Method for improving biological output and polysaccharide content of lucid ganoderma

A technology of biological yield and Ganoderma lucidum polysaccharide, which is applied in the direction of using vectors to introduce foreign genetic material, recombinant DNA technology, etc., to achieve the effect of increasing polysaccharide content, polysaccharide content and biomass, and increasing polysaccharide content

Inactive Publication Date: 2010-06-02
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] So far, according to the principle of metabolic engineering, the method of using genet

Method used

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  • Method for improving biological output and polysaccharide content of lucid ganoderma
  • Method for improving biological output and polysaccharide content of lucid ganoderma
  • Method for improving biological output and polysaccharide content of lucid ganoderma

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Example 1: Agrobacterium-mediated transformation of Zizhi

[0019] (1) Construction of Ganoderma lucidum overexpression vector OsU-gdp mediated by Agrobacterium

[0020] The transformation vector of this embodiment can be selected from the conventional binary expression vector mediated by Agrobacterium, between the two borders of the T-DNA, the promoter expressed in the fungus (the gpd promoter in this embodiment), plus the present invention The target gene (uridine diphosphate glucose pyrophosphorylase gene) and the selectable marker gene (hygromycin resistance gene in this example) can be used to obtain the transformation vector of Ganoderma lucidum mediated by Agrobacterium.

[0021] In this example, the expression vector OsU-gdp was overexpressed in the OsUgp2 driven by the Ubi promoter by using the above-mentioned conventional method, and was used for the expression vector of Agrobacterium-mediated transformation of rice (named OsU-ubi, see the attached figure 1 )...

Embodiment 2

[0035] Example 2 PCR Identification and Southern Identification of Transgenic Zizhi

[0036] (1) Preparation of transgenic Zizhi total DNA (CTAB method)

[0037] Weigh about 0.2 g of vacuum-dried mycelia in a mortar, add liquid nitrogen to grind into powder, transfer to a 2 mL centrifuge tube, add CTAB extract [10 mmol / L Tris-HCL, 2.0% hexadecane trimethylammonium bromide (CTAB), 20mmol / L ethylenediaminetetraacetic acid (EDTA), 1.4mol / LNaCl, pH8.0] 1mL and CTAB precipitation solution (50mmol / L Tris-HCL, 1.0%CTAB, 10mmol / L EDTA, pH 8.0) 100 μL, mix, stand at 65°C for 1 hour, then centrifuge at 12000 rpm for 10 minutes, take the supernatant, add 1 / 10 volume of CTAB / NaCl solution (1.0% CTAB, 0.7mol / L NaCl), mix well, and then add an equal volume of 24:1 chloroform:isoamyl alcohol solution, mix well, extract, shake for a few minutes, centrifuge at 4°C and 8000rpm for 10 minutes, take the supernatant, and add Equal volume of chloroform, shake for 5 minutes, centrifuge at 8000rp...

Embodiment 3

[0059] Example 3 Determination of Transgenic Zizhi Polysaccharide Content and Biomass

[0060] (1) Determination of the extraction content of intracellular and extracellular polysaccharides of Zizhi strain

[0061] Extract intracellular polysaccharides by hot water extraction: Take 0.2g of Ganoderma lucidum powder, extract with 15mL of 85% ethanol at 60°C for 30 minutes, repeat twice to remove interfering components such as monosaccharides, disaccharides, and oligosaccharides . The pretreated bacterial powder was extracted in a boiling water bath for 1 hour, filtered to a volume of 50 mL, and used to determine the polysaccharide content.

[0062] Extract exopolysaccharides by ethanol precipitation method: take 500 μL of fermentation broth, add 1.5 mL of 95% ethanol, let stand overnight at 4°C, centrifuge after about 18 hours, remove the supernatant, wash the precipitate twice with absolute ethanol, and dry in the air Dissolve in 1mLddH 2 O, for the determination of polysacc...

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Abstract

The invention discloses a method for improving biological output and polysaccharide content of lucid ganoderma. The UDP-glucose pyrophosphorylase (OsUgp2) gene derived from rice is connected to an overexpression vector and transferred into the lucid ganoderma by a agrobacterium rhizogenes mediated method; in the obtained transgenosis lucid ganoderma strain, the expression of the OsUgp2 gene is strengthened, and the biological output and the intracellular polysaccharide and extracellular polysaccharide content of the ganoderma sinensis are obviously improved. The invention can effectively improve the biological output and the polysaccharide content in edible and medicinal fungi, such as the lucid ganoderma, and the like, provides a new approach for improving the polysaccharide content of the edible and medicinal fungi and has favorable application and development prospect.

Description

technical field [0001] The invention belongs to the technical field of genetically modified edible fungi. Specifically, it relates to a method for over-expressing the uridine diphosphate glucose pyrophosphorylase gene into the edible fungus Ganoderma lucidum through transgenic technology, thereby increasing the biological yield and polysaccharide content of the Ganoderma lucidum. Background technique [0002] UDP-glucose pyrophosphorylase (UDP-glucose pyrophosphorylase, UGPase, EC 2.7.7.9) is an important enzyme in organisms, which is closely related to carbohydrate metabolism. UGPase can catalyze a reversible reaction dependent on magnesium, the reaction formula is as follows: UTP+G-1-P←→UDPG+PPi. Uridine diphosphate glucose (UDPG) is the main activated form of glucose, which can be used as a donor of glucose groups to participate in sucrose, starch, cellulose, hemicellulose, pectin and glycolipids, glycoproteins, glycogen, β- Synthesis of various carbohydrates including ...

Claims

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Application Information

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IPC IPC(8): C12N15/80
Inventor 穆虹张帆钟威李刚
Owner SOUTH CHINA AGRI UNIV
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