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

A methylglutaryl coenzyme and reductase technology, applied in the fields of oxidoreductase, application, genetic engineering, etc., can solve the problem of isolating and cloning the full-length 3-hydroxy-3-methylglutaryl coenzyme A reductase gene And other issues

Inactive Publication Date: 2008-08-27
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In an analysis of existing literature, "Bioscience reports (biological science reports), 2006, 26 (2): 171-181" reported that -hydroxy-3-methylglutaryl-CoA reductase was cloned from Eucommia ulmoides gene, but so far there has been no literature report on the isolation and cloning of the full-length 3-hydroxy-3-methylglutaryl-CoA reductase gene from the medicinal plant Salvia miltiorrhiza

Method used

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

Examples

Experimental program
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Embodiment 1

[0044] Example 1 Cloning of Salvia miltiorrhiza 3-hydroxy-3-methylglutaryl-CoA reductase gene

[0045] 1. Tissue separation (isolation)

[0046] Salvia miltiorrhiza plants come from Henan, and the young roots are immediately frozen in liquid nitrogen for preservation.

[0047] 2. RNA isolation (RNA isolation)

[0048] 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 (TRlzol 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.

[0049] 3. Cloning of Full-length cDNA

[0050] According to the conserved amino acid sequence of 3-hydroxy-3-methylglutaryl-CoA reductase cloned from Eucommia ulmoides, tobacco and others, degenerate primers were designed, and using the principle...

Embodiment 2

[0058] Example 2 Sequence information and homology analysis of Danshen HMGR gene

[0059] The full-length cDNA of Danshen 3-hydroxy-3-methylglutaryl-CoA reductase of the present invention is 1258bp in length, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at nucleotides 65-1126. According to the full-length cDNA, the amino acid sequence of 3-hydroxy-3-methylglutaryl-CoA reductase in Danshen was deduced, with a total of 565 amino acid residues, a molecular weight of 60.5KD, and a pI of 7.1. See SEQ ID NO.2 for the detailed sequence.

[0060] The full-length cDNA sequence of Danshen 3-hydroxy-3-methylglutaryl-CoA reductase and its encoded protein were published in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundantGenBank CDS translations+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 80% homology with the Andrographis panicula...

Embodiment 3

[0072] Example 3 Prokaryotic expression and purification of Salvia miltiorrhiza 3-hydroxy-3-methylglutaryl-CoA reductase or polypeptide in Escherichia coli

[0073] In this example, the full-length coding sequence or fragment of Danshen SmHMGR was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0074] 1. Construction of prokaryotic expression vector and transformation of Escherichia coli

[0075] According to the nucleotide sequence of Salvia miltiorrhiza SmHMGR, the primers for amplifying the protein coding region were designed, and restriction endonuclease sites were introduced on the forward and reverse primers (this was determined according to 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 SmHMGR gene was cloned into the pET32a(+) vector (Novagen) under the premise of ensuri...

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Abstract

The invention discloses a salvia 3-hydroxy-3-methylglutaryl coenzyme A reductase gene, protein which is encoded by the salvia 3-hydroxy-3-methylglutaryl coenzyme A reductase gene and the use thereof, which fills a gap that the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene is separated and cloned from salvia which is precious traditional Chinese medicine in China. The 3-hydroxy-3-methylglutaryl coenzyme A reductase gene which is provided by the invention has a nucleotide sequence or a homologous sequence which adds, replaces, inserts or losses one or a plurality of nucleotides or allele thereof and the nucleotide sequence which is derived from the 3-hydroxy-3-methylglutaryl coenzyme A reductase 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 reductase gene which is provided by the invention can increase the content of tanshinone which is a terpenes active component in the salvia 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 improving the quality of salvia mi ltiorrhiza and has very good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expressed in salvia miltiorrhiza and its encoded protein and application. Background technique [0002] Cardiovascular and cerebrovascular diseases, diabetes, and cancer are currently the three major types of diseases that threaten humans the most. Among them, cardiovascular and cerebrovascular diseases rank first among these three diseases. According to statistics, about 17 million people die of cardiovascular and cerebrovascular diseases in the world every year, accounting for about 1 / 3 of the total number of deaths in the world; about 2.6 million people die of cardiovascular and cerebrovascular diseases in my country every year, and cardiovascular and cerebrovascular diseases have become a threat The "number one killer" of human health and life. Therefore, active research and development of high-efficiency, low-to...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/63C12N9/02C12N5/10C12N1/19C12N1/21A01H1/00C12R1/19C12R1/645
Inventor 开国银廖攀周伟董彦君周根余
Owner SHANGHAI NORMAL UNIVERSITY
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