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Japan snakeroot strictosidine synthase gene and its coding protein and application

A technology of isosidine and snakeroot, applied to the isosidine synthase gene and its encoded protein and its application field

Inactive Publication Date: 2008-08-27
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 the existing literature, "Plant Cell and Physiology (plant cell and physiology), 2003, 44, 395-403" reported that the isosidine synthase has been cloned from snakeroot grass, but so far there is no Literature report on the isolation and cloning of isosidine synthase gene from the medicinal plant snakeroot japonica

Method used

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  • Japan snakeroot strictosidine synthase gene and its coding protein and application
  • Japan snakeroot strictosidine synthase gene and its coding protein and application
  • Japan snakeroot strictosidine synthase gene and its coding protein and application

Examples

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Embodiment 1

[0034] The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook and other molecular cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer conditions of. Example 1 (Cloning of the isocarin synthase gene of Snakegrass japonica)

[0035] 1. Organization separation (isolation)

[0036] The Snakeroot plant originated from Fujian, and the young roots were taken and immediately placed in liquid nitrogen for cryopreservation.

[0037] 2. RNA isolation (RNA isolation)

[0038] Take part of the tissue and grind it with a mortar, add it to a 1.5mL EP tube containing lysate, 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 total RNA quality, and...

Embodiment 2

[0048] Example 2 (Sequence information and homology analysis of Japanese snakeroot isocarin synthase gene)

[0049] The length of the full-length cDNA of the new snakeroot isocarin synthase of the present invention is 1258 bp, and the detailed sequence is shown in SEQ ID NO. 1, wherein the open reading frame is located at nucleotides 65-1126. According to the full-length cDNA, the amino acid sequence of Snakegrass isoflavin synthase was deduced, with a total of 353 amino acid residues, a molecular weight of 39.3KD, and a pI of 5.7. The detailed sequence is shown in SEQ ID NO.2.

[0050] The full-length cDNA sequence and its encoded protein of Snakegrass isoflavin synthase and its encoded protein were carried out 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 revealed that it has 95% homology with the snakeroot STR gene (GenBank Accession No.AB060341...

Embodiment 3

[0061] Results: In the comparison of 353 amino acids, the two have 94% identity and 96% similarity, respectively. Example 3 (Prokaryotic expression and purification of Japanese snakeroot isocarin synthase or polypeptide in Escherichia coli)

[0062] In this example, a full-length Japanese snakeroot OjSTR coding sequence or fragment was constructed into a commercial protein fusion expression vector to express and purify the recombinant protein.

[0063] 1. Construction of prokaryotic expression vector and transformation of Escherichia coli

[0064] According to the nucleotide sequence of Snakeroot OjSTR, 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 Japanese snakeroot OjSTR gene was cloned into the pET32a(+) v...

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Abstract

The invention discloses a Japan snakeroot strictosidine synthase gene, protein which is encoded by the Japan snakeroot strictosidine synthase gene and the use thereof. The Japan snakeroot strictosidine synthase gene 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 Japan snakeroot strictosidine synthase gene, 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 Japan snakeroot strictosidine synthase gene which is provided by the invention can increase the content of camptothecin which is an anticancer component in resource plants such as Japan snakeroot or camptotheca and the like through the genetic engineering technology and can be used in research and industrialization for increasing the content of the camptothecin through utilizing the transgenic technology, which is helpful for accelerating to solve the problem that the drug resources of the camptothecin are seriously scarce and has very good application prospect.

Description

Technical field [0001] The present invention belongs to the field of biotechnology, and specifically relates to an isocavidin synthase gene expressed in Japanese snakeroot and its encoded protein and application. Background technique [0002] Camptothecin (Camptothecins, CPT) is an active substance originally isolated from China-specific Camptothecin by Wall et al. It is currently one of the most effective natural anticancer drugs. Research has found that CPT exerts its anti-cancer effect by blocking the synthesis of topoisomerase I (topo I). The discovery of this unique anti-cancer mechanism has led to research and research on camptothecin and its analogues. Development boom. At present, 4 kinds of camptothecin derivatives CPT-II (Irinotecan), TPT (Topotecan), 9-AC (9-aminocamptothecin) and 9-NC (9-nitrocamptothecin) synthesized with CPT as the precursor have obtained American food and Drug Administration (FDA) approved for clinical treatment of colon cancer, rectal cancer and r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/63C12N15/82C12N5/10C12N1/19C12N1/21A01H1/00C12R1/19C12R1/645
Inventor 开国银陆杨王伟严向明董彦君周根余
Owner SHANGHAI NORMAL UNIVERSITY
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