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Recombinant plasmid for genetic transformation of pleurotus eryngii and application thereof

A technology of recombinant plasmids and Pleurotus eryngii, which is applied to recombinant plasmids for genetic transformation of Pleurotus eryngii and its application field, can solve the problems of low biotransformation rate, long growth cycle of Pleurotus eryngii, and restrictions on the development and application of Pleurotus eryngii , to achieve the effect of good passage stability and wide application field

Active Publication Date: 2013-04-10
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pleurotus eryngii has a long growth cycle and low biotransformation rate, which limits the development and application of Pleurotus eryngii and its bioactive substances. Therefore, basic research on the genetic development mechanism of Pleurotus eryngii needs to be strengthened urgently

Method used

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  • Recombinant plasmid for genetic transformation of pleurotus eryngii and application thereof
  • Recombinant plasmid for genetic transformation of pleurotus eryngii and application thereof
  • Recombinant plasmid for genetic transformation of pleurotus eryngii and application thereof

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Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1, the construction of recombinant expression vector

[0040] 1. Construction of recombinant plasmid pUC19-Pgpd-Tgpd

[0041] 1. Extract the genomic DNA of Pleurotus eryngii mycelium.

[0042] 2. Cloning of Pleurotus eryngii glyceraldehyde triphosphate dehydrogenase gene promoter (Pgpd)

[0043] (1) With reference to the full sequence design of the gpd gene of shiitake mushroom published in 2000 by Hirano, the primer pair for amplifying the Pleurotus eryngii glyceraldehyde triphosphate dehydrogenase gene promoter is as follows:

[0044] Pgpd-F (upstream primer): 5'-ATTA GCATGCCGAAGTTTGAGGTGGTT-3' (underlined Sph I recognition sequence);

[0045] Pgpd-R (downstream primer): 5'-AA GTC GAC ATTCAAGCAGTCAATGGAT-3' (Sal I recognition sequence is underlined).

[0046] (2) Using the genomic DNA extracted in step 1 as a template, perform PCR amplification with a primer pair composed of Pgpd-F and Pgpd-R to obtain a PCR amplification product (about 1000 bp).

...

Embodiment 2

[0081] Embodiment 2, PEG / CaCl 2 mediated transformation of Pleurotus eryngii protoplasts

[0082] MMC damping fluid is made up of solute and solvent; Said solvent is 50mM maleic acid damping fluid (pH 5.5); Described solute and its concentration in MMC damping fluid are as follows: 0.5M mannitol, 5mM CaCl 2 .

[0083] Liquid MCM medium: Dissolve 2 g of yeast extract, 2 g of tryptone, 20 g of glucose, 0.5 g of magnesium sulfate, 0.5 g of potassium dihydrogen phosphate and 1 g of dipotassium hydrogen phosphate in water and make up to 1 L with water.

[0084] Solid MCM plate: Add agar to the liquid MCM medium to make the concentration 20g / L.

[0085] PTC buffer is made up of solute and solvent; Said solvent is 10mM Tris-HCl buffer (pH7.5); Described solute and its concentration in PTC buffer are as follows: 0.4g / mL PEG3350, 100mM CaCl 2 .

[0086] STC buffer is made up of solute and solvent; Described solvent is 10mM Tris-HCl buffer (pH7.5); Described solute and its concentra...

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Abstract

The invention discloses a recombinant plasmid for genetic transformation of pleurotus eryngii and application thereof and provides a DNA (Deoxyribonucleic Acid) segment and the recombinant plasmid containing the DNA segment, wherein the DNA segment sequentially comprises a promotor shown as a sequence 1 and a terminator shown as a sequence 2 from upstream to downstream. According to a method for genetic transformation of the pleurotus eryngii, which is established by the invention, the foundation for theoretical basis and technical support for fundamental research and molecular breeding of the growth and development mechanism of the pleurotus eryngii is laid, the storage for developing a novel bioreactor is provided and wide application field for developing and utilizing pleurotus eryngiiresources and bioactive substances thereof is opened up.

Description

technical field [0001] The invention relates to a recombinant plasmid used for genetic transformation of Pleurotus eryngii and its application. Background technique [0002] Pleurotus eryngii, also known as Pleurotus eryngii, belongs to Basidiomycotina, Hymenomycetes, Homobasidiomycetidae, Agaricales, Pleurotaceae ( Pleurotaceae), Pleurotus. Pleurotus eryngii is a new species of rare edible fungus that has been successfully developed and cultivated in recent years and is suitable for industrial cultivation. It has a unique flavor and rich nutrition, and can produce a variety of bioactive molecules and enzymes. Body immune function, anti-tumor and antioxidant activity, so Pleurotus eryngii has high edible, medicinal, economic and ecological value, and has a broad market prospect. Pleurotus eryngii has accounted for 5.01% of the total output of edible fungi in the Beijing market. Pleurotus eryngii has a long growth cycle and low biotransformation rate, which limits the deve...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/63C12N15/113A01H15/00
Inventor 许峰刘宇尹永刚王守现赵爽王鹏耿小丽王兰青孟莉莉
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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