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Ganoderma applanatum transgenic method

A technology of transgenic and tree tongue, applied in the field of transgenic to achieve the effect of reducing bacterial contamination

Pending Publication Date: 2022-02-25
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Existing genetic transformation methods of Ganoderma lucidum are not fully applicable to Ganoderma lucidum

Method used

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  • Ganoderma applanatum transgenic method
  • Ganoderma applanatum transgenic method
  • Ganoderma applanatum transgenic method

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Embodiment Construction

[0028] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0029] As shown in the figure, a method for transgenic tree tongue, wherein, at first the culture medium is prepared, including:

[0030] YPG liquid medium: peptone 10g / L, yeast extract powder 5g / L, glucose 20g / L, MgSO 4 0.5g / L, KH 2 PO 4 1g / L;

[0031] YPG solid medium: peptone 10g / L, yeast extract powder 5g / L, glucose 20g / L, MgSO 4 0.5g / L, KH 2 PO 4 1g / L, agar powder 15g / L;

[0032] MYG liquid regeneration medium: glucose 5g / L, maltose 13g / L, yeast powder 4.5g / L, mannitol 109g / L;

[0033] MYG solid regeneration medium: glucose 5g / L, maltose 13g / L, yeast powder 4.5g / L, agar powder 15g / L, mannitol 109g / L;

[0034] The above medium was sterilized at 110°C for 30 min.

[0035] Specific steps are as follows;

[0036] (1) Clone the endogenous glyceraldehyde-3-phosphate dehydrogenase gene promoter (pGPD) of Ganoderma lucidum and the fungal immunomodulatory prote...

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Abstract

The invention discloses a Ganoderma applanatum transgenic method, and belongs to the field of biology. The method comprises the following steps: preparing a culture medium comprising a YPG culture medium and an MYG regeneration culture medium, constructing a recombinant eukaryotic expression plasmid vector pCAMBIA1303-pGPD-FIP-gap, suspending purified 1 * 10<7> Ganoderma applanatum protoplast in 250 [mu]L of osmotic pressure stabilizer, adding 5 [mu]g of plasmid pCAMBIA1303-pGPD-FIP-gap, lightly shaking up, then placing on ice, and carrying out ice bath treatment for 10 min; adding 50 [mu]L of a PEG buffer solution, placing on ice, carrying out ice bath treatment for 30 min, adding 1 mL of the PEG buffer solution, uniformly mixing, and standing at 28 DEG C for 20 min, wherein the PEG buffer solution comprises PEG6000, 15mM CaCl2 and 10mM Tris aqueous solution; and carrying out regeneration culture, hygromycin resistance screening and subculture, and then detecting. The result shows that transformants subjected to hygromycin resistance screening twice are positive, the PCR positive detection result shows that the positive transformants can be obtained by 10-60% of the PEG buffer solution, the efficiency is the best when the 30% of the PEG buffer solution is used for carrying out ganoderma applanatum conversion, 1 [mu[g of plasmid can obtain about 110-120 positive transformants, and the positive transformation rate can reach 95%.

Description

technical field [0001] The invention relates to a transgenic method, in particular to a tree tongue transgenic method. Background technique [0002] Ganoderma lucidum has the functions of clearing heat and reducing inflammation, protecting the liver, treating cardiovascular and cerebrovascular diseases and strengthening the body. In recent years, a variety of medicinal active substances have been found in Ganoderma lucidum, such as polysaccharides, steroids, triterpenes, lipid compounds, amino acids, proteins, phenols and some trace elements. Ganoderma lucidum polysaccharide is an active substance against dyslipidemia and its related complications. At present, the main research of the tree tongue is the in vitro detection of the separation and purification of biologically active substances. There are no reports on molecular biology and genetic transformation. Due to the distant kinship of subgenus Ganoderma and other Ganoderma lucidum genera, the genetic backgrounds are n...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12R1/645
CPCC12N15/80C07K14/375
Inventor 林景卫梁耕源白玉东范天宁陈焕李守坤王浩安郭芡芡申玉华陈罡高英旭范俊岗
Owner SHENYANG AGRI UNIV
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