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Preparation method of agaricus bisporus heme dioxygenase and application thereof

A technology of Agaricus bisporus and dioxygenase, which is applied in the field of genetic engineering of Agaricus bisporus, can solve the problems of low biological activity, not being able to better meet the needs of practical applications, and mildew.

Pending Publication Date: 2020-09-22
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] 1-Octen-3-ol has two optical isomers, the natural (−) isomer has a more typical aroma than the (+) isomer, showing a typical mushroom aroma and high biological activity, while ( +) isomers show a musty smell and low biological activity
The 1-octen-3-ol produced by artificial synthesis is a mixture of two isomers, so it cannot meet the needs of practical applications; the 1-octen-3-ol synthesized naturally by biology is mainly the (−) isomer , is also the commonly used method for the production of 1-octen-3-ol

Method used

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  • Preparation method of agaricus bisporus heme dioxygenase and application thereof
  • Preparation method of agaricus bisporus heme dioxygenase and application thereof
  • Preparation method of agaricus bisporus heme dioxygenase and application thereof

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specific Embodiment

[0066] The present application will be further explained below in conjunction with the accompanying drawings and embodiments. Before introducing specific embodiments, a brief description of the experimental background of some biological materials involved in the following embodiments is as follows.

[0067] biomaterials:

[0068] Agaricus bisporus AS2796 strains were purchased from the Institute of Edible Fungi, Fujian Academy of Agricultural Sciences; PDA medium was used for strain preservation and cultivation;

[0069] Escherichia coli BL21 strain was purchased from Shanghai Qincheng Biotechnology Co., Ltd.; LB medium was used for strain cultivation;

[0070] The prokaryotic expression vector pCold-TF was purchased from Shanghai Beinuo Biotechnology Co., Ltd.

Embodiment 1

[0072] On the basis of previous studies on fungal heme dioxygenase, combined with related sequence comparisons, the inventor speculated that there are similar biological enzymes and metabolic pathways in Agaricus bisporus. In order to further study and determine the characteristics of related biological enzymes in Agaricus bisporus, The inventor first cloned and sequenced the gene sequence of heme dioxygenase in Agaricus bisporus, and the relevant experimental process is briefly introduced as follows in this example.

[0073] (1) Total RNA was extracted and reverse transcribed into cDNA for later use

[0074] Refer to the instructions of the RNAiso Plus Total RNA Extraction Kit (Takara, Dalian, China), and use Agaricus bisporus mycelium (plate culture) as a sample to extract total RNA;

[0075] Subsequently, using Thermo Scientific™ Maxima™ First Strand cDNA Synthesis Kit for RT-qPCR (#K1641) (Thermo Fisher Scientific, Waltham, MA, USA), refer to its instructions to synthesize...

Embodiment 2

[0093] Based on the cloning results of Example 1, in order to further analyze the function of the gene, the inventors performed heterologous expression of the gene in Escherichia coli using the prokaryotic expression vector pCold-TF. The relevant experiments are described in detail as follows.

[0094] (1) Construction of recombinant expression vector

[0095] Using the plasmid pCold-TF as the prokaryotic expression vector, the PCR amplification product of Example 1 and the plasmid pCold-TF were respectively carried out Nde I. Xba I double enzyme digestion, the recovered enzyme digestion products were ligated with T4 DNA ligase to construct the recombinant expression vector pCold-TF-AbD.

[0096] Nde I. Xba For I double enzyme digestion, the 50 μL enzyme digestion system is designed as follows:

[0097] PCR product (or plasmid pCold-TF), 15 μl;

[0098] Nde I, 1 μl;

[0099] Xba I, 1 μl;

[0100] Cutsmart, 5 μl;

[0101] wxya 2 O, 28 μl;

[0102] Digest at 37...

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Abstract

The invention belongs to the technical field of agaricus bisporus genetic engineering, and particularly relates to agaricus bisporus heme dioxygenase and application thereof. A known amino acid sequence of fungal heme dioxygenase is utilized, comparing with an agaricus bisporus genome sequence,and according to the agaricus bisporus heme dioxygenase, the heme dioxygenase of agaricus bisporus is obtained, the heme dioxygenase has a CYP domain. The amino acid sequence homology with the PpoC of aspergillus nidulans is 33%, the similarity is 51%, but the CYP domain of the heme dioxygenase is the same as that of the PpoC, a complete P450 characteristic consensus sequence heme conservative binding region is not contained, it is speculated that the CYP domain of the heme dioxygenase possibly has no function, and the heme dioxygenase is named AbDOX-(CYP). The cloning of the gene and the conversion expression of escherichia coli further show that the heme dioxygenase can catalyze linoleic acid to synthesize 1-octene-3-ol. On the basis of the evidence, a good technical foundation can be laid for further industrial production improvement of the 1-octene-3-ol.

Description

technical field [0001] This application belongs to the technical field of genetic engineering of Agaricus bisporus, and specifically relates to a patent application for Agaricus bisporus heme dioxygenase and its application. Background technique [0002] 1-octen-3-ol (1-octen-3-ol), also known as mushroom alcohol, is a characteristic aroma substance of edible fungi, with green, earthy, waxy, immature fruity and milk flavors, often It is used in various foods including beverages, candies, iced foods, baked foods, condiments and tobacco flavors, especially in foods processed with various edible fungi. In addition, 1-octen-3-ol is also an attractant for mosquitoes and is widely used in the production and application of green mosquito repellent products; other studies have shown that 1-octen-3-ol can affect the germination of various mold spores and the The synthesis of toxin has a strong inhibitory effect, and has a strong inhibitory effect on plant pests, Tribolium castane...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/53C12N15/70C12N9/02C12P7/04
CPCC12N9/0083C12Y114/99003C12N15/70C12P7/04Y02A50/30
Inventor 邱立友刘皓皓高玉千李亚楠戚元成张朝辉
Owner HENAN AGRICULTURAL UNIVERSITY
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