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Salvia 3-hydroxy-3-methylglutaryl A synthase gene and its coding protein and application

A methylglutaryl coenzyme and synthetase technology, applied in the biological field, can solve the problem of isolating and cloning 3-hydroxy-3-methylglutaryl coenzyme A synthetase gene, etc.

Inactive Publication Date: 2010-06-02
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In an analysis of existing literature, "Science Asia (Asian Science) 2002, 28: 29-36" reported that the 3-hydroxy-3-methylglutaryl-CoA synthetase gene was cloned from rubber trees, but so far There is no literature report on the isolation and cloning of 3-hydroxy-3-methylglutaryl-CoA synthetase gene from the medicinal plant Salvia miltiorrhiza

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Cloning of Salvia miltiorrhiza 3-hydroxyl-3-methylglutaryl-CoA synthetase gene

[0042] 1. Tissue separation (isolation)

[0043] Salvia miltiorrhiza plants come from Sichuan, and the young roots are immediately frozen in liquid nitrogen for preservation.

[0044] 2. RNA isolation (RNA isolation)

[0045] Take part of the tissue and grind it with a mortar, add it to a 1.5mL EP tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to 1.5mL EP tube, and extract total RNA (TRIzol Reagents, GIBCO BRL, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0046] 3. Cloning of Full-length cDNA

[0047] According to the conserved amino acid sequences of 3-hydroxy-3-methylglutaryl-CoA synthetases cloned from Brazil rubber, yew and other clones, degenerate primers were designed, and using the prin...

Embodiment 2

[0055] Example 2 Sequence information and homology analysis of Danshen 3-hydroxy-3-methylglutaryl-CoA synthetase gene

[0056] The novel Danshen 3-hydroxy-3-methylglutaryl-CoA synthetase full-length cDNA is 1655 bp in length, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at 118-1500 nucleotides. The amino acid sequence of Danshen 3-hydroxy-3-methylglutaryl-CoA synthetase was deduced according to the full-length cDNA, which has a total of 460 amino acid residues, a molecular weight of 50.6KD, and a pI of 6.04. See SEQ ID NO.2 for the detailed sequence.

[0057] The full-length cDNA sequence of Danshen 3-hydroxy-3-methylglutaryl-CoA synthetase and its encoded protein were published in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt using BLAST program Nucleotide and protein homology searches were carried out in the +Superdate+PIR database, and it was found that it had 79% homology with the...

Embodiment 3

[0157] Example 3 Prokaryotic expression and purification of Salvia miltiorrhiza 3-hydroxy-3-methylglutaryl-CoA synthetase or polypeptide in Escherichia coli

[0158] In this example, the full-length coding sequence or fragment of Salvia miltiorrhiza DsHMGS was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0159] 1. Construction of prokaryotic expression vector and transformation of Escherichia coli

[0160] According to the nucleotide sequence of Danshen DsHMGS, design primers to amplify the protein coding region, and introduce restriction endonuclease sites on the forward and reverse primers (this depends on the selected pET32a(+) vector), so as to construct Expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Danshen DsHMGS gene was cloned into the pET32a(+) vector (Novagen) under the premise of ensuring the correct reading frame. The id...

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PUM

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Abstract

The invention discloses a salvia 3-hydroxy-3-methylglutaryl coenzyme A synthase gene, protein which is encoded by the salvia 3-hydroxy-3-methylglutaryl coenzyme A synthase gene and the use thereof. The 3-hydroxy-3-methylglutaryl coenzyme A synthase gene has a nucleotide sequence or a homologous sequence which adds, replaces, inserts or losses one or a plurality of nucleotides or allele thereof andthe nucleotide sequence which is derived from the 3-hydroxy-3-methylglutaryl coenzyme A synthase gene, which are displayed in the SED ID No.1. The protein which is encoded by the gene has an amino acid sequence or the homologous sequence which adds, replaces, inserts or losses one or a plurality of amino acids, which is displayed in the SEQ ID No.2. The 3-hydroxy-3-methylglutaryl coenzyme A synthase gene which is provided by the invention can increase the content of tanshinone which is a drug used active compound in the resource plants such as salvia and the like through the genetic engineering technology and can be used in research and industrialization for increasing the content of the tanshinone through utilizing the transgenic technology, which is helpful for accelerating to solve theproblem that the drug resources of the camptothecin are seriously scarce and has very good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a 3-hydroxy-3-methylglutaryl coenzyme A synthetase gene expressed in salvia miltiorrhiza and its encoded protein and application. Background technique [0002] Tanshinone (tanshinone) is a class of active substances isolated from the traditional Chinese medicinal material Salvia miltiorrhiza, mainly composed of fat-soluble tanshinone I, tanshinone IIA, dihydrotanshinone, cryptotanshinone and other tanshinones. In clinical medicine, total tanshinone has been widely used in the treatment of suppurative tonsillitis, mastitis, cellulitis, osteomyelitis, inflammation of the external auditory canal, carbuncle, and surgical infection caused by Staphylococcus aureus and its drug-resistant strains, and streptococci. Tanshinone is widely used in clinical medicine, and the current global market demand is huge. Tanshinone in modern industrial production is mainly obtained by extracti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/52C12N15/63C12N9/00C12N5/10C12N1/21C12N1/19A01H1/00C12R1/645C12R1/19
Inventor 开国银王敬陆杨董彦君周根余
Owner SHANGHAI NORMAL UNIVERSITY
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