well as coded protein and application thereof
  • Salviamiltiorrhizabge-cytochrome P450 (SmP450) gene as well as coded protein and application thereof
  • Salviamiltiorrhizabge-cytochrome P450 (SmP450) gene as well as coded protein and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1, the making of Danshen cDNA chip

[0041] 1. Isolation and detection of total RNA from Salvia miltiorrhiza

[0042] Take 2 g of Shaanxi Shangluo Salvia (Salvia Miltiorrhiza Bge) roots, quickly grind them into powder with liquid nitrogen in a mortar, and quickly transfer them to 10 mL of extraction buffer (CTAB (W / V) 2%, Tris-HCl (pH8.0)100mmol·L -1 , EDTA 25mmol·L -1 , NaCl 2.0mol·L -1 , PVP402%, spermidine 0.5g / L, mercaptoethanol 2%), fully shake and mix; extract twice with equal volume of chloroform, and centrifuge at 7500g for 15 minutes. Add 1 / 4 volume of 10M LiCl to the supernatant, mix well and place it at 4°C to precipitate overnight; centrifuge at 7500g for 20 minutes, and use 500 μL SSTE (SDS 0.5%, NaCl 1mol L -1 , Tris-HCl (pH8.0) 10mmol L -1 , EDTA 1mmol·L -1 , dissolved at 65°C for 5 minutes. Extract with an equal volume of chloroform, centrifuge at 13,000g for 5 minutes; add 2 times the volume of absolute ethanol to the supernatant, and ...

Embodiment 2

[0052] Embodiment 2: Cloning of Danshen SmP450 gene

[0053] 1. Chip hybridization analysis

[0054] The hairy roots of Salvia miltiorrhiza were induced by Ri-plasmid transformation by direct infection with Agrobacterium rhizogenes 15834. Well-grown hairy roots of Salvia miltiorrhiza (0.1 g each) were subcultured on 6, 7V solid medium, placed in an incubator at 25°C for dark culture, and harvested at 30 days, 45 days, and 60 days, respectively. The 30-day-old material was used as a reference, and hybridization was carried out with the 45-day-old and 60-day-old material respectively, and was repeated twice. Each group was labeled positively and negatively to eliminate the error of the dye.

[0055] The indirect labeling method is used for probe labeling, including double-stranded cDNA synthesis, T7-mediated RNA in vitro transcription to synthesize cRNA, random primer reverse transcription, and cDNA labeling with KLENOW enzyme.

[0056] (1) Synthesis of double-stranded cDNA: t...

Embodiment 3

[0068] The bioinformatics analysis of embodiment 3, SmP450 gene

[0069] The length of the full-length cDNA of the salvia miltiorrhiza diterpene synthase gene involved in the present invention is 1512bp, and the detailed sequence is shown in sequence 1 in the sequence list, wherein the open reading frame is located at 1-1512bp. The full-length cDNA sequence of Salvia miltiorrhiza was searched for nucleotide homology in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translation+PDB+Swissprot+Superdate+PIR databases with BLAST program. The gene has high homology with P450 in other species at the amino acid level, and also has the characteristic FxxGxRxCxG domain. figure 1 .

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Abstract

The invention discloses a cytochrome P450 (SmP450) gene as well as a coded protease and application thereof. The gene is obtained by clone from salviamiltiorrhizabge by utilizing a cDNA chip technology, which fills the blank of separating and cloning a structure-modification type functional gene which is cytochrome P450 (SmP450) gene from a rare traditional Chinese medicine of salviamiltiorrhizabge. The cytochrome P450 (SmP450) gene has a nucleotide sequence shown as SEQ ID NO.1, or a homological sequence adding, substituting, inserting or lacking one or more nucleotides or allelic genes and derived nucleotide sequence thereof. The protein coded by the cytochrome P450 (SmP450) gene has an amino acid sequence shown as SEQ ID NO.2 or a homological sequence adding, substituting, inserting or lacking one or more amino acids. The cytochrome P450 (SmP450) can improve the content of tanshinone which is diterpene active element in the salviamiltiorrhizabge by the biotechnology, is beneficial to the quality improvement of the salviamiltiorrhizabge, can be used for the breeding selection and has good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and mainly relates to the use of cDNA chip technology to clone various enzyme genes related to the biosynthesis of active components in Danshen and their encoded products and applications, especially to phenolic acids and diterpene quinone compounds, the main active components of Danshen The enzyme gene involved in its structural modification in biosynthesis, its coded product and its application belong to the field of genetic engineering of medicinal plants. Background technique [0002] The formation of active ingredients (secondary metabolites) of medicinal plants is the product of a unique group of 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 hot...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/63C12N5/10C12N15/82
Inventor 黄璐琦戴住波崔光红张夏楠
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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