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Laccase protein gene and cloning and sequencing method thereof

A technology of Antrodia cintrodia and genes, applied in the field of genetic engineering, can solve problems such as the reduction of enzyme activity

Inactive Publication Date: 2015-06-17
SHANGHAI QINSHENGYUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The first type of copper ion (Cu1) can interact with solvents including water, and various copper ion complexing agents can remove it, resulting in a greatly reduced enzyme activity

Method used

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  • Laccase protein gene and cloning and sequencing method thereof
  • Laccase protein gene and cloning and sequencing method thereof
  • Laccase protein gene and cloning and sequencing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Cloning of the cDNA sequence of the Antrodia camphorata Laccase protein gene

[0031] 1. Isolation

[0032]The mycelia of Antrodia cinnamomea in the growth stage were collected from the culture site of Antrodia cinnamomea, No. 20, Yumin Road, Chenjia Town, Chongming District, Shanghai Qinshengyuan Biotechnology Co., Ltd., and were tested with ddH 2 O washing to remove impurities such as culture medium, the tissue samples were quickly frozen in liquid nitrogen and stored at -70°C, and brought back to the laboratory for extraction of total tissue RNA.

[0033] 2. Total RNA isolation (Total RNA isolation)

[0034] (1) Preparation for RNA extraction

[0035] Glass products were soaked in 0.1M NaOH overnight, rinsed repeatedly with tap water, rinsed twice with distilled water, and baked at 180°C for 4 hours.

[0036] Soak the homogenizer and electrophoresis tank with 3% hydrogen peroxide for 20-30 minutes, and then rinse with 0.1% DEPC water. Since the uninactiv...

Embodiment 2

[0058] Example 2: Homology comparison and phylogenetic tree construction of Antrodia camphorata Laccase protein gene coding sequence

[0059] 1. Homology comparison of the coding sequence of Antrodia antrodia Laccase protein gene

[0060] Search and obtain the Laccase protein gene coding protein sequences of 11 species of fungi such as Pleurotus rugosa (AF263467.2), Pleurotus eryngii (FJ231091.1), Agaric (XM_007387099), and Coprinus pubescens (JQ228450.1) on GenBank , together with the sequence of SEQ ID NO.2 obtained by the present invention obtained by cloning sequencing, sequence homology comparison is carried out on the molecular biology software MEGA4 (see attached figure 2 ). The comparison results show that the homology between the sequence of SEQ ID NO.2 and the amino acid sequence of Pleurotus eryngii protein is 90.00%.

[0061] 2. Construction of phylogenetic tree of Laccase protein gene coding sequence

[0062] Search and obtain the coding protein sequences of L...

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Abstract

The invention discloses an Antrodia camphorate <{EN0}>Laccase protein gene and a cloning and sequencing method thereof, belonging to the technical field of molecular biology. The Antrodia camphorate <{EN0}>Laccase protein gene is a gene of laccase; specifically, the nucleotide sequence of the gene is as shown in SEQ ID No. 1, and codes polypeptide with an amino acid sequence as shown in SEQ ID No. 2. The invention further provides a cloning and sequencing method for the Antrodia camphorate <{EN0}>Laccase protein gene. The method comprises the following steps: tissue isolation; isolation of total RNA; and cloning and sequencing of a full-length cDNA sequence. The nucleic acid sequence and variety of the gene can be confirmed by analyzing sequencing results with molecular biology software and constructing a phylogenetic tree.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a Laccase protein gene of Antrodia camphorata and its cloning and sequencing methods. Background technique [0002] Laccase (Laccase) is a kind of glycoprotein, and its peptide chain is generally composed of about 500 amino acids, and the glycoside accounts for 10% to 45% of the whole molecule. The sugar composition includes hexosamine, glucose, mannose, fucose and arabinose, etc. Due to the difference in the sugar groups in the molecule, the molecular weight of laccases will vary greatly with different sources, even laccases from the same source will have different molecular weights. Although the amino acid sequence homology of laccases varies greatly, and generally the homology of laccases from different sources is less than 50%, their catalytic site sequences are still very conserved. Through the study of the crystal structure of laccase protein, it was found tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/10
Inventor 管荣良陈国义裘东辉周龙陈宇王伟萍王芳王静
Owner SHANGHAI QINSHENGYUAN BIOTECH
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