Method for screening oil-producing microalgae through combination inhibitor and plasma mutation

Abstract

The invention provides a method for screening oil-producing microalgae through combined inhibitor synergistic plasma mutagenesis, which comprises the following steps: step 1, culturing a to-be-mutagenized algae solution to a logarithmic phase to obtain an algae solution culture solution, and sequentially carrying out centrifugation and resuspension treatment on the obtained algae solution culture solution to obtain an algae solution; 2, mutagenizing the algae liquid by using plasma to obtain mutagenized algae colonies; step 3, diluting the obtained mutagenic algal colony to obtain diluted algal liquid; step 4, uniformly coating the diluted algae liquid on a solid culture medium containing an inhibitor, and culturing to obtain a mutant algae strain; step 5, screening the obtained mutant algae strains to obtain high-oil-yield mutant algae strains; compared with the existing screening method, the screening method provided by the invention can reduce the workload caused by index test, save the screening time and more effectively screen out the target algal strain.

Description

Technical Field

[0001] The invention belongs to the field of biomass energy, and in particular relates to a method for screening oil-producing microalgae by combining inhibitors and plasma mutagenesis. Background Art

[0002] Compared with traditional fossil energy, biomass energy has unique advantages in environmental protection and economy. Biodiesel prepared from microalgae has similar physical and chemical properties (carbon chain length, energy density, etc.) as fossil fuels in terms of combustion performance, but it is better than fossil fuels in terms of combustion efficiency and reduction of toxic gas emissions such as SOx and NOx, and is more in line with the concept of sustainable development. At present, the use of microalgae to prepare biodiesel still faces many limitations and challenges. The biggest difficulty is that the oil yield of raw algae species is relatively low, resulting in high downstream processing costs (such as purification, conversion, etc.).

[0003] Breeding can effectively improve the oil yield of microalgae and reduce the economic cost of subsequent processing. Plasma mutagenesis breeding is relatively simple in operation and has excellent mutagenesis efficiency. It is a breeding technology with broad application prospects. On the other hand, mutagenesis is random. In the past, the screening of characteristic microalgae mutagenesis included direct indicators (such as oil content, specific growth rate, etc.), direct indicator combination comparison or phenotypic functional screening, which had the problems of large test volume, long screening cycle and low efficiency. In recent years, methods of high-throughput screening using automated equipment have also emerged. Although such methods have fast screening speed and high efficiency, their expensive equipment cost is the biggest obstacle to widespread application. Summary of the invention

[0004] The object of the present invention is to provide a method for screening oil-producing microalgae by combined inhibitor and plasma mutagenesis, which solves the defects of low efficiency or high cost in the existing screening methods for high oil-producing microalgae.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: The present invention provides a method for screening oil-producing microalgae by combined inhibitors and plasma mutagenesis, comprising the following steps: Step 1, culturing the algae solution to be induced to a logarithmic phase to obtain an algae solution culture solution, and sequentially centrifuging and resuspending the obtained algae solution culture solution to obtain an algae solution; Step 2, using plasma to induce mutation in the algae liquid to obtain a mutated algae colony; Step 3, diluting the obtained induced algal colony to obtain a diluted algal liquid; Step 4, evenly coating the obtained diluted algae liquid on a solid culture medium containing an inhibitor for culturing to obtain a mutant algae strain; Step 5, screening out high oil-yielding mutant algae strains from the obtained mutant algae strains; Wherein, the preparation method of the solid culture medium containing the inhibitor is: Iodoacetic acid, quizalofop-p-ethyl and sesamol were added to the liquid medium to form a solid medium containing inhibitors.

[0006] Preferably, in step 1, the concentration of the algae solution is (1-2)×106 cells / mL.

[0007] Preferably, in step 2, the algae liquid is induced to undergo mutation using plasma to obtain a mutated algae colony, and the specific method is: The algae liquid is placed in a plasma reactor, and the algae liquid is induced to undergo mutation for different time periods at a set input voltage, current and air flow rate to obtain initial induced algae liquid corresponding to different time periods; Calculate the lethality of the initial mutagenic algae solution at different times; The induced algal colony is obtained from the initial induced algal solution whose lethality is greater than the set threshold.

[0008] Preferably, in step 3, the obtained mutagenic algae solution is diluted to obtain a diluted algae solution, and the specific method is: The obtained mutant algal colony was placed in a sterilized glycerol protective solution for dark adaptation to obtain a diluted algal solution, wherein the concentration of the diluted algal solution was (0.5-1)×104 cells / mL.

[0009] Preferably, the glycerol protective solution consists of glycerol and BG-11 liquid culture medium, wherein the volume fraction of glycerol in the glycerol protective solution is 10%.

[0010] Preferably, the concentrations of iodoacetic acid, quizalofop-P-ethyl and sesamol after being added to the liquid culture medium are 0.001 mM / L, 0.2 mM / L and 0.12 mM / L, respectively.

[0011] Preferably, in step 5, a high oil-yielding mutant algae strain is obtained by screening the obtained mutant algae strains, and the specific method is: The obtained mutant algae strain was inoculated into BG-11 liquid culture medium and placed in a constant temperature and light incubator for batch culture. During the culture of the mutant algae strain, the three-index screening principle was used to screen out high-oil-yielding mutant algae strains.

[0012] Preferably, the three-index screening principle is: The first screening principle is that the specific growth rate is 10% greater than that of the normal strain, and the relative fluorescence value is 20% greater than that of the normal strain; The second screening principle is that the product of the specific growth rate and the relative fluorescence value is greater than 35% of the control strain.

[0013] An oil-producing microalgae is obtained by screening based on the method.

[0014] A combined inhibitor for screening oil-producing microalgae, comprising iodoacetic acid, quizalofop-p-ethyl and sesamol, wherein the concentrations of iodoacetic acid, quizalofop-p-ethyl and sesamol after being added to a liquid culture medium are 0.001 mM / L, 0.2 mM / L and 0.12 mM / L, respectively.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for screening oil-producing microalgae by combined inhibitor synergistic plasma mutagenesis. The algae liquid is mutated by DBD plasma, and a solid culture medium to which the combined inhibitor is added is used for primary screening and re-screening based on the three-index screening principle. The method can ensure the accuracy of screening while improving the screening efficiency and reducing the testing workload, and has low requirements for screening equipment, so that the invention has wide applicability and can be completed in any laboratory.

[0016] Furthermore, a combination of three enzyme inhibitors, iodoacetic acid, quizalofop-p-ethyl and sesamol, was added to the BG-11 culture medium as a preliminary screening to filter out some algal strains with weak potential for characteristic oil accumulation (algal colonies that can grow on inhibitor solid plates indicate that mutagenesis has increased the activity of the corresponding enzymes, breaking through the inhibition and having strong potential for characteristic oil accumulation).

[0017] Furthermore, the present invention uses DBD plasma to first obtain the optimal mutagenesis time through the lethality, and at the same time determines the concentration of the combination added to the solid screening medium according to the inhibitor liquid culture and solid culture effects, and establishes a combined inhibitor solid culture medium initial screening platform. On this basis, the mutant algae strains at the optimal mutagenesis time are screened for inhibitors and accurately screened for comprehensive indicators, and the final high-oil-yielding mutant algae strains are obtained. Compared with the existing screening methods, this screening method can reduce the workload caused by indicator testing, save screening time, and can more effectively screen out target algae strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 There are three inhibitor action pathways (GAPD: glyceraldehyde 3-phosphate dehydrogenase; ACC: acetyl-CoA carboxylase; ME: malic enzyme); Figure 2 It is the voltage and current waveform and Lissajous figure of plasma discharge end; Figure 3 It is the mutagenesis time-lethality rate change curve; Figure 4 This is the decrease curve of lipid fluorescence of Scenedesmus when three inhibitors were added to liquid culture at different concentrations; Figure 5 It is a verification of the additive effect of the three inhibitors acting together; Figure 6 It is the changing trend of physiological indexes of Scenedesmus in liquid culture with the addition of reference concentration combination inhibitors; Figure 7 It is a comparison of the specific growth rate and lipid fluorescence after algae colony expansion on two solid media; Figure 8 It is the screening effect of adding inhibitors in different concentration ratios on algae strains in solid culture medium. DETAILED DESCRIPTION

[0019] In the following description, specific details such as specific system structures, technologies, etc. are provided for the purpose of illustration rather than limitation, so as to provide a thorough understanding of the embodiments of the present application. However, it should be clear to those skilled in the art that the present application may also be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, circuits, and methods are omitted to prevent unnecessary details from obstructing the description of the present application.

[0020] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of described features, integers, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0021] It should also be understood that the term “and / or” used in the specification and appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0022] As used in the specification and appended claims of this application, the term "if" can be interpreted as "when" or "uponce" or "in response to determining" or "in response to detecting", depending on the context. Similarly, the phrase "if it is determined" or "if [described condition or event] is detected" can be interpreted as meaning "uponce it is determined" or "in response to determining" or "uponce [described condition or event] is detected" or "in response to detecting [described condition or event]", depending on the context.

[0023] In addition, in the description of the present application specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the descriptions and cannot be understood as indicating or implying relative importance.

[0024] References to "one embodiment" or "some embodiments" etc. described in the specification of this application mean that one or more embodiments of the present application include specific features, structures or characteristics described in conjunction with the embodiment. Therefore, the statements "in one embodiment", "in some embodiments", "in some other embodiments", "in some other embodiments", etc. that appear in different places in this specification do not necessarily refer to the same embodiment, but mean "one or more but not all embodiments", unless otherwise specifically emphasized in other ways. The terms "including", "comprising", "having" and their variations all mean "including but not limited to", unless otherwise specifically emphasized in other ways.

[0025] Example 1 The present embodiment provides a combined inhibitor for screening oil-producing microalgae, which consists of iodoacetic acid, quizalofop-P-ethyl and sesamol, wherein the concentrations of iodoacetic acid, quizalofop-P-ethyl and sesamol after being added to BG-11 liquid culture medium are 0.001 mM / L, 0.2 mM / L and 0.12 mM / L, respectively.

[0026] Example 2 The present embodiment provides a method for screening oil-producing microalgae by using a combination of inhibitors and plasma mutagenesis, comprising the following steps: Step 1: Cultivate the algae solution to be induced to the logarithmic phase to obtain an algae solution culture solution, centrifuge the obtained algae solution culture solution, and resuspend it in pure water to obtain an algae solution, and the concentration of the algae solution is (1-2)×10 6 Pieces / mL; Take 200 μL of algae solution to the cap of a sterilized centrifuge tube and put it into a DBD plasma reactor. The algae solution was mutated for different times with the set input voltage, current and ventilation flow rate to determine the optimal mutagenesis time and obtain the mutagenic algae colony corresponding to the optimal mutagenesis time. Step 2: Take 100 μL of the mutagenized algae colony corresponding to the optimal mutagenesis time and transfer it to 20 mL of sterilized glycerol protective solution, and dark-adapt it in the glycerol protective solution for 4 hours to obtain a diluted algae solution. The concentration of the diluted algae solution is (0.5-1)×10 4 Pieces / mL.

[0027] The glycerol protective solution consists of glycerol and BG-11 liquid culture medium, wherein the volume fraction of glycerol in the glycerol protective solution is 10%.

[0028] Step 3, select iodoacetic acid (Iodoacetic acid, glyceraldehyde 3-phosphate dehydrogenase inhibitor), Quizalofop-P-ethyl (Quizalofop-P-ethyl, acetyl CoA carboxylase inhibitor), and Sesamol (Sesamol, malic enzyme inhibitor) and add them to the BG-11 liquid culture medium to form a culture medium mixture, and form a BG-11 solid culture medium containing inhibitors from the culture medium mixture, wherein the concentration of Quizalofop-P-ethyl in the culture medium mixture is 0.2 mM / L, the concentration of iodoacetic acid is 0.001 mM / L, and the concentration of Sesamol is 0.12 mM / L.

[0029] Step 4, take 200 μL of the diluted algae liquid and evenly spread it on the BG-11 solid culture medium containing the inhibitor. After culturing for 15 to 20 days, when the algae colony has clear boundaries and is visually visible, and the diameter is greater than 1 mm, use a sterilized inoculation loop to select mutant algae strains that meet the requirements in diameter and color on the BG-11 solid culture medium containing the inhibitor, and inoculate them in a 6-well plate containing BG-11 liquid culture medium. Then, place the 6-well plate inoculated with the mutant algae strain in a constant temperature and light incubator for batch liquid culture to obtain microalgae corresponding to each batch of culture; Step 5: For each batch of microalgae cultured, the absorbance OD value was measured on the third and eighth days. 680 The specific growth rate and relative fluorescence value of each microalgae were obtained by analyzing the oil fluorescence value after Nile red staining, which were used to evaluate the proliferation speed and oil accumulation of each mutant algae strain, and the three-indicator screening principle was used to accurately screen high-oil-yielding mutant algae strains.

[0030] The three-index screening principle is as follows: The first screening principle is that the specific growth rate is 10% greater than that of the normal strain, and the relative fluorescence value is 20% greater than that of the normal strain; The second screening principle is that the product of the specific growth rate and the relative fluorescence value is greater than 35% of the control strain.

[0031] Example 3 Based on Example 2, this example provides a method for screening oil-producing microalgae by combined inhibitors and plasma mutagenesis. In step 1, the obtained algae culture solution is centrifuged and resuspended with pure water to obtain an algae solution. The specific method is: After centrifugation at 8000 rpm for 10 min, the culture medium in the algae solution was resuspended and washed with pure water to eliminate other substances that interfere with the mutagenesis process and adjust the cell concentration to 10 6 About 1000 cells / mL, which is convenient for algal colony counting in the later stage.

[0032] Take 200 μL of algae solution to the sterilized centrifuge tube cap and put it into the DBD plasma reactor. The algae solution was induced for different time periods with the set input voltage, current and ventilation flow rate to determine the optimal mutation time. The power supply input voltage was controlled to be 80V, the current was 1A, the air flow rate was 3-5L / min, and the mutation time was 0 s, 10 s, 20 s, 30 s, 40 s, 50 s, and 60 s respectively. The specific method is: Set the power supply input voltage to 30 V and the current to 2 A. After preheating for 10 min, adjust the power supply voltage and frequency knobs to an input voltage of 80 V and a current of 1 A. Record the oscilloscope voltage and current waveforms and Lissajous figure data. At this time, the actual discharge end power of the plasma is about 42.90 W.

[0033] Keep the power frequency knob unchanged, return the voltage knob to zero, turn off the power switch, take 200 μL of algae solution to the sterilized centrifuge tube cap and put it into the DBD plasma reactor, turn on the power switch again, and adjust the voltage knob to 80V to start mutagenesis; The algae liquid was induced to mutagenesis by adjusting different mutagenesis times. The algae colonies on the solid culture medium were counted using ImageJ software, taking the 90% lethality of the mutagenesis algae liquid coated on the plate as a reference to determine the optimal mutagenesis time.

[0034] Lethality rate = (number of algae falling on the unmutated coating - number of algae falling on the mutated coating) / number of algae falling on the unmutated coating × 100%.

[0035] Example 4 This embodiment provides a method for screening oil-producing microalgae by combined inhibitors and plasma mutagenesis. This example takes Scenedesmus as an example to explain this method in detail. Scenedesmus in this example was purchased from the freshwater algae seed bank of Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. The DBD plasma mutagenesis system used was purchased from Nanjing Suman Plasma Technology Co., Ltd., and includes the following steps: Step 1. Cultivate Scenedesmus in BG-11 liquid culture medium. Inoculate 200 mL of Scenedesmus algae liquid into a 500 mL conical flask, seal the flask mouth with a sterile film, and place it in a constant temperature and light incubator. Cultivate in semi-continuous culture mode. Average light intensity: 6000 lux, light cycle: 12 L: 12 D, culture temperature: (25±1)℃. Randomly change the position of the conical flask every day and shake it 3-4 times.

[0036] Step 2. Cultivate the algae to be induced to the logarithmic phase, centrifuge and resuspend in sterile water to reduce the interference of residual culture medium, and adjust the concentration to 10 6 200ul of algae solution was placed in a sterilized centrifuge tube cap and placed in a DBD plasma reactor for mutagenesis.

[0037] Step 3. Determination of the optimal mutagenesis time: (1) Control the power input voltage to 80V, the current to 1A, and the air flow rate to 5L / min. In this example, the mutagenesis time is adjusted, and the mutagenesis radiation time of the algae solution is 0 s, 10 s, 20 s, 30 s, 40 s, 50 s, and 60 s. Set the power input voltage to 30V and the current to 2 A. After preheating for 10 min, adjust the power supply voltage and frequency knob to 80V and 1A. Record the voltage and current waveforms and Lissajous graph data on the oscilloscope ( Figure 2 ), at this time, the actual discharge power of the plasma is about 42.90w. Keep the power frequency knob unchanged, return the voltage knob to zero, turn off the power switch, take 200ul of algae solution to the sterilized centrifuge tube cap and put it into the DBD plasma reactor, turn on the power switch again, and adjust the voltage knob to 80v to start mutagenesis. The algae solution is induced by adjusting different mutagenesis times. According to the theory of mutagenesis breeding, it is easier to obtain positive mutations when the lethality is above 90%. Taking the lethality of 90% of the algae solution plate after mutagenesis as a reference, the optimal mutagenesis time is determined to be 30s ( Figure 3 ).

[0038] Lethality rate = (number of algae falling on the unmutated coating - number of algae falling on the mutated coating) / number of algae falling on the unmutated coating × 100% Step 4. Construction of the combination inhibitor screening platform: (1) Three inhibitors were added to BG-11 liquid culture medium to culture Scenedesmus in six-well culture plates. (When studying the effect of adding inhibitors to solid media on microorganisms, inferring or simulating the situation in solid media based on the experimental results of liquid media may have limitations due to differences in material transfer and diffusion. However, when determining the basic effect and concentration range of inhibitors, the experimental results of liquid media can provide a reference.) The effect of a single inhibitor on Scenedesmus at each concentration was observed. On the 10th day of culture, the algae solution was centrifuged and washed, resuspended in pure water and the relative fluorescence value of Nile red staining was measured to obtain the lipid fluorescence decrease curve of Scenedesmus cultured when the three inhibitors were added separately ( Figure 4 ); (2) According to the lipid fluorescence reduction curve, the addition concentrations of the three inhibitors were determined within the range of 20% to 30% of the lipid fluorescence reduction rate (to ensure that the three inhibitors had similar effects on lipid fluorescence): 1 mM quizalofop-p-ethyl, 0.005 mM iodoacetic acid, and 0.6 mM sesamol.

[0039] (3) The three inhibitors were added in combination at the determined concentrations and compared with the sum of the fluorescence reduction rates of the oil when added separately, to verify that the three selected inhibitors have an additive effect on the fluorescence reduction effect of unit volume of oil ( Figure 5 ); (4) The microalgae to be induced to mutagenesis were inoculated into a BG-11 liquid medium containing a reference concentration of a combination of inhibitors, and cultured continuously in a 250 ml conical flask for 30 days, and the changes in physiological indicators such as microalgae biomass, lipids, intracellular proteins, and intracellular polysaccharides were monitored during the period. After 30 days of culture, the microalgae were centrifuged, resuspended three times with pure water to wash away the residual inhibitor interference, and transferred to fresh BG-11 liquid medium for continued culture for 7 days. The above physiological indicators were re-measured to verify that the microalgae had the ability to recover from the inhibitor effect ( Figure 6 ); (5) The reference concentration of the combined inhibitor was added to the BG-11 solid culture medium, and the algae colonies grown after the mutagenic algae solution was applied were cultured in six-well plates, and the algae colonies grown on the ordinary BG-11 solid culture medium were used as controls. The absorbance values ​​on the 3rd and 8th days were measured to calculate the specific growth rate, and the relative fluorescence value of lipids was determined by Nile red staining. SPSS 25 software was used for significant difference analysis, which verified that the algae strains that passed the inhibitor screen had trait recovery ability (P < 0.05) ( Figure 7 ); (6) The reference concentration was scaled and added to the BG-11 solid culture medium. The control group was the BG-11 solid culture medium without the addition of inhibitors. The non-mutagenized algae solution and the 30s mutated algae solution were coated. The optimal concentration of the combined inhibitor added to the solid culture plate was determined to be quizalofop-p-ethyl: iodoacetic acid: sesamol = 0.2 mM: 0.001 mM: 0.12 mM (Table 1) ( Figure 8 ).

[0040] (1) Table 1 Screening rate of algae strains in solid culture medium by adding inhibitors at different concentration ratios

[0041] Note: - screening rate (<1%), + / - screening rate (1– 10%), + screening rate (10– 20%), ++ screening rate (20– 40%), +++ screening rate (40– 60%), ++++ screening rate (>60%). Actual mutagenesis screening procedure: Transfer 100ul of the algae solution induced for 30s into a 10ml plastic centrifuge tube with 5ml sterilized glycerol protective solution, wrap the centrifuge tube with tin foil, and dark adapt for 4h.

[0042] Take 200ul of the diluted algae solution in the glycerol protection solution and add it to a 90 mm BG-11 solid plate containing a combination inhibitor (Quizalofop-p-butyl: iodoacetic acid: sesamol = 0.2mM: 0.001mM: 0.12mM) and evenly spread it with a sterilized triangular coating rod, immediately cover it with a lid and invert it into a constant temperature light incubator for cultivation. The culture conditions are a light cycle of 12h:12h and a culture temperature of 25±1℃.

[0043] After 15 to 20 days of culture, use an inoculation loop to select the initial screening algae colonies with larger diameter and darker color and transfer them to a 6-well plate for liquid culture. The culture conditions are as follows: Average light intensity: 6000 lux, light cycle: 12 L: 12 D, culture temperature: (25±1)℃. To prevent the occurrence of wall adhesion and static sedimentation, the six-well plate was gently shaken twice a day, each time for 1 minute. The absorbance OD was measured on the third and eighth days respectively. 680 And the oil fluorescence value after Nile red staining to obtain the specific growth rate and relative fluorescence value of each mutant algae strain, and use the three-indicator screening principle to screen high oil trait algae strains.

[0044] The three inhibitors were selected based on the following criteria in the mutagenesis screening method: Iodoacetic acid is an inhibitor of the key enzyme in the glycolysis pathway (glyceraldehyde 3-phosphate dehydrogenase), which plays a key role in regulating energy metabolism and cell growth during the growth of microalgae; Quizalofop-P-ethyl is an inhibitor of the key enzyme in the production of fatty acid raw materials (acetyl-CoA carboxylase), which determines the supply capacity of raw materials for fatty acid synthesis; Sesamol is an inhibitor of the enzyme (malic enzyme) that produces the reducing power required for the synthesis of saturated fatty acids. The NADPH generated during the catalysis of this enzyme acts as a reducing power to reduce unsaturated fatty acids to saturated fatty acids, while affecting the supply of pyruvate, a precursor substance for fatty acid synthesis ( Figure 1 ).

[0045] Example 5 An oil-producing microalgae is screened based on the method described in Example 2.

[0046] The embodiments described above are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application, and should all be included in the protection scope of the present application.

Claims

1. A combined inhibitor for screening oil-producing microalgae, characterized in that: Including iodoacetic acid, quizalofop-PhD and sesamol, wherein the concentrations of iodoacetic acid, quizalofop-PhD and sesamol after added into liquid culture medium are 0.001 mM / L, 0.2 mM / L and 0.12 mM / L respectively.

2. A method for screening oil-producing microalgae by combined inhibitors and plasma mutagenesis, characterized in that: The following steps are involved: Step 1, culturing the algae solution to be induced to a logarithmic phase to obtain an algae solution culture solution, and sequentially centrifuging and resuspending the obtained algae solution culture solution to obtain an algae solution; Step 2, using plasma to induce mutation in the algae liquid to obtain a mutated algae colony; Step 3, diluting the obtained induced algal colony to obtain a diluted algal liquid; Step 4, evenly coating the obtained diluted algae liquid on a solid culture medium containing an inhibitor for culturing to obtain a mutant algae strain; Step 5, screening out high oil-yielding mutant algae strains from the obtained mutant algae strains; Wherein, the preparation method of the solid culture medium containing the inhibitor is: Iodoacetic acid, quizalofop-p-ethyl and sesamol were added to the liquid medium to form a solid medium containing inhibitors.

3. The method for screening oil-producing microalgae by combined inhibitor synergistic plasma mutagenesis according to claim 2, characterized in that: In step 1, the concentration of algae solution is (1-2)×10 6 Pieces / mL.

4. The method for screening oil-producing microalgae by combined inhibitors and plasma mutagenesis according to claim 2, characterized in that: In step 2, the algae liquid is mutated by using plasma to obtain a mutated algae colony. The specific method is: The algae liquid is placed in a plasma reactor, and the algae liquid is induced to undergo mutation for different time periods at a set input voltage, current and air flow rate to obtain initial induced algae liquid corresponding to different time periods; Calculate the lethality of the initial mutagenic algae solution at different times; The induced algal colony is obtained from the initial induced algal solution whose lethality is greater than the set threshold.

5. The method for screening oil-producing microalgae by combined inhibitor synergistic plasma mutagenesis according to claim 2, characterized in that: In step 3, the obtained mutagenic algae solution is diluted to obtain a diluted algae solution, and the specific method is: The obtained mutant algal colony was placed in a sterilized glycerol protective solution for dark adaptation to obtain a diluted algal solution, wherein the concentration of the diluted algal solution was (0.5-1)×10 4 Pieces / mL.

6. The method for screening oil-producing microalgae by combined inhibitors and plasma mutagenesis according to claim 5, characterized in that: The glycerol protective solution consists of glycerol and BG-11 liquid culture medium, wherein the volume fraction of glycerol in the glycerol protective solution is 10%.

7. The method for screening oil-producing microalgae by combined inhibitors and plasma mutagenesis according to claim 2, characterized in that: Iodoacetic acid, quizalofop-p-ethyl and sesamol were added to the liquid culture medium at concentrations of 0.001 mM / L, 0.2 mM / L and 0.12 mM / L, respectively.

8. The method for screening oil-producing microalgae by combined inhibitors and plasma mutagenesis according to claim 2, characterized in that: Step 5, screening out high oil-yielding mutant algae strains from the obtained mutant algae strains, the specific method is: The obtained mutant algae strain was inoculated into BG-11 liquid culture medium and placed in a constant temperature and light incubator for batch culture. During the culture of the mutant algae strain, the three-index screening principle was used to screen out high-oil-yielding mutant algae strains.

9. The method for screening oil-producing microalgae by combined inhibitors and plasma mutagenesis according to claim 2, characterized in that: The three-index screening principle is as follows: The first screening principle is that the specific growth rate is 10% greater than that of the normal strain, and the relative fluorescence value is 20% greater than that of the normal strain; The second screening principle is that the product of the specific growth rate and the relative fluorescence value is greater than 35% of the control strain.

10. An oil-producing microalgae, characterized in that: The method is screened based on claim 2.

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