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Taxaceae 3-hydroxy-3-methylpentadiacyl cozymase A synthetic zymoprotein coding sequence

A technology of methylglutaryl coenzyme and coding sequence, which is applied in the fields of molecular biology and genetic engineering, and can solve problems that have not been found in literature reports.

Inactive Publication Date: 2005-02-23
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the analysis of existing documents, "The Plant Journal (Plant Journal), 2000, 22: 415-426" and "Science Asia (Asian Science) 2002, 28: 29-36" etc. successively reported that from rapeseed and rubber tree The 3-hydroxy-3-methylglutaryl coenzyme A synthetase gene has been cloned, but no literature report on the subject of the present invention has been found so far

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] Cloning of tm-Hmgs Protein Gene from Taxus mandia

[0140] 1. Tissue separation (isolation)

[0141] The young leaves of Taxus chinensis (variety "Mandia") were obtained from Southwest Normal University, and the materials were collected and frozen in liquid nitrogen immediately.

[0142] 2. RNA isolation (RNA isolation)

[0143] Take part of the tissue, 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.

[0144] 3. Full-length cloning of the gene (Cloning of Full-length cDNA)

[0145] According to the conserved amino acid sequences of some plant Hmgs, design annexation primers, use the principle of homologous gene cloning, and use t...

Embodiment 2

[0154] Sequence Information and Homology Analysis of Taxus chinensis tm-Hmgs Protein Gene

[0155]The length of the full-length eDNA of the novel yew tm-Hmgs protein of the present invention is 1776 bp, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at 98-1528 nucleotides. The amino acid sequence of Taxus tm-Hmgs protein was deduced according to the full-length cDNA, with a total of 476 amino acid residues, a molecular weight of 52859.09, and a pI of 5.23. See SEQ ID NO.4 for the detailed sequence.

[0156] The full-length eDNA sequence of Taxus mandiae tm-Hmgs protein and its encoded protein were carried out in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR databases using BLAST program Nucleotide and protein homology search, it was found that it has 86% homology at the nucleotide level with Scots pine 3-hydroxy-3-methylglutaryl-CoA synthetase gene (X96386.1) (Supplement...

Embodiment 3

[0158] Prokaryotic Expression and Purification of Taxus tm-Hmgs Protein in Escherichia coli

[0159] In this example, the full-length taxus tm-Hmgs protein coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0160] The yew tm-Hmgs protein polypeptide was prokaryotically expressed in Escherichia coli in the form of fusion protein.

[0161] Construction of prokaryotic expression vector and transformation of Escherichia coli

[0162] According to the amino acid sequence of the yew tm-Hmgs protein, design primers for the protein coding region, and introduce restriction endonuclease sites on the forward and reverse primers (this depends on the selected pET32a(+) vector), so as to construct the expression carrier. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the yew tm-Hmgs protein gene was cloned into the pET32a(+) vector (Novagen) u...

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PUM

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Abstract

A taxaceae 3-hydroxy-3-methyl-diacid coenzyme A synthetic protein coding sequence is disclosed. Separated DNA molecule includes: coding polypeptide ribonucleotide sequence with taxaceae tm-Hmgs protein activity, having at least 70% homology for the ribonucleotide sequence and SEQ ID NO.3 of ribonucleotide sequence from No.98-1528 of ribonucleotide, the ribonucleotide sequence hybridizing with SEQ ID NO.3 of ribonucleotide sequence from No.98-1528 of ribonucleotide under condition of 40-55oC. It can be used to improve content of taxol or its precursor and protect human health.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering. Specifically, the present invention relates to a tm-Hmgs protein expressed in Taxus chinensis (Taxus 3-hydroxy-3-methylglutaryl-CoA synthase protein, Taxusmedia 3-hydroxy-3-methylglutaryl-CoA Synthase, TMHMGS) and its nucleic acid sequence. Background technique [0002] Paclitaxel (trade name: Taxol) is a taxane diterpenoid present in the bark and needles of Taxus spp. in the family Taxaceae. Paclitaxel is currently one of the best natural anticancer drugs certified by the US FDA (1992), and is widely used clinically to treat advanced ovarian cancer, breast cancer and other cancers. At present, the severe shortage of paclitaxel drug sources has become the biggest obstacle restricting the development of paclitaxel-related industries. In recent years, the rapid development and wide application of plant genetic engineering technology has opened up a new way for ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/79
Inventor 开国银苗志奇唐克轩
Owner SHANGHAI JIAO TONG UNIV
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