CN114426995B - Method for improving yield of extracellular polysaccharide of cordyceps militaris by utilizing over-expressed hexokinase gene HK - Google Patents
Method for improving yield of extracellular polysaccharide of cordyceps militaris by utilizing over-expressed hexokinase gene HK Download PDFInfo
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- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
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- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01001—Hexokinase (2.7.1.1)
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Abstract
The invention discloses a method for improving the yield of Cordyceps militaris exopolysaccharide by using over-expressed hexokinase gene HK, which comprises inoculating Cordyceps militaris genetic engineering bacteria containing hexokinase gene HK to a fermentation medium, fermenting and culturing at 22-25 ℃ with a shaking table at 120-180 rpm to obtain fermentation liquor containing Cordyceps militaris exopolysaccharide, and separating and purifying the fermentation liquor to obtain Cordyceps militaris exopolysaccharide. The invention constructs the genetically-stable recombinant cordyceps militaris genetic engineering strain through the over-expression of the genetic engineering technology, compared with a wild strain, the genetically-stable recombinant cordyceps militaris genetic engineering strain has the yield of extracellular polysaccharide of cordyceps militaris in fermentation broth of 1.61 times, and compared with a wild strain, the mutant strain which also over-expresses mannose-1-uridine phosphate transferase gene MPG has the yield of extracellular polysaccharide of 0.91 times, thus laying a foundation for further screening of good varieties of cordyceps militaris.
Description
Field of the art
The invention belongs to the field of cordyceps militaris cultivation, and particularly relates to a method for improving the yield of extracellular polysaccharide of cordyceps militaris by utilizing over-expressed hexokinase gene HK.
(II) background art
Cordyceps militaris (Cordyceps militaris) is also called Cordyceps militaris, is a model species of Cordyceps genus, and has been cultivated in an artificial scale. Cordyceps militaris has various natural active ingredients, such as cordycepin, mannitol, cordyceps polysaccharide and the like, and has high medicinal value. Cordyceps polysaccharide is one of main active ingredients in Cordyceps militaris, and pure product is white flocculent powder, is easily dissolved in water, and has been researched to prove that the Cordyceps polysaccharide has various important biological activities, such as antioxidation, anti-tumor, antibiosis, anti-inflammatory, blood sugar reduction, immunoregulation, kidney protection and the like, so that the Cordyceps polysaccharide has a higher application prospect in medicine production. However, due to environmental limitations, the yield of wild or natural cultured cordyceps militaris cannot meet the production requirements, so that the improvement of the yield of artificially cultured cordyceps militaris on a large scale becomes particularly important.
Hexokinase gene HK and mannose-1-phosphate uridine transferase gene MPG are related enzyme genes in the polysaccharide biosynthesis pathway of Cordyceps militaris, which have been verified in the genomic sequence of Cordyceps militaris. Cloning related enzyme genes and introducing the genes into a wild cordyceps militaris genome, and improving the extracellular polysaccharide yield of the cordyceps militaris by an over-expression method.
(III) summary of the invention
The invention aims to provide a method for improving the yield of extracellular polysaccharide of cordyceps militaris by over-expressing hexokinase gene HK, which obtains a mutant strain of cordyceps militaris by a genetic engineering technology, wherein the yield of extracellular polysaccharide in fermentation broth of the mutant strain is 1.61 times compared with that of a wild strain, and the yield of extracellular polysaccharide in fermentation broth of the mutant strain which is also over-expressing mannose-1-uridine transferase gene MPG is 0.91 times compared with that of the wild strain, thus laying a foundation for further screening good varieties of cordyceps militaris.
The technical scheme adopted by the invention is as follows:
the invention provides a method for improving the output of extracellular polysaccharide of cordyceps militaris by utilizing over-expressed hexokinase gene HK, which is characterized in that the content of extracellular polysaccharide of cordyceps militaris is improved by over-expressing hexokinase gene HK in cordyceps militaris; the nucleotide sequence of the hexokinase gene HK is shown in SEQ ID NO. 1.
Preferably, the method comprises the following steps: constructing a cordyceps militaris genetic engineering bacterium containing a hexokinase gene HK, inoculating the cordyceps militaris genetic engineering bacterium into a fermentation medium, and carrying out shake fermentation culture at a temperature of between 22 and 25 ℃ and at a speed of between 120 and 180rpm (preferably at a temperature of 25 ℃ and at a speed of 180 rpm) to obtain a fermentation broth containing extracellular polysaccharide of cordyceps militaris; the fermentation broth medium comprises the following components: glucose 20g/L, (NH) 4 ) 2 SO 4 7g/L，K 2 HPO 4 ·3H 2 O 0.5g/L，KH 2 PO 4 0.5g/L，MgSO 4 ·7H 2 O0.5 g/L, L-glycine 1g/L, distilled water as solvent, and natural pH.
Further, the cordyceps militaris genetic engineering bacteria are obtained by seamlessly cloning hexokinase gene HK and binary vector pCAMBIA, then connecting to obtain a recombinant vector, then converting the recombinant vector into escherichia coli, extracting plasmids to convert the agrobacterium, and then infecting the cordyceps militaris bacteria by the agrobacterium. The agrobacterium is agrobacterium tumefaciens (Agrobacterium tumefaciens) AGL-1.
Further, the Cordyceps militaris genetic engineering bacteria are constructed according to the following method: (1) The method comprises the steps of (1) performing seamless cloning on a hexokinase gene HK and a linearization binary vector pCAMBIA, then connecting to obtain a recombinant vector, transforming escherichia coli by using the recombinant vector, extracting plasmids to transform agrobacterium, inoculating to an IM culture medium containing 25 mug/mL rifampicin and 50 mug/mL kanamycin, culturing at 28 ℃ for 36h, and screening to obtain an agrobacterium strain containing the hexokinase gene HK; the IM culture medium consists of: KH (KH) 2 PO 4 1.45g/L，K 2 HPO 4 2.05g/L，MgSO 4 ·7H 2 O 0.6g/L，NaCl 0.1g/L，CaCl 2 0.01g/L，FeSO 4 0.001g/L，NH 4 NO 3 0.5g/L, 5mL/L glycerol, 40mL/L MES buffer solution with pH of 5.5 and 1mol/L, 5mL/L microelement stock solution, 2g/L glucose and distilled water as solvent; microelement stock solution composition: znSO (ZnSO) 4 ·7H 2 O100mg/L，CuSO 4 ·5H 2 O 100mg/L，H 3 BO 3 100mg/L，Na 2 MoO 4 ·7H 2 O100mg/L, and water as solvent; (2) Agrobacterium strain containing hexokinase gene HK was inoculated into liquid LB medium containing 50mg/L kanamycin at 28℃and 180rCulturing for 36h by pm shaking; transferring the culture solution into liquid AIM culture medium at a volume concentration of 1%, and culturing at constant temperature of 28deg.C at 180rpm to reach thallus concentration OD 600 =0.60 to 0.80 to obtain agrobacterium liquid; the AIM culture medium is prepared by adding 200 mug/mL of Acetosyringone (AS) into an IM culture medium; (3) Inoculating wild Cordyceps militaris (preferably Cordyceps militarisCM 01) into solid sand culture medium, culturing at 25deg.C under reverse condition in dark place for 7 days, repeatedly washing the surface of the solid culture medium with sterile water on a super clean bench to obtain Cordyceps militaris bacterial suspension, filtering bacterial suspension with 4 layers of sterilized gauze to remove mycelium to obtain Cordyceps militaris spore suspension; solid sand culture medium: glucose 40g/L, peptone 10g/L, agar powder 20g/L, distilled water as solvent, and natural pH; (4) Mixing the agrobacterium tumefaciens bacteria solution in the step (2) with the cordyceps militaris spore suspension in the step (3) in equal volume to obtain mixed bacteria solution, coating the mixed bacteria solution on the upper surface of sterile cellophane placed on the surface of a selective culture medium, performing constant temperature light-proof co-cultivation for 48 hours at 25 ℃, transferring the mixed bacteria solution together with the cellophane to the surface of a new selective culture medium (the coated strain faces upwards, the thickness of the culture medium is about 2 mm), pouring a layer of selective culture medium on the upper surface of the cellophane to completely cover the cellophane (the thickness of the culture medium is about 2 mm), performing constant temperature light-proof cultivation for 4-5 days at 25 ℃, picking hyphae penetrating through the upper selective culture medium as potential positive clones, inoculating the potential positive clones to a Sa solid culture medium containing 200 mug/mL hygromycin B, performing sub-cultivation (preferably transferring cultivation for the next generation after the hyphae grow out, and co-cultivating for five generations) to obtain the cordyceps militaris genetic engineering bacteria containing the hexokinase gene HK which is stable and inherit; the selection medium was an IM medium containing 200. Mu.g/mL hygromycin B, 100. Mu.g/mL ceftriaxone sodium and 50. Mu.g/mL kanamycin.
Further, the concentration of the cordyceps militaris spore suspension is 10 6 ～10 8 /mL, preferably 10 6/ mL。
Further, before the fermentation of the cordyceps militaris genetic engineering bacteria containing the hexokinase gene HK, inoculating the cordyceps militaris genetic engineering bacteria to a solid culture medium containing 200 mug/mL hygromycin B, culturing for 4-5 days at a constant temperature and in a dark place at 25 ℃, and then picking a colony to inoculate to the fermentation culture medium.
Compared with the prior art, the invention has the beneficial effects that: the invention constructs the Cordyceps militaris gene mutant strain which can be stably inherited and over-express the hexokinase gene HK through the genetic engineering technology, and compared with the wild strain, the mutant strain has the extracellular polysaccharide yield of 1.61 times in the fermentation broth, thus laying a foundation for further screening the good variety of the Cordyceps militaris.
(IV) description of the drawings
FIG. 1 shows the PCR results of Agrobacterium colonies containing the HK gene of example 1 and the MPG gene of example 3. Wherein lane M:5000DNA Marker, lane WT: in the control group, lanes 1 and 2 are parallel to the PCR products of the Agrobacterium colonies containing the target gene HK of example 1, and lanes 3 to 7 are parallel to the PCR products of the Agrobacterium colonies containing the target gene MPG of example 3.
FIG. 2 shows the identification results of the hygromycin resistance gene of the recombinant Cordyceps militaris in examples 2 and 3. Wherein lane M:5000DNA Marker, lanes 1-5 are parallel samples of recombinant cordycepin hygromycin resistance gene identification PCR products containing the target gene HK, lanes 6-10 are parallel samples of recombinant cordycepin hygromycin resistance gene identification PCR products containing the target gene MPG, and lane 11 is a control group.
FIG. 3 is a bar graph of extracellular polysaccharide content of wild type Cordyceps militaris strain and recombinant strain containing target genes HK and MPG.
(fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
Culture medium for cordyceps militaris strain
Sand culture medium: glucose 40g/L, peptone 10g/L, agar powder 20g/L, distilled water as solvent, and natural pH, and adding agar powder 20g/L if solid culture medium is to be prepared.
Fermentation medium: glucose 20g/L, (NH) 4 ) 2 SO 4 7g/L，K 2 HPO 4 ·3H 2 O 0.5g/L，KH 2 PO 4 0.5g/L，MgSO 4 ·7H 2 O0.5 g/L, L-glycine 1g/L, distilled water as solvent, and natural pH.
Culture medium for bacterial strains
LB medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl, distilled water as solvent, pH7.0. If a solid culture medium is to be prepared, 20g/L of agar powder is added.
IM medium: KH (KH) 2 PO 4 1.45g/L，K 2 HPO 4 2.05g/L，MgSO 4 ·7H 2 O 0.6g/L，NaCl 0.1g/L，CaCL 2 0.01g/L，FeSO 4 0.001g/L，NH 4 NO 3 0.5g/L, 5mL/L glycerol, 40mL/L MES buffer (1 mol/L, pH 5.5), 5mL/L microelement stock solution, 2g/L glucose, and distilled water as solvent; the trace element stock solution comprises the following components: znSO (ZnSO) 4 ·7H 2 O 100mg/L，CuSO 4 ·5H 2 O100mg/L，H 3 BO 3 100mg/L，Na 2 MoO 4 ·7H 2 O100mg/L, and water as solvent. In addition, the final solubility of glucose in the IM solid culture medium is reduced to 1g/L, and the final solubility of glucose in the IM solid culture medium is reduced to 20g/L.
AIM medium: IM medium containing acetosyringone at a final concentration of 200. Mu. Mol/L.
Selection medium: IM medium containing 200. Mu.g/mL hygromycin B, 100. Mu.g/mL ceftriaxone sodium and 50. Mu.g/mL kanamycin.
EXAMPLE 1 construction of Agrobacterium containing hexokinase Gene HK
1. Construction of recombinant expression vectors
(1) Extracting genomic DNA of cordyceps militaris: wild Cordyceps militaris (Cordyceps militaris) CM01 (purchased from Litsea Corp.) was inoculated into a solid Saccharum medium, cultured in an inverted manner in a constant temperature incubator at 25deg.C for 7d (or in a liquid shake flask of a fermentation medium at 25deg.C and 180rpm for five days, and filtered with four layers of sterilized gauze) to obtain mycelia. Scraping proper amount of mycelium and absorbing water, and adopting a fungus genome DNA rapid extraction kit (purchased from biological engineering (Shanghai) Co., ltd., product number: B518229), extracting genome DNA according to the related operation instructions of the kit, wherein the method comprises the following steps: (1) taking 50-100 mg of fresh fungi or 20mg of dried fruiting bodies or mycelia, fully grinding the fresh fungi or mycelia into powder in liquid nitrogen, putting the powder into a 1.5mL centrifuge tube, sequentially adding 400 mu L Buffer Digestion and 4 mu L of beta-mercaptoethanol, and shaking and uniformly mixing. The cells were completely lysed in a water bath at 65℃for 1 h. (2) 200mL Buffer PF was added and mixed well by inversion and placed in a refrigerator at-20℃for 5min. (3) Centrifuge at 10000rpm for 5min at room temperature, transfer supernatant (500-550. Mu.L) to a new 1.5mL centrifuge tube. (4) Adding isopropyl alcohol with the same volume, reversing for 5-8 times to fully and uniformly mix, and standing at room temperature for 2-3 min. Centrifuge at 10000rpm for 5min at room temperature, discard supernatant. (5) 1mL of 75% ethanol was added, rinsing was reversed for 1-3 min, centrifugation was performed at 10000rpm for 2min, and the supernatant was discarded. (6) Repeating the step (5) once. (7) And the cover is opened and the room temperature is inverted for 5 to 10 minutes until the residual ethanol is completely volatilized. (8) The obtained DNA was dissolved in50 to 100. Mu.L of TE Buffer. The extracted wild Cordyceps militaris DNA can be immediately subjected to the next experiment or stored at-20deg.C.
(2) Amplification of the hexokinase Gene HK: the wild cordyceps militaris DNA extracted in the step (1) is subjected to PCR amplification by using a PCR reaction system of the primers HK-F/HK-R in the table 1 to obtain a target gene fragment, namely a hexokinase gene HK (the nucleotide is shown as SEQ ID NO. 1) on the genomic DNA of the cordyceps militaris. PCR amplification procedure: pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, annealing at 62℃for 30s, extension at 72℃for 1min for 50s,3 cycles, denaturation at 94℃for 30s, annealing at 70℃for 30s, extension at 72℃for 1min for 50s,27 cycles, deletion at 72℃for 10min, and low-temperature storage at 4 ℃.
SEQ ID NO.1
ATGGTTGGCCTAGGTCCTCGCGCTCCGCCCTCTAGAAAAGGTACGCCCCGGATACTGCTACTACGGCGGAAAAATATTGAATCGAACTTTGTCAACGTCTTTTCACCGATGCAAGACGCTCCTGGGATTGTGACGACCATCCGCTGCTGCACTAGAAGCTAACAACCACTGAGGGTCGAAAGCCGATGTTCCCAAGGACACCATGGAGGCGATCCGGAACATTGAGGAGCTCTTTACGGTCGACACTGCCAAGCTCAAGGCCATCACAGAGCACTTCGTCTCCGAGCTTACCAAGGGTTTGTGCCGCGCCCGAATTAAACCAGCCAGATTGCGCCAACCAACACCAGCCACTAACCGTCTTCTCAGGCCTCACCAAAGAGGGTGGCAGCATTGTAAGCATATATGAATCTACACCAACTACTGTAACTCAGGCTAACAGATCTCCAGCCCATGAACCCTACTTGGGTCATGTCGTATCCCGATGGCAATGAGACTGGCACCTTCCTCGCCCTCGACATGGGCGGCACCAACCTGCGCGTCTGCCAAATCACCCTTACAGATAAGAGGTCCGAGTTTGACATTATCCAGTCCAAGTACAAGATGCCTGAAGAGCTCAAGACTGGCAAGAGCGATGAGCTGTGGGAGTACATTGCCGACTGTCTCCACCAGTTCCTCGAAACCCACCATGGTGACACCAAGAACATTGGCACCTTGCCCTTGGGGTTCACCTTTTCTTACCCCGCTACCCAGAACTACATTGACGCGGGTATCTTGCAGCGATGGACCAAGGGCTTTGACATTGACGGCGTCGAGGGCCAGGATGTCGTTCCCATGTTCGAGGCTGCTCTTGCCAAGCGTGTATGTCTAATATGCCTCACTCTCTCGTACTCTGGTAGCTTGCTGACCTGTATGTAGGGTGTTCCCATTCAACTGACTGCTCTTATTAATGACACCACCGGAACGCTCATGGCCTCGGCCTATACTGACCCTACCATGAAGATTGGCTGCATCTTCGGCACCGGCTGCAACGCCGCGTACGTCGAACACTGCGGTTCGATCCCCAAGCTTGCCGACATGGGTCTACCTGATGACATGGAAATGGCCATCAACTGCGAGTGGGGTGCCTTTGACAATGAGCACAAGATTCTACCCCGTACCAAGTACGATGTCATCATTGATGAGGAGTCACCCCGCCCTGGCCAACAGGCGTTTGAGAAAATGATTGCAGGTCTCTACCTTGGCGAGATTTTCCGCCTTGCCCTTGTCGATCTGCATAACGAAAAGCACCTCTTTCAGGGACAGGACATCAACTGTCTGCGCAAGGCCTACAGCCTCGACGCTTCGTTCCTTTCGGCTATTGAGGAGTGAGTCACATCGTTCTGCTACTGAGGCATGCCGTCTAACTACCGGGCTCCAGGGATCCTTTCGAGAACTTACAGGAGACGTTCGACCTGTTTGCAAACAAGTTGCAAATCAATGTCACCGGCCCCGAGCTTGAGCTGATCCGCCGCCTCGCTGAGCTGATCGGTACCCGCGCCGCCCGTCTCGCTTCCTGCGGTGTCGCTGCCATTTGCAAGAAGAAGAACTTCAAGACCTGCCACGTCGGTGCCGACGGCTCCGTCTTCAATAAGTACCCTCACTTCAAGGCTCGTGGAGCGCAGGCCCTGAAGGAGATTCTTGACTGGCCCGACAAGACTGATGCCAAAGCCGAGGACCCGGTTGAAATTCTTGCCGCCGAAGACGGCAGTGGTGTCGGTGCCGCTCTGATTGCCGCCTTGACCCTGAAGCGTGCGCAGCAGGGCAACCTGGCGGGCATTCTTCACCCGGAGCGCTTCAAATAG 1841
TABLE 1 hexokinase Gene HK amplification primers
TABLE 2 amplification PCR reaction System
(3) Recombinant expression vectors
Linearization vector pCAMBIA: the Vector pCAMBIA (commercially available) was amplified by PCR using the primers Vector-F and Vector-R of Table 3 according to the PCR reaction system of Table 4. PCR amplification procedure: pre-denaturation at 94 ℃ for 5min, cycle number 1; denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 6min, cycle number 25; the extension is carried out for 10min at 72 ℃ and the cycle number is 1. The amplified product was then digested with 1. Mu.L of DpnI enzyme in a water bath at 37℃for 2 hours to remove the remaining template DNA, and heated in a water bath at 80℃for 15 minutes to inactivate the DpnI enzyme. And (3) carrying out 1% agarose gel electrophoresis identification on the obtained product, and primarily judging whether the sequence is amplified successfully or not according to the length of the strip. The PCR amplified product was gel-recovered using a SanPrep column type DNA gel recovery kit (available from Shanghai Biotechnology Co., ltd., product number: B518131), and the DNA solution of the linearized vector pCAMBIA was obtained by operating according to the specific procedure of the gel recovery kit and stored at-20 ℃.
TABLE 3 vector linearization primers
TABLE 4 linearization PCR reaction System
Recombinant expression vector: by usingUniSeamless Cloning and Assembly Kit (Beijing full gold Biotechnology Co., ltd., catalog number: CU 101-02), hexokinase gene HK (Inserts) was gently mixed with linearization vector pCAMBIA (Linearized vector) and 2xAssembly Mix according to Table 5, and reacted at 50℃for 15 minutes. After the reaction is finished, the centrifuge tube is placed on ice for cooling for a few seconds to obtain the recombinant expression vector, and the recombinant expression vector is stored at-20 ℃ or directly used for transformation.
TABLE 5 seamless cloning reaction System
2. Transformation of E.coli DH 5. Alpha
Adding 6 mu L of the recombinant expression vector constructed in the step (3) in the step (1) into 100 mu L of escherichia coli DH5 alpha competence, and standing on ice for 30min; heat shock is carried out for 30s in a water bath at the temperature of 42 ℃, and the mixture is cooled for 2min on ice; adding 800 mu LLB liquid culture medium into competence, and shake culturing at 37 ℃ and 180rpm for 1h; centrifuging at 3500rpm for 10min, discarding supernatant on a super clean bench, and reserving 100 mu L of bacterial liquid; the resuspended bacteria liquid is spread in LB medium and cultured for 24h at 37 ℃. Single colonies were picked and PCR verified with primers Ide-F/Ide-R of Table 6, and the reaction system (Table 7) was used to check if the transformation was successful.
TABLE 6 colony PCR primers
TABLE 7 colony PCR reaction System
Amplification procedure for PCR: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s, extension at 72 ℃ for 1min, and cycle number for 30; the deficiency is made up for 10min at 72 ℃. According to the result of 1% agarose gel electrophoresis identification, whether the clone is positive or not can be preliminarily judged, and whether the transformation is successful or not is checked.
The E.coli positive clone was inoculated into LB medium, plasmid DNA was extracted with SanPrep column type plasmid DNA miniprep kit (available from Biotechnology (Shanghai) Co., ltd., product No. B518131), and the obtained DNA was subjected to 35. Mu.L of ddH 2 O is dissolved. The extracted DNA can be immediately subjected to the next experiment or stored at-20 ℃.
3. Transformation of Agrobacterium tumefaciens
Adding 5 mu L of the plasmid DNA solution extracted in the step 2 into 100 mu L of Agrobacterium tumefaciens AGL-1 (purchased from Shanghai Jiqi biotechnology Co., ltd.) to be competent, freezing with liquid nitrogen for 8s, and carrying out water bath at 37 ℃ for 5min; 800 mu L of LB liquid medium is added into the competence, and shake culture is carried out for 1h at 28 ℃ and 180 rpm; centrifuging at 3500rpm for 10min, discarding supernatant on a super clean bench, and reserving 100 mu L of bacterial liquid; the re-suspension bacteria liquid is coated on IM culture medium containing 25 mug/mL rifampicin and 50 mug/mL kanamycin, cultured for 36h at 28 ℃, and single colony is selected to be agrobacterium containing hexokinase gene HK.
Single colonies were picked and identified by colony PCR using primers Ide-F/Ide-R of Table 6, the PCR reaction system is shown in Table 7, and the PCR amplification procedure is as follows: pre-denaturation at 94 ℃ for 5min, cycle number 1; denaturation at 94℃for 30s, annealing at 64℃for 30s, extension at 72℃for 1min, cycle number 30; the extension is carried out for 10min at 72 ℃ and the cycle number is 1. According to the result of 1% agarose gel electrophoresis identification, whether the recombinant engineering bacteria are successfully transformed can be judged, the result is shown in a figure 1, and lanes 1-2 are parallel samples of the PCR products of the agrobacterium colony containing the target gene HK.
EXAMPLE 2 transformation of homologous recombinant vector in Cordyceps militaris
1. Agrobacterium tumefaciens mediated genetic transformation of filamentous fungi
Single colonies of example 1, step 3, were picked and inoculated into 3mL of liquid LB medium containing 50mg/L kanamycin (Kan), and shake-cultured at 28℃for 36h at 180 rpm. Transferring the culture solution into a liquid AIM culture medium at a volume concentration of 1%, and culturing at constant temperature of 28 ℃ at 180rpm for 6-8 h until the concentration of the bacterial cells OD 600 The agrobacteria liquid was obtained =0.80.
2. Cordyceps militaris spore suspension
Inoculating wild Cordyceps militaris CM01 into a solid sand culture medium, culturing in an inverted manner at a constant temperature of 25 ℃ for 7 days (spore suspension is prepared by adopting inclined planes or solid culture generally, liquid culture conditions are unfavorable for producing spores or the concentration of spores is low), and repeatedly flushing the surface of the solid culture medium with sterile water on an ultra-clean workbench to obtain Cordyceps militaris bacterial suspension. Filtering the bacterial suspension with 4 layers of sterilized gauze to remove mycelium, thus obtaining Cordyceps militaris spore suspension. And (3) taking a trace amount of cordyceps militaris spore suspension, and determining the concentration of cordyceps militaris spores in the spore suspension by using a blood cell counting plate.
3. Transformation
200. Mu.L of the solution prepared in step 2 was concentrated to 10 6 Cordyceps militaris/mLSpore suspension and OD prepared in step 1 600 Equal volume mixing of the agrobacterium solution is equal to 0.80, and mixed bacterial solution is obtained.
The method comprises the steps of tightly attaching sterile cellophane to the surface of a solid culture medium of Sa, uniformly coating mixed bacterial liquid on the surface of the sterile cellophane, carrying out constant-temperature light-shielding co-cultivation for 48 hours at 25 ℃, transferring the mixed bacterial liquid to the surface of a new selective culture medium (the coated bacterial surface faces upwards, the thickness of the culture medium is about 2 mm), pouring a layer of selective culture medium on the surface of the cellophane to completely cover the cellophane (the thickness of the culture medium is about 2 mm), carrying out constant-temperature light-shielding cultivation for 4-5 days at 25 ℃, picking hypha penetrating through the upper selective culture medium as a potential positive clone, inoculating the hypha to the Sa solid culture medium containing 200 mu g/mL hygromycin B, carrying out constant-temperature light-shielding cultivation for 4-5 days at 25 ℃, transferring the hypha to cultivate the next generation, and carrying out co-cultivation for five generations to obtain a recombinant Cordyceps militaris hygromycin resistant strain containing a hexokinase gene HK (shown in SEQ ID NO. 1).
4. Cordyceps militaris hygromycin resistance strain gene identification
Extracting genome DNA of recombinant Cordyceps militaris hygromycin resistant strain, and dissolving the obtained DNA with 50-100 mu L TE Buffer. The extracted DNA can be immediately subjected to the next experiment or stored at-20 ℃. Taking genomic DNA of the extracted Cordyceps militaris hygromycin resistant strain, and carrying out PCR verification by using hph-F/hph-R primers to check whether the transformation is successful. The primers for positive PCR identification are shown in Table 8, and the reaction system is shown in Table 9. According to the result of 1% agarose gel electrophoresis identification, whether the clone is positive or not can be judged, and the result is shown in a figure 2, and lanes 1-5 are parallel samples of the PCR product identified by the recombinant cordycepin resistance gene of the target gene HK. And (5) preserving the transformant with positive identification result.
TABLE 8 hygromycin identification primers
TABLE 9 hygromycin identification PCR reaction System
Amplification procedure for PCR: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 60℃for 30s, elongation at 72℃for 30s, cycle number 30; final extension at 72℃for 10min.
5. Polysaccharide content detection of Cordyceps militaris hygromycin resistant strain
The recombinant cordyceps militaris hygromycin resistant strain prepared by the wild cordyceps militaris CM01 and the recombinant cordyceps militaris hygromycin resistant strain prepared by the method of the step 3 are inoculated to a Sa solid culture medium containing 200 mug/mL hygromycin B, the culture is carried out for 4-5 days at the constant temperature and the dark place, the bacterial blocks with the same size are picked and inoculated to a fermentation culture medium, and the culture is carried out for 7 days at the constant temperature and the 180 rpm.
10. Mu.L of the fermentation broth was diluted to 2mL with deionized water, and the total sugar content was measured by the sulfuric acid-phenol method (concentrated sulfuric acid 5mL,5% phenol 1mL, sample 1 mL) and the measurement was repeated three times.
100 μl of the fermentation broth was diluted to 500 μl with deionized water, and the glucose residue concentration was measured with a biosensing analyzer, and the measurement was repeated three times. The total sugar content minus the glucose residue content gave the Cordyceps militaris exopolysaccharide content, which is shown in Table 10 and FIG. 3.
Table 10 extracellular polysaccharide content of wild type Cordyceps militaris and Cordyceps militaris hygromycin resistant Strain
Example 3 construction of Cordyceps militaris engineering bacterium containing mannose-1-phosphate uridine transferase MPG
The mannose-1-phosphouridine transferase gene PGM (SEQ ID NO. 2) on the genomic DNA of Cordyceps militaris was obtained by amplification with the primers MPG-F/MPG-R of Table 11, usingUniSeamless Cloning and Assembly Kit (Beijing full gold biotechnology Co., ltd., catalog number: CU 101-02) the target gene fragment was linked to plasmid pCAMBIA to give recombinant vector pCAMBIA-PgpdA-MPG-Tcbh1-hph-PtrpC.
TABLE 11 mannose-1-phosphate uridine transferase MPG amplification primers
Other operations are the same as in examples 1 and 2, recombinant Cordyceps militaris engineering bacteria containing the gene MPG are obtained, genomic DNA of the extracted Cordyceps militaris hygromycin resistant strain is taken, PCR verification is carried out by using primers hph-F/hph-R in table 8, whether the transformation is successful is checked, the result is shown in figure 2, lanes 6-10 are parallel samples of PCR product identification for the recombinant Cordyceps militaris hygromycin resistant gene of the target gene MPG, and transformants with positive identification results are stored; the total sugar content and the glucose residue concentration of the transgenic strain were measured, and the total sugar content was subtracted from the glucose residue content to obtain the Cordyceps polysaccharide content, which is shown in Table 10 and FIG. 3. The results show that not all enzyme gene overexpression related to cordyceps militaris polysaccharide biosynthesis can improve extracellular polysaccharide yield.
SEQ ID NO.2
ATGGCGTCCGTTTCCGAGTCGGCTCTGGTCGCCATTGCCGCCCAGCTCAGCCAGGTCATGGGCGCTCTCGAGAAGCAGGTGGGTTGCGTCTTATGTACTTTGTTTGTGCCGCCGAGGGGTTGTCGGAGTCGCTTCGCCGTTGCGCGCCGTGCCAGCCCTACGCGCGCGCAGGTCTCAATGGCGAGATGAACTAATGTGCCATAGAATTCCGAGCTCTCTGAGCTTCGTGTCGAGTGCTCTGCTCTGCGCACCCAGAATACCAACATGGAGCGCCTCCTCCAGGATAACGTGGTAGGTGATGCCCAACTCCTTTCTCGATATTGAGGCTCCACTCGTCAATTCCAGCTGGGCCTGGATTGTCTGCACCTCACTCATTGAGCGAGTGTAGACTGCATCCCACAGGAATCAATCGCACATGCAGTCGCTCTCGCTTATACTCGGCCTATTCATGATCCATTCTCCCCTGCTGCCTCATACTGATCCGTCACAGCACAAGTACGACAACCGCAACCTCAGCCTCTCCCACCCCTCCCCGGGCCTTGAGACCTCGTCGCCTTTCCTCTCCGCCACCTCTCTCCCCCGCGTGGCCTCTACTCTCACCTTTAACCCTCTTGGTGATCAGCCTCTTTTCACCACCGCCTCCCCCGCCTATGAAATTCCCGGCTTCTATGTCGTCATTCCCGCTGGTGGTGCTGGTACCCGTCTCTGGCCCCTGTCCCGCAAGAACCATCCCAAGTTCCTGCTCGATGTCTCGCTTTCAGGCCGCTCCATGCTGCAGTCTACCTGGAACCGTCTTGTTCCCCTGACTGGTTCCTCTCGCATGACTGTCGTCGCCGGTCCTGCTCACTCCGAGGCCATCAAGGGCCAGCTCCCCCAGCTCGAGGACAACAACCTGTTCACTGAGCCTGGTCCCAAGGACTCCATGGCCGCCATTGGTCTCGCCGCCGCCATTCTCGCCGAGCGTGATCCCGATGCCATTATTGGTTCCTTCGCCGCCGATCACATGATTTCCGGTGAAGATGCTTTCCTCGGCGCCGTCCGTGAAGCTGCCAACGTCGCCAAGGCTGGCTATCTCGTTACTATTGGTATCGCGCCGTCTCACCCTGCGACCGGCTTCGGTTACGTACAGCTCGGAAAGGCCCTCAACGGCCACAACGCTCCCACCGCCCGTCTCGTCAGCTCCTTCAAGGAGAAGCCCGATGCCCGCACCGCCGCCAAGTACCTCGCCTCCGGTGAGTACCGCTGGAACGCTGGTATGTTCGTCACGCGCGCAGCTACCCTCATGGAGCTTGTACTAGAGCACCGCCCCGAGATCCACGCTGGTCTCGTCAAAATCGCCAAGGTCTGGGACAACGAGCCTGAGCGTCTTGCTGTTCTCGCCGAGATTTGGCCTACTCTCGAGACAATTGCCATTGACCATGCTATTGCGGAGCCTGCCGCTGCCGAGGGTAAGGTTGCCGTCATTCCCGCCACTTTCGGTTGGGACGATGTAGGTGACTTTTCGTCCCTTGCTGAAATGCTTCCCGCCGAAGCCAACTCCCCTCGCGTTCTCGGTGACAGCGATCTCGTCGTTTCCGAGCAGACCTTTGGTGGTATCATTGTTCCCGGTTCCGGCCGTCTTGTTGCTTGCCTGGGTGTTGACGATCTGGTCATCGTCGATCTGCCCGATGCGCTCATGATCACCACCCGCGCTCGTTCGCAAGAAGTCAAGACGCTTGTGAAGAAGGCCCAGCGGGCTGGTTTCGGGAGCCTTATGTAG 1759
Sequence listing
<110> Zhejiang university of industry
<120> method for improving extracellular polysaccharide yield of Cordyceps militaris by using over-expressed hexokinase gene HK
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1841
<212> DNA
<213> Unknown (Unknown)
<400> 1
atggttggcc taggtcctcg cgctccgccc tctagaaaag gtacgccccg gatactgcta 60
ctacggcgga aaaatattga atcgaacttt gtcaacgtct tttcaccgat gcaagacgct 120
cctgggattg tgacgaccat ccgctgctgc actagaagct aacaaccact gagggtcgaa 180
agccgatgtt cccaaggaca ccatggaggc gatccggaac attgaggagc tctttacggt 240
cgacactgcc aagctcaagg ccatcacaga gcacttcgtc tccgagctta ccaagggttt 300
gtgccgcgcc cgaattaaac cagccagatt gcgccaacca acaccagcca ctaaccgtct 360
tctcaggcct caccaaagag ggtggcagca ttgtaagcat atatgaatct acaccaacta 420
ctgtaactca ggctaacaga tctccagccc atgaacccta cttgggtcat gtcgtatccc 480
gatggcaatg agactggcac cttcctcgcc ctcgacatgg gcggcaccaa cctgcgcgtc 540
tgccaaatca cccttacaga taagaggtcc gagtttgaca ttatccagtc caagtacaag 600
atgcctgaag agctcaagac tggcaagagc gatgagctgt gggagtacat tgccgactgt 660
ctccaccagt tcctcgaaac ccaccatggt gacaccaaga acattggcac cttgcccttg 720
gggttcacct tttcttaccc cgctacccag aactacattg acgcgggtat cttgcagcga 780
tggaccaagg gctttgacat tgacggcgtc gagggccagg atgtcgttcc catgttcgag 840
gctgctcttg ccaagcgtgt atgtctaata tgcctcactc tctcgtactc tggtagcttg 900
ctgacctgta tgtagggtgt tcccattcaa ctgactgctc ttattaatga caccaccgga 960
acgctcatgg cctcggccta tactgaccct accatgaaga ttggctgcat cttcggcacc 1020
ggctgcaacg ccgcgtacgt cgaacactgc ggttcgatcc ccaagcttgc cgacatgggt 1080
ctacctgatg acatggaaat ggccatcaac tgcgagtggg gtgcctttga caatgagcac 1140
aagattctac cccgtaccaa gtacgatgtc atcattgatg aggagtcacc ccgccctggc 1200
caacaggcgt ttgagaaaat gattgcaggt ctctaccttg gcgagatttt ccgccttgcc 1260
cttgtcgatc tgcataacga aaagcacctc tttcagggac aggacatcaa ctgtctgcgc 1320
aaggcctaca gcctcgacgc ttcgttcctt tcggctattg aggagtgagt cacatcgttc 1380
tgctactgag gcatgccgtc taactaccgg gctccaggga tcctttcgag aacttacagg 1440
agacgttcga cctgtttgca aacaagttgc aaatcaatgt caccggcccc gagcttgagc 1500
tgatccgccg cctcgctgag ctgatcggta cccgcgccgc ccgtctcgct tcctgcggtg 1560
tcgctgccat ttgcaagaag aagaacttca agacctgcca cgtcggtgcc gacggctccg 1620
tcttcaataa gtaccctcac ttcaaggctc gtggagcgca ggccctgaag gagattcttg 1680
actggcccga caagactgat gccaaagccg aggacccggt tgaaattctt gccgccgaag 1740
acggcagtgg tgtcggtgcc gctctgattg ccgccttgac cctgaagcgt gcgcagcagg 1800
gcaacctggc gggcattctt cacccggagc gcttcaaata g 1841
<210> 2
<211> 1759
<212> DNA
<213> Unknown (Unknown)
<400> 2
atggcgtccg tttccgagtc ggctctggtc gccattgccg cccagctcag ccaggtcatg 60
ggcgctctcg agaagcaggt gggttgcgtc ttatgtactt tgtttgtgcc gccgaggggt 120
tgtcggagtc gcttcgccgt tgcgcgccgt gccagcccta cgcgcgcgca ggtctcaatg 180
gcgagatgaa ctaatgtgcc atagaattcc gagctctctg agcttcgtgt cgagtgctct 240
gctctgcgca cccagaatac caacatggag cgcctcctcc aggataacgt ggtaggtgat 300
gcccaactcc tttctcgata ttgaggctcc actcgtcaat tccagctggg cctggattgt 360
ctgcacctca ctcattgagc gagtgtagac tgcatcccac aggaatcaat cgcacatgca 420
gtcgctctcg cttatactcg gcctattcat gatccattct cccctgctgc ctcatactga 480
tccgtcacag cacaagtacg acaaccgcaa cctcagcctc tcccacccct ccccgggcct 540
tgagacctcg tcgcctttcc tctccgccac ctctctcccc cgcgtggcct ctactctcac 600
ctttaaccct cttggtgatc agcctctttt caccaccgcc tcccccgcct atgaaattcc 660
cggcttctat gtcgtcattc ccgctggtgg tgctggtacc cgtctctggc ccctgtcccg 720
caagaaccat cccaagttcc tgctcgatgt ctcgctttca ggccgctcca tgctgcagtc 780
tacctggaac cgtcttgttc ccctgactgg ttcctctcgc atgactgtcg tcgccggtcc 840
tgctcactcc gaggccatca agggccagct cccccagctc gaggacaaca acctgttcac 900
tgagcctggt cccaaggact ccatggccgc cattggtctc gccgccgcca ttctcgccga 960
gcgtgatccc gatgccatta ttggttcctt cgccgccgat cacatgattt ccggtgaaga 1020
tgctttcctc ggcgccgtcc gtgaagctgc caacgtcgcc aaggctggct atctcgttac 1080
tattggtatc gcgccgtctc accctgcgac cggcttcggt tacgtacagc tcggaaaggc 1140
cctcaacggc cacaacgctc ccaccgcccg tctcgtcagc tccttcaagg agaagcccga 1200
tgcccgcacc gccgccaagt acctcgcctc cggtgagtac cgctggaacg ctggtatgtt 1260
cgtcacgcgc gcagctaccc tcatggagct tgtactagag caccgccccg agatccacgc 1320
tggtctcgtc aaaatcgcca aggtctggga caacgagcct gagcgtcttg ctgttctcgc 1380
cgagatttgg cctactctcg agacaattgc cattgaccat gctattgcgg agcctgccgc 1440
tgccgagggt aaggttgccg tcattcccgc cactttcggt tgggacgatg taggtgactt 1500
ttcgtccctt gctgaaatgc ttcccgccga agccaactcc cctcgcgttc tcggtgacag 1560
cgatctcgtc gtttccgagc agacctttgg tggtatcatt gttcccggtt ccggccgtct 1620
tgttgcttgc ctgggtgttg acgatctggt catcgtcgat ctgcccgatg cgctcatgat 1680
caccacccgc gctcgttcgc aagaagtcaa gacgcttgtg aagaaggccc agcgggctgg 1740
tttcgggagc cttatgtag 1759
Claims (7)
1. A method for improving the yield of extracellular polysaccharide of cordyceps militaris by using over-expressed hexokinase gene HK is characterized in that the method is that the content of extracellular polysaccharide of cordyceps militaris is improved by over-expressing hexokinase gene HK in cordyceps militaris gene; the nucleotide sequence of the hexokinase gene HK is shown in SEQ ID NO. 1.
2. The method of claim 1, whereinThe method is characterized in that: constructing cordyceps militaris genetic engineering bacteria containing hexokinase gene HK, inoculating the cordyceps militaris genetic engineering bacteria into a fermentation medium, and carrying out shake fermentation culture at a temperature of 22-25 ℃ and at a speed of 120-180 rpm to obtain fermentation liquor containing cordyceps militaris extracellular polysaccharide; the fermentation broth medium comprises the following components: glucose 20g/L, (NH) 4 ) 2 SO 4 7g/L，K 2 HPO 4 ·3H 2 O 0.5g/L，KH 2 PO 4 0.5g/L，MgSO 4 ·7H 2 O0.5 g/L, L-glycine 1g/L, distilled water as solvent, and natural pH.
3. The method of claim 2, wherein the Cordyceps militaris genetic engineering bacteria are obtained by seamlessly cloning and then connecting a hexokinase gene HK and a binary vector pCAMBIA to obtain a recombinant vector, and then transforming the recombinant vector into agrobacterium; and then the Cordyceps militaris is infected by agrobacterium.
4. A method according to claim 3, wherein the agrobacterium is agrobacterium tumefaciens (Agrobacterium tumefaciens) AGL-1.
5. The method of claim 3, wherein the genetically engineered Cordyceps militaris is constructed as follows: (1) The hexokinase gene HK and the binary vector pCAMBIA are subjected to seamless cloning and then are connected to obtain a recombinant vector, agrobacterium is transformed, the recombinant vector is inoculated into an IM culture medium containing 25 mu g/mL rifampicin and 50 mu g/mL kanamycin, the culture is carried out for 36h at 28 ℃, and the agrobacterium strain containing the hexokinase gene HK is obtained through screening; the IM medium: KH (KH) 2 PO 4 1.45g/L，K 2 HPO 4 2.05g/L，MgSO 4 ·7H 2 O 0.6g/L，NaCl 0.1g/L，CaCl 2 0.01g/L，FeSO 4 0.001g/L，NH 4 NO 3 0.5g/L, 5mL/L glycerol, 40mL/L MES buffer solution with pH of 5.5 and 1mol/L, 5mL/L microelement stock solution, 2g/L glucose and distilled water as solvent; microelement stock solution composition: znSO (ZnSO) 4 ·7H 2 O100mg/L，CuSO 4 ·5H 2 O 100mg/L，H 3 BO 3 100mg/L，Na 2 MoO 4 ·7H 2 O100mg/L, and water as solvent; (2) Inoculating an agrobacterium strain containing hexokinase gene HK into a liquid LB culture medium containing 50mg/L kanamycin, and shake culturing at 28 ℃ and 180rpm for 36 hours; transferring the culture solution into liquid AIM culture medium at a volume concentration of 1%, and culturing at constant temperature of 28deg.C at 180rpm to reach thallus concentration OD 600 =0.60 to 0.80 to obtain agrobacterium liquid; the AIM culture medium is prepared by adding 200 mug/mL acetosyringone into an IM culture medium; (3) Inoculating wild Cordyceps militaris to a solid sand culture medium, culturing for 7 days under the condition of constant temperature and light shielding, repeatedly flushing the surface of the solid culture medium with sterile water on an ultra-clean workbench to obtain Cordyceps militaris bacterial suspension, and filtering the bacterial suspension to remove mycelia to obtain Cordyceps militaris spore suspension; solid sand culture medium: glucose 40g/L, peptone 10g/L, agar powder 20g/L, agar powder 20g/L, distilled water as solvent, and natural pH; (4) Mixing the agrobacterium tumefaciens bacteria liquid in the step (2) with the cordyceps militaris spore suspension in the step (3) in equal volume to obtain mixed bacteria liquid, coating the mixed bacteria liquid on the upper surface of sterile cellophane placed on the surface of a selective culture medium, performing constant-temperature light-shielding co-cultivation for 48 hours at 25 ℃, transferring the mixed bacteria liquid together with the cellophane onto the surface of a new selective culture medium, pouring a layer of selective culture medium on the upper surface of the cellophane, and performing constant-temperature light-shielding cultivation for 4-5 days at 25 ℃; selecting hypha penetrating through an upper layer selection culture medium, inoculating the hypha into a Sa solid culture medium containing 200 mug/mL hygromycin B, culturing for 4-5 days at a constant temperature and in a dark place at 25 ℃, and continuing subculture to obtain Cordyceps militaris genetic engineering bacteria containing hexokinase gene HK; the selection medium was an IM medium containing 200. Mu.g/mL hygromycin B, 100. Mu.g/mL ceftriaxone sodium and 50. Mu.g/mL kanamycin.
6. The method of claim 5, wherein the concentration of the Cordyceps militaris spore suspension is 10 6 ~10 8 /mL。
7. The method of claim 2, wherein the cordyceps militaris genetically engineered strain containing hexokinase gene HK is inoculated to a solid culture medium containing 200 mug/mL hygromycin B before fermentation, cultured for 4-5 d at a constant temperature and in a dark place at 25 ℃, and then colonies are picked and inoculated to the fermentation medium.
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