Camptotheca acuminata 3-hydroxy-3-methylglutaryl A synthase gene and its coding protein and application

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

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

[0004] In the analysis of existing literature, "The Planta (Plant Journal), 2005, 221 (4): 502-12." etc. cloned 3-hydroxy-3-methylglutaryl-C

Method used

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  • Camptotheca acuminata 3-hydroxy-3-methylglutaryl A synthase gene and its coding protein and application
  • Camptotheca acuminata 3-hydroxy-3-methylglutaryl A synthase gene and its coding protein and application
  • Camptotheca acuminata 3-hydroxy-3-methylglutaryl A synthase gene and its coding protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 (cloning of camptotree 3-hydroxyl-3-methylglutaryl-CoA synthetase gene)

[0046] 1. Tissue separation (isolation)

[0047] Campylodendron cypress plants were sourced from the campus of Shanghai Normal University, and the young leaves were immediately frozen in liquid nitrogen for storage.

[0048] 2. RNA isolation (RNA isolation)

[0049] 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.

[0050] 3. Full-length cloning of the gene (Cloning of Fu11-length cDNA)

[0051] According to the conserved amino acid sequence of HMGS of camptophylla and other plant species, degenerate primers were designe...

Embodiment 2

[0059] Example 2 (Sequence information and homology analysis of camptotree 3-hydroxy-3-methylglutaryl-CoA synthetase gene)

[0060] The length of the novel full-length 3-hydroxy-3-methylglutaryl-CoA synthetase cDNA of the present invention is 1801 bp, and the detailed sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at 153-1568 nucleotides. The amino acid sequence of camptotheca 3-hydroxy-3-methylglutaryl-CoA synthetase was deduced according to the full-length cDNA, with a total of 471 amino acid residues, a molecular weight of 52.2KD, and a pI of 6.04. For the detailed sequence, see SEQ ID NO.2 .

[0061] The full-length cDNA sequence of camptotheca 3-hydroxy-3-methylglutaryl-CoA synthetase and its encoded protein were published in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundantGenBank CDS translations+PDB+ using BLAST program Nucleotide and protein homology searches were carried out in the SwissProt+Superdate+PIR database, and it was found t...

Embodiment 3

[0073] Example 3 (Prokaryotic expression and purification of camptotheca 3-hydroxy-3-methylglutaryl-CoA synthetase or polypeptide in Escherichia coli)

[0074] In this example, the full-length camptotheca 3-hydroxy-3-methylglutaryl-CoA synthetase coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0075] 1. Construction of prokaryotic expression vector and transformation of Escherichia coli

[0076] According to the nucleotide sequence of camptotheca 3-hydroxy-3-methylglutaryl-CoA synthetase, the primers for amplifying the protein coding region were designed, and restriction endonuclease sites ( This depends on the selected pET32a (+) vector), in order to construct the expression vector. Using the amplification product obtained in Example 1 as a template, after PCR amplification, the camptotheca 3-hydroxy-3-methylglutaryl-CoA synthetase gene was cloned into pET32a ( +) Veh...

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Abstract

The invention discloses a camptotheca 3-hydroxy-3-methylglutaryl coenzyme A synthase gene, protein which is encoded by the camptotheca 3-hydroxy-3-methylglutaryl coenzyme A synthase gene and the use thereof. The 3-hydroxy-3-methylglutaryl coenzyme A 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 3-hydroxy-3-methylglutaryl coenzyme A 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 3-hydroxy-3-methylglutaryl coenzyme A synthase gene which is provided by the invention can increase the content of camptothecin alkaloid in plants such as camptotheca and the like through the genetic engineering technology and can be used in research and industrialization for increasing the content of the camptothecin alkaloid through utilizing the transgenic technology.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering. Specifically, the present invention relates to a CaHMGS protein (Camptotheca 3-hydroxy-3-methylglutaryl-CoA synthase, Camptotheca acumulate 3-hydroxy-3-methylglutaryl-CoA Synthase, CaHMGS) expressed in camptotheca And its nucleic acid sequence and application. Background technique [0002] Camptothecin (Camptothecins, CPT) is an active substance isolated from Camptothecus endemic to China by Wall et al. It is one of the most effective natural anticancer drugs at present. Studies have found that CPT exerts its anticancer effect by blocking the synthesis of topoisomerase I (topoisomerase I, topo I). Development boom. At present, there are 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, which have been approved by the American Food and Drug ...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N15/63C12N15/82C12N9/00C12N5/10C12N1/21C12N1/19A01H1/00C12R1/19C12R1/645
Inventor 开国银王伟陆杨张冉董彦君周根余
Owner SHANGHAI NORMAL UNIVERSITY
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