Salvia 1-deoxidation xylulose-5-phosphate synthase gene 1 and its coding protein and application

A deoxyxylulose and phosphate synthase technology, applied in the biological field, can solve the problem of isolating and cloning 1-deoxyxylulose-5-phosphate synthase gene 1

Inactive Publication Date: 2008-08-27
SHANGHAI NORMAL UNIVERSITY
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Problems solved by technology

[0004] In the analysis of the existing literature, "The Plant Journal (Plant Magazine) 2000, 22: 503-513" reported that the 1-deoxyxylulose-5-phosphate synthase gene was isolated and cloned from tomato, but it has not been There is a literature report on the isolation and cloning of 1-deoxyxylulose-5-phosphate synthase gene 1 from the unique medicinal plant Salvia miltiorrhiza in my country

Method used

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  • Salvia 1-deoxidation xylulose-5-phosphate synthase gene 1 and its coding protein and application
  • Salvia 1-deoxidation xylulose-5-phosphate synthase gene 1 and its coding protein and application
  • Salvia 1-deoxidation xylulose-5-phosphate synthase gene 1 and its coding protein and application

Examples

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Embodiment 1

[0043] Example 1 (cloning of Salvia miltiorrhiza 1-deoxyxylulose-5-phosphate synthase gene 1)

[0044] 1. Tissue separation (isolation)

[0045] Salvia miltiorrhiza plants come from Henan, and the young roots are immediately frozen in liquid nitrogen for preservation.

[0046] 2. RNA isolation (RNA isolation)

[0047] 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.

[0048] 3. Cloning of Full-length cDNA

[0049] According to the conserved amino acid sequences of DXS genes such as snapdragon, peppermint, etc., degenerate primers were designed, and using the principle of homologous gene cloning, the Smart-RACE...

Embodiment 2

[0056] Example 2 (Sequence information and homology analysis of Salvia miltiorrhiza 1-deoxyxylulose-5-phosphate synthase gene 1)

[0057] The full-length cDNA of the new Salvia miltiorrhiza 1-deoxyxylulose-5-phosphate synthase gene 1 of the present invention has a length of 2519bp, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at nucleotides 294-2438. The amino acid sequence of 1-deoxyxylulose-5-phosphate synthase gene 1 was deduced based on the full-length cDNA, which has a total of 714 amino acid residues, a molecular weight of 175.89KD, and a pI of 4.91. See SEQ ID NO.2 for the detailed sequence. The full-length cDNA sequence of Salvia miltiorrhiza 1-deoxyxylulose-5-phosphate synthase gene 1 and its encoded protein were published in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt using BLAST program Nucleotide and protein homology searches were carried out in the +Superdate+PIR datab...

Embodiment 3

[0068] Example 3 (Prokaryotic expression and purification of Danshen 1-deoxyxylulose-5-phosphate synthase 1 or polypeptide in Escherichia coli)

[0069] In this example, the full-length coding sequence or fragment of Salvia miltiorrhiza 1-deoxyxylulose-5-phosphate synthase gene 1 was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0070] 1. Construction of prokaryotic expression vector and transformation of Escherichia coli

[0071] According to the nucleotide sequence of Danshen DXS1, 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 DXS gene 1 was cloned into the pET32a(+) vector (Novagen) under the premise of ensuring the correct r...

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Abstract

The invention discloses a salvia 1-deoxidation xylulose-5-phosphat synthase gene1, protein which is encoded by the salvia 1-deoxidation xylulose-5-phosphat synthase gene1 and the use thereof, which fills a gap that the 1-deoxidation xylulose-5-phosphat synthase gene1 is separated and cloned from salvia which is precious traditional Chinese medicine in China. The 1-deoxidation xylulose-5-phosphat synthase gene1 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 1-deoxidation xylulose-5-phosphat synthase gene1, 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 1-deoxidation xylulose-5-phosphat synthase gene1 which is provided by the invention has prominent effect of increasing the content of tanshinone in salvia through the technology of excessive expression of the gene and can be widely applied in improving the quality of salvia mi ltiorrhiza, which has very good application prospect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to 1-deoxyxylulose-5-phosphate synthase gene 1 expressed in salvia miltiorrhiza and its encoded protein and application. Background technique [0002] Salvia miltiorrhiza Bunge is a traditional Chinese medicine in my country. Modern pharmacological studies have shown that Danshen has a significant effect on the cardiovascular system and blood system. A variety of compound preparations based on Danshen, such as Compound Danshen Injection, Compound Danshen Tablets, Compound Danshen Capsules, and Compound Danshen Dropping Pills (applied to the US FDA as therapeutic drugs in 1997), have been widely used in clinical treatment of heart disease. Vascular disease, kidney disease, liver disease and anti-infection etc. However, due to the long growth cycle of Salvia miltiorrhiza (more than 2 years), under the traditional cultivation mode, it faces many disadvantages such as serious ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/63C12N9/00C12N5/10C12N1/19C12N1/21A01H1/00A01H5/06C12R1/19C12R1/645
Inventor 开国银王敬张林董彦君周根余
Owner SHANGHAI NORMAL UNIVERSITY
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