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Inonotus obliquus 3-hydroxy-3-methylglutaryl CoA reductase gene, protein of code thereof and application thereof

A technology of methylglutaryl coenzyme and fibrillar bacteria, which is applied in the fields of genetic engineering and molecular biology, and can solve the problems that have not yet been discovered.

Active Publication Date: 2012-01-04
广西五利通生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the analysis of existing literature, "Biosci.Biotechnol.Biochem., 2008, 72(5): 1333-1339" reported that 3-hydroxy-3-methylglutaryl-CoA reduced Enzyme gene, but have not found so far the bibliographical report identical with subject matter of the present invention

Method used

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  • Inonotus obliquus 3-hydroxy-3-methylglutaryl CoA reductase gene, protein of code thereof and application thereof
  • Inonotus obliquus 3-hydroxy-3-methylglutaryl CoA reductase gene, protein of code thereof and application thereof
  • Inonotus obliquus 3-hydroxy-3-methylglutaryl CoA reductase gene, protein of code thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 (cloning of the 3-hydroxyl-3-methylglutaryl-CoA reductase gene of P. obliquus)

[0045] 1. Tissue separation (isolation)

[0046] The Mycelium obliquus was obtained from Jiangsu Provincial Key Laboratory of Medicinal Plant Biotechnology, and the mycelia were collected in liquid nitrogen and immediately frozen in liquid nitrogen.

[0047] 2. RNA isolation (RNA isolation)

[0048] Take the frozen mycelium, grind it with a mortar, add it to a 1.5mL DS tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to a 1.5mL DS 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.

[0049] 3. Cloning of Full-length cDNA

[0050] According to the conserved amino acid sequence of HMGR in some fungi, degenerate primers were designed, and using the pri...

Embodiment 2

[0058] Example 2 (Sequence Information and Homology Analysis of the 3-Hydroxy-3-Methylglutaryl-CoA Reductase Gene of P. obliquus)

[0059] The length of the coding region of the novel 3-hydroxy-3-methylglutaryl-CoA reductase gene of the present invention is 4146 bp, and the detailed sequence is shown in SEQ ID NO.1. According to the sequence of the full-length coding region, the amino acid sequence of 3-hydroxy-3-methylglutaryl-CoA reductase of Inocellus obliquus was deduced, with a total of 1381 amino acid residues, a molecular weight of 147.44kDa, and a pI of 8.92. See SEQ ID NO.2 for the detailed sequence.

[0060] The full-length cDNA sequence and its encoded protein of 3-hydroxy-3-methylglutaryl-CoA reductase of Poreophora obliquus were published in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations by BLAST program Nucleotide and protein homology search in +PDB+SwissProt+Superdate+PIR database, it was found that it is related to Ganoderma luci...

Embodiment 3

[0199] Example 3 (Prokaryotic expression and purification of Poreophora obliquus 3-hydroxy-3-methylglutaryl-CoA reductase or polypeptide in Escherichia coli)

[0200] In this example, the full-length coding sequence or fragment of the P. obliquus 3-hydroxy-3-methylglutaryl-CoA reductase gene was constructed into a commercially available protein fusion expression vector to express and Purify the recombinant protein.

[0201] 1. Construction of prokaryotic expression vector and transformation of Escherichia coli

[0202] According to the amino acid sequence of the 3-hydroxyl-3-methylglutaryl-CoA reductase of Poriocellus obliquus, the primers of the protein coding region were designed, and restriction endonuclease sites were introduced on the forward and reverse primers (this is based on the The selected pET32a (+) vector depends), so as to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the 3-hydroxy-3-...

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Abstract

The invention discloses an inonotus obliquus 3-hydroxy-3-methylglutaryl CoA reductase gene, protein of a code thereof and an application thereof. A squalene synthase gene provided in the inonotus obliquus 3-hydroxy-3-methylglutaryl CoA reductase gene has nucleotide sequences indicated by SEQ ID NO. 1 or homologous sequences for adding, replacing, inserting or missing one or a plurality of nucleotides or nucleotide sequences derived by an allele of the nucleotide and the nucleotide. The protein of the code of the gene has amino acid sequences indicated by SEQ ID NO.2 or homologous sequences for adding, replacing, inserting or missing one or a plurality of amino acids. The inonotus obliquus 3-hydroxy-3-methylglutaryl CoA reductase gene can improve the content of inonotus alcohol in inonotusobliquus by utilizing the gene engineering technology and also can be used in the research of improving the content of the inonotus alcohol in the inonotus obliquus by utilizing the transgenic technology and the industrialization. A plurality of secondary metabolites have huge application value clinically and help to protect the healthy growth of people. Therefore, the inonotus obliquus 3-hydroxy-3-methylglutaryl CoA reductase gene, the protein of the code thereof and the application thereof have huge application prospect.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering. Specifically, the present invention relates to a kind of IoHMGR albumen (Inonotus obliquus 3-hydroxy-3-methylglutaryl-CoA reductase protein, Inonotus obliquus3-hydroxy-3-methylglutaryl) expressed in Inonotus obliquus -CoA reductase, IoHMGR) and nucleic acid sequence thereof, are 3-hydroxyl-3-methylglutaryl coenzyme A reductase gene and its encoded protein and application of Polypores obliquus. Background technique [0002] Inonotus obliquus (Pers.: Fr.) J.Schroet., commonly known as chaga, Inonotus obliquus, Chaga, belongs to Basidiomycota, Hymeno-mycetes , Aphyllophorales, Hymenochaetaceae, and Inonotus, mainly distributed in 45°-50° north latitude, and mainly grown in Jilin, Heilongjiang, Tibet, Qinghai, etc. On birch trees in high-altitude mountainous areas, prominent black tumor-like sclerotias are formed. As a medicinal fungus, P. obliquus has anti-tumor...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/63C12N15/80C12N1/15C12R1/645
Inventor 曹小迎蒋继宏陈凤美冯友建孙勇郑竹君李长根魏江春王力
Owner 广西五利通生物科技有限公司
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