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Scopoloa acutangula tropinone reductase II gene and its coding protein and application

A reductase and tropinone technology, applied in the fields of oxidoreductase, application, genetic engineering, etc., can solve the problem of unisolated and cloned tropinone reductase II

Inactive Publication Date: 2008-04-30
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the analysis of existing documents, "Plant Physiology (Plant Physiology), 1993, 103, 1465-1466" reported that the tropinone reductase II gene was cloned from scopolamine, but so far there is no specific drug from Yunnan, my country. Literature Report on Isolation and Cloning of Tropinone Reductase II from Three-thirds of Plants

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1 (cloning of three-point tritropine ketone reductase II gene)

[0037] 1. Tissue separation (isolation)

[0038] Three-thirds of the plants come from Lijiang, Yunnan, and the young roots are immediately frozen in liquid nitrogen for preservation.

[0039] 2. RNA isolation (RNA isolation)

[0040] 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.

[0041] 3. Cloning of Full-length cDNA

[0042] According to the conserved sequence of TRII amino acids of Scopolamine and other Solanaceae plants, degenerate primers were designed, and using the principle of homologous gene cloning, the Smart-RACE m...

Embodiment 2

[0050] Example 2 (Sequence information and homology analysis of the three-point tritropine ketone reductase II gene)

[0051] The length of the novel full-length cDNA of tripartite ketone reductase II of the present invention is 1097bp, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at 92-874 nucleotides. According to the full-length cDNA deduced the amino acid sequence of tritropine reductase II, a total of 260 amino acid residues, molecular weight 28.291KD, pI 5.35, detailed sequence see SEQ ID NO.2.

[0052] The full-length cDNA sequence of tritropinone reductase II and its encoded protein were carried out in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR databases using the BLAST program Nucleotide and protein homology search, it was found that it has 92% homology (see Table 2) with Scopolonia TRII gene (GenBank Accession No.L20485); The amino acid residues 1-260 o...

Embodiment 3

[0157] Embodiment 3 (prokaryotic expression and purification of tritropin ketone reductase II or polypeptide in Escherichia coli)

[0158] In this example, the full-length three-thirds AaTRII coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0159] 1. Construction of prokaryotic expression vector and transformation of Escherichia coli

[0160] According to the nucleotide sequence of three-thirds AaTRII, design primers for amplifying the protein coding region, and introduce restriction endonuclease 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 AaTRII gene was cloned into the pET32a(+) vector (Novagen) under the premise of ensuring the correct reading frame. The ident...

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PUM

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Abstract

The invention discloses anisodus tropinone reductase II gene and the protein and the use of the code thereof, and fills the blank of separating and cloning the tropinone reductase II gene from unique medicinal plants in Yunnan, China. The anisodus tropinone reductase II gene provided by the invention has nucleotide sequence shown in SEQ ID No.1, and the protein of the gene code has amino acid sequence shown in SEQ ID No.2. The anisodus tropinone reductase II gene provided by the invention has prominent effect for improving the content of tropane alkaloid in anisodus plants, etc. through an antisense gene technology, thereby being widely applied to the qauality improvement of resource plants producing tropane alkaloid.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, relates to the tropinone reductase II gene expressed in Sanfensan and its encoded protein and application. Background technique [0002] Hyoscyamine alkaloids such as hyoscyamine and scopolamine are mainly extracted from plants of the family Solanaceae such as belladonna, datura, hyoscyamine and Anisodus scutangulus. An anticholinergic drug that acts on the parasympathetic nervous system and has anesthesia, antispasmodic and analgesic functions. 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. [0003] Three-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/82A01H1/06
Inventor 开国银李礼王敬王伟陆杨周根余
Owner SHANGHAI NORMAL UNIVERSITY
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