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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: 2010-06-09
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 (Cloning of Tripartite 1,4-Butanediamine-Nitrogen-Methyltransferase Gene 1)

[0041] 1. Organization separation (isolation)

[0042] Three-thirds of the plants were from Lijiang, Yunnan, and the young and tender roots were immediately placed in liquid nitrogen for cryopreservation.

[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 containing the lysis solution, shake it enough, 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.

[0045] 3. Cloning of Full-length cDNA

[0046] According to the PMT amino acid conservative sequence of Hyoscyamine and other Solanaceae plants, degenerate primers were designed, using the principle of homologous gene cloning, an...

Embodiment 2

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

[0055] The length of the full-length cDNA of the novel three-thirds 1,4-butanediamine-nitrogen-methyltransferase gene 1 of the present invention is 1353 bp, and the detailed sequence is shown in SEQ ID NO. Glycidic acid. The amino acid sequence of three-thirds 1,4-butanediamine-nitrogen-methyltransferase was deduced from the full-length cDNA. It has a total of 338 amino acid residues, a molecular weight of 37.223KD, and a pI of 5.50. The detailed sequence is shown in SEQ ID NO.2. .

[0056] The full-length cDNA sequence of three-thirds of 1,4-butanediamine-nitrogen-methyltransferase gene 1 and its encoded protein were used BLAST program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations The nucleotide and protein homology search in the +PDB+SwissProt+Superdate+PIR database showed that it has 98% homology with the PMT...

Embodiment 3

[0147] Example 3 (Third-thirds of 1,4-butanediamine-nitrogen-methyltransferase 1 or polypeptides were prokaryotic expression and purification in E. coli)

[0148] In this example, the full-length three-thirds AaPMT1 coding sequence or fragment was constructed into a commercial protein fusion expression vector to express and purify the recombinant protein.

[0149] 1. Construction of prokaryotic expression vector and transformation of Escherichia coli

[0150] According to the nucleotide sequence of three-point three AaPMT1, 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 AaPMT1 gene was cloned into the pET32a(+) vector (Novagen) under the premise of ensuring the correct reading frame. The identified ...

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PUM

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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-methyltransferasegene1, 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 aminoacids, 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 present invention belongs to the field of biotechnology. Specifically, it relates to 1,4-butanediamine-nitrogen-methyltransferase gene 1 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 thirds, etc. They act on the parasympathetic nerves in medical applications. The systemic anticholinergic drugs have the functions of anesthesia, antispasmodic and pain relief. 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. [00...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/63C12N15/82C12N9/10C12N5/10C12N1/19C12N1/21A01H1/00C12R1/645C12R1/19
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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