Method for cloning of pleurotus djamor HP1 laccase gene and dye decoloration of recombinase

A cloning method, the technology of Pleurotus ostreatus, applied in genetic engineering, plant genetic improvement, chemical instruments and methods, etc., can solve problems such as high cost, low enzyme activity, and no effect

Inactive Publication Date: 2013-06-26
QIQIHAR UNIVERSITY
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Problems solved by technology

[0004] The purpose of the present invention is to solve the existing problems in the prior art, that is, white rot fungi contain the most laccase, but the growth period of white rot fungi is long and the enzyme activity is low, which is not conducive to in...

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  • Method for cloning of pleurotus djamor HP1 laccase gene and dye decoloration of recombinase
  • Method for cloning of pleurotus djamor HP1 laccase gene and dye decoloration of recombinase
  • Method for cloning of pleurotus djamor HP1 laccase gene and dye decoloration of recombinase

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[0041] The present invention will be described in further detail below in conjunction with the accompanying drawings: the present embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation is provided, but the protection scope of the present invention is not limited to the following embodiments.

[0042] 1. Total RNA detection

[0043] After extracting the total RNA of Pleurotus ostreatus HP1 mycelium, it was detected by 1% agarose gel electrophoresis, and the results showed that the bands of 28S rRNA and 18S rRNA were clear, and the brightness of the former was about twice that of the latter, indicating that the integrity of the extracted RNA was better ; tested by a nucleic acid detector, 260 / 280, 260 / 230 were 2.07 and 2.37, respectively, indicating a high purity. The extracted RNA can be used in follow-up experiments (such as figure 1 shown).

[0044] 2. Cloning and sequence analysis of laccase cDNA of Pleurotu...

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Abstract

The invention provides a method for the cloning of a pleurotus djamor HP1 laccase gene and the dye decoloration of recombinase. According to the invention, a pair of degenerate primers is designed according to a conserved region of several white-rot fungi laccase gene copper ion binding site amino acids, a laccase cDNA gene core segment is amplified through RT-PCR, and finally a laccase cDNA full-length sequence is amplified by a 3'/5' RACE technology. The pleurotus djamor cDNA is connected with an expression vector pPICZB to construct a recombinant plasmid pPICZB/lac, and the pichiapastoris SMD1168H is converted by an electric conversion method to obtain an engineering strain. The purified laccase is obtained by DEAE-sepharose CL-6B ion exchange column chromatography, polyethylene glycol concentration and SephadexG-75 gel filtration chromatography. The unique degradation ability of the purified laccase against alizarin red can be applied to the wastewater treatment after dye pollution.

Description

technical field [0001] The invention relates to a novel laccase gene. By combining RT-PCR and RACE technology, the full-length sequence of cDNA and Genomic DNA encoding the laccase gene is obtained from the strain for the first time, and the obtained laccase cDNA is combined with an expression vector pPICZB was connected to obtain the recombinant expression vector pPICZB / lac, which was transformed into Pichia pastoris (P.pastoris) SMD1168H cells by electroporation, and used DEAE-Sepharose CL-6B ion exchange column chromatography, polyethylene glycol concentration and SephadexG-75 gel Purified laccase was obtained by filtration column method, and the purified laccase was used to decolorize dyes with different chemical structures at a final concentration of 50 mg / L. That is, the cloning, heterologous expression, purification of crude enzyme solution and dye decolorization method of purified laccase gene of Pleurotus ostreatus HP1. Background technique [0002] Laccase (Laccas...

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Application Information

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IPC IPC(8): C12N15/10C12N15/53C12N15/81C12N9/02C02F3/00
Inventor 郑苗苗邵淑丽池玉杰
Owner QIQIHAR UNIVERSITY
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