CN110527630B

CN110527630B - Aleurites lutescens mutant strain bred by ARTP mutagenesis technology and application thereof

Info

Publication number: CN110527630B
Application number: CN201910438160.7A
Authority: CN
Inventors: 柳志强; 蒋欢; 张博; 郑裕国
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2019-05-24
Filing date: 2019-05-24
Publication date: 2021-04-20
Anticipated expiration: 2039-05-24
Also published as: CN110527630A

Abstract

The invention relates to a gibberellic disease mutant of granary-GA-347 (Fusarium fujikuroi GA-347) bred by an ARTP mutagenesis technology and application thereof. The strain is preserved in China center for type culture Collection with the address: wuhan university, Wuhan, China, zip code 430072, preservation number: CCTCC NO: m2019378, date of deposit 2019, 5 months and 20 days. The strain does not produce conidium in the growth process, is inoculated into a liquid seed culture medium, is transferred into a fermentation culture medium after cells grow mature, greatly improves the fermentation level, and has the gibberellin yield reaching 22 times that of wild bacteria, which is the international leader. Gibberellin is secreted extracellularly and can be directly obtained from fermentation liquor in a gathering way, which is beneficial to solid-liquid separation and increases the extraction yield. Meanwhile, the utilization rate of equipment and raw materials is improved, the production cost is greatly reduced, and the method has obvious economic and social benefits.

Description

Aleurites lutescens mutant strain bred by ARTP mutagenesis technology and application thereof

(I) technical field

The invention relates to a gibberella barnacii mutant strain bred by an ARTP mutagenesis technology and application thereof.

(II) background of the invention

Gibberellin is used as a natural plant growth regulator, has various biological functions on plant growth, can regulate the processes of plant rooting, germination, fruiting and the like, and can stimulate the growth and development of crops such as rice, cotton, hemp and the like. Therefore, the method is widely used for the seed production of hybrid rice, the fruit protection of oranges and tangerines, the boll protection of cotton, the growth of vegetables and young forests, and has great economic and social benefits for promoting agricultural production, particularly grain production. In the beer industry, it can accelerate the germination of barley, make the malt germinate uniformly, and simultaneously can raise the activity of starch bacteria, amylase and some protease so as to raise the quality of malt. The main active substance of gibberellins is GA₃、GA₄、GA₇And GA₉Etc. wherein GA₃Is the most used finished product of gibberellin. In 1999, international market demand was over 250 tons worth about $ 10 billion; 23 production enterprises registered in 2000 years in China have annual output of about 150 tons, the output value is about 10 billion yuan, and a large number of exports exist. In addition, GA₄And GA₇The usable area of the mixture is also expanding continuously.

Gibberellin GA₃Is a diterpenoid acid with a molecular formula C₁₉H₂₂O₆Molecular weight of 346.37, melting point of 233-phosphate buffer pH 6.2, insoluble in water and ether.

The plasma is a substance fourth state coexisting with the solid state, the liquid state and the gas state of the substance, is an ionized gas state with approximately equal positive ion and electron density, and has the characteristics of conductivity, luminescence, active chemical property and the like. The atmospheric pressure injection frequency glow discharge plasma of the present bare metal electrode structure is a new ion source. The method is mainly applied to disinfection and sterilization. Based on the principle of the plasma, atmospheric pressure room temperature plasma (ARTP) is developed by Qingtian wood biotechnology limited, and the ARTP has the characteristics of low jet temperature, uniform discharge and abundant chemical particles which can cause genetic material mutation.

In 1970, high-yield gibberellin varieties 4303 are screened in China, but the high-yield gibberellin varieties have no spore production capability and are multi-core hyphae, so that continuous breeding is very difficult. The current production titer is in a state of lingering, and some production strains are degenerated. The yield of the gibberella is retained after long-term physical and chemical mutagenesis, the chemical mutagenesis has the defects of environmental pollution, operation danger and the like, for example, nitrosoguanidine as a mutagen for breeding microorganisms has the characteristics of flammability, toxicity, carcinogenic possibility, irritation and the like, the operation danger is generated in experiments, and the tolerance to organisms can be generated by using the same mutagen for a long time. Physical mutagens have the advantages of environmental protection, small pollution and the like, for example, ultraviolet mutagenesis is widely applied, but the genetic stability of a mutagenized strain is poor. Therefore, new mutagenesis approaches are needed to improve strain yield and genetic stability.

Disclosure of the invention

The invention aims to provide a gibberella granatum mutant strain bred by an ARTP mutagenesis technology and application thereof, wherein the strain is named as GA-347 and can be applied to industrial production in a large scale.

The technical scheme adopted by the invention is as follows:

a gibberellic disease mutant strain GA-347(Fusarium fujikuroi GA-347) bred by an ARTP mutagenesis technology is deposited in China center for type culture Collection, and the address is as follows: wuhan university, Wuhan, China, zip code 430072, preservation number: CCTCC NO: m2019378, date of deposit 2019, 5 months and 20 days.

The process for obtaining the strain of the invention is as follows:

1. the specific method refers to the research of high-efficiency transformation method of exogenous gene of Gibberella protoplast such as Gibberella cinnabarina (proceedings of Zhejiang university, 2013, 41(5), 482-

2. ARTP mutagenesis to obtain the rice bakanae disease GA-347.

The invention provides a method for obtaining high-yield GA by preparing a Gibberella fujikuroi protoplast, carrying out ARTP mutagenesis and screening 3000 mutant strains₃And (3) strain. The method is ion mutagenesis, obviously improves the tolerance of the strain generated after multiple physicochemical mutagenesis, and provides economic benefit for industrial production.

The 18s rDNA sequence of the rice bakanae disease GA-347 is shown in SEQ ID NO. 1.

The invention also relates to application of the bakanae disease of rice GA-347 in preparation of gibberellin through microbial fermentation.

Specifically, the bakanae disease of rice GA-347 is used for preparing gibberellin GA by microbial fermentation₃。

Specifically, the application is as follows: inoculating the bakanae disease oryzae GA-347 to a fermentation culture medium, and performing fermentation culture for 12-48 h under the conditions of 25-30 ℃ (preferably 28 ℃) and 200-300 rpm (preferably 250rpm) to obtain GA containing gibberellin₃The fermentation liquor of (1) is separated and purified to obtain gibberellin GA₃。

The fermentation medium comprises the following components: 60-90 g/L of corn starch, 70-100 g/L of rice flour, 3-7 g/L of soybean flour, 3-7 g/L of peanut powder and K₂SO₄ 0.3～0.7g/L，KH₂PO₄0.3-0.7 g/L, water as solvent, natural pH, and sterilizing at 121 ℃ for 30 min.

Generally, the rice bakanae disease GA-347 is firstly inoculated to a slant culture medium (cultured for 4-7 days at 25-30 ℃), the activated strain is inoculated to a seed culture medium, a seed solution is obtained by seed culture (cultured overnight at 25-30 ℃ and 250rpm), and then the seed solution is inoculated to a fermentation culture medium, wherein the slant culture medium comprises the following components: 150-200 g/L of potato, 10-30 g/L of cane sugar, 0.1-0.3 g/L of magnesium sulfate, 0.1-0.3 g/L of calcium carbonate and 20g/L of agar, wherein the pH is natural, and the potato is sterilized for 30min at 115 ℃; the seed culture medium comprises the following components: 10-30 g/L of corn starch, 10-30 g/L of cane sugar, 10-30 g/L of peanut powder, 10-30 g/L of soybean meal, 0.5-1.5 g/L of monopotassium phosphate and 0.5-1.5 g/L of magnesium sulfate, wherein the pH is natural, and the sterilization is carried out for 30min at 121 ℃.

The invention has the following beneficial effects:

1. the invention provides a gibberella bardawil mutant strain for high yield of gibberellin, wherein conidia are not generated in the growth process of the strain.

2. The invention provides gibberellin GA₃The fermentation method of (1), which employs GA that produces gibberellin₃The strain is inoculated into a liquid seed culture medium, and is transferred into a fermentation culture medium after cells grow mature, so that the fermentation level is greatly improved, the yield of the gibberellin reaches 22 times of that of wild bacteria, and the strain is the international leader. Gibberellin is secreted extracellularly and can be directly obtained from fermentation liquor in a gathering way, which is beneficial to solid-liquid separation and increases the extraction yield. Meanwhile, the utilization rate of equipment and raw materials is improved, the production cost is greatly reduced, and the method has obvious economic and social benefits.

(IV) description of the drawings

FIG. 1 is the electrophoresis diagram of GA-347 strain 18S rDNA sequence PCR amplified argrose.

FIG. 2 is a phylogenetic tree of GA-347 strain.

FIG. 3 shows the mycelium morphology under electron microscope observation.

FIG. 4 shows GA in fermentation broth₃The content was varied.

FIG. 5 is a graph showing pH change in fermentation broth.

FIG. 6 shows the change in dry weight of GA-347 fermentation.

FIG. 7 is GA₃High Performance Liquid Chromatography (HPLC) detection standard curve.

FIG. 8 is a graph showing the effect of different fermentation temperatures on the dry weight of the hyphae of strain GA-347.

FIG. 9 shows the effect of ARTP and UV mutagenesis on genetic stability of GA-347.

FIG. 10 shows the effect of ARTP and UV mutagenesis on the hyphal dry weight of the strain.

FIG. 11 shows the effect of ARTP and UV mutagenesis on the pH of the strain.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1: preparation of wild Alternaria lutea protoplast

(1) Preparing a bevel: inoculating the wild Alternaria barnacii strain (laboratory preservation) into a slant culture medium, culturing at 28 ℃ for 3-7 days, and transferring to a seed solution after aerial hyphae grow over the slant.

(2) Preparing a seed solution:

and (2) selecting a small piece of the bacteria in the step (1), inoculating the small piece of bacteria into a seed culture medium, and culturing at 28 ℃ and 250rpm for 48 hours to obtain a seed solution. The seed culture medium is prepared by the following method: 20g/L of corn starch, 20g/L of cane sugar, 20g/L of peanut powder, 20g/L of soybean powder, 1.0g/L of monopotassium phosphate and 1.0g/L of magnesium sulfate, wherein the solvent is tap water, the pH is natural, and the sterilization is carried out for 30min at 121 ℃.

(3) Fermentation culture

And (3) shaking a flask with the specification of 250mL, filling 40mL of fermentation medium, inoculating a seed solution with the volume concentration of 1-4% during fermentation, and carrying out fermentation culture at 28 ℃ and 250rpm for 168 h. The fermentation medium comprises the following components: 80g/L of corn starch, 80g/L of rice flour, 5g/L of soybean flour, 5g/L of peanut powder and K₂SO₄ 0.5g/L，KH₂PO₄0.5g/L, the solvent is tap water, the pH is natural, and sterilization is carried out for 30min at 121 ℃.

(4) Protoplast preparation

A small pellet was inoculated from the slant into YEPD liquid medium (yeast extract 0.3%, peptone 1%, glucose 2%) and cultured at 28 ℃ and 180rpm for 48 hours. Adding 2ml of the bacterial liquid into a sterile Eppendorf tube, and sequentially adding sterile water and 1M MgSO₄Each wash was performed once. Dipping a small amount of hyphae into 1.5mL of wall-breaking buffer (1M MgSO 2) which has been subjected to filtration sterilization by using a sterilized gun head₄Total enzyme concentration 15mg/mL, wherein drylass: yatalase ═ 3:7), the mixture was blown uniformly and placed in a 30 ℃ water bath shaker to react for 45 minutes (the protoplast production rate was 95% or more). Centrifuging at 5000rpm for 10min, transferring 0.75mL of supernatant to another centrifuge tube, and adding equal amount of sterileWater, mix well, centrifuge at 6000rpm for 5 minutes, discard the supernatant. The cell pellet was suspended with 1mL of 1M sorbitol and protoplasts were successfully prepared. After microscopic examination, the protoplast can be recovered by diluting the protoplast with proper times and plating the protoplast on MYG culture medium (maltose 0.5%, glucose 1%, yeast powder 0.5%, sucrose 5M, and agar 2%). The control was suspended in sterile water, diluted and plated. The number of protoplasts contained per unit volume was measured by counting on a hemocytometer. And diluting the protoplast solution to 10⁶-10⁷one/mL.

Example 2: high yield GA₃Screening of mutant strains

(1) Inoculating wild type Gibberella fujikuroi to a slant culture medium, culturing at 28 ℃ for 3-7 days, inoculating a small bacterium block from the slant to a YEPD liquid culture medium (yeast extract is 0.3%, peptone is 1%, and glucose is 2%), washing with sterile water and saline buffer solution once, adding a small amount of hypha into a wall breaking buffer solution, placing in a 30 ℃ water bath shaking table for reaction for 45 minutes, centrifuging at 3000rpm for 10 minutes, discarding supernatant, washing with sterile water once, and adding the buffer solution for suspension.

(2) The ARTP mutagenesis method: coating the activated strain in the step (1) on a MYG solid culture medium (maltose 0.5%, yeast powder 0.5%, glucose 1%, sucrose 5M and agar 2%), culturing at 28 ℃ for 3-7 days until a bacterial colony grows out, preparing protoplast through the step (1), and controlling the bacterial count to be about 10⁷one/mL.

Before the ARTP instrument is used, a high-pressure input end of a pressure reducing valve needs to be connected to an outlet of a gas cylinder, a low-pressure output end of the ARTP instrument is connected to an air inlet of the instrument through an organ, the air tightness is checked, a cold water shortage circulator is started to cool, an operation chamber disinfection program is started, an operation chamber door is opened after disinfection to pull out a manual rotating sample carrying platform, a sample slide to be processed is placed in a circular groove above the manual rotating sample carrying platform through tweezers, the carrying platform is clamped after restoring the original position, the operation chamber door is closed, the power is set to be 120W, an air flow 10SLM (selective laser) is adopted, and 0s, 10 s. And (3) starting to process the sample by an electric shock starting button, turning off the radio frequency power supply, opening the operation chamber, putting the slide glass into an EP (EP) tube containing 1mL of sterile physiological saline, screwing a cover, and marking. Dilution coated 3 dishes per gradient.

Table 1: mutagenesis procedure by ARTP mutagenesis method

For the high-producing strain obtained by each round of mutagenesis, mutagenesis was performed again as the original strain in accordance with the method described above. Final screening to obtain GA₃The mutant strain GA-347 with the yield of 2-4 g/L is named as rice bakanae disease GA-347(Fusarium fujikuroi GA-347), is preserved in China Center for Type Culture Collection (CCTCCNO), and has the preservation date of 2019, 5 and 20 months and the preservation number of CCTCCNO: m2019378, the preservation address is Wuhan university, Wuhan, China, zip code 430072.

The present invention includes, but is not limited to, one of the mutagenesis methods described above.

The preparation of MYG solid medium comprises the following steps: maltose 0.5%, yeast powder 0.5%, glucose 1%, sucrose 5M, agar 2%, and tap water as solvent, with natural pH, and sterilizing at 121 deg.C for 20 min.

Preparation of YEPD culture medium: yeast extract 0.3%, peptone 1%, glucose 2%, and solvent tap water, with natural pH, sterilizing at 121 deg.C for 20 min.

Example 3: 18S rDNA molecular identification

(1) DNA extraction: taking hyphae for centrifugation, taking a small amount of hyphae, putting the hyphae into a 1.5mL EP tube, adding 978 mu L of sodium phosphate buffer, and suspending thalli; transferring the bacterial suspension into a lysine Matrix E Tube, adding 112 mu L MT Buffer, and uniformly mixing; using MP

-24 homogeneous disruptor disruption of cells with speed set at 6.0 and time set at 40 s; centrifuging at 12000rpm for 10min to remove sample debris; transferring the supernatant into a new EP tube, adding 250 microliter PPS reagent, holding the centrifuge tube, shaking for 10 times to mix uniformly, and centrifuging at 12000rpm for 5 min; transferring the supernatant to a 10mL clean centrifuge tube, adding 1mL Binding Matrix subspension, inverting the centrifuge tube for 2min, standing for 3min to attach DNA to the Binding Matrix, andwaiting for the silica matrix to precipitate; carefully removing 500. mu.L of supernatant (to avoid sucking out the precipitate), mixing the remaining supernatant with the precipitate, sucking about 600. mu.L of the mixture, transferring the mixture into a SPIN Filter, centrifuging at 12000rpm for 1min, discarding the filtrate, transferring the remaining liquid to the SPIN Filter, and centrifuging to discard the filtrate; adding 500 μ L SEWS-M solution, centrifuging at 12000rpm for 1min, discarding the filtrate, centrifuging the SPIN Filter for 2min, transferring the SPIN Filter to a new catch tube, and drying at room temperature for 5 min; adding 50 μ L DES solution, standing at room temperature for 1min, and centrifuging at 12000rpm for 1min, wherein the filtrate in catch tube is the genome of the extracted strain.

(2) PCR amplification and sequence analysis of fungal 18S rDNA: fungus ribosome ITS rDNA universal primer

The primers used were as follows:

TABLE 2

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

Wherein the cloning of the PCR system: add 10 XTaq Buffer (Mg +) 5. mu.L, add 10mM dNTPs 1. mu.L, Taq DNA Polymerase 0.5. mu.L, ITS1 and ITS4 primers 0.5. mu.L each, template 1.5. mu.L, make up deionized water to 50. mu.L.

Wherein the cloning PCR procedure: denaturation at 95 ℃ for 45s, annealing at 55-60 ℃ for 30s, and extension at 72 ℃ for 45min for 35 cycles. And finally extension at 72 ℃ for 30 s.

(3) 5 mu L of PCR amplification product is taken for agarose gel electrophoresis experiment and observed and analyzed by a gel imager. If the band is clear and error-free (about 500 bp), the PCR product is recovered by gel. The recovered products were then verified by agarose gel electrophoresis (FIG. 1) using 5. mu.L each, and if the bands were clear and of correct size, the recovered samples were used for sequencing. Sequencing is completed by Hangzhou Optingke Cathi Biotechnology Limited, and sequencing primers are the same as PCR primers. Sequence alignment was performed using the NCBI blast database.

Sequencing was performed by Hangzhou Zhikexi Biotechnology Limited, with the following results:

construction of phylogenetic trees: the 18S rDNA sequence of the GA-347 strain was subjected to Blast analysis with the nucleic acid sequence in the NCBI database to call out a nucleic acid sequence having a high correlation with the sequence, which was automatically analyzed by alignment by cluster using MEGA5 software, and then a phylogenetic tree was generated by 1000 repetitions using MEGA5 software (see FIG. 2). The results showed that GA-347 strain has 99% homology to Fusarium fujikuroi AB725604 and 99% support on the same branch of the evolutionary tree.

Example 4: physiological and biochemical identification

Carbon source utilization was determined using a biolog (ff) automated identification system. Since morphological characteristics and culture characteristics of the test strain are similar to those of fungi, the present experiment uses FF-identified microplates to determine the carbon source utilization of the test strain.

1. Preparing protoplasts:

(1) a small pellet was inoculated from the slant into YEPD liquid medium and cultured at 28 ℃ and 180rpm for 48 hours.

(2) Adding 2ml of the bacterial liquid into a sterile Eppendorf tube, and sequentially adding sterile water and 1M MgSO₄Each wash was performed once. Dipping a small amount of hypha into 1.5mL of wall-breaking buffer solution which is filtered and sterilized by a sterilized gun head, blowing and beating uniformly, and placing in a water bath shaking table at 30 ℃ for reaction for 45 minutes. Centrifuging at 5000rpm for 10min, transferring 0.75mL of supernatant to another centrifuge tube, adding equal amount of sterile water, mixing well, centrifuging at 6000rpm for 5min, and discarding supernatant. The cell pellet was suspended well with 1mL of 1M sorbitol.

2. FF micro-plating:

(1) pouring the prepared bacterial suspension into a sample adding groove, and inoculating the bacterial suspension into 96 holes of a micro-plate by using an eight-channel electric pipettor;

(2) the microplate was identified using FF with an inoculum size of 100. mu.L/well.

3. FF micro-plate culture:

the FF identification micro-plate inoculated with the filamentous fungi is cultured in air at 26 mu for 24h, 48h, 72h, 96h, 168h and 240h, and the culture environment is not excessively humid.

4. FF microplate reading and result preservation:

(1) opening a 'MicroLog' application program, inputting a user name and a password, and clicking 'OK' to enter a main interface;

(2) entering a 'Setup' interface, clicking 'initialize reader', carrying out initialization setting, and clicking 'Read' when a red 'ComNot Open' key on the interface is changed into a green 'Ready';

(3) after entering a 'Read' interface, selecting a Reader mode-Reader, and if Manual reading is adopted, entering a Manual mode (Manual); inputting a Data storage Name and a storage address after the Data File Name; clicking the 'Read New Plate' to select the type of the microplate and the culture time, and selecting the type of the filamentous fungi in a pull-down menu of 'Strain type';

(4) placing the microplate on a reading instrument bracket, closing a reading instrument cover, and preparing for reading;

(5) pressing the "Read Next" key starts the reading.

(6) The results are saved in PDF format.

The metabolism of the strains on 95 carbon sources was investigated using a Biolog automated microbial identification system: inoculating the strain to PDA plate culture medium, culturing at 28 deg.C for 5 days, washing thallus on the plate with sterile cotton swab, mixing with inoculating solution (FF-IF), making into bacterial suspension, and adjusting to 75% T/FF (reference value: 75% +/-3%) with turbidimeter. The bacterial suspensions were added to each well of a Biolog FF microwell assay plate using an 8-well electric applicator, 100 μ l per well. The microwell assay plates were placed in a 28 ℃ incubator and read on a Biolog reader after 24h, 48h, 72h, 96h, 168h, and 240h incubation, respectively. The data of each time point read by the Biolog reading instrument are comprehensively considered, and the result of 24h identification is given.

Physiological and biochemical identification is carried out on wild strains according to the method, a Biolog automatic microorganism identification system is utilized to investigate the metabolism condition of the strains on 95 carbon sources, each time point data read by a Biolog reading instrument is comprehensively considered, and 24h identification results are given, and are shown in table 3.

Table 3: ability of strain GA-347 to utilize 95 carbon sources on Biolog FF plates

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

Example 5: high yield GA₃Observing strain by electron microscope

(1) And (3) strain culture: selecting slant strain preservation liquid by using an inoculating loop, marking out a PDA flat plate, culturing for 5d at 28 ℃, selecting a well grown strain block, inoculating into a seed culture medium, culturing for 2d at 250rpm and 28 ℃, and waiting for treatment.

(2) And (3) thallus treatment: (a) 1mL of the hypha seed solution was dispensed into 2.5% glutaraldehyde and left overnight in a refrigerator at 4 ℃. (b) The overnight cells were centrifuged at 4000-. (c) Adding phosphate buffer solution, sucking out buffer solution every 15min, and repeating the steps for three times at random. (d) Adding osmate, and standing for 1.5 h. (e) And c, repeating the step b. (f) Adding 30%, 50%, 70%, 80%, 90%, 95% ethanol, and sucking out ethanol every 15 min. (g) Adding absolute ethyl alcohol, storing, and observing by SEM.

GA-347 strain cultured on potato Plates (PDA) at 28 ℃ for 5 days was treated as described above and analyzed by SEM scanning electron microscopy, the results of which are shown in FIG. 3. An SEM scanning electron microscope with the model number of SU-8010 is adopted, the magnification in the figure is 1000 times, and the morphological characteristics of the strains observed under the SEM scanning electron microscope are that the whole hyphae are smooth and dense, are criss-cross and have a nodular structure locally.

Example 6: mutant strain GA-347 for producing GA by fermentation₃

(1) Preparing a bevel: the gibberellin-producing mutant strains of gibberellin-producing fungi of the present invention prepared in example 1 were inoculated into a slant culture medium and cultured at 28 ℃ for 3-7 days until aerial hyphae were grown over the slant and were transferred to the seed solution.

(2) Preparing a seed solution:

and (2) selecting a small piece of the bacteria in the step (1), inoculating the small piece of bacteria into a seed culture medium, and culturing at 28 ℃ and 250rpm for 48 hours to obtain a seed solution.

The seed culture medium is prepared by the following method: 20g/L of corn starch, 20g/L of cane sugar, 20g/L of peanut powder, 20g/L of soybean powder, 1.0g/L of monopotassium phosphate and 1.0g/L of magnesium sulfate, wherein the solvent is water, the pH is natural, and the sterilization is carried out for 30min at 121 ℃.

(3) Fermentation culture

And (3) shaking a flask with the specification of 250mL, filling 40mL of fermentation medium, inoculating a seed solution according to the volume concentration of 1-4% during fermentation, and carrying out fermentation culture at 28 ℃ and 250rpm for 168 h. During the fermentation process, GA in the fermentation liquor₃The content changes are shown in FIG. 4, and the pH changes in the fermentation broth are shown in FIG. 5; the mutant bacteria dry weight change is shown in FIG. 6.

The fermentation medium comprises the following components: 80g/L of corn starch, 80g/L of rice flour, 6g/L of soybean meal, 5g/L of peanut powder and K₂SO₄0.5g/L，KH₂PO₄0.5g/L, the solvent is tap water, the pH is natural, and sterilization is carried out for 30min at 121 ℃.

The mutant strain is produced by shake flask fermentation, and is detected according to the method of example 7, and GA in the obtained fermentation liquid₃The content is 2235 mg/L.

Example 7: GA₃HPLC detection method of

The fermentation broth prepared in example 6 was centrifuged at 12000rpm for 5min, and the supernatant was filtered through a 0.45 μm organic membrane and then detected by High Performance Liquid Chromatography (HPLC).

The detection method comprises the following steps: the chromatographic column is C18 column (150 × 4.6mm), the column temperature is 32 deg.C, the flow rate is 0.6mL/min, the sample volume is 20 μ L, the chromatographic retention time is 30min, and the detection wavelength is 210 nm. GA₃The peak-off time of (1) was 14 min.

The preparation method of the mobile phase comprises the following steps: 0.5mL of phosphoric acid was diluted with water to 1L, and 600mL of the solution was mixed with 400mL of methanol;

GA₃the yield calculation method comprises the following steps: GA purchase from BBI Life Sciences₃The standard (c) was prepared by using methanol to prepare GA at different concentrations (0mg/L, 200mg/L, 400mg/L, 800mg/L, 1000mg/L)₃Standard solutions, respectively detecting peak area of the above standard solutions by HPLC, and determining peak area and GA₃The concentration of the standard solution was calculated as Y-983.5X-9.6508 and R2-0.996 (wherein Y is GA)₃X is the peak area). The GA of unknown concentration₃The sample was tested by HPLC to obtain a peak area, which was substituted into the above formula of the standard curve to obtain the concentration, and the results of the standard curve are shown in FIG. 7.

Example 8: temperature change to hyphal dry weight

Samples were obtained from the fermentation flasks at 24-hour intervals for analysis of cell mass (dry cell weight [ DCW ]), as in example 6, except that the fermentation temperature in example 5 was changed to 25 deg.C, 28 deg.C, 30 deg.C, 32 deg.C, and 37 deg.C, respectively. As a result, FIG. 8, DCW was performed by using 1mL of the initial fermentation medium as a control, taking another 1mL of the fermentation sample, centrifuging at 12000rpm for 5 minutes, washing twice with 1mL of ultrapure water, resuspending the mycelium pellet and centrifuging at 12000rpm for 5 minutes. Finally, the tube containing the washed mycelium pellets was dried at 80 ℃ to constant weight, and the difference in dry weight of the fermentation sample from the control was taken as the dry weight of the mycelium, and the results are shown in FIG. 7. The fermentation temperature is optimized at 28 deg.C, and the maximum dry weight of mycelia is about 36g/L in 120 hr. At 37 deg.C, the dry weight of mycelia is lowest, which is not favorable for growth.

Example 9: effect of different mutagenesis modalities on the genetic stability of the Strain

Passage comparison is carried out on ARTP and high-yield strain subjected to ultraviolet mutagenesis, and 10 generations of GA are subjected₃As a result of examining the productivity, as shown in FIG. 9, the highly productive strain GA-347 induced by ARTP had better genetic stability.

Example 10: effect of different mutagenesis modalities on the Dry weight of the hyphae of the Strain

As in example 8Method for measuring ultraviolet mutation high-yield GA₃The same procedure as in example 6 was repeated except that the dry weight of the mycelia of the strain and GA-347 was changed, and the results are shown in FIG. 10. in 168 hours during the fermentation, the dry weight curves of the mycelia of both strains tended to rise before 120 hours and the dry weight of the mycelia of GA-347 was higher than that of the UV-induced strain, the mycelia of the mutant strain GA-347 accumulated rapidly before 72 hours and became in the growth period, the growth started to slow down to reach the stationary phase after 72 hours, the growth reached the maximum value after 120 hours, and the mycelia began to degrade partially and the cells became in the death period after 120 hours.

Example 11: effect of different mutagenesis modalities on the pH of the Strain

During the fermentation process, the pH shows a downward trend, and the pH is reduced due to the continuous formation of acidic fermentation products during the fermentation process. The initial pH of the fermentation medium is consistent, and the pH curve of the mutant strain GA-347 in the fermentation process is integrally higher than that of the UV mutant strain. Referring to FIG. 10, after 120 hours, the dry weight of the mycelia decreased, and the pH of the mutant strain GA-347 was 4 or less, which was not favorable for the growth of the cells.

Sequence listing

<110> Zhejiang industrial university

<120> A gibberella barnacle mutant strain bred by ARTP mutagenesis technology and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 584

<212> DNA

<213> Fusarium fujikuroi

<400> 1

gggaagtaaa aggtcgtaac aaggtctccg ttggtgaacc agcggaggga tcattaccga 60

gtttacaact cccaaacccc tgtgaacata ccaattgttg cctcggcgga tcagcccgct 120

cccggtaaaa cgggacggcc cgccagagga cccctaaact ctgtttctat atgtaacttc 180

tgagtaaaac cataaataaa tcaaaacttt caacaacgga tctcttggtt ctggcatcga 240

tgaagaacgc agcaaaatgc gataagtaat gtgaattgca gaattcagtg aatcatcgaa 300

tctttgaacg cacattgcgc ccgccagtat tctggcgggc atgcctgttc gagcgtcatt 360

tcaaccctca agccctcggg tttggtgttg gggatcggcg agcccttgcg gcaagccggc 420

cccgaaatct agtggcggtc tcgctgcagc ttccattgcg tagtagtaaa accctcgcaa 480

ctggtacgcg gcgcggccaa gccgttaaac ccccaacttc tgaatgttga cctcggatca 540

ggtaggaata cccgctgaac ttaagcatat caataagcgg agga 584

<210> 2

<211> 19

<212> DNA

<213> Unknown (Unknown)

<400> 2

tccgtaggtg aacctgcgg 19

<210> 3

<211> 20

<212> DNA

<213> Unknown (Unknown)

<400> 3

tcctccgctt attgatatgc 20

Claims

1. A strain of Gibberella fujikuroi, (A) and (B)Fusariumfujikuroi) GA-347, deposited in the China center for type culture Collection, address: wuhan university, Wuhan, China, zip code 430072, preservation number: CCTCC NO: m2019378, date of deposit 2019, 5 months and 20 days.

2. Preparation of gibberellin GA-347 by microbial fermentation of the gibberellic disease of claim 1, wherein the gibberellin GA-347 is prepared by microbial fermentation of the gibberellin GA-347₃The use of (1).

3. The use according to claim 2, characterized in that the use is: inoculating the gibberellin GA-347 to a fermentation culture medium, and performing fermentation culture for 12-48 h at 25-30 ℃ and 200-300 rpm to obtain GA containing gibberellin₃The fermentation liquor of (1) is separated and purifiedObtaining gibberellin GA₃。

4. Use according to claim 3, characterized in that the fermentation medium consists of: 60-90 g/L of corn starch, 70-100 g/L of rice flour, 3-7 g/L of soybean flour, 3-7 g/L of peanut powder and K₂SO₄ 0.3~0.7 g/L，KH₂PO₄0.3-0.7 g/L, water as solvent, natural pH, and sterilizing at 121 ℃ for 30 min.

5. The use of claim 3, wherein the gibberellic disease GA-347 is inoculated into a slant culture medium, the activated strain is inoculated into a seed culture medium, a seed solution is obtained by seed culture, and then the seed solution is inoculated into a fermentation culture medium, wherein the slant culture medium comprises: 150-200 g/L of potato, 10-30 g/L of cane sugar, 0.1-0.3 g/L of magnesium sulfate, 0.1-0.3 g/L of calcium carbonate and 20g/L of agar, wherein the pH is natural, and the potato is sterilized for 30min at 115 ℃; the seed culture medium comprises the following components: 10-30 g/L of corn starch, 10-30 g/L of cane sugar, 10-30 g/L of peanut powder, 10-30 g/L of soybean meal, 0.5-1.5 g/L of monopotassium phosphate and 0.5-1.5 g/L of magnesium sulfate, wherein the pH is natural, and the sterilization is carried out for 30min at 121 ℃.