Scopolia acutangula hyoscyamine 6 Beta-hydroxylase gene, protein encoded thereby and application thereof

A scopolamine, hydroxylase technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of low effective components of scopolamine and the like

Inactive Publication Date: 2009-09-23
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is to provide a kind of hyoscyamine 6β-hydroxylase gene and its coded protein and its use, so as to fill in the fact that hyoscyamine has not been isolated and cloned from the unique medicinal plant Sanfensan in Yunnan, my country. The blank of 6β-hydroxylase gene can overcome the defect of low active ingredient of scopolamine in three-thirds

Method used

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  • Scopolia acutangula hyoscyamine 6 Beta-hydroxylase gene, protein encoded thereby and application thereof
  • Scopolia acutangula hyoscyamine 6 Beta-hydroxylase gene, protein encoded thereby and application thereof
  • Scopolia acutangula hyoscyamine 6 Beta-hydroxylase gene, protein encoded thereby and application thereof

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0045] Cloning of Sanfen 6β-Hydroxylase Gene

[0046] 1. Tissue separation (isolation)

[0047] Three-thirds of the plants come from Lijiang, Yunnan, and the young roots are immediately frozen in liquid nitrogen for preservation.

[0048] 2. RNA isolation (RNA isolation)

[0049] 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 (TRIzolReagents, 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.

[0050] 3. Cloning of Full-length cDNA

[0051] According to the conserved sequence of H6H amino acid of scopolamine and other Solanaceae plants, degenerate primers were designed, and using the principle of homologous gene cloning, the Smart-RACE method (Clonetech kit) was used for full...

Embodiment 2

[0060] Sequence information and homology analysis of Sanfen 3 hyoscyamine 6β-hydroxylase gene

[0061] The length of the novel full-length cDNA of three-part scopolamine 6β-hydroxylase of the present invention is 1364bp, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at nucleotides 125-1159. The amino acid sequence of three-part scopolamine 6β-hydroxylase was deduced according to the full-length cDNA, with a total of 344 amino acid residues, a molecular weight of 38.769KD, and a pl of 5.09. See SEQ ID NO.2 for the detailed sequence.

[0062] The full-length cDNA sequence and its encoded protein of three-part scopolamine 6β-hydroxylase were stored in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using BLAST program Nucleotide and protein homology searches were carried out, and it was found that it had 92% homology (see Table 2) with scopolamine H6H gene (GenBank...

Embodiment 3

[0091] Prokaryotic expression and purification of three-part hyoscypolamine 6β-hydroxylase or polypeptide in Escherichia coli

[0092] In this example, the full-length three-thirds AaH6H coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0093] Construction of prokaryotic expression vector and transformation of Escherichia coli

[0094] According to the nucleotide sequence of three-thirds AaH6H, design primers for amplifying the protein coding region, and introduce restriction enzyme sites on the forward and reverse primers respectively (this depends on the selected pET32a(+) vector), In order to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the three-thirds AaH6H gene was cloned into the pET32a(+) vector (Novagen) under the premise of ensuring the correct reading frame. The identified expression...

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Abstract

The invention discloses a scopolia acutangula hyoscyamine 6 Beta-hydroxylase gene which has a nucleotide sequence shown by SEQ ID NO.1, and also discloses a hyoscyamine 6 Beta-hydroxylase protein encoded by the SEQ ID NO.1 or containing an amino acid sequence shown by SEQ ID NO.2. In addition, the invention also discloses the application of the hyoscyamine 6 Beta-hydroxylase gene in expressing a eukaryotic cell in scopolia acutangula and improving the scopolamine content in transgenic rooting. The invention obviously improves the content of effective components in plant resources and can be used for improving the quality of traditional Chinese medicinal materials such as the scopolia acutangula, and the like.

Description

technical field [0001] The invention belongs to the field of biological technology, in particular to hyoscyamine 6β-hydroxylase gene and protein expressed in Sanfensan and application thereof. Background technique [0002] Tropane alkaloids are mainly extracted from Solanaceae plants such as hyoscyamine and Anisodus acutangulus. They are anticholinergic drugs acting on the parasympathetic nervous system in medical terms, and have the functions of anesthesia, spasmolysis and pain relief. In addition, it also has the effect of improving microcirculation, and can be used clinically to treat diseases of microcirculation disorders. Thanks to the efforts of Chinese scholars, the clinical application of tropane alkaloids has been widely used in internal medicine, surgery, obstetrics and gynecology, neurology, dermatology, otolaryngology, etc., and can treat more than 100 diseases, and the market demand is very huge. Hyoscyamine (racemate atropine) and scopolamine are two structura...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N15/29C12N9/00C12N15/82C12N1/21C12N5/10A01H4/00
Inventor 开国银陈军峰李礼周伟周根余
Owner SHANGHAI NORMAL UNIVERSITY
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