CN110042061B - High yield gibberellin GA3Gibberella fujikuroi mutant strain and application thereof - Google Patents

Abstract

The invention discloses a mutant strain of Gibberella fujikura with high production of gibberellin GA ₃ - rice bakanae GA-251 and its application; , deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, zip code 430072, preservation number: CCTCC NO: M 2019201, preservation date March 25, 2019. The strain was obtained by compound mutagenesis of protoplasts with lithium chloride and cobalt 60 radiation, and the gibberellin GA ₃ production of this strain was increased by 20%. The method has the advantages of high titer, easy extraction of the final product, etc., and overcomes the shortcomings of low fermentation titer and high cost of the original process.

Description

Gibberella fujikura mutant strain with high production of gibberellin GA3 and its application

(1) Technical field

The present invention relates to a high-yielding gibberellin GA ₃ mutant strain of Gibberella fujikura - rice bakanae pathogen GA-251 and its application.

(2) Background technology

Gibberellins (GAs) are plant hormones widely used in agriculture, forestry, horticulture, food brewing and many other fields. The preparation methods mainly include plant extraction method, chemical method and microbial fermentation method. However, the microbial fermentation method has the advantages of low production cost and low environmental pollution, and has broad industrial application prospects. Mycin was first discovered by Japanese phytopathologist Kenkichi Sawada in his study of rice bakanae disease, and was later confirmed by his colleague Eiichi Kurosawa, who published a paper in 1926 showing that the symptoms of the disease could be Using sterile fungal cultures to replicate, he found that the secretions also stimulated the growth of several seedlings other than rice. This landmark publication was followed by numerous reports on the properties of the secreted substance, and in 1935, chemist Teijiro Yabuta, a professor of agricultural chemistry at the University of Tokyo, obtained a purified sample with high biological activity, called gibberellin. Gibberellin can promote seed germination, plant growth and increase the yield of vegetables and fruits; it can be used as a plant growth regulator to promote crop growth and development; it has estrogen-like activity and can promote scalp blood circulation in hair products.

Gibberellin GA ₃ is a diterpenoid acid, molecular formula C ₁₉ H ₂₂ O ₆ , molecular weight 346.37, melting point 233-235°C, easily soluble in alcohol, acetone, ethyl acetate, sodium bicarbonate solution and pH 6.2 phosphate buffer, insoluble in water and ether.

GAs biosynthesis starts from GGDP via isopentenyl diphosphate (IPP), and after two cyclization steps from GGDP via Cuban pyrophosphate (CPP), kaurene is produced. Kaurenoic acid is produced by oxidation of kaurenol and kaurial at C-19, which is post-oxidatively contracted to form GA ₁₂ -aldehyde. GA ₁₂ -aldehyde is hydroxylated to GA ₁₄ -aldehyde, which is then oxidized at the C-7 position to form GA ₁₄ , which is converted to GA ₄ by oxidation, and GA ₄ is _desaturated to GA ₇ , which is then converted to GA ₃ by hydroxylation .

Since 1970, a high-yielding variety "4303" has been screened in my country. However, the strain has no spore-producing ability and is polykaryotic, making it very difficult to continue breeding. The current production titer shows a stagnant state, and some production strains are degraded. China's invention patent (publication number CN104892554A) describes the preparation method of gibberellin GA ₃ , but has not reported the high-yielding gibberellic acid Gibberella fujikura and its application, the traditional strain has been 1.8g～2g in output Wandering between, to be further improved.

(3) Contents of the invention

The purpose of the present invention is to provide a high-yielding gibberellin GA ₃ mutant of Gibberella fujikura - rice bakanae GA-251 and its application, the strain can be applied to industrial production, and has more High protein-producing capacity can provide profits for industrial production, and less impurities in the later stage of fermentation can reduce the extraction and separation work.

The technical scheme adopted in the present invention is:

A high-yielding gibberellin GA ₃ mutant of Gibberella fujikura - bakanae GA-251 (Fusarium fujikuroi GA-251), deposited in the China Center for Type Culture Collection, Address: Wuhan University, Wuhan, China, zip code 430072, Deposit number: CCTCC NO: M 2019201, deposit date March 25, 2019.

The obtaining process of the bacterial strain of the present invention is as follows:

1. Preparation of protoplasts. For the specific method, refer to Zhu Tingheng et al. Research on efficient transformation of exogenous genes from Gibberella sp. (Journal of Zhejiang University of Technology, 2013, 41(5), 482-485)

2. Gibberella fujikura GA-251 was obtained by cobalt 60-γ-ray (Co ⁶⁰ γ-ray), lithium chloride single-factor mutagenesis and compound mutagenesis.

The 18s rDNA sequence of the rice bakanae pathogen GA-251 is shown in SEQ ID NO.1.

The invention also relates to the application of the rice bakanae pathogen GA-251 in the preparation of gibberellins by microbial fermentation.

Specifically, the rice bakanae GA-251 is used for microbial fermentation to prepare gibberellin GA ₃ .

Specifically, the application is as follows: inoculating the rice bakanae GA-25 into a fermentation medium, fermenting and culturing it at 20-30° C. and 200-300 rpm for 12-48 hours, to obtain a fermentation broth containing gibberellin GA ₃ , the fermentation broth was separated and purified to obtain gibberellin GA ₃ .

The fermentation medium is composed as follows: corn starch 60-90 g/L, rice flour 70-100 g/L, soybean flour 3-7 g/L, peanut flour 3-7 g/L, K ₂ SO ₄ 0.3-0.7 g/L, KH ₂ PO ₄ 0.3～0.7g/L, solvent is water, pH is natural, sterilization at 121 degrees for 30min.

Preferably, the rice bakanae GA-25 is first inoculated into a slant medium, the activated strain is inoculated into a seed medium, and the seed liquid is obtained by seed culture, and then inoculated into a fermentation medium, and the slant medium is composed as follows: potato 150-200 g/L, sucrose 10-30 g/L, magnesium sulfate 0.1-0.3 g/L, calcium carbonate 0.1-0.3 g/L, agar 20 g/L, natural pH, sterilized at 115°C for 30 minutes; the seed medium The composition is as follows: corn starch 10-30g/L, sucrose 10-30g/L, peanut flour 10-30g/L, soybean flour 10-30g/L, potassium dihydrogen phosphate 0.5-1.5g/L, magnesium sulfate 0.5-1.5 g/L, natural pH, sterilized at 121°C for 30min.

The beneficial effects of the present invention are mainly reflected in:

1. The present invention provides a Gibberella fujikura mutant with high gibberellin production, which does not produce conidia in the growth process of the bacterial strain;

2. The present invention provides a fermentation method for gibberellin GA ₃ , which can produce gibberellin GA ₃ ; the strain of the present invention is inoculated into a liquid seed medium, and after the cells grow and mature, it is transferred to a fermentation medium Among them, the fermentation level has been greatly improved, and the yield of gibberellin has reached 21 times that of wild bacteria, leading the world. Gibberellin is secreted extracellularly and can be directly obtained from the fermentation broth, which is beneficial to solid-liquid separation and increases the extraction yield. At the same time, it improves the utilization rate of equipment and raw materials, greatly reduces the production cost, and has significant economic and social benefits;

3. The present invention can be implemented on existing pharmaceutical production equipment without additional investment.

(4) Description of drawings

Fig. 1 is the argrose electrophoresis picture of the PCR amplification of the 18S rDNA sequence of the strain.

Figure 2 shows the morphology of the mycelium under electron microscope observation.

Figure 3 shows the change of GA3 content in the fermentation broth.

Figure 4 shows the pH changes in the fermentation broth.

Figure 5 is the change in dry weight of GA-251 fermentation.

Figure 6 is a standard curve for GA3 high performance liquid chromatography (HPLC) detection.

Figure 7 shows the effects of different fermentation temperatures on the production of strain GA-251GA3

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but the protection scope of the present invention is not limited to this:

Example 1: Preparation of wild Gibberella fujikura protoplasts

(1) Preparation of the slant: Inoculate the wild Gibberella fujikura strain into the slant medium, cultivate at 28°C for 3-7 days, and transfer to the seed solution when the aerial mycelium is covered with the slant.

(2) Preparation of seed liquid:

Pick a small piece of bacteria in step (1) and inoculate it into a seed medium, and cultivate at 28° C. and 250 rpm for 48 hours to obtain a seed liquid. The seed culture medium is prepared by the following method: corn starch 20g/L, sucrose 20g/L, peanut flour 20g/L, soybean flour 20g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 1.0g/L, the solvent is Tap water, natural pH, sterilized at 121°C for 30min.

(3) Fermentation culture

A 250mL shake flask was filled with 40mL fermentation medium, and the seed liquid was inoculated at a concentration of 1-4% by volume during fermentation, and the fermentation was cultured at 28°C and 250rpm for 168h. Fermentation medium composition: corn starch 80g/L, rice flour 80g/L, soybean flour 5g/L, peanut flour 5g/L, K ₂ SO ₄ 0.5g/L, KH ₂ PO ₄ 0.5g/L, the solvent is tap water, Natural pH, sterilized at 121 degrees for 30 minutes.

(4) Protoplast preparation

A small colony was inoculated from the slant to YEPD liquid medium (0.3% yeast extract, 1% peptone, 2% glucose, and the solvent was water), and cultured at 28° C. and 180 rpm for 48 hours. Take 2ml of bacterial solution and add it to a sterile Eppendorf tube, and wash it once with sterile water and 1M MgSO4 successively. Dip a small amount of mycelium with a sterilized pipette tip into 1.5 mL of filter-sterilized wall-breaking buffer (1M MgSO4, total enzyme concentration of 15 mg/mL, where Dryslase: Yatalase=3:7), pipette evenly, and place it in The reaction was carried out in a water bath shaker at 30°C for 45 minutes (the yield of protoplasts was over 95%). Centrifuge at 5000rpm for 10 minutes, transfer 0.75 mL of the supernatant to another centrifuge tube, add an equal amount of sterile water, mix well, centrifuge at 6000rpm for 5 minutes, and discard the supernatant. The above cell pellets were fully suspended with 1 mL of 1 M sorbitol, and protoplasts were successfully prepared. After microscopic examination, it can be diluted with appropriate multiples and plated on MYG medium (maltose 0.5%, glucose 1%, yeast powder 0.5%, sucrose 5M, agar 2%, solvent is water) for protoplast recovery. Controls were suspended and diluted with sterile water and plated. The number of protoplasts contained in a unit volume was measured by counting with a hemocytometer. And the protoplast solution was diluted to 10 ⁶ -10 ⁷ /mL.

Example 2: Screening of high-yielding GA ₃ mutants

(1) Inoculate the wild-type Gibberella fujikura into the slant medium, cultivate at 28°C for 3 to 7 days, and inoculate a small colony from the slant to YEPD liquid medium (0.3% yeast extract, 1% peptone, 2% glucose) , the solvent is water), wash once with sterile water and salt buffer, add a small amount of mycelium to the wall-breaking buffer, place it in a water bath shaker at 30 °C for 45 minutes, centrifuge at 3000 rpm for 10 minutes, discard the supernatant and wash with sterile water Add buffer suspension after one time.

(2) Co ⁶⁰ mutagenesis method: The strain activated in step (1) was spread on MYG solid medium (maltose 0.5%, yeast powder 0.5%, glucose 1%, sucrose 5M, agar 2%), and cultured at 28°C for 3 -7 days to grow colonies, prepare protoplasts by step ( ¹ ), control the number of bacteria to be about 107/mL, and use different doses (0Gy, 200Gy, 400Gy, 600Gy, 800Gy and 1000Gy) Co ⁶⁰ pairs of protoplasts body for mutagenesis. The protoplasts mutagenized by Co ⁶⁰ were diluted with physiological saline to 10 ^-5 and then plated on MYG plate medium and cultured at 30°C for 5 days. Fermentation, the fermentation broth was collected and the yield was detected by HPLC until a high-yielding GA ₃ mutant was obtained. The number of mutations, mutation rate and lethality are shown in Table 1.

Table 1: Co ⁶⁰ Mutagenesis Method Mutagenesis Process

(3) LiCl-Co ⁶⁰ mutagenesis method: put 5 mL of the protoplast suspension prepared in step (1) into a sterile ep tube, and after step (2), use LiCl for treatment, treatment method: per 1 mL of bacterial cells The suspension was stirred with 1 M sorbitol buffer solution containing 1 mg/mL lithium chloride (LiCl) for 0.5 h, centrifuged at 6000 rpm for 5 min, the cells were collected and washed with sterile water for 3 times, and then resuspended and coated in MYG solid medium. Cultivated at 28°C, initially screened the colony of large bacteria and strains with dense aerial silk, picked a single colony, fermented, and collected the fermentation broth to detect the yield by HPLC until a high-yielding GA ₃ mutant was obtained. The number of mutations, mutation rate and lethality are shown in Table 2.

Table 2: UV-nitrosoguanidine composite mutagenesis process

The high-yielding strains obtained in each round of mutagenesis were re-used as initial strains and subjected to compound mutagenesis according to the above method. The mutant strain GA-251 with a GA ₃ yield of 2-4 g/L was finally screened and named as Fusarium fujikuroi GA-251, which was deposited in the China Type Culture Collection on March 25, 2019. The deposit number is CCTCCNO: M 2019201, and the deposit address is Wuhan University, Wuhan, China, zip code 430072.

The present invention includes but is not limited to the above three mutagenesis methods.

The preparation of MYG solid medium: maltose 0.5%, yeast powder 0.5%, glucose 1%, sucrose 5M, agar 2%, the solvent is tap water, the pH is natural, and sterilized at 121 degrees for 20 minutes.

Preparation of YEPD medium: 0.3% yeast extract, 1% peptone, 2% glucose, tap water as the solvent, natural pH, sterilization at 121 degrees for 20 minutes.

Example 3: 18S rDNA molecular identification

均质破碎仪破碎细胞，速度设定为6.0，时间设定为40s；12000rpm离心Lysing Matrix E Tube 10min，去除样品碎屑；将上清液转移至新的EP管中，加入250μL PPS试剂，手持离心管晃动10次以混匀，12000rpm离心5min；将上清转移至10mL干净离心管中，加入1mL Binding Matrix suspension，将离心管上下颠倒2min，静置3min，使DNA附着于 Binding Matrix上，并等待二氧化硅基质沉淀；小心移除500μL上清 (避免吸出沉淀)，将剩余的上清液与沉淀物重新混匀，吸取约600μL 混合液移入SPIN Filter中，12000rpm离心1min，弃滤液，再将剩余液体都转移至SPIN Filter，离心弃滤液；加500μLSEWS-M溶液，12000 rpm离心1min弃滤液，再将SPIN Filter离心2min，将SPIN Filter转移至新的catch tube，敞开室温干燥5min；加入50μL DES溶液，室温静置1min，并12000rpm离心1min，catch tube中的滤液即为所提取的菌种的基因组。(1) DNA extraction: centrifuge the mycelium, put a small amount into a 1.5mL EP tube, add 978μL sodiumphosphate buffer, and suspend the bacteria; transfer the bacterial suspension to a Lysing Matrix E Tube, add 112μL MTBuffer and mix; use MP

The cells were broken by a homogenizer, the speed was set to 6.0, and the time was set to 40 s; the Lysing Matrix E Tube was centrifuged at 12,000 rpm for 10 min to remove sample debris; the supernatant was transferred to a new EP tube, 250 μL of PPS reagent was added, and the hand held The centrifuge tube was shaken 10 times to mix, and centrifuged at 12,000 rpm for 5 minutes; the supernatant was transferred to a 10 mL clean centrifuge tube, 1 mL of Binding Matrix suspension was added, the centrifuge tube was inverted for 2 minutes, and allowed to stand for 3 minutes to allow the DNA to adhere to the Binding Matrix. Wait for the silica matrix to settle; carefully remove 500 μL of the supernatant (avoid aspiration of the precipitate), re-mix the remaining supernatant with the precipitate, pipette about 600 μL of the mixture into the SPIN Filter, centrifuge at 12,000 rpm for 1 min, discard the filtrate, and then Transfer the remaining liquid to the SPIN Filter, and centrifuge to discard the filtrate; add 500 μL SEWS-M solution, centrifuge at 12,000 rpm for 1 min to discard the filtrate, then centrifuge the SPIN Filter for 2 min, transfer the SPIN Filter to a new catch tube, and dry it at room temperature for 5 min; add 50 μL The DES solution was allowed to stand at room temperature for 1 min, and centrifuged at 12,000 rpm for 1 min. The filtrate in the catch tube was the genome of the extracted strain.

(2) PCR amplification and sequence analysis of fungal 18S rDNA: universal primers for fungal ribosomal ITS rDNA

The primers used are as follows:

ITS1 5’-TCCGTAGGTGAACCTGCGG-3’ ITS4 5’-TCCTCCGCTTATTGATATGC-3’

Clone PCR system: add 5 μL of 10×Taq Buffer (Mg+), add 1 μL of 10 mM dNTPs, 0.5 μL of Taq DNA Polymerase, 0.5 μL of ITS1 and ITS4 primers, 1.5 μL of template, and make up to 50 μL of deionized water.

The cloning PCR program was as follows: denaturation at 95°C for 45s, annealing at 55-60°C for 30s, and extension at 72°C for 45 min, a total of 35 cycles. A final extension at 72°C for 30s.

(3) Take 5 μL of PCR amplification products, conduct agarose gel electrophoresis experiment, and observe and analyze with a gel imager. If the band is clear and the size is correct (about 500bp), the PCR product is recovered by gel. Take 5 μL of each recovered product for verification by agarose gel electrophoresis. If the band is clear and the size is correct, the recovered sample can be used for sequencing. Sequencing was completed by Hangzhou Qingke Zixi Biotechnology Co., Ltd., and the sequencing primers were the same as PCR primers. Sequence alignments were performed using the NCBI blast (http://blast.ncbi.nlm.nih.gov/) database.

The sequencing was completed by Hangzhou Qingke Zixi Biotechnology Co., Ltd. The results are as follows (SEQ ID NO.1):

ggtccggccgggcctttccctctgtggaaccccatgcccttcactgggtgtggcggggaa 60

acaggacttttactgtgaaaaaattagagtgctccaggcaggcctatgctcgaatacatt 120

agcatggaataatagaataggacgtgtggttctattttgttggtttctaggaccgccgta 180

atgattaatagggacagtcgggggcatcagtattcaattgtcagaggtgaaattcttgga 240

tttattgaagactaactactgcgaaagcatttgccaaggatgttttcattaatcaggaac 300

gaaagttaggggatcgaagacgatcagataccgtcgtagtcttaaccataaactatgccg 360

actagggatcggacggtgttattttttgacccgttcggcaccttacgagaaatcaaagtg 420

cttgggctccagggggagtatggtcgcaaggctgaaacttaaagaaattgacggaagggc 480

accaccaggggtggagcctgcggcttaatt510

Similarity analysis between the obtained sequence and the data stored in genBank found that the microorganism identified in this experiment has the highest homology with Fusarium fujikuroi (homology, 99%/510bp, based on 18S rDNA), according to the principle of molecular genetic identification of microorganisms, based on 18S The homology of the rDNA sequence was higher than 99%, and the identified bacteria basically belonged to the control bacteria.

Example 4: Physiological and biochemical identification

The biolog automatic microbial identification system was used to investigate the metabolism of the strains to 95 carbon sources: the strains were inoculated into the PDA plate medium, cultured at a constant temperature of 28 °C for 5 d, the bacteria on the plate were washed with a sterile cotton swab, and the inoculum (FF -IF) mixed to prepare a bacterial suspension, which was adjusted to 75% T/FF with a turbidimeter (reference value: 75% ± 3%). The bacterial suspension was added to each well of the Biolog FF microwell identification plate with an 8-well electric pipette, 100 μl per well. The microwell identification plate was placed in a 28°C incubator, and the results were read on a Biolog reader after culturing for 24h, 48h, 72h, 96h, 168h and 240h, respectively. Considering the data of each time point read by the Biolog reader, the 24h identification result is given.

Physiological and biochemical identification of wild strains was carried out according to the above method, and the metabolism of the strains to 95 carbon sources was investigated by using the Biolog automatic microbial identification system. The 24-hour identification results were given comprehensively considering the data at each time point read by the Biolog reader, as shown in the table. 3.

Table 3: The ability of strain GA-251 to utilize 95 carbon sources on Biolog FF plates

Notes:+,positive;-,negative;B,borderline

Example 5: Electron microscope observation of high-yielding GA3 strain

(1) Bacterial culture: use the inoculation loop to pick the slanted surface preservation strain solution, streak the PDA plate, cultivate at 28°C for 5 days, pick the grown strain block and insert it into the seed medium, cultivate at 250rpm, 28°C for 2d, wait for deal with.

(2) Bacteria treatment: (a) 1 mL of mycelial seed solution was dispensed into 2.5% glutaraldehyde, and placed in a refrigerator at 4°C overnight. (b) Centrifuge the overnight cells at 4000-5000 rpm for 2 min on the second day. (c) Add phosphate buffer solution and aspirate the buffer solution every 15 minutes, try not to aspirate the bacterial cells, and the amount of buffer solution is arbitrary, and repeat three times. (d) Add osmic acid and let stand for 1.5h. (e) Repeat step b. (f) Add 30%, 50%, 70%, 80%, 90%, 95% ethanol, and suck out the ethanol every 15 minutes. (g) Add absolute ethanol, save, and observe by SEM.

The GA-251 strain cultured at 28° C. for 5 days on a potato plate (PDA) was treated according to the method described above, and SEM scanning electron microscope analysis was performed, and the results are shown in Figure 2 . The model is SU-8010SEM scanning electron microscope, and the magnification in the figure is 1000 times. The morphological characteristics of the strain observed under the scanning electron microscope are smooth, dense and criss-crossed hyphae.

Example 6: Fermentation of mutant strain GA-251 to produce GA3

(1) Preparation of slant: The mutant strain of Gibberella fujikura with high gibberellin production prepared in Example 1 was inoculated into the slant medium, cultivated at 28° C. for 3-7 days, and transferred when the aerial mycelium covered the slant. to the seed solution.

(2) Preparation of seed liquid:

Pick a small piece of bacteria in step (1) and inoculate it into a seed medium, and cultivate at 28° C. and 250 rpm for 48 hours to obtain a seed liquid.

The seed culture medium is prepared by the following method: corn starch 20g/L, sucrose 20g/L, peanut flour 20g/L, soybean flour 20g/L, potassium dihydrogen phosphate 1.0g/L, magnesium sulfate 1.0g/L, the solvent is Water, natural pH, sterilized at 121°C for 30min.

(3) Fermentation culture

A 250mL shake flask was filled with 40mL fermentation medium, and the seed liquid was inoculated at a concentration of 1-4% by volume during fermentation, and the fermentation was cultured at 28°C and 250rpm for 168h. During the fermentation process, the changes of GA ₃ content in the fermentation broth are shown in Figure 3, the pH changes in the fermentation broth are shown in Figure 4; the dry weight changes of mutant bacteria are shown in Figure 5.

Fermentation medium composition: corn starch 80g/L, rice flour 80g/L, soybean flour 6g/L, peanut flour 5g/L, K ₂ SO ₄ 0.5g/L, KH ₂ PO ₄ 0.5g/L, the solvent is tap water , natural pH, sterilized at 121 degrees for 30 minutes.

The above-mentioned mutant bacteria were produced by shaking flask fermentation and detected according to the method of Example 7, and the GA ₃ content in the obtained fermentation broth was 2100 mg/L.

Example 7: HPLC detection method of GA ₃

Take the fermentation broth prepared by the method in Example 6, centrifuge the fermentation broth at 12,000 rpm for 5 min, take the supernatant and filter it with a 0.45 μm organic filter and then detect by high performance liquid chromatography (HPLC).

Detection method: The chromatographic column is a C18 column (150 × 4.6 mm), the column temperature is 32 °C, the flow rate is 0.6 mL/min, the injection volume is 20 μL, the chromatographic retention time is 30 min, and the detection wavelength is 210 nm. The peak time of GA ₃ was 14 minutes.

The mobile phase preparation method: 0.5mL of phosphoric acid is diluted to 1L with water, and 600mL of this solution is mixed with 400mL of methanol;

GA ₃ yield calculation method: buy GA ₃ standard from BBI Life Sciences, prepare GA ₃ standard solutions of different concentrations (0mg/L, 200mg/L, 400mg/L, 800mg/L, 1000mg/L) with methanol, Detect the peak area of the above standard solution by HPLC respectively, and calculate the standard curve according to the peak area and the concentration of the GA ₃ standard solution as Y=28123X-50.41, R = 0.999 (wherein Y is the concentration of GA ₃ , and X is the peak area) . The GA ₃ sample of unknown concentration can be detected by HPLC to obtain a peak area, which is brought into the above standard curve formula to obtain the concentration. The result of the standard curve is shown in Figure 6.

Example 8: Optimization of fermentation temperature of shake flask fermentation system

The fermentation temperature in Example 5 was changed to 25°C, 28°C, 30°C, 32°C, and 37°C, respectively. Other operations were the same as those in Example 6. The fermentation broth GA ₃ was detected according to the detection method of Example 7. The results are shown in Figure 7 .

The best fermentation temperature was 28℃, and the highest yield was about 2.1g/L in 168 hours. When the temperature exceeds 30°C, the GA ₃ yield is lower than that of the optimum temperature.

sequence listing

<110> Zhejiang University of Technology

<120> Gibberella fujikura mutant strain with high gibberellin GA<sub>3<sub> and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 510

<212> DNA

<213> Fusarium fujikuroi

<400> 1

ggtccggccg ggcctttccc tctgtggaac cccatgccct tcactgggtg tggcggggaa 60

acaggacttt tactgtgaaa aaattagagt gctccaggca ggcctatgct cgaatacatt 120

agcatggaat aatagaatag gacgtgtggt tctattttgt tggtttctag gaccgccgta 180

atgattaata gggacagtcg ggggcatcag tattcaattg tcagaggtga aattcttgga 240

tttattgaag actaactact gcgaaagcat ttgccaagga tgttttcatt aatcaggaac 300

gaaagttagg ggatcgaaga cgatcagata ccgtcgtagt cttaaccata aactatgccg 360

actagggatc ggacggtgtt attttttgac ccgttcggca ccttacgaga aatcaaagtg 420

cttgggctcc agggggagta tggtcgcaag gctgaaactt aaagaaattg acggaagggc 480

accaccaggg gtggagcctg cggcttaatt 510

Claims (6)

1. A mutant strain of Gibberella fujikura with high production of gibberellin GA ₃ - rice bakanae GA-251 (Fusarium fujikuroi GA-251), deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, zip code 430072, deposit number: CCTCC NO: M 2019201, deposit date March 25, 2019. 2 . The rice bakanae pathogen GA-251 according to claim 1 , wherein the 18s rDNA sequence of the rice bakanae pathogen GA-251 is shown in SEQ ID NO.1. 3 . 3. The application of the rice bakanae bacterium GA-251 according to claim 1 in the preparation of gibberellin GA ₃ by microbial fermentation. 4. The application according to claim 3, characterized in that the application is: inoculating the bakanae bacterium GA-25 into a fermentation medium, and fermenting and culturing for 168h at 28°C and 250rpm to obtain gibberellin-containing The fermentation broth of GA ₃ is separated and purified to obtain gibberellin GA ₃ . 5. application as claimed in claim 4 is characterized in that described fermentation medium is composed as follows: corn starch 60～90g/L, rice flour 70～100g/L, soybean flour 3～7g/L, peanut flour 3～7g /L, K ₂ SO ₄ 0.3～0.7g/L, KH ₂ PO ₄ 0.3～0.7g/L, solvent is water, pH is natural, sterilized at 121 degrees for 30 minutes. 6. application as claimed in claim 4, it is characterized in that described rice bakanae GA-25 is first inoculated to slant medium, the bacterial strain after activation is inoculated to seed medium through seed culture to obtain seed liquid and then inoculated to fermentation culture The composition of the slant medium is as follows: potato 150-200g/L, sucrose 10-30g/L, magnesium sulfate 0.1-0.3g/L, calcium carbonate 0.1-0.3g/L, agar 20g/L, natural pH, Sterilize at 115°C for 30min; the seed medium is composed as follows: corn starch 10-30g/L, sucrose 10-30g/L, peanut flour 10-30g/L, soybean flour 10-30g/L, potassium dihydrogen phosphate 0.5 ～1.5g/L, magnesium sulfate 0.5～1.5g/L, natural pH, sterilized at 121℃ for 30min.

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