Lechang michelia 3-hydroxy-3-methy glutaryl CoA reductase protein coding sequence

A technology of methylglutaryl coenzyme and reductase, applied in the fields of molecular biology and genetic engineering

Inactive Publication Date: 2008-07-30
XUZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Before the publication of the present invention, there has not been any disclosure or report of the Lechang Michelle Mc-Hmgr protein sequence and its nucleic acid sequence mentioned in this patent application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] Cloning of the Mc-Hmgr Protein Gene of Lechang Miracle

[0136] 1. Tissue separation (isolation)

[0137] The young leaves of Lechang Michelia came from Jiangsu Provincial Key Laboratory of Medicinal Plant Biotechnology. After the materials were collected, they were immediately frozen in liquid nitrogen for preservation.

[0138] 2. RNA isolation (RNA isolation)

[0139] 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, GIBCOBRL, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0140] 3. Cloning of Full-length cDNA

[0141] According to the conserved amino acid sequence of Hmgr in some plants, design annexation primers, use the principle of homologous gene cloning...

Embodiment 2

[0149] Sequence Information and Homology Analysis of the Mc-Hmgr Protein Gene of Lechang Michelia

[0150] The full-length cDNA of the new Lechang Michelle Mc-Hmgr protein of the present invention is 2229bp in length, and the detailed sequence is shown in SEQID NO.3, wherein the open reading frame is located at nucleotides 56-1726. The amino acid sequence of Lechang Michelle Mc-Hmgr protein was deduced according to the full-length cDNA, which has a total of 556 amino acid residues, a molecular weight of 59293.71, and a pI of 8.56. See SEQ ID NO.4 for the detailed sequence.

[0151]The full-length cDNA sequence of Lechang Michelle Mc-Hmgr protein and its encoded protein were analyzed by BLAST program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database Acid and protein homology search, it was found that it has a high homology with the 3-hydroxy-3-methylglutaryl-CoA reductase gene (AH429388) of Hevea parahevea at ...

Embodiment 3

[0153] Prokaryotic Expression and Purification of Lechang Michelle Mc-Hmgr Protein in Escherichia coli

[0154] In this example, the full-length Lechang Michelia Mc-Hmgr protein coding sequence or fragment was constructed into a commercially available protein fusion expression vector to express and purify the recombinant protein.

[0155] The Lechang michelia Mc-Hmgr protein polypeptide was prokaryotically expressed in the form of fusion protein in Escherichia coli.

[0156] Construction of prokaryotic expression vector and transformation of Escherichia coli

[0157] According to the amino acid sequence of Lechang michelia Mc-Hmgr 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 Expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the Lechang Michelia, Mc-Hmgr protein ...

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PUM

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Abstract

The Lechang michelia Mc-Hmgr protein coding sequence belongs to the field of gene engineering. The separated DNA includes nucleotide sequence coding polypeptide with Lechang michelia Mc-Hmgr protein activity, and the nucleotide sequence has at least 70 % homolog with the nucleotide sequence of sites 56-1726 in SEQ ID No. 3 or can hybridize with the nucleotide sequence of sites 56-1726 in SEQ ID No. 3 at 40-55 deg.c. The present invention is one kind of 3-hydroxy-3-methyl glutaryl coenzyme A reductase capable of raising the content of secondry metabolite or its precursor in Lechang michelia. The secondry metabolite has the functions of regulating blood pressure of human body in bi-direction, lowering cholesterol content, preventing cardiac and cerebral angioscerosis, etc.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering. Specifically, the present invention relates to a Mc-Hmgr protein expressed in Michelia michelia (Michelia michelia 3-hydroxy-3-methylglutaryl-CoA reductase protein, Michelia chapensis 3-hydroxy-3-methylglutaryl- CoA Reductase, McHMGR) and its nucleic acid sequence. Background technique [0002] Lechang Michelia (Michelia chapensis), also known as Jinglie Bailan, is an evergreen tree of the Magnoliaceae Michelia genus. The tree is tall and straight, with beautiful shape and fragrant flowers. . But its chemical composition has not been reported. Plants of the same genus, white orchid, mountain magnolia, yellow myangui, Yunnan michelia, etc. can be used as medicine. White orchid flowers have the effect of relieving cough and turbidity, and can treat chronic bronchitis, prostatitis and other diseases. The flowers of magnolia have anti-inflammatory and heat-clea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/79C12N9/02A01H5/00
Inventor 蒋继宏曹小迎陈凤美孙勇开国银刘群
Owner XUZHOU NORMAL UNIVERSITY
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