Salvia miltrorrhiza squalene synthase (SmSQS) gene and encoding protein and application thereof

A technology of squalene synthase and alkene synthase, applied in the field of biological and medicinal plant genetic engineering

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

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Problems solved by technology

Before the present invention was published, there was no disclosure or report of the Danshen terpenoid enzyme gene and its amino acid sequence mentioned in this patent application

Method used

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  • Salvia miltrorrhiza squalene synthase (SmSQS) gene and encoding protein and application thereof
  • Salvia miltrorrhiza squalene synthase (SmSQS) gene and encoding protein and application thereof
  • Salvia miltrorrhiza squalene synthase (SmSQS) gene and encoding protein and application thereof

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Experimental program
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Effect test

Embodiment 1

[0040] Embodiment 1, Danshen squalene synthase (SmSQS) gene cloning

[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 su...

Embodiment 2

[0051] The bioinformatics analysis of embodiment 2, SmSQS gene

[0052]The length of the full-length cDNA of the salvia miltiorrhiza diterpene synthase gene involved in the present invention is 1457 bp, and the detailed sequence is shown in sequence 1 in the sequence list, wherein the open reading frame is located at 120-1360 bp. 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 using BLAST program. The gene has high homology with squalene synthase in other species at the amino acid level, and has a typical DXXDD domain of terpenoid enzymes, figure 1 .

Embodiment 3

[0053] Example 3, Functional Verification of Salvia Squalene Synthase

[0054] 1. Expression vector construction

[0055] F-A DNA preparation containing Xho I and Nco I restriction sites

[0056] Using the plasmid containing the full-length PSS cDNA as a template, FNco: 5′-GG CCATGGGGAGTTTACGTGCGATTTTGAG-3′ and RXho: 5′-GCCTCGAGTTAGACGGCTCTCTTTGGTGCAAAG-3′ as primers, PCR was performed with Pyrobest DNA Polymerase high-fidelity enzyme to obtain the target F-A DNA. PCR reaction conditions: 94°C for 4min, 1cycle; 94°C for 35sec; 59°C for 30sec; 72°C for 2min, 32cycles; 72°C for 8min, 1cycle; stop at 4°C. The product was separated by 1% agarose gel electrophoresis, recovered by cutting the gel, and stored at -20°C.

[0057] Digestion and Ligation Reaction F-A DNA and pET-32a(+) Vector were digested with Xho I and Nco I respectively, and recovered. Double digestion reaction system: Xho I, 1 μL; Nco I, 1 μL; 10×K buffer, 2 μL; BSA (0.01%), 2 μL; F-A or pET-32a(+), 1 μg; add deio...

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Abstract

The invention discloses a squalene synthase gene and an encoding protease and application thereof. The gene is cloned from salvia miltrorrhiza by the homology cloning technology for the first time, and fills a gap of cloning and separating the squalene synthase gene from the traditional precious Chinese herbal medicine salvia miltrorrhiza in China. The provided squalene synthase gene has a nucleotide sequence shown in SEQ ID NO.1, a homologous sequence in which one or more nucleotides are added, substituted, inserted or deleted or a nucleotide sequence of allele thereof and derivatives. The protein encoded by the gene has an amino acid sequence shown in SEQ ID NO.2, or a homologous sequence in which one or more amino acids are added, substituted, inserted or deleted. The provided squalene synthase gene can improve the content of triterpenoid saponin components in plants through biotechnology, and can improve the content of diterpene (tanshinone) substances in the salvia miltrorrhiza by the RNAi technology simultaneously. The squalene synthase gene has good application prospect on research and industrialization of medicinal plant secondary metabolic engineering.

Description

technical field [0001] The invention belongs to the field of biotechnology, mainly relates to the use of homologous cloning technology to obtain terpenoid enzyme genes in Salvia miltiorrhiza and their coded products and applications, in particular to the biosynthesis of terpene enzyme genes with pharmacologically active ingredients and their coded products and applications, belonging to pharmaceutical In the field of plant genetic engineering. 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 synthesis of medicinal plants with unique characteristics and broad application prospects has gradually become a research hotspot. It provides a theoretical basis for the biosynthetic pathway and its regulation mechanism...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/63C12N1/21C12N5/10C12N15/82C12R1/19
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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