Sweet protein and application thereof in improving sweetness of cordyceps militaris sporocarp

Abstract

The invention discloses sweet protein and application thereof in improving sweetness of cordyceps militaris sporocarp, and relates to the technical field of edible mushrooms. The sweet protein disclosed by the invention is shown as SEQ ID NO.1 or SEQ ID NO.2, and the sweet protein is introduced into a cordyceps militaris strain, so that the sweetness of a sporocarp of the cordyceps militaris strain can be improved, the sweetness and flavor of the cordyceps militaris can be effectively improved, and the requirements of people on healthy foods can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of edible fungi, in particular to a sweet protein and application thereof in improving the sweetness of Cordyceps militaris fruiting bodies. Background Art

[0002] With the improvement of public health awareness and the increasing demand for healthy food, reducing the sugar content of products while maintaining sweetness has become an urgent need.

[0003] Cordyceps militaris (Cordyceps militaris L. Link) is a fungus with important medicinal value. It is rich in bioactive ingredients such as cordycepin and cordyceps polysaccharides, and has multiple biological activities such as antioxidant, antibacterial, anti-inflammatory, and immunomodulatory. It has been widely used in traditional Chinese medicine. Currently, people's consumption of Cordyceps militaris mainly stays in the consumption of fresh and dried fruiting bodies. However, most existing Cordyceps militaris fruiting bodies lack sweetness.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The present invention aims to provide a sweet protein and its application in improving the sweetness of Cordyceps militaris fruiting bodies. The Cordyceps militaris fruiting bodies provided by the present invention have a high sweetness, effectively improve the sweetness and flavor of Cordyceps militaris, and can meet people's demand for healthy food.

[0006] The present invention is achieved in that:

[0007] In a first aspect, the present invention provides a sweet protein, the amino acid sequence of which is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0008] Natural sweet protein is high in sweetness, low in calories and non-toxic, and can meet the pursuit of healthy, low-calorie food.

[0009] SEQ ID NO. 1 shows thaumatin, a sweet protein discovered and identified in the West African plant Thaumatococcus danielli. It is a mixture of six similar proteins with a molecular weight of 22 kDa and a sweetness threshold of 1.1 mg / kg. Its sweetness is 1,600 times that of sucrose, with some studies claiming it is 3,000 times sweeter. With an isoelectric point of 12, it is an alkaline protein and exhibits strong thermal stability under acidic conditions. It remains stable even after heating at 100°C for several hours at a pH below 5.5.

[0010] SEQ ID NO. 2 shows miraculin, also known as miraculin or miraculin. It was isolated from the fruit of the West African plant Richadella dulcifa Baehni and has a molecular weight of 28 kDa. Miraculin is a single-chain polypeptide consisting of 191 amino acids with an isoelectric point of 9. It is stable below 100°C and within a pH range of 3 to 12. It is a flavor-modifying protein that binds to and activates sweet taste receptors (T1R2 / T1R3). Upon encountering sour substances, it alters the human taste perception, converting acidic taste into sweetness. The sweetening effect depends on the acidity and pH, and this taste-modulating effect can last for 1 to 2 hours.

[0011] The present invention applies the above two proteins to the cultivation of Cordyceps militaris for the first time to increase the sweetness of its fruiting body, greatly improving the sweetness and flavor of Cordyceps militaris and effectively meeting people's demand for healthy food.

[0012] In another aspect, the present invention provides an isolated nucleic acid molecule encoding the sweet protein as described above.

[0013] "Nucleic acid molecules," "polynucleotides," "nucleic acid sequences," "nucleotide sequences," or "nucleic acids" of the present invention are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers that optionally contain synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single-letter names as follows: "A" for adenosine or deoxyadenosine (RNA or DNA, respectively), "C" for cytidine or deoxycytidine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A or C or T, "D" for A, T, or G, "I" for inosine, and "N" for any nucleotide.

[0014] Optionally, in some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.3 or SEQ ID NO.4.

[0015] SEQ ID NO. 3 and SEQ ID NO. 4 are codon-optimized sequences, which have higher protein expression efficiency in the Cordyceps militaris strain CM01.

[0016] In another aspect, the present invention provides a vector comprising the nucleic acid molecule described above.

[0017] The "vector" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be an RNA (such as mRNA) that can be translated, for example, an RNA generated by in vitro transcription. In some embodiments, the vector backbone of the present invention is PKD2, such as Figure 1 shown.

[0018] Transformation of host cells with vectors can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, a step well known in the art. Another method is to use MgCl2, or Agrobacterium transformation can be used in addition. If necessary, transformation can also be carried out using the electroporation method. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0019] In another aspect, the present invention provides a recombinant cell comprising the above-mentioned nucleic acid molecule or the above-mentioned vector.

[0020] Recombinant cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as animal cells. Representative examples include Escherichia coli, Streptomyces, Agrobacterium; fungal cells, such as yeast; and animal cells.

[0021] In another aspect, the present invention provides use of the sweet protein, nucleic acid molecule, vector, or recombinant cell described above in improving the sweetness of Cordyceps militaris fruiting bodies.

[0022] The present invention applies the above two sweet proteins to the cultivation of Cordyceps militaris for the first time to enhance the sweetness of its fruiting bodies, greatly improving the sweetness and flavor of Cordyceps militaris and effectively meeting people's demand for healthy food.

[0023] On the other hand, the present invention provides a method for improving the sweetness of Cordyceps militaris fruiting bodies, comprising: introducing the nucleic acid molecule or the vector described above into a Cordyceps militaris strain to express the sweet protein described above.

[0024] Optionally, in some embodiments of the present invention, the Cordyceps militaris strain is a CM01 strain.

[0025] In another aspect, the present invention provides a Cordyceps militaris strain comprising the nucleic acid molecule or the vector as described above.

[0026] The present invention applies the above two sweet proteins to the cultivation of Cordyceps militaris for the first time. The strain obtained by cultivation can develop and grow normally, and the sweet protein of its fruiting body can be successfully expressed, with obvious sweetness.

[0027] In another aspect, the present invention provides a use of the Cordyceps militaris strain as described above in improving the sweetness of Cordyceps militaris fruiting bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 Schematic diagram of the vector structure in Example 1 (a-PKD2-SOD-Tha-HPH, b-PKD2-SOD-Mir-HPH).

[0030] Figure 2 The colony PCR verification results of the plasmid vector in Example 1 (a-transformed Thaumatin gene: Maker size 2000, fragment length 1200, b-transformed Miraculin gene: Maker size 2000, fragment length 700).

[0031] Figure 3 The colony PCR verification results of the Cordyceps militaris strain transformants in Example 2 (channels 1-4 are for verifying Thaumatin transformants, channels 5-7 are for verifying Miraculin transformants, and channel 8 is the position of the GFP band, which is 700 bp in size).

[0032] Figure 4 These are the results of Western blot detection of GFP protein expressed by the transformants in Example 3 (a-thaumatin gene-transformant, b-miraculin gene-transformant).

[0033] Figure 5 This is a heat map of the amino acid content of the Thaumatin gene transformants in Example 4.

[0034] Figure 6 These are the results of amino acid concentration testing of the two transformants in Example 4 (in the figure: Thaumatin - transformant transformed with the Thaumatin gene; Miraculin - transformant transformed with the Miraculin gene, the same applies hereinafter).

[0035] Figure 7 The spore germination rate test results of the two transformant strains in Example 5 are shown.

[0036] Figure 8These are the results of mycelial diameter measurement of the two transformant strains in Example 6.

[0037] Figure 9 The spore production results of the two transformant strains in Example 7 are shown.

[0038] Figure 10 The results of photographing the germination of the fruiting bodies of the two transformant strains in Example 8 were obtained.

[0039] Figure 11 These are the test results of the weight loss rate of the fruiting bodies of the two transformant strains in Example 9. DETAILED DESCRIPTION

[0040] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0041] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0042] Example 1

[0043] Construction of gene editing vectors using homologous recombination technology

[0044] 1. Artificially synthesize the sweet protein gene sequence and optimize it according to the codon preference of Cordyceps militaris. Based on the sequence, primers are designed and the target gene sequence with specific sticky ends is obtained by PCR amplification.

[0045] Thaumatin protein was synthesized in this example, and its amino acid sequence is shown in SEQ ID NO.1; the corresponding coding gene sequence is shown in SEQ ID NO.3.

[0046] Simultaneously, the miraculin protein was synthesized in this example, and its amino acid sequence is shown in SEQ ID NO.2; the corresponding coding gene sequence is shown in SEQ ID NO.4.

[0047] SEQ ID NO. 3 and SEQ ID NO. 4 are codon-optimized sequences, which have higher protein expression efficiency in the Cordyceps militaris strain CM01.

[0048] SEQ ID NO.1:

[0049] MAATTCFFFLFPFLLLLTLSRAATFEIVNRCSYTVWAAASKGDAALDAGGRQLNSGESWTINVEPGTKGGKIWARTDCYFDDSGRGICRTGDCGGLLQCKRFGRPPTTLAEFSLNQYGKDYIDISNIKGFNVPMDFSPTTRGCRGVRCAADIVGQCPAKLKAPGGGCNDACTVFQTSEYCCTTGKCGPTEYSRFFKRLCPDAFSYVLDKPTTVTCPGSSNYRVTFCPTALELEDE

[0050] SEQ ID NO.2:

[0051] MKELTMLSLSFFFVSALLAAAANPLLSAADSAPNPVLDIDGEKLRTGTNYYIVPVLRDHGGGLTVSATTPNGTFVCPPRVVQTRKEVDHDRPLAFFPENPKEDVVRVSTDLNINFSAFMPCRWTSSTVWRLDKYDESTGQYFVTIGGVKGNPGPETISSWFKIEEFCGSGFYKLVFCPTVCGSCKVKCGDVGIYIDQKGRRRLALSDKPFAFEFNKTVYF

[0052] SEQ ID NO.3:

[0053] atggctgccaccacttgctttttcttcctctttccatttctcctcctcctcaccctctctcgagccgctacctttgaaattgtcaaccgttgctcttacacagtttgggcagcagcttctaaaggcgacgcagctcttgacgcaggcggtcgtcaacttaactcaggcgaatcttggaccattaacgtcgaaccaggcacaaaaggcggtaagatttgggctcgcaccgattgctacttcgacgattcaggtcgaggtatttgccgtacaggcgattgcggcggtcttcttcagtgcaaacgttttggtcgtccaccaacaacacttgctgagttctccctcaaccaatacggcaaggactacatcgacatctccaacatcaagggcttcaacgtcccaatggacttctctccaaccacacgaggttgccgaggcgttcgttgcgcagcagatattgttggccagtgcccagctaagctcaaggctccaggcggcggttgcaacgacgcttgcacagtctttcagacctccgagtattgctgcacaacaggtaagtgcggtccaacagagtactctcgtttcttcaagcgcctttgcccagacgctttttcttacgtcctcgacaagccaacaaccgttacttgcccaggctcttctaactaccgagtcaccttttgcccaacagctcttgaactcgaagacgagtaa

[0054] SEQ ID NO.4:

[0055] atgaaggaactcaccatgctctctctctccttcttcttcgtctccgctctccttgccgctgcagctaatccacttctttccgcagcagattctgctccaaacccagttctcgacattgacggcgaaaagcttcgtacaggcacaaactactacattgtcccagtt cttcgagatcacggcggcggtcttacagtttccgctacaacacctaacggcacatttgtttgcccaccacgagttgtccagacacgtaaggaagtcgatcacgatcgtccactcgccttcttcccagaaaacccaaaggaagacgttgttcgcgtctctacagacc tcaacatcaacttctccgccttcatgccttgccgatggacatcttctaccgtttggcgtctcgataagtacgacgaatccacaggccaatacttcgtcacaattggcggcgttaagggtaacccaggtccagaaaccatttcctcttggttcaagatcgaagagtt ttgcggctccggtttctacaagctcgtcttttgccccaccgtttgcggttcttgcaaagttaagtgcggcgacgtcggcatttacattgaccagaagggtcgtcgtcgtcttgctctctccgataagccatttgccttcgagttcaacaagaccgtctacttctaa

[0056] 2. Enzyme Digestion and Transformation

[0057] The gene insertion position was determined to be after the SOD promoter, the restriction enzyme cutting site was Xmal I, and the target gene and the linearized vector were connected using ligase.

[0058] Take 10 μl of the ligation product and transform it into the colon competent Top10. The transformation method is the 45s heat shock method. The specific method is as follows:

[0059] Take 100 μL of E. coli Top10 competent cells, add 10 μL of ligation product, and place on ice for 25 minutes; place in a 42°C water bath for 45 seconds, and place on ice for 2 minutes; add to 500 μL of LB non-resistant liquid medium, and culture at 37°C, 200 rpm for 1 hour; then spread the bacterial liquid on LB solid medium containing kanamycin resistance and culture overnight.

[0060] 3. Recombinant Vector Verification

[0061] Eight single clones were selected for colony PCR verification, and two positive clones were selected. The plasmids were extracted using a plasmid miniprep kit and sent for sequencing. The sequencing results were analyzed using Snapgene software to verify and confirm that the recombinant vector had been successfully constructed.

[0062] The vector into which the Thaumatin gene was transferred was named PKD2-SOD-Tha-HPH, and its map is shown in Figure 1 The verification results are shown as Figure 2 -a as shown.

[0063] The vector into which the Miraculin gene was transferred was named PKD2-SOD-Mir-HPH. The verification results were as follows: Figure 2 -b as shown.

[0064] Example 2

[0065] Genetic transformation of Cordyceps militaris

[0066] 1. Plasmid Transformation into Agrobacterium

[0067] Take 100 μL of Agrobacterium tumefaciens AGL1 competent cells, add 1-2 ng of plasmid (PKD2-SOD-Tha-HPH or PKD2-SOD-Mir-HPH), and place on ice for 25 minutes. Quickly place the mixture in liquid nitrogen and freeze for 1-2 minutes. Incubate in a 37°C water bath for 5 minutes, then place on ice for 5 minutes. Add the mixture to 400 μL of LB non-resistant liquid medium and incubate at 28°C at 200 rpm for 4-6 hours. Then, spread the bacterial solution on LB solid medium containing kanamycin- and rifampicin-resistant strains and incubate for 2 days. Synthesize the following detection primers for PCR amplification and verify the recombinant vector's integration into the competent cells by agarose gel electrophoresis.

[0068] 2. Genetic transformation of Cordyceps militaris

[0069] (1) A single colony of Agrobacterium tumefaciens AGL-1 that was verified to contain the target plasmid was picked from the LB plate and inoculated into 5 mL of LB liquid medium containing 50 μg / mL kanamycin and carboxybenzone, and cultured at 220 rpm and 28°C overnight.

[0070] (2) Centrifuge at 5000 rpm for 2 min to collect the cells, resuspend them in an equal volume of IM liquid induction medium, and transfer an appropriate amount of the resuspension to 5 mL of IM liquid induction medium to adjust the OD600 to 0.2-0.3.

[0071] (3) Incubate at 28°C, 220 rpm, and shake for 4-6 h until the bacterial concentration reaches OD600 = 0.5-0.8.

[0072] (4) Collecting Cordyceps militaris blastospores: In a cleanroom, the mycelium cultured in SDB medium at 25°C, 150 rpm, and a shaker for 4 days was filtered with a sterile non-woven fabric, and the collected blastospores were stored at 4°C for use.

[0073] (5) Take the above Agrobacterium solution and dilute it to 1×10 6 100 μL of each spore suspension of 100 μg / mL was mixed thoroughly and spread on IM plates, and cultured at 28°C for 2 days.

[0074] (6) Add 2 mL of 0.05% Tween 20 solution to the above co-culture plate and gently scrape the co-culture with a spreader to mix.

[0075] (7) Spread 200 μL of the washed mixture evenly on M-100 medium containing 300 μg / mL cephalosporin and 250 μg / mL phosphinothricin, and culture at 25°C for 5-6 days until transformants appear.

[0076] (8) Pick the transformants and place them on the same screening medium. When the colony diameter grows to about 2 cm, extract a small amount of the transformant genome for PCR verification. The verification results are as follows: Figure 4 -a (transformant transformed with Thaumatin gene), Figure 4 -b (transformants transformed with the Miraculin gene).

[0077] Example 3

[0078] Positive strain detection

[0079] (1) Preparation of experimental materials

[0080] Biological sample acquisition:

[0081] 14-day-old mycelia of the transformants were quickly ground into a powder under liquid nitrogen. An appropriate amount of protein extraction buffer was then added, mixed thoroughly, and extracted with shaking at 4°C for 30 minutes. After extraction, the mixture was centrifuged at 12,000 rpm at 4°C for 15 minutes, and the supernatant was collected as a protein sample.

[0082] Antibody preparation:

[0083] Purchase a specific primary antibody against GFP. This primary antibody has high specificity and sensitivity. It is usually derived from a rabbit polyclonal antibody and has undergone rigorous quality verification. It can accurately recognize the unique antigenic epitope of the GFP protein.

[0084] The matching secondary antibody is anti-rabbit IgG and labeled with HRP (horseradish peroxidase) to ensure the efficiency of subsequent signal detection. The dilution factor and other parameters are determined according to the secondary antibody product instructions and combined with preliminary experimental optimization.

[0085] Other ingredients:

[0086] Materials required for SDS-PAGE gel preparation: acrylamide, bisacrylamide, Tris base, SDS, ammonium persulfate, TEMED, etc. Gels of different concentrations are prepared according to conventional formulas and experimental requirements. Generally, a gel concentration of 12%-15% is more suitable for GFP (molecular weight approximately 27 kDa) to ensure effective separation of proteins during electrophoresis.

[0087] Transfer materials: Use a 0.22μm pore size PVDF membrane with transfer buffer (such as 25mM Tris, 192mM glycine, 20% methanol). At the same time, prepare filter paper, sponge and other items needed to construct the transfer "sandwich" structure.

[0088] Immunoreaction buffer: The blocking buffer is prepared by dissolving 5% skim milk powder or BSA (bovine serum albumin) in TBST buffer (25mM Tris, 192mM glycine, 0.1% SDS, 0.05% Tween-20). The primary and secondary antibody dilutions also use TBST buffer, and the dilution ratio is optimized based on the antibody titer and preliminary research.

[0089] (2) Experimental operation process

[0090] Protein sample processing:

[0091] The protein sample obtained above was mixed with an appropriate amount of 5× SDS loading buffer (containing 250 mM Tris-HCl pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, and 5% β-mercaptoethanol) and heated in a boiling water bath at 100°C for 5 minutes to fully denature the protein to ensure that the electrophoretic mobility was only related to the protein molecular weight.

[0092] Electrophoresis:

[0093] Carefully load the denatured protein sample into the pre-prepared SDS-PAGE gel loading wells and add running buffer (e.g., Tris-glycine buffer, composed of 25 mM Tris, 192 mM glycine, and 0.1% SDS). Perform electrophoresis in constant voltage mode with a starting voltage of 80 V. After the bromophenol blue indicator migrates to the separating gel, increase the voltage to 120 V. Continue electrophoresis for approximately 1.5-2 hours, until the bromophenol blue indicator approaches the bottom edge of the gel, to ensure accurate separation of proteins in the gel based on their molecular weight.

[0094] Transfer:

[0095] After electrophoresis, the electrophoresis apparatus was quickly disassembled, the gel gently removed, and the transfer apparatus precisely assembled according to the standard "sponge-filter paper-gel-membrane-filter paper-sponge" sandwich structure. The gel, 0.22μm pore size PVDF membrane, and filter paper were tightly fitted together, ensuring that no bubbles remained to prevent the transfer. The assembled transfer apparatus was placed in an ice bath and transferred using a constant current setting of 300mA for approximately 60-90 minutes to ensure efficient and complete transfer of the GFP protein from the gel to the PVDF membrane.

[0096] Immune response:

[0097] After transfer, place the PVDF membrane in a blocking solution containing 5% skim milk powder and incubate it with slow shaking at room temperature for 1 hour to block nonspecific binding sites on the membrane and reduce background noise.

[0098] After blocking, place the membrane in a GFP-specific primary antibody solution diluted to an appropriate concentration with blocking solution and incubate overnight at 4°C to ensure that the primary antibody is fully bound to the GFP protein on the membrane.

[0099] The next day, the membrane was removed and washed 3-5 times with TBST buffer at room temperature for 5-10 minutes each time to remove unbound primary antibody.

[0100] Next, place the membrane in a secondary antibody solution that matches the primary antibody and is diluted with blocking solution (the dilution factor is usually 1:5000-1:10000), and incubate slowly with shaking at room temperature for 1 hour to allow the secondary antibody to bind to the primary antibody and achieve signal amplification.

[0101] Wash the membrane again with TBST buffer 3-5 times, 5-10 minutes each time, to completely remove unbound secondary antibody.

[0102] Detection and analysis:

[0103] After the above treatment, the membrane is developed with ECL chemiluminescent reagent. The membrane is placed in a dark box to allow the membrane to fully contact the chemiluminescent reagent. The operation is carried out according to the reagent instructions. Then, the protein bands on the membrane are exposed using X-ray film (or chemiluminescent imager). The exposure time is determined by optimizing multiple experiments based on the signal intensity and is generally within the range of 30 seconds to 5 minutes. A clear image of the GFP protein band is obtained by exposure, such as Figure 4 As shown, GFP protein was successfully expressed in both transformants, indicating that Thaumatin and Miraculin proteins were expressed in the strain.

[0104] Example 4

[0105] Comparison of amino acid content detection

[0106] (1) Metabolite extraction:

[0107] The sample was ground with liquid nitrogen at 60 Hz for 50 s; a certain amount of sample was weighed and placed in a 2 mL EP tube, 2 small steel balls were added, 1000 μL of extraction solution (methanol: acetonitrile: water, v:v:v = 2:2:1, containing an isotope internal standard mixture, pre-cooled at -40°C) was added, and vortexed for 30 s; the sample was ground at 35 Hz for 4 min, and ultrasonicated in an ice-water bath for 5 min; the grinding and ultrasonication steps were repeated 3 times; the sample solution was allowed to stand at -40°C for 1 h; the sample was centrifuged at 4°C, 12000 rpm (centrifugal force 13800 (×g), radius 8.6 cm) for 15 min; 100 μL of the supernatant was taken and rotary evaporated to dryness; it was reconstituted with 100 μL of 50% methanol water, 100 μL of derivatization agent was added, 50 μL of 1 M NaHCO3 was added, and vortexed; derivatized in a 40°C water bath for 1 h, removed and cooled to room temperature, and 50 μL of 2 M HCl, rotary evaporate to dryness; re-dissolve in 200 μL methanol and load onto the instrument.

[0108] (2) On-machine testing:

[0109] Mobile phase conditions:

[0110] The target compounds were separated chromatographically using a Thermo Vanquish UHPLC System (Thermo Fisher) on a Waters ACQUITY UPLC BEH C18 column (100 × 2.1 mm, 1.7 μM, USA). The HPLC phase A consisted of 5 mM ammonium acetate, and the HPLC phase B consisted of acetonitrile. The column oven temperature was 45°C, the sample tray was set to 4°C, and the injection volume was 2 μL.

[0111] Mass spectrometry conditions:

[0112] Mass spectrometric analysis was performed using a Thermo Altis TSQ Plus Mass Spectrometer triple quadrupole mass spectrometer equipped with an ESI electrospray ionization source in multiple reaction monitoring (MRM) mode. Source parameters were as follows: Spray Voltage = -3300 V, Sheath Gas = 40 Arb, Aux Gas = 10 Arb, Sweep Gas = 1 Arb, Ion Transfer Tube Temp = 325°C, Vaporizer Temp = 350°C.

[0113] Prior to UHPLC-MS / MS analysis, a standard solution of the target compound was introduced into the mass spectrometer. For each target compound, several precursor ion-daughter ion transitions with the highest signal intensity were selected, and their MRM parameters were optimized. The transition with the best response was selected for quantitative analysis, while the remaining transitions were used for qualitative analysis of the target compound.

[0114] In this example, all target compound quantitative analysis was performed using Skyline, and all data acquisition was performed using Xcalibur (4.4.16.14, Thermo Fisher). Figure 5 、 Figure 6 As shown, the two transformant strains had higher amino acid contents than the CM01 strain.

[0115] (3) Method detection limit and quantification limit

[0116] The calibration solutions were serially diluted two-fold and analyzed by UHPLC-MRM-MS. The limits of detection and quantification (LOQs) were calculated based on the signal-to-noise ratio. The lowest limit of detection (LLOD) was defined as the compound concentration at a signal-to-noise ratio of 3, and the lowest limit of quantification (LLOQ) was defined as the compound concentration at a signal-to-noise ratio of 10 (according to the US FDA guideline for bioanalytical method validation).

[0117] Example 5

[0118] Determination of Cordyceps militaris spore germination rate

[0119] The specific steps are as follows:

[0120] 1) The transformant strain is activated, and after 5 days, the activated bacteria are obtained;

[0121] 2) The activated cells were inoculated into SDB liquid medium containing 40 g / L glucose, 10 g / L peptone, and 10 g / L yeast extract powder, and fermented in a shaking incubator at 25°C and 150 rpm for 6 days. The cells were then filtered through a non-woven fabric to remove the cells and obtain a spore solution.

[0122] 2) Add SDB liquid medium to the spore solution to make the volume 5mL, dilute the spore concentration to 105 / mL, and ferment in a shaking incubator at 25℃ and 150rpm for 4h, 8h, and 12h. Calculate the spore germination rate. The specific results are as follows: Figure 7 As shown, it can be seen that the spore germination rate of the transformant strain is higher.

[0123] Example 6

[0124] Determination of Cordyceps militaris mycelium diameter

[0125] The obtained spore solution was diluted to 10 5 / mL, take 100uL and spot it in the center of the PDA plate, Cm01 was the control group; the mycelial diameters were compared after 21 days.

[0126] The test results are as follows Figure 8 , it can be seen that the mycelial diameter of the transformant strain is larger than that of the wild-type CM01 strain.

[0127] Example 7

[0128] Determination of spore production of Cordyceps militaris

[0129] The specific steps are as follows:

[0130] 1) Measure the diameter of the plate cultured for 21 days in the previous example and calculate the mycelial surface area;

[0131] 2) Elute the plate with 0.05% Tween 20 and count the spores;

[0132] 3) Spore production = number of spores / hyphae surface area.

[0133] The test results are as follows Figure 9 , it can be seen that the spore production of the transformant strain is greater than that of the wild-type CM01 strain.

[0134] Example 8

[0135] Cordyceps militaris fruiting body germination

[0136] The obtained spore solution was diluted to 10 5 / mL, take 2mL and evenly spread it in the wheat culture medium cultivation bottle. Each bottle of wheat culture medium consists of 25g wheat, 2.5g silkworm pupa powder, and 2.5g soybean meal powder. CM01 is the control group; the growth status of the fruiting body is observed after 50 days.

[0137] The test results are as follows Figure 10 , it can be seen that the fruiting body growth of the transformant strain is greater than that of the wild-type CM01 strain.

[0138] Example 9

[0139] Fruiting body weight loss rate detection

[0140] Prepare a calibrated weighing device and, in a suitable environment with constant temperature and humidity, no strong ventilation or direct sunlight, clean the excess moisture and impurities on the surface of the CM01 and transformed fruiting bodies, then measure and record the initial mass. Store the fruiting bodies in a refrigerated storage at 4°C, and measure the mass again under the same conditions every three days. Finally, calculate the weight loss rate according to the formula "weight loss rate (%) = [(initial mass - mass after storage) / initial mass] × 100%". The calculation result is as follows: Figure 11 It can be seen that the fruiting body weight loss rate of the transformant strain is lower than that of the wild-type CM01 strain.

[0141] It can be seen from the above examples that:

[0142] 1. The Escherichia coli and Agrobacterium tumefaciens recombinant vectors carrying sweet protein genes (Miraculin or Thaumatin) obtained through homologous recombination technology provide core genetic tools for subsequent gene transformation and trait improvement of Cordyceps militaris.

[0143] 2. Through a series of rigorous and innovative genetic transformation techniques, we successfully obtained Cordyceps militaris transformants capable of expressing thaumatin.

[0144] 3. Rigorous experimental measurements and data analysis revealed that the total content of 20 essential amino acids in transformants incorporating the sweet protein gene (miraculin or thaumatin) was significantly higher than that in wild-type CM01 fruiting bodies. This indicates that the genetic transformation technology and the introduction of the sweet protein gene (miraculin or thaumatin) described herein have a positive impact on amino acid metabolism in the transformants, resulting in higher total amino acid accumulation.

[0145] 4. Transformants expressing the sweet protein gene (miraculin or thaumatin) demonstrated superiority in various basic morphological evaluations. Compared to wild-type CM01, transformants exhibited a higher spore germination rate, faster mycelial growth, and higher conidia production. Fruiting bodies of the transformants exhibited superior growth, with fuller morphology and greater numbers. Post-harvest, the transformants exhibited a superior weight loss rate, reflecting their improved ability to maintain quality during storage.

[0146] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A sweet protein, characterized in that Its amino acid sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. An isolated nucleic acid molecule, characterized in that It encodes the sweet protein as claimed in claim 1.

3. The nucleic acid molecule according to claim 3, characterized in that Its nucleotide sequence is shown in SEQ ID NO.3 or SEQ ID NO.

4.

4. A carrier, characterized in that It contains the nucleic acid molecule according to claim 2.

5. A recombinant cell, characterized in that It contains the nucleic acid molecule according to claim 2 or 3, or the vector according to claim 4.

6. Use of the sweet protein according to claim 1, the nucleic acid molecule according to claim 2 or 3, the vector according to claim 4, or the recombinant cell according to claim 5 in improving the sweetness of Cordyceps militaris fruiting bodies.

7. A method for improving the sweetness of Cordyceps militaris fruiting body, characterized in that: It includes: The nucleic acid molecule according to claim 2 or 3, or the vector according to claim 4, is introduced into a Cordyceps militaris strain to express the sweet protein according to claim 1.

8. The method according to claim 7, characterized in that The Cordyceps militaris strain is the CM01 strain.

9. A Cordyceps militaris strain, characterized in that: It contains the nucleic acid molecule according to claim 2 or 3, or the vector according to claim 4.

10. Use of the Cordyceps militaris strain according to claim 9 in improving the sweetness of Cordyceps militaris fruiting bodies.

Images