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A kind of Ganoderma lucidum polysaccharide high-yield engineering strain rmust and its construction method

An engineering strain, Ganoderma lucidum polysaccharide technology, applied in the field of genetic engineering and metabolic engineering, can solve the problem of low production of Ganoderma lucidum polysaccharides

Inactive Publication Date: 2015-12-09
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to solve the problem that the wild-type ganoderma lucidum strain itself produces relatively low ganoderma polysaccharides, and provide a high-yield ganoderma polysaccharide strain, which is a ganoderma lucidum with high yield of ganoderma polysaccharides ( Ganodermal lucidum ) engineering bacteria Rmust, which was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on September 18, 2013, address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCCNO.8258

Method used

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  • A kind of Ganoderma lucidum polysaccharide high-yield engineering strain rmust and its construction method
  • A kind of Ganoderma lucidum polysaccharide high-yield engineering strain rmust and its construction method
  • A kind of Ganoderma lucidum polysaccharide high-yield engineering strain rmust and its construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: Construction of pJW-EXP vector

[0044] 1. Extraction of Ganoderma lucidum genomic DNA

[0045] Weigh about 0.2 g of freeze-dried wild-type Ganoderma lucidum (CCGMC5.616) mycelium and grind it into powder in liquid nitrogen, and transfer the powder into 1.5 mL of CTAB (cetyltrimethyl bromide) preheated at 65 °C Ammonium) in the extraction buffer, keep warm at 65°C for 30min, then centrifuge at 10,000g for 20min at 4°C, take the supernatant and add an equal volume of chloroform:isoamyl alcohol (24:1) mixture, shake gently for more than 30min, Centrifuge at 10,000g for 20min at 4°C; transfer the supernatant into a 1.5mL centrifuge tube, add 2 / 3 volume of isopropanol pre-cooled at -20°C, shake gently for 5min, remove the DNA with a glass rod, and use 75 % ethanol was washed 2-3 times, dried at room temperature, dissolved in an appropriate amount of TE containing 20 μg / mL RNase, digested RNA at 37°C for 30 min, and then the genomic DNA of Ganoderma lucidum ...

Embodiment 2

[0061] Embodiment 2: Construction of pJW-EXP-tPGM vector

[0062] 1. Cloning of PGM gene

[0063] Using Ganoderma lucidum genomic DNA as a template, the PGM gene was obtained by PCR with primers PGM-Nhe-F: 5'-GCTAGCATGTCGTACCAGGTCAAGGAG-3' and PGM-Sma-R: 5'-GGGCCCCTACGTGATGACGGTCGGC-3'; the PCR conditions were: 95°C for 10 min, 95°C for 30s, 58°C for 30s, 72°C for 2min, 72°C for 10min (see image 3 ).

[0064] 2. Insert the PGM gene into the pJW-EXP vector

[0065] The pJW-EXP vector was double-digested with SmaI and NheI, and the digested fragment was recovered. Using T4 ligase at 16°C, the PGM gene was inserted between NheI and SmaI of the pJW-EXP vector to obtain pJW-EXP- tPGM vector (see Figure 4 ).

Embodiment 3

[0066] Example 3: Transformation of pJW-EXP-tPGM into wild-type Ganoderma lucidum cells by PEG-mediated protoplast fusion

[0067] 1. Preparation and transformation of Ganoderma lucidum protoplasts

[0068] First, wild-type Ganoderma lucidum (CCGMC5.616) mycelia were prepared into protoplasts with lysozyme, and then Ganoderma lucidum protoplasts were suspended in 100 μL of STC (0.55M sorbitol, 10mM CaCl 2 , 10mM Tris-HCl buffer, pH 7.5), then add 1μg of plasmid DNA and PTC buffer (60% PEG4000 (W / V), 10mM Tris-HCl buffer, pH 7.5, 50mM CaCl 2 ); incubate on ice for 10 min, then add 1 mL of PTC buffer to mix well and incubate at room temperature for 20 min; use 10 mL of melted CYM solid medium to mix with the transformed protoplasts, pour the plate and add carboxin to make the final concentration of carboxin 2 μg / L; Several single colonies can grow after cultured at 30°C for 10 days.

[0069] 2. Subculture on plates containing carboxin resistance

[0070] Transfer a single...

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Abstract

The invention discloses a high-yield ganoderan engineering strain Rmust and a construction method thereof. A collection number of the ganoderan high-yield engineering strain Rmust collected in China General Microbiological Culture Collection Center (CGMCC) is CGMCCNO.8258. The high-yield ganoderan lucid ganoderma engineering strain Rmust provided by the invention is obtained by selecting a lucid ganoderma strong promoter P-(i)gpd( / i) to over-express and code a PGM (phosphoglucomutase)-gene. Shake flask fermentation experiment indicates that the content of ganoderan generated by converter strain is 1.6 times the content of wild type strain (WT strain) on the condition that cell growth of the ganoderan high-yield engineering strain Rmust is not affected, wherein the amount (mg / g) of the ganoderan produced by 1g of dry mycelium is improved to 236.95mg / g + / -1.28mg / g from 145.9 mg / g + / -1.65mg / g, and therefore, the high-yield strain can be applied as the engineering strain for producing the ganoderan and has wide application prospect.

Description

technical field [0001] The invention belongs to the field of genetic engineering and metabolic engineering, and specifically relates to a method for overexpressing the PGM (glucose phosphomutase) gene related to the synthesis of ganoderma polysaccharides in the ganoderma lucidum metabolic pathway through genetic engineering, and constructs a high-yield project of ganoderma polysaccharides strain. Background technique [0002] Ganoderma lucidum ( Ganodermal lucidum ) is Basidiomycetes, Polyporaceae, Ganoderma lucidum fungus. There are more than 20 kinds of ganoderma fungus in our country, including red ganoderma, yellow ganoderma, purple ganoderma, black ganoderma, thin-cover ganoderma, tree tongue and so on. Ganoderma lucidum has been used as medicine in my country for more than two thousand years. Ganoderma lucidum has significant curative effects on enhancing human immunity, regulating blood sugar, controlling blood pressure, assisting tumor radiotherapy and chemotherap...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C12N15/80C12N15/61C12R1/645
Inventor 徐军伟任梦飞纪森林贺宜龙周降生
Owner KUNMING UNIV OF SCI & TECH
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