Flammulina velutipes fatty acid synthase gene FfFAS and application thereof

Abstract

The invention discloses a flammulina velutipes fatty acid synthase gene FfFAS and an encoded protein thereof, and provides application of the flammulina velutipes fatty acid synthase gene FfFAS, the FfFAS gene is overexpressed in flammulina velutipes, and the growth speed of flammulina velutipes hyphae can be slowed down, hypha tip branches can be increased, and cell elongation can be blocked. The FfFAS gene disclosed by the invention can be used for remarkably inhibiting the cell elongation of the flammulina velutipes under the irradiation of blue light, and can be applied to controlling the elongation of the stipe of the flammulina velutipes and keeping the uniformity in production.

Description

Technical Field

[0001] The present invention belongs to the field of edible mushroom genetic engineering, and specifically relates to the Flammulina filiformis fatty acid synthase gene FfFAS and its application. Background Art

[0002] Flammulina filiformis is the first edible mushroom variety to achieve industrialized production, and it is also the industrialized variety with the largest daily output and annual output over the years. The differentiation of the Flammulina filiformis fruiting body requires light stimulation, and the elongation of the stipe requires light regulation. As early as 1990, there were reports in China on using light to regulate the fruiting of Flammulina filiformis. After a large number of practices, it was proved that the light quality that plays an important role is blue light. Practical experience shows that blue light has a significant effect on the development and morphogenesis of the Flammulina filiformis fruiting body, which is mainly manifested in the following: under dark conditions, the stipes of Flammulina filiformis growing out of the cultivation bottle mouth are uneven in height. If blue light irradiation is given at a certain development stage (generally 9 - 25 days after scratching the bacteria), the mushrooms growing faster and in the upper layer receiving the blue light will have their growth inhibited, the stipes will become shorter, while the mushrooms growing slower and in the lower layer shielded from the blue light will grow normally. This makes the neatness of the whole bunch of Flammulina filiformis better and the yield increases. Therefore, regulating the stipe length of Flammulina filiformis by blue light in production is an important means to ensure the commercial traits of Flammulina filiformis and is a key technology commonly used. However, the current blue light irradiation can only give regulation from the surface and has not been deeply studied at the gene level.

[0003] Therefore, it is necessary to conduct in - depth research on the mechanism of blue - light regulation of Flammulina filiformis stipe elongation. Summary of the Invention

[0004] One object of the present invention is to provide a Flammulina filiformis fatty acid synthase gene FfFAS, and its sequence is as shown in SEQ NO.1.

[0005] The Flammulina filiformis fatty acid synthase FfFAS gene is cloned from the Flammulina filiformis genome by the RACE technique;

[0006] The present invention also provides a Flammulina filiformis fatty acid synthase gene FfFAS protein, which is the encoded protein of the above - mentioned Flammulina filiformis fatty acid synthase FfFAS gene, and its sequence is as shown in SEQ NO.2.

[0007] Another object of the present invention is to provide the application of the above - mentioned Flammulina filiformis fatty acid synthase gene FfFAS, specifically to over - express it in Flammulina filiformis; to regulate the elongation of hyphae and stipes under blue - light conditions;

[0008] More specifically, the FfFAS gene is recombined into the pCAMBIA1300 vector by the double - digestion method, and the FfFAS gene is transformed into the Flammulina filiformis gene to over - express the FfFAS gene in Flammulina filiformis.

[0009] The mycelial growth rate of Flammulina filiformis strains overexpressing the FfFAS gene slows down. Under blue light conditions, the number of branches at the mycelial tip increases, the distance between cell septa at the mycelial tip becomes shorter, and cell elongation is inhibited.

[0010] The nucleotide sequence of the FfFAS gene is shown in SEQ ID NO.1, with a sequence length of 1158bp, and the encoded amino acid sequence is shown in SEQ ID NO.2, with a length of 385 amino acids.

[0011] Overexpression of the Flammulina filiformis fatty acid synthase gene FfFAS gene in Flammulina filiformis, the specific method comprises the following steps:

[0012] (1) Obtaining the full-length sequence of the FfFAS gene containing restriction sites: Using the Flammulina filiformis FfFAS gene sequence obtained by RACE technology as a template, and using the primer pair of SEQ NO.5 and SEQ NO.6 to amplify the FfFAS gene sequence;

[0013] Forward primer Ff-F: CGC GGATCC ATGATTCCGGTTATCTGCCA (SEQ NO.5);

[0014] Reverse primer Ff-R: CCG GAATTC CTAGTAATATGTACGGACGT (SEQ NO.6);

[0015] The underlined sequences respectively represent the restriction sites BamH I and EcoR I;

[0016] (2) Construction of the target recombinant vector pCAMBIA1300-FfFAS

[0017] Recombine the FfFAS gene onto the pCAMBIA1300 vector using double digestion technology;

[0018] Extract the plasmid of the bacterial liquid with correct sequencing in step (1), use the restriction endonucleases BamH I and EcoR I to digest the FfFAS plasmid and the pCAMBIA1300 vector respectively, and ligate the digested fragments with T4 ligase to form the target vector pCAMBIA1300-FfFAS.

[0019] (3) Preparation of Agrobacterium infection solution

[0020] ① After the target vector pCAMBIA1300-FfCry was propagated in competent Escherichia coli cells, it was transferred to Agrobacterium tumefaciens EHA105. The Agrobacterium tumefaciens carrying the target vector was inoculated into 1 mL of LB (containing 50 mg / L rifampicin and 100 mg / L kanamycin), and cultured at 28 °C and 200 r / min until OD600 = 0.5 - 0.6.

[0021] The formula of LB medium: Tryptone 10 g / L, Yeast extract 5 g / L, NaCl 10 g / L.

[0022] ② Take 200 - 500 μL of the above Agrobacterium tumefaciens bacterial solution and inoculate it into a new 5 mL of LB (containing 50 mg / L rifampicin and 100 mg / L kanamycin), and culture at 28 °C and 200 r / min until OD600 = 0.5 - 0.6;

[0023] ③ Take 2 - 3 mL of the bacterial solution in step ②, centrifuge at 3000 rpm for 1 min, discard the supernatant, resuspend the precipitate in 5 mL of induction medium added with AS, and culture at 28 °C and 200 r / min until OD600 = 0.5 - 0.6. The prepared Agrobacterium tumefaciens infection solution should be used immediately for transformation.

[0024] The formula of the induction medium is as follows: 1 mL of K-buffer; 2 mL of M-N solution; 0.1 mL of 1% CaCl2; 1 mL of 0.01% FeSO4; 0.25 mL of 20% NH4NO3; 0.5 mL of Spore elements; 1 mL of 50% glycerol; 4 mL of 1 mol / L pH 5.3 MES (morpholineethanesulfonic acid); 0.5 mL of 2 mol / L glucose; made up to 100 mL with sterile ddH2O.

[0025] The induction medium added with AS is the induction medium added with acetosyringone (AS), and the addition concentration of AS is 200 μmol / L.

[0026] (4) Agrobacterium tumefaciens infects the millet - Flammulina velutipes mycelium substrate

[0027] ① Preparation of the Flammulina velutipes receptor strain:

[0028] Wash the millet clean, soak it in distilled water for 20 minutes until the millet is slightly soft, spread it out on a clean gauze, and absorb the moisture; weigh 30 g, put it into a 250 mL Erlenmeyer flask, and sterilize it by high temperature and high pressure (120 °C, 60 minutes);

[0029] The mycelium of Flammulina velutipes Dan3 cultured on PDA medium for 7 days, together with the medium (50 mm × 50 mm), was picked into a homogenizer, PDB medium was added, and it was intermittently broken for 30 s. 8 - 12 ml of the above liquid mycelium was inoculated into the millet medium, and cultured at 25 °C for 7 - 10 days. During this period, it was shaken three times a day (10 s each time in the morning, noon, and evening), until the mycelium of Flammulina velutipes grew on the surface of the millet grains, and the millet grain-Flammulina velutipes mycelium complex was obtained.

[0030] ② Agrobacterium tumefaciens infection of the millet grain-Flammulina velutipes mycelium matrix

[0031] The cultured millet grain-Flammulina velutipes mycelium matrix was added to a small glass test tube, induction medium was added, ultrasonic treatment was carried out for 1 min (frequency 40 KHz, power 160 W), and it was left standing for 10 min, then the supernatant was aspirated off;

[0032] The prepared Agrobacterium tumefaciens infection solution was added, ultrasonic treatment was carried out for 10 s (frequency 40 KHz, power 160 W), and it was left standing for infection for 20 min, then the excess bacterial solution was aspirated off, and it was left standing and cultured at 25 °C for 48 - 72 hours. During this period, it was shaken well 2 times a day;

[0033] The single millet grains were transferred to the PDA primary screening medium (hygromycin Hyg 8 mg / L and cefotaxime sodium cef 400 mg / L were added to the PDA medium), and cultured at 25 °C for 10 days;

[0034] The mycelium growing around the millet grains was inoculated onto the PDA secondary screening medium (Hyg 12 mg / L and cef 400 mg / L were added to the PDA medium). An uninfected mycelium block was set in the middle of the plate as a control, and it was cultured at 25 °C;

[0035] The putative transformants that could grow mycelium on the secondary screening medium were transferred to the PDB medium, and cultured in the dark with shaking in a flask at 23 °C - 25 °C. After 3 d - 4 d, the mycelium was collected; The genomic DNA of the above mycelium was extracted by the CTAB method, and the concentration and purity of the total genomic DNA were detected by agarose gel electrophoresis, and the concentration of the sample DNA was adjusted to be the same; PCR amplification of the marker gene hygromycin Hyg was carried out on the above extracted DNA;

[0036] The transformants with correct sequencing results were regarded as positive transformants;

[0037] The positive transformants were transferred to a new PDA plate. A cover glass was obliquely inserted 1 cm away from the inoculation block. The plate was placed in a blue light incubator and cultured at 25 °C. After the mycelium climbed onto the cover glass, the cover glass was taken out under sterile conditions and the growth of the mycelium tip was observed under a microscope (20×10) ( Figure 5 ), and the cell wall staining was observed under a fluorescence microscope, and the cell length was calculated according to the cell wall spacing ( Figure 6 ).

[0038] Collect the mycelium samples of positive transformants after blue light irradiation. For the collected samples, total RNA extraction and the synthesis of the first cDNA strand are carried out. The specific method is shown in step (1). Analyze the expression of the FfFAS gene in the transformants by the Real-time PCR method.

[0039] The results show that in the transformants with overexpression of the FfFAS gene, the branches at the hyphal tips increase and the cell elongation is blocked, indicating that the FfFAS gene plays a role in regulating cell elongation and controlling hyphal tip branching.

[0040] The present invention also provides a recombinant vector pCAMBIA1300-FfFAS containing the Flammulina filiformis fatty acid synthase gene FfFAS;

[0041] The present invention also provides a Flammulina filiformis transformant containing the recombinant Flammulina filiformis fatty acid synthase gene FfFAS.

[0042] The FfFAS gene of the present invention can regulate the cell elongation of Flammulina filiformis and play an important role in regulating hyphal elongation and stipe elongation in production.

[0043] The Flammulina filiformis fatty acid synthase protein FfFAS gene of the present invention can play an important role in regulating hyphal elongation in production. Specifically: Under dark conditions, the stipes of Flammulina filiformis growing out of the cultivation bottle mouth are uneven in height. If blue light irradiation is given at a certain development stage (generally 9 - 25 days after raking the bacteria), the Flammulina filiformis mushrooms growing faster and located in the upper layer receive blue light and their growth is inhibited, and the stipes become shorter, while the mushrooms growing slower and located in the lower layer are shielded from blue light and grow normally, thus making the neatness of the whole bunch of Flammulina filiformis better and the yield increased. And FfFAS is proved in the present invention to inhibit cell elongation after blue light irradiation and play an important role in regulating hyphal elongation (stipe elongation). In addition, the Flammulina filiformis FfFAS gene provided by the present invention can also be used for genetic improvement in Flammulina filiformis breeding.

[0044] For the Flammulina filiformis FfFAS gene provided by the present invention, a Flammulina filiformis strain with overexpression of the FfFAS gene is obtained by using the mature Agrobacterium-mediated method with millet grains as the culture medium in this laboratory. Under the condition of blue light irradiation, the overexpression transformants have more branches at the hyphal tips, shorter cell septum spacing, and blocked cell elongation. The present invention provides a candidate gene for regulating the hyphal elongation of Flammulina filiformis and has application prospects in regulating the hyphal elongation of Flammulina filiformis. Description of the Drawings

[0045] Figure 1 RACE amplification electrophoresis diagram

[0046] M. 2000bp Marker; 1 and 2 represent the amplified bands using 5`RACE cDNA as template; 3 and 4 represent the amplified bands using 3`RACE cDNA as template

[0047] Figure 2 SDS-PAGE electrophoresis pattern of the heterologous expression product of FfFAS

[0048] M. DNA molecular weight standard; 1 and 2 represent non-induced empty pET-32a; 3 and 4 represent non-induced recombinant protein pET-32a-FfFAS; 5 and 6 represent the supernatant of non-induced empty pET-32a induced by 0.3M IPTG; 7 and 8 represent the precipitate of non-induced empty pET-32a induced by 0.3M IPTG; 9 and 10 represent the supernatant of recombinant protein pET-32a-FfFAS induced by 0.3M IPTG; 11 and 12 represent the precipitate of recombinant protein pET-32a-FfFAS induced by 0.3M IPTG

[0049] Figure 3 Expression level of FfFAS gene in overexpression transformants

[0050] Figure 4 The mycelial growth rate of Flammulina velutipes overexpressing FfFAS gene slows down

[0051] Figure 5 The mycelial tip branches of Flammulina velutipes overexpressing FfFAS gene increase under blue light conditions

[0052] A represents the negative control Dan3 under dark conditions, B represents the negative control Dan3 under blue light conditions, C represents the overexpression transformant OE39 under dark conditions, and D represents the overexpression transformant OE39 under blue light conditions.

[0053] Figure 6 The distance between cell septa at the mycelial tip of Flammulina velutipes overexpressing FfFAS gene becomes shorter under blue light conditions, and cell elongation is inhibited.

[0054] Among them Figure 3 、 4 In 5 and 6, Dan3 represents the negative control, and OE + number represents the overexpression transformant (different numbers represent different transformants) Detailed implementation methods

[0055] The following examples are further explanations of the present invention rather than limitations thereof.

[0056] The synthesis and sequencing of nucleotide sequences in the following examples were all completed by Sangon Biotech (Shanghai) Co., Ltd.

[0057] The reagents or materials used in the following examples are all commercially available products unless otherwise specified.

[0058] SEQ NO.1 FfFAS gene sequence

[0059] ATGATTCCGGTTATCTGCCAATGCGACGTATGCACCCAAGTCATCCCGGAGGATAA

[0060] ACCAAGAGTCCATTGCCTGACTTGCTCTAATTACGACCTCTGTGCGAATTGTGCTC

[0061] TCGGCGGTAAAGCTAACGGGGACCACAATCCGGCTCATCCAACCCAGATCTACTC

[0062] GATCAGTGGAGGTCGTCAAACCATCCCTTGTGTATCTGACGGTCTTTCGTTGACGT

[0063] ACGCCACGCCAGCCGCACCAAGTCCAGCTGTATCCTCGAGATCTCCTCCTCCACTC

[0064] CCCAATCGTAACTCCGTACCCCCTCCACTCCCTCCTAGGACATCCAGTGGTAGCAA

[0065] TGCTTCACGTCGCCCACTATCTACTAGCGGTCCTTCTCCTTCATACGATAGTTCCCC

[0066] TCCGCGTCCTCGTGCAGCGCCGCCTCCACCACCTGGACAGTGGGGCTTTTTGTTCG

[0067] ATTACATGGACGATAGCGGTGGTCGCGACGTCACCCCACTCGGTGAAATGGTCTTC

[0068] GAAGCCATCTTCTCCTATCTGGACACGTCCCGCTCTGGATACCTCGCCCCAGAAGC

[0069] CTACTCCCGCTTTTTGGATGATCAGGGATACCAGGGCCAGGAGAACGTTTGGAGA

[0070] AATAATCTGAAGCCAGACATGGTGTACGGACAGTCGAAAGAGTCTGTCGCTGACA

[0071] AGTCTCTGAAGAATGCGTACGACCTTTTCTCGATTGAGCATATCCTCAAGGATAGA

[0072] CCCCGCGCGCCAGGGCCCCCAAATCCCTCTATATCGTCGCAGATGTTCTCCTTGTTT

[0073] GGTGTCACCCCGACGCCTCTAACAGGAAGCGTCGCAGGGAGCAAGATGCCTCTGC

[0074] TCACGCTCAAAGGGTTCATGGACATAACGGCGGTCGAATTGTTAGCGGACCCATCT

[0075] ACGGGGTGGGGAAATCTATCTCGCGTACTGAAGAAATACCAACTCCCTGCGTTGA

[0076] GGGGATACTCAGACCTTCCGAGGAGTGTATTGCCTGAGTATCCGGACGAGAGGAC

[0077] GGTTGACAGAGTAGCGAGGTTGACGGCGTTCCAGCAGAGGAAGGATCAAGAAAT

[0078] GTTGGCGGCGGCGATGGTGCAGGCTGAACTGCAACGTCAAGGGCAACAGGCTGCA

[0079] GTTGATTTAGTATCTGACTATAGATACGTCCGTACATATTACTAG

[0080] The protein expressed by SEQ NO.2 FfFAS gene

[0081] MIPVICQCDVCTQVIPEDKPRVHCLTCSNYDLCANCALGGKANGDHNPAHPTQIYSISG

[0082] GRQTIPCVSDGLSLTYATPAAPSPAVSSRSPPPLPNRNSVPPPLPPRTSSGSNASRRPLSTS

[0083] GPSPSYDSSPPRPRAAPPPPPGQWGFLFDYMDDSGGRDVTPLGEMVFEAIFSYLDTSRS

[0084] GYLAPEAYSRFLDDQGYQGQENVWRNNLKPDMVYGQSKESVADKSLKNAYDLFSIEH

[0085] ILKDRPRAPGPPNPSISSQMFSLFGVTPTPLTGSVAGSKMPLLTLKGFMDITAVELLADPS

[0086] TGWGNLSRVLKKYQLPALRGYSDLPRSVLPEYPDERTVDRVARLTAFQQRKDQEMLA

[0087] AAMVQAELQRQGQQAAVDLVSDYRYVRTYY

[0088] Example 1: RACE cloning of the fatty acid synthase FfFAS gene from Flammulina filiformis

[0089] (1) The FfFAS gene is an interacting protein gene of the FfCry-DASH gene in the authorized patent ZL201710198507.6. A partial sequence of the FfFAS gene was obtained by yeast two-hybrid technology. Based on this partial sequence,

[0090] 3' RACE primers and 5' RACE primers for the FfFAS gene were designed:

[0091] 3' RACE primer: ATGATTCCGGTTATCTGCCAATGCGACG (SEQ NO.3)

[0092] 5' RACE primer: CATCCAAAAAGCGGGAGTAGGCTTCTGG (SEQ NO.4)

[0093] (2) Total RNA extraction from Flammulina filiformis mycelium and synthesis of the first cDNA strand: Using a plant RNA extraction kit (Takara Codo no. 9769), total RNA of Flammulina filiformis (strain Dan3, from the preservation center of the Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences) mycelium was extracted according to the kit instructions. The RNA with qualified quality detection was used with PrimeScript TMThe first strand of cDNA was synthesized using the RT reagent Kit with gDNA Eraser (Takara Codo no. RR047) and stored at -20°C for later use.

[0094] (3) Amplify the FfFAS gene with reference to the RACE kit instructions:

[0095] ① Prepare the RNA denaturation and annealing reaction solution as shown in Table 1:

[0096] Table 1 Preparation of RNA denaturation and annealing reaction solution

[0097]

[0098]

[0099] ② Prepare the reverse transcription reaction solution according to Table 2 below:

[0100] Table 2 Preparation of reverse transcription reaction solution

[0101]

[0102] ③ Add the reverse transcription reaction solution to the annealed reagent (Table 3) to obtain 3`RACE cDNA and 5`RACE cDNA respectively.

[0103] Table 3 Reaction preparation of 3`RACE cDNA and 5`RACE cDNA

[0104]

[0105] ④ Add 3`RACE cDNA and 5`RACE cDNA to the RACE amplification reaction system (Table 4):

[0106] Table 4 Preparation of RACE amplification reaction solution

[0107]

[0108] After amplification, use 1% agarose electrophoresis gel, voltage 120V, for 30 min to test the RACE amplification products ( Figure 1 ), cut the gel and recover, sequence, and splice into the complete FAS sequence.

[0109] Example 2: Prokaryotic expression analysis of the FfFAS gene in Flammulina filiformis

[0110] (1) Obtaining the FfFAS gene sequence containing restriction enzyme sites:

[0111] ①Synthesis of primers for Flammulina filiformis FfFAS gene: Based on the full-length sequence information of FfFAS obtained by the RACE technique in Example 1, restriction enzyme primers were designed:

[0112] Forward primer Ff-F: CGC GGATCC ATGATTCCGGTTATCTGCCA (SEQ NO.5);

[0113] Reverse primer Ff-R: CCG GAATTC CTAGTAATATGTACGGACGT (SEQ NO.6);

[0114] The underlined sequences represent the restriction enzyme sites BamH I and EcoR I respectively.

[0115] ②Amplification and detection of FfFAS gene containing restriction enzyme sites: Using the FfFAS sequence obtained by the RACE technique as a template, and using primers SEQ NO.5 and SEQ NO.6, the gene sequence was amplified using HSDNA Polymerase (Takara high-fidelity enzyme, and the PCR amplification method was according to the kit instructions). After detecting the target band by electrophoresis, the PCR product was recovered.

[0116] (2) Double digestion of prokaryotic expression vector: The plasmid pET-32a was extracted using a plasmid DNA extraction kit and subjected to double digestion with BamH I and EcoR I. Digestion system: Buffer, 5ul; DNA, 2ul (total 1000ng); enzyme, 2.5ul; water, 40.5ul. Reaction program: 30°C, water bath for 1h. The digested product was run on a gel to verify the band and the gel was recovered.

[0117] (3) Obtaining of recombinant plasmid: The seamless cloning kit was used to ligate the digested and recovered products of the restriction enzyme and the PCR product. Reaction system: Digested product of plasmid pET-32a, 1.5ul; Digested product of target fragment FfFAS1, 1.5ul; 2xClonExpressmix, 5ul; dd water, 2ul. Reaction program: 50°C, 5min; 4°C, hold.

[0118] 10ul of the recombinant product was added to 100ul of DH5α and left standing on ice for 30min. Heat shock at 42°C in a water bath for 45s and cool on ice for 3min. Add 700ul of LB (without antibiotics), shake the bacteria at 37°C and 200rpm for 1h. Pipette 40ul and spread it on an LB (containing Kan) plate. Incubate overnight at 37°C in an inverted position. Pick 15 monoclonal colonies, add 1ml of LB (containing Kan), shake the bacteria at 37°C and 200rpm for 12h. Select the bacterial solution with completely correct sequencing bases for amplification and extract the plasmid to obtain the recombinant plasmid pET-32a-FfFAS.

[0119] Take out the competent cells E. coli BL21(DE3) from -50 °C and quickly place them on ice to melt for 5 min. Add 2.5 μl of the recombinant plasmid, gently mix well, and let it stand on ice for 30 min. Heat shock in a 42 °C water bath for 45 s, then quickly place it on ice and let it stand for 5 min. Add 500 μl of LB without antibiotics, incubate at 37 °C and 200 rpm for 1 h. Pipette 40 μl of the bacterial solution and spread it on the LB (containing Kan) solid medium. Invert the plate and incubate it overnight in a 37 °C incubator. Pick a single clone and add 1 ml of LB (containing Kan), incubate at 37 °C and 200 rpm for 12 h.

[0120] (4) Induction expression and purification of protein FfFAS:

[0121] Inoculate the bacterial solution obtained in the previous step into 50 ml of LB (containing Kan) at a ratio of 1-2%. Incubate at 37 °C and 200 rpm until the OD 600 value reaches 0.5-0.8. Add IPTG to a final concentration of 0.3 M, incubate at 16 °C and 120 rpm for 12 h. Centrifuge at 4 °C and 3000 g for 15 min, discard the filtrate, and collect the bacterial cells. Resuspend the bacterial cells with 0.1 mol / L PBS at pH 7.4 for washing, and centrifuge at 5000 g for 15 min. Resuspend with the protein lysis solution, perform ultrasonic lysis, and centrifuge at 5000 g for 15 min. Pipette the supernatant into a new centrifuge tube and resuspend the precipitate with the protein lysis solution. Take 20 μl of the collected protein supernatant and precipitate respectively, mix well with 5 μl of 5×loading buffer, boil for 10 min, and perform SDS-PAGE gel electrophoresis at 180 v for 45 min. After completing the electrophoresis, peel off the gel from between the two glass plates, put it into a box and pour in the Coomassie brilliant blue protein staining solution, boil in water for 2 min, and incubate on a shaker for 2 h. After staining, add clear water and wash overnight on a shaker until the bands are clear and take a picture ( Figure 2 ). Through SDS-PAGE gel electrophoresis analysis of the recombinant protein pET-32a-FfFAS, it was found that an obvious specific target band appeared at about 60 kDa (including the size of the tagged protein) in the precipitate, which was consistent with the theoretical analysis amount of the target protein expression frame.

[0122] Example 3: Overexpression of the Flammulina velutipes fatty acid synthase FfFAS gene in the Flammulina velutipes Dan3 strain (1) Obtaining the FfFAS gene sequence containing restriction sites:

[0123] ① Synthesis of primers for the Flammulina velutipes FfFAS gene: Based on the full-length sequence information of FfFAS obtained by the RACE technique, design restriction primers:

[0124] Forward primer Ff-F: CGC GGATCC ATGATTCCGGTTATCTGCCA (SEQ NO.5);

[0125] Reverse primer Ff-R: CCG GAATTC CTAGTAATATGTACGGACGT (SEQ NO.6);

[0126] The underlined sequences respectively represent the restriction enzyme sites BamH I and EcoR I.

[0127] ② Amplification and detection of the FfFAS gene containing restriction enzyme sites: Using the FfFAS sequence obtained by the RACE technology in Example 1 as a template, and using the primers of the FfFAS gene of SEQ NO.5 and SEQ NO.6, adopt HS DNA Polymerase (Takara high-fidelity enzyme, the PCR amplification method is according to the kit instructions) to amplify the gene sequence. After detecting the target band by electrophoresis, recover the PCR product and ligate it to the cloning vector ( Cloning Vector, Beijing TransGen Biotech Co., Ltd.). The ligation product is transformed into Escherichia coli competent cells (Trans1-T1 Phage Resistant Chemically Competent Cell, Beijing TransGen Biotech Co., Ltd.), and after detecting positive colonies, further sequencing verification is carried out.

[0128] ③ Result analysis: The sequencing results show that the cDNA sequence of the FfFAS gene is as shown in SEQ NO.1, with a sequence length of 1158 bp; the encoded amino acid sequence is as shown in SEQ NO.2, with a sequence length of 385 aa.

[0129] (2) Construction of the target recombinant vector pCAMBIA1300-FfFAS

[0130] Use double digestion technology to recombine the FfFAS gene onto the pCAMBIA1300 vector (Changsha Yingrun Biotechnology Co., Ltd.).

[0131] Extract the plasmid of the bacterial solution with correct sequencing in step (1), and use the restriction endonucleases BamH I and EcoR I (Thermo Fisher Scientific (China) Co., Ltd.) to digest the FfFAS plasmid and the pCAMBIA1300 vector respectively. The digested fragments are ligated by T4 ligase (T4 DNA Ligase, Beijing TransGen Biotech Co., Ltd.) to form the target vector pCAMBIA1300-FfFAS.

[0132] (3) Preparation of Agrobacterium infection solution

[0133] ① After the target vector pCAMBIA1300-FfCry was propagated in competent Escherichia coli cells, it was transferred to Agrobacterium tumefaciens EHA105 (Shanghai Weidi Biotechnology Co., Ltd.). The Agrobacterium tumefaciens carrying the target vector was inoculated into 1 mL of LB (containing 50 mg / L rifampicin and 100 mg / L kanamycin), and cultured at 28 °C and 200 r / min until OD600 = 0.5 - 0.6.

[0134] The formula of LB medium: Tryptone 10 g / L, Yeast extract 5 g / L, NaCl 10 g / L.

[0135] ② Take 200 - 500 μL of the above Agrobacterium tumefaciens liquid and inoculate it into a new 5 mL of LB (containing 50 mg / L rifampicin and 100 mg / L kanamycin), and culture at 28 °C and 200 r / min until OD600 = 0.5 - 0.6;

[0136] ③ Take 2 - 3 mL of the bacterial liquid in step 2, centrifuge at 3000 rpm for 1 min, discard the supernatant, resuspend the precipitate in 5 mL of induction medium added with AS, and culture at 28 °C and 200 r / min until OD600 = 0.5 - 0.6. The prepared Agrobacterium tumefaciens infection solution should be used immediately for transformation.

[0137] The formula of the induction medium is as follows: K-buffer 1 mL; M-N solution 2 mL; 1% CaCl2 0.1 mL; 0.01% FeSO4 1 mL; 20% NH4NO3 0.25 mL; Spore elements 0.5 mL; 50% glycerol 1 mL; 1 mol / L pH 5.3 MES (morpholineethanesulfonic acid) 4 mL; 2 mol / L glucose 0.5 mL; Sterile ddH2O is made up to 100 mL.

[0138] The composition of K-buffer: K2HPO4 20 g, KH2PO4 14.5 g, adjust the pH value to 7.0 with KOH, and make up to 100 mL with sterile ddH2O.

[0139] The composition of M-N solution: MgSO4·7H2O 3 g, NaCl 1.5 g, make up to 100 mL with sterile ddH2O.

[0140] The composition of the spore elements is as follows: 500 mg / L of ZnSO4·7H2O, 500 mg / L of CuSO4·5H2O, 500 mg / L of H3BO3, 500 mg / L of MnSO4·H2O, 500 mg / L of NaMoO4·2H2O. The five solutions are mixed evenly in equal volumes, filtered and sterilized, and stored at 4°C.

[0141] The induction medium added with AS is prepared by adding acetosyringone (AS) to the induction medium. The added concentration of AS is 200 μmol / L, and it is added freshly before use. AS cannot be repeatedly frozen and thawed, and AS is purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0142] (4) Agrobacterium tumefaciens infection of millet grain-Flammulina velutipes mycelium substrate

[0143] ① Preparation of the Flammulina velutipes receptor strain:

[0144] Wash ordinary commercially available millet clean, soak it in distilled water for 20 minutes until the millet becomes slightly soft, spread it out on a clean gauze, and absorb the moisture; weigh 30 g, put it into a 250 ml Erlenmeyer flask, and sterilize it by high temperature and high pressure (120°C, 60 minutes).

[0145] Pick the Flammulina velutipes Dan3 mycelium cultured on the PDA medium for 7 days, together with the medium (50 mm × 50 mm), into a homogenizer, add 100 ml of PDB medium (PDB powder is purchased from BD Company, USA), and intermittently break it for 30 s.

[0146] Take 8 - 12 ml of the above liquid mycelium and inoculate it into the millet grain medium, culture it at 25°C for 7 - 10 days, shake it three times a day during this period (10 s each time in the morning, noon, and evening), and culture until Flammulina velutipes mycelium grows on the surface of the grains to obtain the millet grain-Flammulina velutipes mycelium complex.

[0147] ② Agrobacterium tumefaciens infection of millet grain-Flammulina velutipes mycelium substrate and its verification

[0148] Take about 1 g of the cultured millet and add it to small glass test tubes (10 tubes in total), add 1 - 1.5 ml of the induction medium, ultrasonicate it for 1 min (frequency 40 KHz, power 160 W) with a dual-frequency ultrasonic cleaner from Shanghai Kedao Ultrasonic Instrument Co., Ltd., let it stand for 10 min, and suck off the supernatant;

[0149] Add 1.5 ml of the prepared Agrobacterium tumefaciens infection solution, ultrasonicate it for 10 s (frequency 40 KHz, power 160 W) with a dual-frequency ultrasonic cleaner from Shanghai Kedao Ultrasonic Instrument Co., Ltd., let it stand for infection for 20 min, suck off the excess bacterial liquid, and let it stand and culture at 25°C for 48 - 72 hours, shake it twice a day during this period;

[0150] Transfer single millet grains into the PDA primary screening medium (PDA medium supplemented with 8 mg / L hygromycin Hyg and 400 mg / L cefotaxime sodium cef) (hygromycin and cefotaxime sodium: Sangon Biotech (Shanghai) Co., Ltd.). Inoculate 25 grains on each plate and culture at 25 °C for 10 days;

[0151] Inoculate the mycelium growing around the millet grains onto the PDA secondary screening medium (PDA medium supplemented with 12 mg / L Hyg and 400 mg / L cef). Set an uninfected mycelium block in the middle of the plate as a control and culture at 25 °C;

[0152] Transfer the putative transformants that can grow mycelium on the secondary screening medium into PDB medium and culture them in the dark on a shaker at 23 °C - 25 °C. After 3 - 4 days, collect the mycelium; Extract the genomic DNA of the above mycelium by the CTAB method, and detect the concentration and purity of the total genomic DNA by agarose gel electrophoresis, and adjust the concentration of the sample DNA to be consistent; Perform PCR amplification of the hygromycin Hyg marker gene on the DNA extracted above;

[0153] The PCR amplification system is as follows: The total volume is 20 μL, including: 2 μL of 10×PCR buffer, 2 μL of 25 mmol / L MgCl2, 0.4 μL of 10 mmol / L dNTP, 0.2 μL of 5 U / μL Taq DNA polymerase, 1 μL each of the forward and reverse primers of 10 μmol / L Hyg, 2 μL of the template DNA with a concentration of 20 ng - 30 ng / μL, and 11.4 μL of ddH2O;

[0154] PCR reaction conditions: 94 °C for 5 min; 94 °C for 30 s, 56 °C for 40 s, 72 °C for 30 s, 30 cycles; 72 °C for 8 min. The hygromycin primers used are: Hyg-F: GATGTTGGCGACCTCGTATT; Hyg-R: TCGTTATGTTTATCGGCACTTT;

[0155] Send the PCR product to Sangon Biotech (Shanghai) Co., Ltd. for sequencing verification;

[0156] The transformants with correct sequencing results are regarded as positive transformants and named OE1, OE2, OE3...

[0157] Transfer the positive transformants onto a new PDA plate. Insert a cover glass obliquely 1 cm away from the inoculation block. Place the plate in a blue light incubator and culture at 25 °C. After the mycelium climbs onto the cover glass, take out the cover glass under sterile conditions and observe the growth of the mycelium tip under a microscope (20×10) ( Figure 5 ), observe the cell wall staining under a fluorescence microscope, and calculate the cell length based on the cell wall spacing (Figure 6 ). The growth rate results of the FfFAS overexpression transformants are shown in Figure 4 .

[0158] Collect the mycelial samples of the positive transformants after blue light irradiation, extract the total RNA of the collected samples and synthesize the first strand of cDNA. The specific method is shown in step (1). Analyze the expression of the FfFAS gene in the transformants by Real-time PCR (the results are shown in Figure 3 ).

[0159] Result analysis:

[0160] In the transformants overexpressing the FfFAS gene, the branches at the hyphal tips increase ( Figure 5 ), and the cell elongation is inhibited ( Figure 6 ). It shows that the FfFAS gene plays a role in regulating cell elongation and controlling hyphal tip branching, etc.

Claims

1. Flammulina velutipes fatty acid synthase gene FfFAS, characterized in that Its sequence is shown in SEQ NO.

1.

2. Flammulina velutipes fatty acid synthase gene FfFAS protein, characterized in that Its sequence is shown in SEQ NO.

2.

3. The use of the Flammulina velutipes fatty acid synthase gene FfFAS as claimed in claim 1, characterized in that The application is to over-express the FfFAS gene in Flammulina velutipes: the FfFAS gene is recombined into the pCAMBIA1300 vector by a double enzyme digestion method, and the FfFAS gene is transformed into the Flammulina velutipes gene, so that the FfFAS gene is over-expressed in Flammulina velutipes; The nucleotide sequence of the FfFAS gene is shown as SEQ ID NO.1, and the encoded amino acid sequence is shown as SEQ ID NO.

2.

4. The use of Flammulina velutipes fatty acid synthase gene FfFAS according to claim 3, wherein the Flammulina velutipes fatty acid synthase gene FfFAS is overexpressed in Flammulina velutipes, specifically comprising the following steps: (1) Obtaining the full-length sequence of the FfFAS gene including restriction sites: The FfFAS gene sequence of Flammulina velutipes obtained by RACE technology was used as a template and the primer pair of SEQ NO.5 and SEQ NO.6 was used to amplify the FfFAS gene sequence; Forward primer Ff-F: CGC GGATCC ATGATTCCGGTTATCTGCCA(SEQ NO.5); Reverse primer Ff-R: CCG GAATTC CTAGTAATATGTACGGACGT(SEQ NO.6); (2) Construction of the target recombinant vector pCAMBIA1300-FfFAS The FfFAS gene was recombined into the pCAMBIA1300 vector using double restriction enzyme digestion technology; (3) Preparation of Agrobacterium infection solution ① The target vector pCAMBIA1300-FfCry was transformed into competent E. coli cells and then transferred to EHA105 Agrobacterium; the Agrobacterium carrying the target vector was inoculated in LB and cultured at 28°C and 200r / min until OD600 = 0.5-0.6; ② Take the above Agrobacterium bacterial solution and inoculate it into LB containing 50 mg / L rifampicin and 100 mg / L kanamycin, and culture at 28°C and 200 r / min until OD600 = 0.5-0.6; ③ Take the bacterial solution in step ②, centrifuge at 3000rpm, discard the supernatant, resuspend the precipitate in the induction medium added with AS, and culture at 28℃, 200r / min until OD600=0.5-0.6; (4) Agrobacterium infection of millet-Flammulina velutipes mycelium matrix ① Preparation of Flammulina velutipes receptor strain: The millet is cleaned, put into a triangular bottle, and sterilized at high temperature and high pressure; the Flammulina velutipes hyphae cultured on the PDA medium for 7 days are picked up together with the medium into a homogenizer, and the PDB medium is added, and the millet is broken intermittently; 8-12 ml of the above liquid hyphae are inoculated into the millet grain medium, and cultured at 25° C. for 7-10 days until Flammulina velutipes hyphae grow on the surface of the rice grains, thereby obtaining a millet grain-Flammulina velutipes hyphae complex; ② Agrobacterium infection of millet grain-Flammulina velutipes mycelium complex Take the cultured millet grain-Flammulina velutipes mycelium complex and put it into a glass test tube, add the induction medium, use ultrasound for 1 minute, let it stand for 10 minutes, and suck off the supernatant; Add the prepared Agrobacterium infection solution, sonicate for 10 seconds, let it stand for infection for 20 minutes, remove the excess bacterial solution, and let it stand for 48-72 hours at 25°C; The millet-Flammulina velutipes mycelium complex was transferred into PDA primary screening medium containing 8 mg / L Hyg and 400 mg / L cef and cultured at 25°C for 10 days; The hyphae grown around the millet grains were inoculated into PDA rescreening medium containing 12 mg / L Hyg and 400 mg / L cef and cultured at 25°C for 10 days; The proposed transformants that can grow hyphae on the rescreened culture medium are inoculated into PDB culture medium, cultured in a light-proof shaking flask at 23°C-25°C, and the hyphae are collected after 3d-4d; the genomic DNA of the hyphae is extracted, and the total genomic DNA concentration and purity are detected by agarose gel electrophoresis, and the concentration of the sample DNA is adjusted to be consistent; the marker gene hygromycin Hyg is amplified by PCR on the extracted DNA; the transformants with correct sequencing results are regarded as positive transformants.

5. Use of the Flammulina velutipes fatty acid synthase protein FfFAS gene according to claim 3 or 4, wherein the Flammulina velutipes is Flammulina velutipes Dan3 strain.

6. The destination vector pCAMBIA1300-FfFAS prepared by using the Flammulina velutipes fatty acid synthase protein FfFAS gene as described in any one of claims 3 to 5.

7. A Flammulina velutipes transformant with overexpression of the FfFAS gene obtained by using the Flammulina velutipes fatty acid synthase protein FfFAS gene as described in any one of claims 3 to 5.

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