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

A technology of reductase and tropinone, applied in oxidoreductase, application, genetic engineering and other directions, can solve the problem of unisolated clones

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 literature, although "The Proceedings of the National Academy of Sciences of the United States of America (National Academy of Sciences Progress) 1993, 90, 9591-9595" reported that tropinone was cloned from Datura reductase I gene, but so far there has not been any literature report on the isolation and cloning of tropinone reductase I from Sanfensan, a unique medicinal plant in Yunnan, my country

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 (Cloning of Tripartite Tropinone Reductase I Gene)

[0038] 1. Organization separation (isolation)

[0039] Three-thirds of the plants are from Lijiang, Yunnan, and the young and tender roots are immediately placed in liquid nitrogen for cryopreservation.

[0040] 2. RNA isolation (RNA isolation)

[0041] Take part of the tissue and grind it with a mortar, add a 1.5mL EP tube containing lysate, shake it well, and then move it into a glass homogenizer. After homogenization, transfer to a 1.5mL EP tube to extract total RNA (TRIzol Reagents, GIBCO BRL, USA). Use formaldehyde denaturing gel electrophoresis to identify the quality of total RNA, and then measure the RNA content on a spectrophotometer.

[0042] 3. Cloning of Full-length cDNA

[0043] According to the TRI amino acid conservative sequences of Hyoscyamus scoparia and other Solanaceae plants, degenerate primers were designed, using the principle of homologous gene cloning, and the Smart-RACE method (Clonetec...

Embodiment 2

[0051] Example 2 (Sequence information and homology analysis of the three-part tritropine reductase I gene)

[0052] The length of the full-length cDNA of the novel three-part tritropine reductase I of the present invention is 1168 bp, and the detailed sequence is shown in SEQ ID NO. 1, wherein the open reading frame is located at nucleotides 70-891. According to the full-length cDNA, the amino acid sequence of three-part tropine reductase I was deduced, with a total of 273 amino acid residues, a molecular weight of 29.463KD, and a pI of 6.45. The detailed sequence is shown in SEQ ID NO.2.

[0053] The full-length cDNA sequence of three-thirds tropine ketone reductase I and its encoded protein were performed in the Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database using BLAST program Nucleotide and protein homology search showed that it has 94% homology with Scopolitan TRI (GenBank Accession No. D88156) (see Table 2)...

Embodiment 3

[0153] Results: In the comparison of 273 amino acids, the two have 90% identity and 96% similarity respectively. Example 3 (Prokaryotic expression and purification of one-third tropine reductase I or polypeptide in E. coli)

[0154] In this example, the full-length three-thirds AaTRI coding sequence or fragment was constructed into a commercial protein fusion expression vector to express and purify the recombinant protein.

[0155] 1. Construction of prokaryotic expression vector and transformation of Escherichia coli

[0156] According to the nucleotide sequence of three-point three AaTRI, design primers to amplify the protein coding region, and introduce restriction endonuclease sites on the positive and negative primers (this depends on the selected pET32a(+) vector), In order to construct an expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the three-thirds AaTRI gene was cloned into the pET32a(+) vector (Novagen) unde...

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PUM

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Abstract

The invention discloses anisodus tropinone reductase I gene and the protein and the use of the code thereof, and fills the blank of separating and cloning the tropinone reductase I 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 I gene provided by the invention has prominent effect for improving the content of tropane alkaloid in anisodus plants, etc. through an gene engineering technology, thereby being widely applied to the qauality improvement of resource plants producing tropane alkaloid.

Description

Technical field [0001] The present invention belongs to the field of biotechnology. Specifically, it relates to a tropine reductase I gene expressed in three-thirds and its encoded protein and applications. Background technique [0002] Scopolane alkaloids such as hyoscyamine and scopolamine are mainly extracted from solanaceous plants such as belladonna, datura, scopole and Anisodus scutangulus. They are used in medical applications. An anticholinergic drug that acts on the parasympathetic nervous system. It has the functions of anesthesia, antispasmodic and analgesic. In addition, it also has the effect of improving microcirculation, and can be used clinically to treat microcirculation disorders. Due to the efforts of Chinese scholars, the clinical application of scopolane alkaloids is widespread in internal medicine, surgery, obstetrics and gynecology, neurology, dermatology, otolaryngology, etc. It can treat more than 100 diseases, and the market demand is very huge. [0003]...

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 开国银李礼张林王敬王伟陆杨周根余
Owner SHANGHAI NORMAL UNIVERSITY
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