Scopoloa acutangula 1,4-tetramethylenediamine-nitrogen-methyltransferase 1 and its coding protein and application

A technology of methyltransferase and butanediamine, applied in 1 field, can solve problems such as unseparated clones

Inactive Publication Date: 2008-08-27
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the existing literature "Plant Cell Physiol (Plant Cell Physiology) 1999,40 (3): 289-297" has reported cloning of 1,4-butanediamine-nitrogen-methyltransferase gene from Belladonna, so far There has not been any literature report on the isolation and cloning of 1,4-butanediamine-nitrogen-methyltransferase gene from Sanfensan, a unique medicinal plant in Yunnan, my country

Method used

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  • Scopoloa acutangula 1,4-tetramethylenediamine-nitrogen-methyltransferase 1 and its coding protein and application
  • Scopoloa acutangula 1,4-tetramethylenediamine-nitrogen-methyltransferase 1 and its coding protein and application
  • Scopoloa acutangula 1,4-tetramethylenediamine-nitrogen-methyltransferase 1 and its coding protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 (cloning of three points three tor 1,4-butanediamine-nitrogen-methyltransferase gene 1)

[0041] 1. Tissue separation (isolation)

[0042] Three-thirds of the plants come from Lijiang, Yunnan, and the young roots are immediately frozen in liquid nitrogen for preservation.

[0043] 2. RNA isolation (RNA isolation)

[0044] 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.

[0045] 3. Cloning of Full-length cDNA

[0046] According to the PMT amino acid conserved sequence of Scopolamine and other Solanaceae plants, degenerate primers were designed, and using the principle of homologous gene clonin...

Embodiment 2

[0054] Example 2 (Sequence information and homology analysis of three-point three 1,4-butanediamine-nitrogen-methyltransferase gene 1)

[0055] The length of the full-length cDNA of the new three-thirds 1,4-butanediamine-nitrogen-methyltransferase gene 1 of the present invention is 1353bp, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at the core of position 117-1133 glycosides. According to the full-length cDNA, the amino acid sequence of three-point 1,4-butanediamine-nitrogen-methyltransferase is deduced, with a total of 338 amino acid residues, a molecular weight of 37.223KD, and a pI of 5.50. For the detailed sequence, see SEQ ID NO.2 .

[0056] The full-length cDNA sequence of three-thirds 1,4-butanediamine-nitrogen-methyltransferase gene 1 and its encoded protein were published in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations using BLAST program Nucleotide and protein homology search in +PD...

Embodiment 3

[0069] Example 3 (Prokaryotic expression and purification of three-thirds 1,4-butanediamine-nitrogen-methyltransferase 1 or polypeptide in Escherichia coli)

[0070] In this example, the full-length three-thirds AaPMT1 coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0071] 1. Construction of prokaryotic expression vector and transformation of Escherichia coli

[0072] According to the nucleotide sequence of three-thirds of AaPMT1, design primers to amplify 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 AaPMT1 gene was cloned into the pET32a(+) vector (Novagen) under the premise of ensuring the correct reading frame. Th...

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Abstract

The invention discloses a radix anisodii acutanguli 1,4-putrescine-nitrogen-methyltransferase gene1, protein which is encoded by the radix anisodii acutanguli 1,4-putrescine-nitrogen-methyltransferase gene1, and the use thereof, which fills a gap that the 1,4-putrescine-nitrogen-methyltransferase gene is separated and cloned from radix anisodii acutanguli which is a specific medicinal plant in Yunnan, China. The radix anisodii acutanguli 1,4-putrescine-nitrogen-methyltransferase gene1 which is provided by the invention has a nucleotide sequence or a homologous sequence which adds, replaces, inserts or losses one or a plurality of nucleotides or allele thereof and the nucleotide sequence which is derived from the radix anisodii acutanguli 1,4-putrescine-nitrogen-methyltransferase gene1, which are displayed in the SED ID No.1. The protein which is encoded by the gene has an amino acid sequence or the homologous sequence which adds, replaces, inserts or losses one or a plurality of amino acids, which is displayed in the SEQ ID No.2. The 1,4-putrescine-nitrogen-methyltransferase gene which is provided by the invention has prominent effect of increasing the content of tropane alkaloid in plants such as the radix anisodii acutanguli and the like through the genetic engineering technology and can be widely applied in improving the quality of resource plants which produce tropane alkaloid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to 1,4-butanediamine-nitrogen-methyltransferase gene 1 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 three-point three, etc., and act on parasympathetic nerves in medical aspects A systemic anticholinergic drug with 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/63C12N15/82C12N9/10C12N5/10C12N1/19C12N1/21A01H1/00C12R1/19C12R1/645
Inventor 开国银张艳陈军峰张林董彦君周根余
Owner SHANGHAI NORMAL UNIVERSITY
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