CN110885816A - Method for mutagenizing and screening microalgae with high oil yield by ARTP - Google Patents

Abstract

The invention discloses a method for using ARTP to mutagenize and screen microalgae with high oil yield, which mainly includes two processes: (1) using ARTP to mutagenize the microalgae, and adjusting the effect of the mutagenesis time on the mutagenesis effect , and determine the optimal mutagenesis time based on the lethality; (2) carry out gradient dilution of the mutagenized algal liquid at each mutagenesis time (can count efficiently and accurately). Spread evenly on solid medium and grow upside down in a lighted incubator. After culturing for 10 days, select algal colonies with dark colors and larger diameters and inoculate them into the liquid medium. By measuring the absorbance of algal fluid and the fluorescence value of neutral oil after Nile red staining, the cell proliferation rate of microalgae can be mastered. According to the situation of oil accumulation, through the comprehensive screening of two basic indicators and the product of the two, the mutant algae strains with high oil production can be better screened, and the possibility of biodiesel production by microalgae can be further improved.

Description

A kind of method utilizing ARTP mutagenesis and screening high oil-yielding microalgae

technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for utilizing ARTP to mutagenize and screen microalgae with high oil yield.

Background technique

Due to the non-renewable nature of fossil fuels and the impact of their combustion on the environment, clean fuels and renewable new energy have attracted widespread attention around the world today. Biomass energy is the most common renewable energy on earth. It is an energy form that stores solar energy in the form of chemical energy through plant photosynthesis, which is called green energy. Biodiesel refers to fatty acid methyl or ethyl esters formed by transesterification of oils and fats from living organisms. It is a biomass liquid fuel and has become a hot spot in biomass energy research in recent years.

At present, the key problem restricting the development of biodiesel is the serious shortage of raw materials. Microalgae are considered to be the most potential biological resources to replace traditional fossil fuels due to their many advantages, such as many species, fast growth rate, strong carbon sequestration ability, and low growth environment requirements. . However, the biomass and oil content of different algae species are different, and the biomass yield and oil content are not positively correlated. Low trend; in addition, under environmental stress, Chlorella biomass showed lower levels, but higher lipid content. Therefore, single selection of mutant algal strains with high biomass or high oil content cannot fully exploit the advantages of microalgal biomass energy.

SUMMARY OF THE INVENTION

In view of the above technical problems, the purpose of the present invention is to provide a method for using ARTP to mutagenize and screen microalgae with high oil yield.

The present invention adopts following technical scheme to realize:

A method for using ARTP to mutagenize and screen microalgae with high oil yield. In the method, algal liquid is first subjected to ARTP mutagenesis to obtain mutants of various traits, and then algal strains with target traits-high oil yield are obtained by screening, specifically including the following step:

(1) the microalgae algal liquid of the algal liquid to be mutagenized to the logarithmic phase;

(2) Determination of plasma mutagenesis parameter information, including voltage, current, ventilation flow and mutagenesis distance;

(3) Set different mutagenesis times to mutagenize the microalgae liquid;

(4) Transfer the mutagenized algal liquid to a pre-sterilized mixed solution containing 10% glycerol and culture solution, so that the final concentration of glycerol is 5%, and the culture solution is hereinafter referred to as BG- 11;

(5) the algal liquid is diluted and evenly coated on the solid medium, and a blank control is made with the unmutated algal liquid coating plate;

(6) Invert cultivation observation in a constant temperature light incubator, after culturing to 10d, count the mutagenized algal strains and the control algal strains on the solid medium respectively and determine the ARTP mutagenesis under each mutagenesis time. fatality rate;

(7) Use the sterilized inoculation loop to select mutant algae strains with larger diameter and darker color, and inoculate them into 6-well culture plates containing BG-11 medium, and place them in a constant temperature light incubator for batches nourish;

(8) Measure the absorbance OD680 of the microalgae cultured in the 6-well plate on the third day and the eighth day and the oil fluorescence value after Nile red staining, to obtain the specific growth rate and relative growth rate of each mutant algal strain. The fluorescence value is used to evaluate the proliferation speed and oil accumulation of each mutant algal strain;

(9) A screening principle of three indicators was established, and mutants with high oil yield were obtained by taking the specific growth rate, the relative fluorescence value and the product of the specific growth rate and the relative fluorescence value as the screening indicators;

(10) It is determined that the specific growth rate is greater than 10% of the normal strain, and the relative fluorescence value is greater than 20% of the normal strain as the first screening principle; the second screening is that the product of the specific growth rate and the relative fluorescence value is greater than 35% of the control strain In principle, trapping and screening high specific growth rate mutants or high relative fluorescence value mutants;

(11) Subculture the screened dominant algal strains, measure the changes in specific growth rate and relative fluorescence value, and use SPSS data statistical analysis to determine whether the difference in subculture is significant. Genetically stable expression of high lipid genes;

(12) Determine algal strains with high oil production by combinatorial screening, and conduct oil gene expression assays, and finally determine mutants with high oil gene expression and expand them for subsequent research on biodiesel production.

A further improvement of the present invention is that the method for pretreatment of algal liquid in step (1) is as follows: the optimum mutagenesis phase of the microalgae is logarithmic phase, the cell concentration is 10 ⁶ cells/ml, and the cell viability under this concentration is stronger and higher Mutation is prone to occur; 5 mL of medium is mixed with 5 mL of 10% glycerol in the same volume, and the final concentration of glycerol is 5%, as a protective nutrient solution after mutagenesis.

A further improvement of the present invention is that in step (2), the mutagenizer controls the power supply input voltage of 120V, the current of 1A, the incoming air flow is 5L/min, and the mutagenesis distance is adjusted to 2mm.

A further improvement of the present invention is that in step (3), the treatment time for mutagenizing the algal liquid is 0s, 10s, 20s, 30s, 40s, 50s, 60s, 65s, and 70s; The stage, and the algae liquid to be mutagenized is stored with a sterilized cover in advance and placed on the stage, and the distance between the cover and the outlet of the plasma device is adjusted to 2mm; the plasma voltage is set to 120V, the current is 1A, The air flow was 5L/min and the pressure was stabilized for 10min; the algal liquid was mutagenized by adjusting different mutagenesis times, and the optimal mutagenesis time was determined with the lethality rate above 95% as a reference.

A further improvement of the present invention is that in step (4), the mutagenized algal liquid is required to be transferred into a mixed solution of glycerol and BG-11 medium to protect the mutant; 5 mL of sterilized BG-11 medium is prepared separately. And 5 mL of 10% sterilized glycerol solution by volume was added to a 50 mL plastic centrifuge tube, so that the final volume fraction of glycerol was 5%.

A further improvement of the present invention is that in step (5), the specific operation of diluting and coating the plate is as follows: take 1 mL of the mutagenized algal liquid, that is, a mixed solution containing 5% glycerol and BG-11 medium, and add it to 9 mL of the mixed solution that contains Dilute it in a mixed solution of 5% glycerol and BG-11 medium; then take 200 μL of the diluted algal liquid and add it to a 90mm solid plate containing BG-11 medium and spread it evenly with a sterilized triangular coating rod. Immediately cover the lid and put it upside down into a constant temperature light incubator for cultivation; under each mutagenesis condition, coat three plates as a parallel, and use the algal liquid without mutagenesis as a blank control.

A further improvement of the present invention is that in steps (6) and (7), both solid culture and 6-well plate culture are required to be cultured in a constant temperature illumination incubator, the illumination period is 12h:12h, and the culture temperature is 25±1°C; the solid culture is 10d Then, count the algae in the mutagenesis group and the control group, calculate the lethality rate and draw the lethality curve, and calculate the lethality rate according to the following formula:

Lethality = (the number of algal colonies in the non-mutated group - the number of algal colonies in the mutagenic group)/the number of algal colonies in the non-mutated group × 100%

Taking the lethality rate of more than 95% as the judgment index of the best mutagenesis time, the mutant algae strains with larger diameter and darker color were selected with a sterilized inoculation ring for the grown mutant algae strains and put them in a 6-well plate for liquid. nourish.

A further improvement of the present invention is that in step (8) and step (9), the specific growth rate and relative fluorescence value are determined by the absorbance value measured at 680 nm using an ultraviolet spectrophotometer and the fluorescence value of the mutant algal liquid stained with Nile red, respectively. Measure the fluorescence value under the emission light wavelength of 570nm under the spectrophotometer, which is determined by the excitation light wavelength of 480 and the emission wavelength range of 500-650nm, and adopts the 6-well plate culture method in step (8) to realize the rapid cultivation of mutant algae, Shorten the overall mutagenesis time.

A further improvement of the present invention is that the first screening principle considered in step (10) can screen the dominant algal strains with both fast growth rate and oil accumulation ability, that is, two dominant traits; and through the second screening principle The single dominant trait of the dominant mutants with high growth rate or high oil accumulation was obtained; all high oil mutants were obtained through two screening principles.

A further improvement of the present invention is that the genetic stability considered in step (11) is based on whether the difference in specific growth rate and relative fluorescence value between generations is significant and whether the screening conditions can be maintained;

The high-oil-producing mutant algal strain obtained in step (12) is expanded and cultured, and after the periodical culture is completed, the algal fluid is collected by centrifugation and stored at -80° C. RT-qPCR technology was used to determine the expression of related genes, and finally to verify and determine the oil-producing ability of the high-oil-producing mutants.

The present invention at least has the following beneficial technical effects:

In order to better reflect the oil production capacity of microalgae, considering both high biomass and high oil content is the most effective indicator to evaluate the high oil production capacity of microalgae. In the present invention, by performing ARTP mutagenesis on the algal fluid and rapidly culturing in a 6-well plate, a comprehensive screening method based on the specific growth rate, the relative oil fluorescence and the comprehensive index of the product of the specific growth rate and the relative oil fluorescence is established as the leak-trap screening. In principle, the mutant algal strains with higher oil production were screened out. Whether it is possible to increase the oil production of microalgae and reduce the cost of products, the key lies in whether it can ensure high biomass and increase the oil content of microalgae, so that the oil production of microalgae can reach a high level in a short time. The algal mutation breeding technology plays an increasingly important role in the field of biomass energy. The mutagenic algal strain obtained by the screening of the present invention can be used in the following aspects:

(1) It can quickly and efficiently screen mutant algal strains with high oil production, which has great advantages in mutation breeding;

(2) Algal strains with different characters such as high biomass, high oil content and high biomass and high oil content can be obtained by using the principle of combination screening, which can be used for different purposes.

To sum up, the present invention firstly determines the mutagenesis parameters of atmospheric pressure and room temperature plasma, changes the mutagenesis time to mutagenize the microalgae, and obtains the optimal mutagenesis time through the lethality rate. The mutant algal strains were screened by comprehensive indicators, that is, the specific growth rate and the relative fluorescence value were the priority screening principle, and the product of the specific growth rate and the relative fluorescence value was the leak-trap screening principle, and finally high-yielding mutant algal strains were obtained. With some screening methods, all mutant algal strains with high oil-yielding traits can be obtained by this method, which greatly improves the acquisition rate of high-oil-yielding mutant algal strains and can more effectively screen out target algal strains.

Description of drawings

Fig. 1 is a schematic diagram of the lethality rate under different mutagenesis time;

Figure 2 is a schematic diagram of the screening of high-fat mutants;

Figure 3 shows the genetic stability of high-oil mutants (1-AT60-5 represents the first generation of the fifth strain under the condition of 60s, which is similar to other mutants);

Figure 4 is a graph showing the expression of oil genes accD and dgat7566.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and embodiments,

The invention provides a method for using ARTP to mutagenize and screen microalgae with high oil yield, and establishes a high-efficiency screening method for high oil-yielding algae strain by using ARTP. The algal liquid is firstly mutated by ARTP to obtain mutants of various traits, and then an algal strain with a target trait-high oil yield is obtained by an efficient screening method, which specifically includes the following steps:

(1) Select the algae liquid of suitable concentration to carry out mutagenesis, that is, the algae liquid is cultured to logarithmic phase, and the concentration is kept at about 10 ⁶ /mL; the method of algae liquid pretreatment in this step is: the optimal mutagenesis of microalgae The phase is logarithmic phase. At this time, the cell viability is strong, and the cell concentration is about 10 ⁶ cells/ml. Take 0.1 mL of the algal fluid in this period for mutagenesis; 5 mL of medium and 5 mL of 10% glycerol are mixed, and the final concentration of glycerol is 5 % as a protective solution after mutagenesis.

(2) The determination of the basic parameters of the plasma and the stability of the device before the mutagenesis need to be ventilated and energized; in this step, the mutagenizer controls the power supply input voltage of 120V, the current of 1A, the incoming air flow rate is 5L/min, and the mutagenesis distance is adjusted to 2mm . The instrument needs to be warmed up and ventilated for 10 minutes before mutagenesis.

(3) Take time as the variable of the mutagenesis process, and mutagenize the algae; in this step, the specific operation of mutagenizing the algae liquid is as follows: the treatment time for mutagenizing the algae liquid is 0s, 10s, 20s, 30s, 40s, 50s, 60s, 65s, 70s. Before mutagenesis, wipe the stage under the plasma with alcohol, and use a pre-sterilized plastic cover with a diameter of 1 cm to store the algal liquid to be mutagenized and place it on the stage, and adjust the distance between the cover and the outlet of the plasma device. The distance between them is 2mm. The plasma voltage was set to 120V, the current was 1A, the air flow rate was 5L/min and the voltage was stabilized for 10min. The algal fluid was mutagenized by adjusting different mutagenesis times, and the optimal mutagenesis time was determined with the lethality rate above 95% as a reference.

(4) Use 5% glycerol medium (BG-11) to protect the mutagenized mutants and stabilize them for 3 hours; this step requires that the mutagenized algal fluid be transferred to BG containing 5% glycerol In the -11 medium mixed solution, high concentrations of glycerol can protect the generated mutants to maintain the current mutant traits.

(5) after the mutagenized algal liquid and the unmutated original algal strain are diluted ¹⁰ times, evenly spread on the solid medium in a sterile environment; in this step, the specific operation of the dilution coating plate is: Transfer 100 μL of the transformed algal liquid together with the sterilized cover to 10 mL of mixed nutrient solution (BG-11 medium containing 5% glycerol) and place it in a light incubator for 3 hours, and then take the stabilized 1 mL of mutagenized algal liquid and add it to the Dilute to 9 mL of mixed solution (BG-11 medium containing 5% glycerol), and dilute 10 ³ times before and after. Finally, 200 μL of the diluted mutant algal liquid was added to a 90 mm solid plate containing BG-11 medium and spread evenly with a sterilized triangular coating rod. Immediately cover the lid and put it upside down into a constant temperature light incubator for cultivation. Three plates were plated in parallel for each mutagenesis condition, and the unmutated algal fluid was used as a blank control.

(6) culturing the coated solid and inverting the culture in a constant temperature light incubator for observation, after culturing for 10d, count the algal colonies grown in the mutagenesis group and the control group respectively and calculate the lethality under each mutagenesis time;

(7) Use the sterilized inoculation loop to select the algal strain with a larger diameter and a darker color for the growth of the mutagenized algae colonies, inoculate the algal strains into a 6-well plate and culture and screen with a liquid medium; steps (6) and (7) Medium requires solid culture and 6-well plate culture to be cultured in a constant temperature light incubator, the light cycle is 12h:12h, and the culture temperature is 25±1℃. After 10 days of solid culture, obvious algal colonies were formed, and the algal colonies were counted for the mutagenesis group and the control group, and the lethality at each mutagenesis time was calculated and the lethality curve was drawn. Calculate the lethality according to the following formula:

Lethality = (the number of algal colonies in the control group - the number of algal colonies in the mutagenesis group)/the number of algal colonies in the non-mutated group × 100%

The best mutagenesis time was judged by the lethality rate above 95%. For the grown mutant algal strains, use a sterilized inoculation loop to select mutant algal strains with larger diameters and darker colors into a 6-well plate for liquid culture.

(8) Measure the absorbance OD680 of the microalgae cultured in the 6-well plate on the third day and the eighth day and the oil fluorescence value after Nile red staining, to obtain the specific growth rate and relative growth rate of each mutant algal strain. The fluorescence value is used to evaluate the proliferation speed and oil accumulation of each mutant algal strain;

(9) A screening principle of three indicators is established, and a screening method for mutants with high oil yield is obtained by taking the specific growth rate, the relative fluorescence value and the product of the specific growth rate and the relative fluorescence value as the screening indicators; step (8) In (9), the specific growth rate and relative fluorescence value of algal cells are required to be measured by UV spectrophotometer at 680nm, respectively, and the oil fluorescence before and after the mutant algal liquid is stained with Nile red is measured under a fluorescence spectrophotometer, That is, the fluorescence value at the emission wavelength of 570 nm was measured (excitation wavelength 480, emission wavelength range 500-650 nm).

(10) It is determined that the specific growth rate is greater than 10% of the normal strain, and the relative fluorescence value is greater than 20% of the normal strain as the first screening principle; the second screening is that the product of the specific growth rate and the relative fluorescence value is greater than 35% of the control strain In principle, the main trap screen for high specific growth rate mutants or high relative fluorescence value mutants. The first screening principle considered in this step can screen the dominant algal strains with both fast growth rate and oil accumulation ability, that is, two dominant traits; and the second screening principle to obtain high growth rate or high oil accumulation. Single-dominant traits of dominant mutants. All high-oil mutants can be obtained by two screening principles.

(11) Subculture the screened dominant algal strains according to the combined screening principle in step (10), measure the changes in specific growth rate and relative fluorescence value, and use SPSS data statistical analysis to determine whether the difference in subculture is significant, and the difference is not significant. Significantly, the subculture characters are relatively stable, and the genetically stable expression of the high-oil gene is satisfied. The genetic stability considered in this step is based on whether the differences in specific growth rates and relative fluorescence values between generations are significant and whether the screening conditions can be maintained.

(12) Determine algal strains with high oil production by combinatorial screening, and conduct oil gene expression assays, and finally determine mutants with high oil gene expression and expand them for subsequent research on biodiesel production. The high-yielding oil-producing mutant algal strain obtained in this step is expanded and cultured, and after the cycle is cultured, the algal fluid is collected by centrifugation and stored at -80°C. The gene sequences related to the regulation of oil synthesis were obtained from the gene bank, and the expression levels of the related genes were determined by RT-qPCR technology to finally verify and determine the oil-producing ability of the high-oil-producing mutants.

Example

This embodiment takes Scenedesmus as an example to describe the method in detail. Scenedesmus in this embodiment was purchased from the freshwater algae seed bank of Wuhan Institute of Hydrobiology, Chinese Academy of Sciences. The ARTP mutagenesis system used was purchased from Nanjing Suman Plasma Technology Co., Ltd.

1. Use BG11 medium to cultivate the algae, inoculate 200mL of Scenedesmus liquid in a 500mL triangular conical flask, seal the bottle mouth with a sterile film, place it in a constant temperature light incubator, and cultivate in a semi-continuous culture mode. Average light intensity: 80 μmol·m ^-1 ·s ^-1 , light cycle: 12L:12D, culture temperature: (25±1)°C. Randomly change the position of the conical flask and shake it 3-4 times a day.

2. After culturing to the logarithmic phase, the cell concentration is about 10 ⁶ cells/ml. At this time, the cell viability is strong and it is more suitable for mutagenesis. Take 0.1 mL of algae liquid and put it in a sterilized plastic cover for mutagenesis.

3. Prepare 5mL sterile BG-11 medium and 5mL sterile glycerol solution with a volume fraction of 10% and add them to a 50mL plastic centrifuge tube, so that the final volume fraction of glycerol is 5%, as the mutant protection solution after mutagenesis and nutrient solution;

4. The specific steps of the mutagenesis operation:

(1) The input voltage of the control power supply of the mutagenizer is set to 120V, the current is 1A, the incoming air flow is 5L/min and the voltage is stabilized for 10min.

(2) In this example, the mutagenesis time is adjusted as an example, which are 0s, 10s, 20s, 30s, 40s, 50s, 60s, 65s, and 70s. Before the mutagenesis, wipe the stage under the plasma with alcohol, and use A plastic lid with a diameter of 1 cm that has been sterilized in advance stores the algal liquid to be mutagenized and placed on the stage, and adjusts the distance between the lid and the outlet of the plasma device to be 2 mm. The algal fluid was mutagenized by adjusting different mutagenesis times, and the optimal mutagenesis time was determined with the lethality rate above 95% as a reference.

(3) The mutagenized algal liquid was transferred to BG11 medium containing 5% glycerol (5 mL of BG11 medium + 5 mL of 10% glycerol solution), resuspended and stabilized in a light incubator for 3 hours.

(4) 1 mL of suspended algal liquid (mixed solution containing 5% glycerol and BG-11 medium) was added to 9 mL of mixed solution (BG-11 medium containing 5% glycerol) for dilution. Then take 200 μL of the diluted algal liquid and add it to a 90 mm solid plate containing BG-11 medium and spread it evenly with a sterilized triangular coating rod. Immediately cover the lid and put it upside down into a constant temperature light incubator for cultivation. The culture conditions were a light cycle of 12h:12h and a culture temperature of 25±1°C. Three plates were plated in parallel for each mutagenic condition, and the unmutated algal fluid was used as a blank control.

(5) After culturing for 10 days, count the algal colonies in the mutagenesis group and the control group, calculate the lethality and draw a lethal curve, and calculate the lethality according to the following formula:

Lethality = (the number of algal colonies in the non-mutated group - the number of algal colonies in the mutagenic group)/the number of algal colonies in the non-mutated group × 100%

According to the theory of mutagenesis breeding, it is easier to obtain forward mutations when the lethality is above 95%. The mutant algae colonies with larger diameter and darker color were selected with an inoculation loop and transferred to a 6-well plate for liquid culture. Culture conditions: average light intensity: 80μmol·m ^-1 ·s ^-1 , light cycle: 12L:12D, culture Temperature: (25±1)℃. In order to prevent the occurrence of such phenomena as sticking to the wall and stagnation, the six-well plate was slightly shaken twice a day for 1 minute each time.

Finally, 60s and 65s were determined as the best mutagenesis time (the lethality rate reached more than 95%, and the lethality rate reached 100% after 70s).

(5) on the third day and the eighth day, carry out absorbance OD680 and the oil fluorescence value after dyeing with Nile red to monitor, OD680nm is the absorbance value that ultraviolet spectrophotometer measures when wavelength 680nm, and the relative fluorescence value measuring method is, Take 2 mL of algae liquid, add 1 mL of 25% dimethyl sulfoxide, and make two groups at the same time (one group is blank), ultrasonically (500KW) for 3 minutes, take one group and add 50μL of Nile red reagent (100mg/ L), after being protected from light for 15 min, the fluorescence value at the emission wavelength of 570 nm was measured under the fluorescence spectrophotometer (excitation light wavelength 480, emission wavelength range 500-650 nm). In this way, the specific growth rate and relative fluorescence value of each mutant algal strain were obtained, which were used to evaluate the cell proliferation speed and oil accumulation of each mutant algal strain;

(6) The specific screening principle is that the specific growth rate is greater than 10% of the normal strain, and the relative fluorescence value is greater than 20% of the normal strain as the first screening principle (preferred); the product value of the specific growth rate and the relative fluorescence value is greater than that of the normal strain. 35% is the principle of trapping and screening, and this condition is mainly for trapping and screening mutants with high specific growth rate or high relative fluorescence value. During the screening process, AT named mutants, and the mutants were distinguished by mutagenesis time. For example, AT60-1 represented the first algal strain selected under 60s. The mutant algal strains screened by the above principles were inoculated into 6-well plates and subcultured under the same conditions.

(7) Measure the changes in the specific growth rate and relative fluorescence value of Scenedesmus in the 6-well plate, and use SPSS data statistical software to analyze whether the difference in subculture is significant (based on satisfying the screening conditions). The culture characters are relatively stable, and the genetically stable expression of the high-oil gene is satisfied. As shown in Figure 3:

(8) After determining the oil production stability of each mutant strain, expand the culture of each dominant mutant strain (cultivated in a 250 mL conical flask, and the culture conditions remain unchanged). Store in -80°C refrigerator. With reference to the sequences of the lipid genes accD and dgat7566 that regulate lipid anabolism in gen bank (https://www.ncbi.nlm.nih.gov/), the expression of each lipid gene was determined by q-PCR technology. As shown in Figure 4:

After measuring the oil gene expression, it was found that the oil gene expression of the mutant strain was much higher than that of the normal strain, and the high-yielding oil mutants could be obtained by the above screening.

The inventors screened out a dominant mutant algal strain with stable genetic performance and oil yield of 343 mg/L by the above method, which is 117% higher than the normal strain, greatly improving the possibility of microalgal biomass for biodiesel production. As shown in Table 1:

Table 1 Comparison of biomass and oil accumulation of mutants

Claims (10)

1. A method for mutagenesis and screening microalgae with high oil yield by using ARTP is characterized in that in the method, algae liquid is firstly mutagenized by ARTP to obtain various character mutants, and then screened to obtain algae strains with target characters and high oil yield, and the method specifically comprises the following steps:

(1) carrying out mutagenesis on the microalgae solution to be subjected to mutagenesis to obtain microalgae solution in a logarithmic phase;

(2) determining plasma mutagenesis parameter information, including voltage, current, ventilation flow and mutagenesis distance;

(3) setting different mutagenesis time to carry out mutagenesis on the microalgae solution;

(4) transferring the mutagenized algae liquid to a pre-prepared sterilized mixed solution containing 10% of glycerol and a culture solution to ensure that the final concentration of the glycerol is 5%, wherein the culture solution is abbreviated as BG-11 hereinafter;

(5) diluting the algae liquid, uniformly coating the algae liquid on a solid culture medium, and coating a flat plate with the non-mutagenized algae liquid as a blank control;

(6) inversely culturing and observing in a constant-temperature illumination incubator, after culturing for 10 days, respectively counting the mutagenic algae strains and the control algae strains on the solid culture medium, and determining the lethality after ARTP mutagenesis at each mutagenesis time;

(7) selecting mutant algae strains with larger diameter and darker color by using the sterilized inoculating loop, inoculating the mutant algae strains into a 6-hole culture plate containing BG-11 culture medium, and placing the mutant algae strains in a constant-temperature illumination incubator for batch culture;

(8) respectively measuring the absorbance OD680 of the microalgae cultured in the 6-hole plate on the third day and the eighth day and the fluorescent value of the grease after nile red staining to obtain the specific growth rate and the relative fluorescent value of each mutant strain, and evaluating the proliferation speed and the grease accumulation condition of each mutant strain;

(9) establishing a screening principle of three indexes, and obtaining a mutant with high grease yield by taking a specific growth rate, a relative fluorescence value and a product value of the specific growth rate and the relative fluorescence value as screening indexes;

(10) determining that the specific growth rate is greater than 10% of that of a normal plant and the relative fluorescence value is greater than 20% of that of the normal plant as a first screening principle; the high specific growth rate mutant or the high relative fluorescence value mutant is screened in a leak-repairing mode according to a second screening principle that the product value of the specific growth rate and the relative fluorescence value is larger than 35% of that of a control strain;

(11) subculturing the screened dominant algae, determining the change of the specific growth rate and the relative fluorescence value of the dominant algae, and determining whether the subculturing difference is obvious or not by utilizing SPSS data statistical analysis, wherein the subculturing character is stable if the difference is not obvious, and the genetic stable expression of the high-fat gene is met;

(12) determining algae strains with high grease yield by a combined screening mode, determining grease gene expression, finally determining mutants with high grease gene expression, and carrying out expanded culture on the mutants so as to carry out subsequent research on preparation of biodiesel.

2. The method for mutagenizing and screening microalgae with high oil yield by ARTP according to claim 1, wherein the pretreatment of the microalgae solution in step (1) comprises: the optimum mutagenesis phase of the microalgae is logarithmic phase, and the cell concentration is 10⁶Cell viability at this concentration is strong and more prone to mutation; and 5mL of medium was mixed with the same volume of 5mL of 10% glycerol, with a final concentration of 5% glycerol, as a protective nutrient solution after mutagenesis.

3. The method for mutagenizing and screening microalgae with high oil production rate by ARTP according to claim 1, wherein in the step (2), the mutagenizing instrument controls the power input voltage to 120V and the current to 1A, the flow rate of the introduced air is 5L/min, and the mutagenizing distance is adjusted to 2 mm.

4. The method for mutagenizing and screening microalgae with high oil yield by ARTP according to claim 1, wherein the mutagenizing treatment time for the microalgae solution in the step (3) is 0s, 10s, 20s, 30s, 40s, 50s, 60s, 65s, 70 s; wiping an objective table below the plasma with alcohol before mutagenesis, storing algae liquid to be mutagenized by using a pre-sterilized cover, placing the algae liquid on the objective table, and adjusting the distance between the cover and an emergent port of the plasma device to be 2 mm; setting the plasma voltage to be 120V and the current to be 1A, wherein the introduced air flow is 5L/min and the voltage is stabilized for 10 min; and (3) carrying out mutagenesis on the algae liquid by adjusting different mutagenesis time, and determining the optimal mutagenesis time by taking the lethality rate of more than 95% as a reference.

5. The method for mutagenizing and screening microalgae with high oil yield by ARTP according to claim 1, wherein the mutagenized microalgae liquid in step (4) is transferred to the mixed solution of glycerol and BG-11 culture medium to protect the mutants; 5mL of sterilized BG-11 medium and 5mL of a 10% volume fraction sterilized glycerol solution were prepared and added to a 50mL plastic centrifuge tube, respectively, to give a final volume fraction of 5% glycerol.

6. The method for mutagenizing and screening microalgae with high oil production rate by ARTP according to claim 1, wherein the dilution coating plate in the step (5) is operated as follows: adding 1mL of mutagenized algae solution, namely a mixed solution containing 5% of glycerol and a BG-11 culture medium, into 9mL of mixed solution, namely a mixed solution containing 5% of glycerol and a BG-11 culture medium, and diluting; adding 200 μ L of diluted algae solution onto 90mm solid plate containing BG-11 culture medium, uniformly coating with sterilized triangular coating rod, immediately covering with a cover, and inversely placing into a constant temperature illumination incubator for culture; three plates were coated as replicates for each mutagenic condition and a blank was made with non-mutagenized algal solution.

7. The method for mutagenizing and screening microalgae with high oil yield by ARTP according to claim 1, wherein the solid culture and the 6-well plate culture in steps (6) and (7) are both cultured in a constant-temperature illumination incubator with an illumination period of 12h:12h and a culture temperature of 25 +/-1 ℃; after solid culture for 10 days, counting algae colonies of the mutagenesis group and the control group, calculating the lethality rate, drawing a lethality curve, and calculating the lethality rate according to the following formula:

lethality rate ═ number of non-mutagenized group algae colonies)/number of non-mutagenized group algae colonies × 100%

Selecting the mutant algae with larger diameter and darker color from the grown mutant algae by using a sterilized inoculating loop to perform liquid culture in a 6-well plate by taking the lethality of more than 95 percent as a judgment index of the optimal mutagenesis time.

8. The method as claimed in claim 1, wherein the specific growth rate and the relative fluorescence in step (8) and step (9) are determined by measuring the absorbance at 680nm with UV spectrophotometer and the fluorescence at 570nm with Nile red staining mutant algae solution with fluorescence spectrophotometer, which is calculated from the excitation light wavelength 480 and the emission wavelength range 500 and 650nm, respectively, and the step (8) adopts 6-well plate culture method to realize rapid culture of mutant algae, thereby shortening the whole mutagenesis time.

9. The method for mutagenizing and screening microalgae with high oil yield by ARTP according to claim 1, wherein the first screening principle considered in step (10) can screen mutation of dominant algae strain with both rapid growth rate and oil accumulation ability, i.e. having two dominant traits; and the single dominant character of the dominant mutant with high growth rate or high grease accumulation is obtained through a second screening principle; all high oil mutants were obtained by two screening principles.

10. The method for mutagenizing and screening microalgae with ARTP and having high oil production rate as claimed in claim 1, wherein the genetic stability considered in the step (11) is based on the requirement of whether the difference of specific growth rate and relative fluorescence value between generations is significant and the screening condition can be maintained;

carrying out amplification culture on the high-yield oil mutant algae strain obtained in the step (12), and after the periodic culture is finished, collecting algae liquid by using a centrifugal method and storing at-80 ℃; gene sequences for relevant regulation and control of oil synthesis are obtained through gene bank, expression quantity of relevant genes is measured by RT-qPCR technology, and finally oil production capacity of the high oil production mutant strain is verified and determined.

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