CN104403955A - Engineering strain Rmust-UDP for high yielding of Ganoderma lucidum polysaccharides, and its construction method - Google Patents

Abstract

The invention discloses an engineering strain Rmust-UDP for high yielding of Ganoderma lucidum polysaccharides, and its construction method. The engineering strain Rmust-UDP is preserved in China General Microbiological Culture Collection Center with the preservation number of CGMCCNO.9324. The engineering strain Rmust-UDP for high yielding of Ganoderma lucidum polysaccharides is obtained through over-expressing gene encoding UDP-glucose pyrophosphorylase (UDP) by a Mythic Fungus strong promoter P-gpd; and shaking bottle fermentation experiments show that the yield of Ganoderma lucidum intracellular polysaccharides yielded by the above UPD converter strain without cell growth influences is 23.47mg/100mg and is 53.6% higher than the yield (5.28mg/100mg) obtained by using wild strain (WT strain), and the content of Ganoderma lucidum extracellular polysaccharides yielded by the UPD converter strain is 1.66g/L and is 36.1% higher than the content (1.22g/L) obtained by using the wild strain (WT strain). The high yielding strain can be used an engineering strain for producing Ganoderma lucidum polysaccharides, and has a wide application prospect.

Description

A kind of ganoderan high-yielding engineering bacterial strain Rmust-UDP and construction process thereof

Technical field

The invention belongs to genetically engineered and metabolic engineering field, be specifically related to a kind of by the method for genetically engineered to UDPG pyrophosphorylase (UDP) gene overexpression of ganoderan synthesis relevant in glossy ganoderma pathways metabolism, construct the high-yielding engineering bacterial strain of a ganoderan.

Background technology

Glossy ganoderma ( ganoderma lucidum) be Basidiomycetes, polyporaceae, Ganoderma fungi.Ganoderma has kind more than 20 in China, comprises red sesame, Huang Zhi, purple sesame, black sesame, Ganoderma capuse, artist's conk etc.China application glossy ganoderma has the history of more than 2,000 year as medicine.Glossy ganoderma, for enhancing body immunity, regulates blood sugar, controls blood pressure, adjuvant therapy chemicotherapy, liver protecting, promotes that the aspects such as sleep all have significant curative effect.Due to the pharmaceutical use that it is special, the analysis of effective component of glossy ganoderma and pharmaceutical research have caused international extensive concern, especially in Japan, and the U.S., the countries such as Korea S.At present, the research of glossy ganoderma has been deep into molecular level, and some monographs are published in succession, describes biological characteristics, cultivation technique, the situation such as pharmacotoxicological effect and clinical application of glossy ganoderma from different angles.

Ganoderan is the new endogenous activity material of one extracted from glossy ganoderma, chemical composition contained by it can significantly improve cytophagous phagocytic activity, strengthen humoral immunization and cellular immune function, can also Superoxide Dismutase Activity of Erythrocytes be improved, to human body, there is several functions.

UDPG pyrophosphorylase (UDP) gene is the gene of an outbalance in ganoderan route of synthesis.In cell, glycogenolysis can produce a large amount of free Cori ester, and UDPG pyrophosphorylase major catalytic Cori ester becomes UDPG, and UDPG is the donor of glycosyl in glycogen biosynthesizing.This is the first step reaction of Glycogen synthesis.Various polysaccharide is just created through a series of reaction again after Glycogen synthesis.Therefore the synthesis of UDPG pyrophosphorylase gene pairs glycogen has important effect.The route of synthesis of ganoderan is as follows:

Along with the raising of people's living standard, the incidence also cumulative year after year of the diseases such as various cancers.So people also more and more come extensively the concern of health.Glossy ganoderma is as a kind of good Chinese medicine to raising body immunity, and anti-oxidant, antitumor, anti-ageing aspect of waiting for a long time has consequence.

Because wild Ganoderma growth cycle is long, the large and wild Ganoderma resource-constrained by such environmental effects, Ganoderma lucidum mycelium is trained the important method into producing active substance.Along with the continuous increase to ganoderma active material requisite, the output how improving glossy ganoderma and the effective constituent increasing glossy ganoderma more and more cause the concern of people.Liquid submerged fermentation technology is the important means of carrying out fast industrialization production.Now commercially available ganoderan mainly obtains from the mycelium that Ganoderma sporophore and liquid submerged fermentation obtain.Because during wild Ganoderma liquid submerged fermentation, the content of ganoderan in glossy ganoderma cell is lower, separation and purification is also more difficult, limits the activity of ganoderan and Study on mechanism and widespread use thereof.In recent years, genetically engineered and metabolic engineering develop rapidly, become the important means of modern molecular breeding.Transformed the genome of bacterial strain by molecular cloning and gene splicing equimolecular biological method, thus the content improving activeconstituents is more and more subject to the favor of scholars.The output how improving ganoderan from molecular level is the technical problem being badly in need of at present solving.

Summary of the invention

The object of the invention is to solve wild-type ganoderma strain capable self and produce the lower problem of ganoderan, a kind of ganoderan superior strain is provided, this bacterial strain be high yield ganoderan glossy ganoderma ( ganoderma lucidum) engineering bacteria Rmust-UDP, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on June 23rd, 2014, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC NO. 9324.

The construction process of ganoderan high-yielding engineering bacterial strain of the present invention is as follows:

Be initial vector with PMD19-T and use the strong promoter P-of glossy ganoderma itself gpd, terminator T- sdhB, cbxgene, cbxgene (having carboxin resistance) is the resistant gene from glossy ganoderma itself, is characterized in that the transformation efficiency of homology marker gene in the transformation system of mushroom class basidiomycetes is higher, can genetic stability, strong resistance;

1, by glossy ganoderma promotor P- gpdwith terminator T- sdhBbe connected with PMD19-T, by glossy ganoderma cbxresistant gene is inserted into psti restriction enzyme site, thus be built into pJW-EXP carrier; It has strong promoter and the terminator of glossy ganoderma, and has the resistance of glossy ganoderma itself; Wherein increase glossy ganoderma strong promoter P- gpdprimer sequence be:

P- gpd-F：5’-TCCAAAGCCGCTCTCATGGCATGGCAC- 3’ ，

P- gpd-R：5’-GCTAGCGTTGAGAGGGGATGAAGAGTGAGTAAGAAG- 3’；

Amplification glossy ganoderma sdhBthe primer sequence of gene terminator is:

T- sdhB-F: 5’-ATGAGCGGGTCAGAGAGT- 3’，

T- sdhB-R : 5’-TGCTCTATGTCTTGCCTTGT- 3’；

Amplification glossy ganoderma cbxthe primer of resistant gene is:

cbx-F：5’-TCTGCTCTTCCCGATTGCTGCATTTGT-3’，

cbx-R：5’-CTATGTCTTGCCTTGTCTCGCGTCAACC-3’；

2, in ganoderan route of synthesis, key enzyme UDPG pyrophosphorylase (UDP) clones from glossy ganoderma genome that (primer used is UDP- nhe-F and UDP- sma-R), and be inserted in pJW-EXP carrier, obtain pJW-EXP-UDP carrier; Primer is UDP- nhe-F:5 '-GCTAGCATGCCCTCCGACTCCCTCAT-3 ' and UDP- sma-R:5 '-GGGCCCTTACAACTCCTGCAGATACCG-3 '; UDP gene nucleotide series is as shown in SEQ ID NO:1, and the aminoacid sequence of its coding is as shown in SEQ ID NO:2;

3, mediated the method for protoplast fusion by PEG, pJW-EXP-UDP is transformed in wild-type glossy ganoderma cell, screens glossy ganoderma transformant using carboxin as resistance, the CYM flat board containing carboxin resistance filters out transformant;

4, transformant is carried out Secondary Culture in the CYM flat board of carboxin resistance;

5, the liquid culture of glossy ganoderma cell:

PDA substratum (g/L): glucose 10, agar 20, magnesium sulfate 1.5, potassium primary phosphate 3, VITMAIN B1 0.05 and the murphy juice prepared.

The preparation method of murphy juice: 200 grams of fresh potatoes of peeling are cut into small pieces, add deionized water 1.0 L and boil 30 minutes, by eight layers of filtered through gauze, get filtrate, for the preparation of PDA substratum.

Seed culture medium (g/L): glucose 35, peptone 5, yeast extract paste 2.5, potassium primary phosphate 1, magnesium sulfate 0.5 and VITMAIN B1 0.05.

Fermention medium (g/L): peptone 5, yeast extract paste 5, potassium primary phosphate 1.0, magnesium sulfate 0.5, VITMAIN B1 0.05, lactose 35, initial pH 5.5.

CYM substratum (g/L): glucose 20, maltose 10, yeast powder 2, peptone 2, MgSO ₄0.5, KH ₂pO ₄4.6, agar 10.

Slant culture: inoculation mycelia is 28 DEG C of cultivation 5-7 days in murphy juice-glucose-agar (PDA) inclined-plane.

First order seed is cultivated: in 250 mL shaking flasks, add 40 mL substratum and 10 mL mycelium suspensions (obtaining from an inclined-plane), and at 30 DEG C, cultivates 5 days under 120 rpm.

Secondary seed is cultivated: in 250 ml shaking flasks, add 45 mL substratum and 5 mL first order seed nutrient solutions (about 500 mg DW/L, one-level culture granulated glass sphere smashes rear inoculation), at 30 DEG C, cultivates 2 days under 120 rpm.

Fermentation culture: add 45 mL fermention mediums and 5 mL secondary seed fermented liquids (about 500 ~ 600 mg DW/L, secondary culture granulated glass sphere smashes rear inoculation) in 250 ml shaking flasks, at 30 DEG C, cultivate under 120 rpm.

6, the separation and Extraction of ganoderan: take stem cell powder 100 mg, adds 1 M sodium hydroxide 3 mL, after 60 DEG C of hydrolysis 1 h, measures intracellular polyse content with the vitriol oil-phynol method.

Advantage of the present invention and technique effect:

Shown by medicine bottle fermenting experiment, this ganoderan high-yielding engineering bacterial strain is (under the condition that culture temperature, substratum composition, inoculum size are identical with training method) in the impregnable situation of its leavening property, the content of UDP transformant bacterial strain product glossy ganoderma intracellular polyse is the content that 23.47 mg/100mg improve 53.6%, UDP transformant bacterial strain product glossy ganoderma exocellular polysaccharide than the content (15.28 mg/100mg) of wild type strain (WT bacterial strain) is that 1.66 g/L improve 36.1% than the content (1.22 g/L) of wild type strain (WT bacterial strain).Under square one, the engineering strain using the present invention to build can save labor force, shortens the production cycle, is lowered into product cost.Therefore, this superior strain as the engineering strain producing ganoderan, can be applicable to suitability for industrialized production, is with a wide range of applications.

Accompanying drawing explanation

Fig. 1 is glossy ganoderma genome electrophorogram of the present invention; In figure: G is glossy ganoderma genome, M is λ-Hind III digest DNA Marker;

Fig. 2 is pJW-EXP carrier structure schematic diagram of the present invention;

Fig. 3 is the UDP gene electrophorogram that increases in the present invention; In figure: M is the nucleic acid standards (Takara) of DL2000; U is UDP gene;

Fig. 4 is pJW-EXP-UDP carrier structure schematic diagram in the present invention;

Fig. 5 is the electrophorogram verifying UDP positive transformant in the present invention; In figure: M is the nucleic acid standards (Takara) of DL2000, and P is positive control, and UDP is the positive transformant turning UDP gene, and WT is wild type strain, and N is negative control.

Embodiment

Below by embodiment, the present invention is described in further detail, but content of the present invention is not limited thereto, method operating all according to a conventional method if no special instructions in the present embodiment, agents useful for same employing conventional reagent if no special instructions or the reagent configured according to a conventional method.

the structure of embodiment 1:pJW-EXP carrier

1, the extraction of glossy ganoderma genomic dna

Take mycelium grind into powder in liquid nitrogen of about 0.2 g freeze-drying wild-type glossy ganoderma (CCGMC 5.0616), powder is proceeded to the CTAB(cetyl trimethylammonium bromide of 1.5 mL through 65 DEG C of preheatings) extract in damping fluid, 65 DEG C of insulation 30 min, then at 4 DEG C, centrifugal 20 min of 10000 g, get supernatant liquor and add isopyknic chloroform: the mixture of primary isoamyl alcohol (24:1), shakes up 30 more than min gently, at 4 DEG C, centrifugal 20 min of 10000 g; After supernatant liquor being moved into 1.5 mL centrifuge tubes, add the Virahol of 2/3 volume through-20 DEG C of precoolings, shake 5 min gently, after pulling DNA out with glass stick, washing with alcohol with 75% 2-3 time, after room temperature airing, be dissolved in the appropriate TE containing 20 μ g/mL RNase, after 37 DEG C of digestion RNA 30 min, glossy ganoderma genomic dna can be arrived.Agarose gel electrophoresis result shows: the genome of glossy ganoderma is single band, and size is in 10000 more than bp (see figure 1)s.

2, the clone of glossy ganoderma strong promoter

With glossy ganoderma genomic dna for template, use P- gpd-F, P- gpd-R carries out pcr amplification as primer, and amplification obtains the promotor P-of glossy ganoderma gpd, primer sequence is as follows:

P- gpd-F：5’-TCCAAAGCCGCTCTCATGGCATGGCAC-3’，

P- gpd-R：5’-GCTAGCGTTGAGAGGGGGATGAAGAGTGAGTAAGAAG-3’；

PCR condition is as follows: 95 DEG C of 10 min, 95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 90 s, 72 DEG C of 10 min.

3, promotor P- gpdbe connected with PMD19-T

By the promotor P-after clone gpdcarry out glue recovery, recovery product is connected at 16 DEG C with PMD19-T T4 ligase enzyme, obtains PMD19-T-P intermediate carrier;

4, the clone of glossy ganoderma terminator

With glossy ganoderma genomic dna for template, use primer

T- sdhB-F:5 '-ATGAGCGGGTCAGAGAGT-3 ' and

T- sdhB-R:5 '-TGCTCTATGTCTTGCCTTGT-3 ' increases, and obtains the sequence of 440bp;

PCR condition is: 95 DEG C of 10 min, 95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 30 s, 72 DEG C of 10 min;

5, terminator T- sdhBbe connected with PMD19-T-P

By the terminator T-after clone sdhBglue reclaims, and PMD19-T-P intermediate carrier Sac I single endonuclease digestion, PCR reclaims digestion products, then fills restriction enzyme site with T4 polysaccharase, and then carries out blunt end cloning with T4 ligase enzyme at 16 DEG C, obtains PMD19-T-P-T intermediate carrier;

6, cbxthe clone of gene

With glossy ganoderma genomic dna for template, use primer cbx-F:5 '-TCTGCTCTTCCCGATTGCTGCATTTGT-3 ' and cbx-R:5 '-CTATGTCTTGCCTTGTCTCGCGTCAACC-3 ' carries out the sdhB gene that PCR obtains glossy ganoderma; PCR condition is: 95 DEG C of 10 min, 95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 90 s, 72 DEG C of 10 min; Bamboo product a pair rite-directed mutagenesis primer sdhB-MR:5'-GAAGATCGTG agGCAGCGGTATAGGC-3'(wherein afor mutational site) and sdhB-MF:5'-GCCTATACCGCTGCC tcACGATCTTC-3'(wherein tfor mutational site).First round PCR primer cbx-F and sdhB-MR increases, and PCR condition is: 95 DEG C of 10 min, 95 DEG C of 30 s, 58 DEG C of 30 s, 72 DEG C of 2 min 20 s, and 72 DEG C of 10 min, obtains fragment sdhB1; Second takes turns PCR primer cbx-R and sdhB-MF increases, and PCR condition is: 95 DEG C of 10 min, 95 DEG C of 30 s, 55 DEG C of 30 s, 72 DEG C of 1 min 20 s, and 72 DEG C of 10 min, obtains fragment sdhB2; After obtaining respective segments, adopt the method for over-lap PCR that two fragments are connected.The primer of third round over-lap PCR is cbx-F and cbx-R, amplification condition is: 94 DEG C of sex change 10 min, 66 DEG C of annealing 30 s, and 72 DEG C extend 4 min, totally 35 circulations, and last 72 DEG C extend 10 min.Finally obtain 3171 bp sudden change glossy ganoderma sdhB gene order (after rite-directed mutagenesis to carboxin microbiotic produce resistance, we are defined as the sdhB gene after rite-directed mutagenesis cbx).After sequencing, confirm that corresponding site suddenlys change.

7, by sudden change cbxgene is inserted into PMD19-T-P-T's psti restriction enzyme site

PMD19-T-P-T carrier is used psti enzyme at 37 DEG C, single endonuclease digestion 2 h, reclaim enzyme cut after fragment, fill restriction enzyme site with T4 polysaccharase, then use T4 ligase enzyme handle at 16 DEG C cbxgene is connected with the fragment after recovery, namely obtains pJW-EXP carrier (see figure 2).

the structure of embodiment 2:pJW-EXP-UDP carrier

1, the clone of UDP gene

With glossy ganoderma genomic dna for template, use primer

UDP- nhe-F:5 '-GCTAGCATGCCCTCCGACTCCCTC-3 ' and

UDP- sma-R:5 '-GGGCCCTTACAACTCCTGCAGATACCG-3 ' carries out PCR and obtains UDP gene; PCR condition is: 95 DEG C of 10 min, 95 DEG C of 30 s, 56 DEG C of 30 s, 72 DEG C of 2 min, and 72 DEG C of 10 min(is shown in Fig. 3).

2, UDP gene is inserted into pJW-EXP carrier

PJW-EXP carrier is used smai and nhei double digestion, reclaim enzyme cut after fragment, with T4 ligase enzyme at 16 DEG C, SQS gene is inserted into pJW-EXP carrier nhei and smanamely pJW-EXP-UDP carrier (see figure 4) is obtained between I.

embodiment 3: the method mediating protoplast fusion by PEG, is transformed into pJW-EXP-UDP in wild-type glossy ganoderma cell

1, the preparation of glossy ganoderma protoplastis and conversion

First with lywallzyme, wild-type Ganoderma lucidum mycelium is prepared into protoplastis, then glossy ganoderma protoplastis is suspended in the sorbyl alcohol of the STC(0.55 M of 100 μ L, the CaCl of 10 mM ₂, the Tris-HCl damping fluid of 10 mM, pH is 7.5) in, then adding plasmid DNA and the PTC damping fluid (PEG4000(W/V of 60%) of 1 μ g, the Tris-HCl damping fluid of 10 mM, pH is the CaCl of 7.5,50 mM ₂); Cultivate 10 min on ice, then the PTC damping fluid adding 1 mL mixes and at incubated at room temperature 20 min; The CYM solid medium melted with 10 mL mixes to be down flat plate and to add carboxin with the protoplastis after conversion makes the final concentration of carboxin be 2 mg/L; Cultivate at 30 DEG C after 10 days and can grow some single bacterium colonies.

2, Secondary Culture on the flat board containing carboxin resistance

Single colony lift in the CYM flat board of the carboxin containing 2 mg/L, Secondary Culture about 7 days at 30 DEG C; Go down to posterity in resistant panel through 3 times and can obtain stable transformant, extract the glossy ganoderma genome after transforming by CTAB method, concrete operation step is shown in embodiment 1 step 1.Respectively with transform after glossy ganoderma genome, pJW-EXP-UDP plasmid (positive control), wild-type (WT) glossy ganoderma genome, water (negative control) for template, use

UDP- gpd-F:5 '-GATGTCGGTTGGGGTATTGTG-3 ' and

UDP- gpd-R:5 '-GATGTCCGCCTTTGTCTTGTC-3 ' carries out PCR, PCR condition as checking primer: 95 DEG C of 10 min, 95 DEG C of 30 s, 57 DEG C of 30 s, 72 DEG C of 1 min 50s, and 72 DEG C of 10 min(is shown in Fig. 5).Due to P- gpdlink together in fungus expression vector pJW-EXP-UDP with UDP gene, with UDP- gpd-F(is positioned at glossy ganoderma gpdin the promotor of gene) and UDP- gpd-R(is positioned on glossy ganoderma UDP gene) for primer and genomic dna are that template carries out PCR, the bacterial strain that can amplify about 1.66 kb bands can think to have imported the transgenic lucid ganoderma of UDP gene.As shown in Figure 5, the band of about 1.66 kb can be amplified from transgenic strain and positive control, and in wild-type, do not occur this band, only occur some non-specific bands.Result shows: UDP gene has been incorporated on glossy ganoderma genome really.

3, slant culture

Positive transformant is transferred to (carboxin containing 2 mg/L) in solid PDA medium, at 28 DEG C, cultivate 5-7 days.

4, first order seed is cultivated

In 250 mL shaking flasks, add 40 mL seed culture mediums and 10 mL mycelium suspensions (obtaining from an inclined-plane), and at 28 DEG C, cultivate 5 days under 120 rpm.

5, secondary seed is cultivated

In 250 mL shaking flasks, add 45 mL seed culture mediums and 5 mL first order seed nutrient solutions (about 500 mg DW/L, one-level culture granulated glass sphere smashes rear inoculation), at 28 DEG C, cultivate 2 days under 120 rpm.

6, fermentation culture

In 250 mL shaking flasks, add 45 mL fermention mediums and 5 mL secondary seed fermented liquids (about 500 ~ 600 mg DW/L, secondary culture granulated glass sphere smashes rear inoculation), at 28 DEG C, cultivate under 120 rpm.

7, the separation and extraction of ganoderan

Take stem cell powder 100 mg, add 1 M sodium hydroxide 3 mL, after 60 DEG C of hydrolysis 1 h, measure intracellular polyse content with the vitriol oil-phynol method.

By the mensuration of the content of wild-type and UDP bacterial strain ganoderan, result shows: shown by medicine bottle fermenting experiment, this bacterium is in the impregnable situation of its leavening property, the content of UDP transformant bacterial strain product glossy ganoderma intracellular polyse is the content that 23.47 mg/100mg improve 53.6%, UDP transformant bacterial strain product glossy ganoderma exocellular polysaccharide than the content (15.28 mg/100mg) of wild type strain (WT bacterial strain) is that 1.66 g/L improve 36.1% than the content (1.22 g/L) of wild type strain (WT bacterial strain).Therefore, this superior strain as the engineering strain producing ganoderan, can be with a wide range of applications.

Sequence table

 

<110> Kunming University of Science and Technology

<120> ganoderan high-yielding engineering bacterial strain Rmust-UDP and construction process thereof

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<170> PatentIn version 3.5

 

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tccaaagccg ctctcatggc atggcac 27

 

<210> 4

<211> 37

<212> DNA

<213> artificial sequence

 

<400> 4

gctagcgttg agagggggat gaagagtgag taagaag 37

 

<210> 5

<211> 18

<212> DNA

<213> artificial sequence

 

<400> 5

atgagcgggt cagagagt 18

 

<210> 6

<211> 20

<212> DNA

<213> artificial sequence

 

<400> 6

tgctctatgt cttgccttgt 20

 

<210> 7

<211> 27

<212> DNA

<213> artificial sequence

 

<400> 7

tctgctcttc ccgattgctg catttgt 27

 

<210> 8

<211> 28

<212> DNA

<213> artificial sequence

 

<400> 8

ctatgtcttg ccttgtctcg cgtcaacc 28

 

<210> 9

<211> 26

<212> DNA

<213> artificial sequence

 

<400> 9

gaagatcgtg aggcagcggt ataggc 26

 

<210> 10

<211> 26

<212> DNA

<213> artificial sequence

 

<400> 10

gcctataccg ctgcctcacg atcttc 26

 

<210> 11

<211> 24

<212> DNA

<213> artificial sequence

 

<400> 11

gctagcatgc cctccgactc cctc 24

 

<210> 12

<211> 27

<212> DNA

<213> artificial sequence

 

<400> 12

gggcccttac aactcctgca gataccg 27

 

<210> 13

<211> 21

<212> DNA

<213> artificial sequence

 

<400> 13

gatgtcggtt ggggtattgt g 21

 

<210> 14

<211> 21

<212> DNA

<213> artificial sequence

 

<400> 14

gatgtccgcc tttgtcttgt c 21

 

Claims (2)

1. a ganoderan high-yielding engineering bacterial strain Rmust-UDP, it is CGMCC NO. 9324 at the deposit number at China Committee for Culture Collection of Microorganisms's common micro-organisms center.

2. the construction process of ganoderan high-yielding engineering bacterial strain Rmust-UDP described in claim 1, it is characterized in that: object fragment being connected with expression vector obtains recombinant expression plasmid pJW-EXP-UDP, the recombinant expression plasmid development of evil in febrile disease is entered in glossy ganoderma by the method for the protoplast fusion then mediated by PEG by recombinant expression plasmid, thus obtains the engineering strain of high yield ganoderan;

Wherein said object fragment is UDPG pyrophosphorylase gene, and its nucleotide sequence is as shown in SEQ ID NO:1, and the aminoacid sequence of its coding is as shown in SEQ ID NO:2.