Geranylgeranyl diphosphate synthase gene in salvia root, and encoding protein and use thereof

A kind of technology of geranyl pyrophosphate and geranyl, applied in biological field

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 full-length

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 (Cloning of Salvia geranyl geranyl geranyl pyrophosphate synthase gene)

[0035] 1. Tissue isolation (isolation)

[0036] Salvia miltiorrhiza plants originated from Henan, and the young roots were immediately placed in liquid nitrogen for cryopreservation.

[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 containing the lysate, shake it sufficiently, and then transfer it into a glass homogenizer. After homogenization, it was transferred to a 1.5 mL EP tube, and total RNA was extracted (TRIzol Reagents, GIBCO BRL, USA). The total RNA quality 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 sequence of geranylgeranyl pyrophosphate synthase from Taxus mandiensis, wild tobacco and other cloned geranylgeranyl pyrophosphat...

Embodiment 2

[0048] Example 2 (sequence information and homology analysis of Salvia miltiorrhiza GGPPS gene)

[0049] The full-length cDNA of the Danshen geranylgeranyl pyrophosphate synthase of the present invention is 1095bp in length, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at nucleotides 73-1167. The amino acid sequence of Danshen geranylgeranyl pyrophosphate synthase was deduced according to the full-length cDNA, which has a total of 364 amino acid residues, a molecular weight of 39.04KDa, and a pI of 5.68. See SEQ ID NO.2 for the detailed sequence.

[0050] The full-length cDNA sequence of Danshen geranylgeranyl pyrophosphate synthase and its encoded protein were published in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate Nucleotide and protein homology searches were carried out in the +PIR database, and it was found that it had 72% homology (see Table 2) with wild tobacco GGP...

Embodiment 3

[0118] Example 3 (Prokaryotic expression and purification of Salvia geranium geranyl geranyl pyrophosphate synthase or polypeptide in Escherichia coli)

[0119] In this example, the full-length Salvia miltiorrhiza SmGGPPS gene coding sequence or fragment was constructed into a commercial protein fusion expression vector to express and purify the recombinant protein.

[0120] 1. Construction of prokaryotic expression vector and transformation of E. coli

[0121] According to the nucleotide sequence of Salvia miltiorrhiza SmGGPPS, primers for amplifying the protein coding region were designed, and restriction endonuclease sites were introduced into the forward and reverse primers respectively (this depends on the selected pET28a(+) vector), so as to construct Expression vector. Using the amplification product obtained in Example 1 as a template, after PCR amplification, the Salvia miltiorrhiza SmGGPPS gene was cloned into pET28a(+) vector (Novagen) under the premise of ensuring...

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Abstract

The invention discloses a Red sage root Geranyl geraniol-based pyrophosphoric acid synthase gene and its encoded protein and the application thereof, which fills the blank of separating the Geranyl geraniol-based pyrophosphoric acid synthase gene from the traditional Chinese herb red sage root. The Geranyl geraniol-based pyrophosphoric acid synthase gene provided by the present invention has the nucleotide sequence No.73-1167 shown by the SEQ ID No.1; 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 inventive Geranyl geraniol-based pyrophosphoric acid synthase gene can improve the terpenes active component tanshinone in the red sage root, which helps to improve the quality of red sage root herbs and has good application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a geranylgeranyl pyrophosphate synthetase gene expressed in salvia miltiorrhiza and its encoded protein and application. Background technique [0002] Cardiovascular and cerebrovascular diseases are currently one of the three major diseases that threaten humans the most, and are at the top of 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-toxic and cheap clinical drugs for the treatment of cardiovascular and cerebrovascular ...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N9/00C12N15/63C12N15/82C12N1/19C12N1/21C12N5/10A01H5/00
Inventor 开国银廖攀张林周伟董彦君
Owner SHANGHAI NORMAL UNIVERSITY
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