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Salvia 1-deoxy-D-xylulose-5-phosphate synthetase II gene, and encoding protein and use thereof

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

Inactive Publication Date: 2009-07-08
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no literature report on the isolation and cloning of the 1-deoxyxylulose-5-phosphate synthase II gene from the medicinal plant Salvia miltiorrhiza

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 (cloning of Salvia miltiorrhiza 1-deoxyxylulose-5-phosphate synthase II gene)

[0035] 1. Tissue separation (isolation)

[0036] Salvia miltiorrhiza plants come from Sichuan, and the young roots are immediately frozen in liquid nitrogen for preservation.

[0037] 2. RNA isolation (RNA isolation)

[0038] 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.

[0039] 3. Cloning of Full-length cDNA

[0040] 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-R...

Embodiment 2

[0048] Example 2 (Sequence information and homology analysis of Salvia miltiorrhiza 1-deoxyxylulose-5-phosphate synthase II)

[0049]The length of the full-length cDNA of Salvia miltiorrhiza 1-deoxyxylulose-5-phosphate synthase II of the present invention is 2522bp, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at nucleotides 100-2274. The amino acid sequence of 1-deoxyxylulose-5-phosphate synthetase II was deduced according to the full-length cDNA, with a total of 724 amino acid residues, a molecular weight of 78.2KD, and a pI of 6.44. See SEQ ID NO.2 for the detailed sequence. The full-length cDNA sequence of Salvia miltiorrhiza 1-deoxyxylulose-5-phosphate synthase II and its encoded protein were published in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBankCDS translations+PDB+SwissProt+Superdate Nucleotide and protein homology searches were carried out in the +PIR database, and it was found that it had 83% homology...

Embodiment 3

[0204] Example 3 (Prokaryotic expression and purification of Salvia miltiorrhiza 1-deoxyxylulose-5-phosphate synthetase II or polypeptide in Escherichia coli)

[0205] In this example, the full-length coding sequence or fragment of Danshen 1-deoxyxylulose-5-phosphate synthase II gene was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0206] 1. Construction of prokaryotic expression vector and transformation of Escherichia coli

[0207] According to the nucleotide sequence of Danshen DXS2, 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 DXS2 gene was cloned into the pET32a(+) vector (Novagen) under the premise of ensuring the correc...

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PUM

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Abstract

The invention discloses a red sage root 1-deoxy-xylulose-5-phosphate synthase II gene and its encoded proteins and applications, which fills the blank of separating the 1-deoxy-xylulose-5-phosphate synthase II gene from the traditional Chinese herb red sage root. The 1-deoxy-xylulose-5-phosphate synthase II gene provided by the present invention has the nucleotide sequence shown by the SEQ ID No.1 or homology sequence with one or a plurality of added, replaced, inserted or deleted nucleotide and its allelomorphic gene as well as it derived nucleotide sequence; the encoded protein has amino acid sequence shown in SEQ ID No.2 or r homology sequence with one or a plurality of added, replaced, inserted or deleted nucleotide. The 1-deoxy-xylulose-5-phosphate synthase II gene plays a significant role for improving the tanshinone content in the red sage root by a gene excess expression technique.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to 1-deoxyxylulose-5-phosphate synthetase II gene 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 Salvia miltiorrhiza has significant effects 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 su...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/63C12N15/82C12N1/21C12N1/19C12N5/10A01H5/00
Inventor 开国银王敬廖攀张林许辉
Owner SHANGHAI NORMAL UNIVERSITY
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