CN110885816A - Method for mutagenizing and screening microalgae with high oil yield by ARTP - Google Patents
Method for mutagenizing and screening microalgae with high oil yield by ARTP Download PDFInfo
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Abstract
The invention discloses a method for mutagenizing and screening microalgae with high oil yield by utilizing ARTP, which mainly comprises two processes: (1) performing mutagenesis treatment on the microalgae by using ARTP, and determining the optimal mutagenesis time by adjusting the influence of the mutagenesis time on the mutagenesis effect and taking the lethality as a basis; (2) the algae liquid after mutagenesis at each mutagenesis time is subjected to gradient dilution (the algae liquid can be efficiently and accurately counted). Uniformly coating on a solid culture medium and performing inverted culture in a light incubator. After 10 days of culture, selecting algae colonies with deep color and large diameter, inoculating the algae colonies into a liquid culture medium, determining the absorbance of an algae solution and the fluorescence value of neutral oil dyed by Nile red to master the cell proliferation rate and the oil accumulation condition of the microalgae, and comprehensively screening by the product of two basic indexes and the product of the two basic indexes to better screen out mutagenic algae strains with high oil yield and further improve the possibility of preparing biodiesel by the microalgae.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for mutagenizing and screening microalgae with high oil yield by using ARTP.
Background
Due to the non-renewable nature of fossil fuels, and the environmental impact of their combustion, clean fuels and new renewable energy sources are gaining wide attention in countries of the world today. Biomass energy is one of the most common renewable energy sources on earth, and is a form of energy in which solar energy is stored in the form of chemical energy in organisms through plant photosynthesis, which is called green energy. Biodiesel refers to fatty acid methyl ester or ethyl ester formed by transesterification of grease from organisms, is a biomass liquid fuel, and has become a hotspot in biomass energy research in recent years.
At present, the key problem restricting the development of biodiesel is that the raw materials are seriously insufficient, and microalgae has the advantages of multiple types, high growth rate, strong carbon fixation capacity, low requirement on growth environment and the like, so that the microalgae is considered as a biological resource with the most potential to replace the traditional fossil fuel. However, the biomass and the oil content of different algae species are different, the biomass yield and the oil content are not positively correlated, for example, the oil content of Scenedesmus tetrandrus shows a lower trend although the biomass can reach a high level in the culture process; in addition, chlorella biomass showed lower levels but higher oil content under environmental stress. Therefore, the single selection of the mutant strain with high biomass or high oil content cannot fully exert the advantages of the microalgae biomass energy.
Disclosure of Invention
In view of the above problems, the present invention provides a method for mutagenizing ARTP and screening microalgae with high oil yield.
The invention is realized by adopting the following technical scheme:
a method for inducing and screening microalgae with high oil yield by using ARTP comprises the steps of firstly obtaining various character mutants by inducing the algae liquid by the ARTP, and then obtaining algae strains with target characters and high oil yield by screening, wherein the method comprises the following specific 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.
The further improvement of the invention is that the method for pretreating the algae liquid in the step (1) comprises the following steps: 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.
The further improvement of the invention is that in the step (2), the mutagenic instrument controls the input voltage of the power supply to be 120V and the current to be 1A, the introduced air flow is 5L/min, and the mutagenic distance is adjusted to be 2 mm.
The invention further improves that the processing time for mutagenizing the algae liquid in the step (3) is 0s, 10s, 20s, 30s, 40s, 50s, 60s, 65s and 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.
The invention has the further improvement that the mutagenized algae liquid is required to be transferred into the mixed solution of glycerol and BG-11 culture medium in the step (4) to protect the mutant; 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.
In a further improvement of the present invention, the dilution coating flat plate in the step (5) is operated as: 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.
The invention has the further improvement that in the steps (6) and (7), the solid culture and the 6-hole plate culture are both required to be cultured in a constant-temperature illumination incubator, the illumination period is 12h:12h, and the culture temperature is 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.
The invention is further improved in that the specific growth rate and the relative fluorescence value in the step (8) and the step (9) are respectively determined by an absorbance value measured by an ultraviolet spectrophotometer at 680nm and a fluorescence value measured by a nile red mutant algae liquid staining under a fluorescence spectrophotometer at an emission wavelength of 570nm, wherein the fluorescence values are determined by calculation of an excitation light wavelength of 480 and an emission wavelength range of 500-650nm, and the step (8) adopts a 6-well plate culture method to realize rapid culture of the mutant algae, thereby shortening the whole mutagenesis time.
The further improvement of the invention is that the first screening principle considered in the step (10) can screen mutation of dominant algae strains with rapid growth speed and grease accumulation capacity, namely, the dominant algae strains have 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.
A further improvement of the invention is that the genetic stability considered in step (11) is based on the requirement whether the specific growth rate and relative fluorescence value differences between generations are significant and the screening conditions 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.
The invention has at least the following beneficial technical effects:
in order to better reflect the oil production capacity of the microalgae, the most effective index for evaluating the high oil production capacity of the microalgae is considered by simultaneously considering high biomass and high oil content. According to the invention, the algae liquid is subjected to ARTP mutagenesis and is subjected to 6-hole plate rapid culture, and a comprehensive principle that the specific growth rate, the relative grease fluorescence basic screening principle and the product of the specific growth rate and the relative grease fluorescence are taken as the leak-repairing screening principle is established, so that the mutant algae strains with high oil yield are screened. Whether the oil yield of the microalgae can be improved and the product cost can be reduced or not is crucial in that whether the oil content of the microalgae can be improved while high biomass is ensured can be selected, so that the oil yield of the microalgae can reach a higher level in a short time, and therefore, the microalgae mutation breeding technology becomes more and more important in the field of biomass energy. The mutant strain obtained by screening can be used for the following aspects:
(1) the mutant strain with high grease yield can be quickly and efficiently screened, and the mutant strain has great advantages in mutation breeding;
(2) the combined screening principle can be used for obtaining the algae strains with different properties such as high biomass, high grease content, high biomass, high grease content and the like, and can be used for different purposes.
In summary, the present invention firstly determines the normal pressure room temperature plasma mutagenesis parameters to mutate the microalgae by changing the mutagenesis time, obtains the optimal mutagenesis time through the lethality rate, and on this basis, performs comprehensive index screening on the mutant algae strains under the optimal mutagenesis time, i.e. the product of the specific growth rate and the relative fluorescence value is the preferential screening principle, and the product of the specific growth rate and the relative fluorescence value is the leak-repairing screening principle, and finally obtains the high oil yield mutant algae strains.
Drawings
FIG. 1 is a graph showing lethality at different mutagenesis times;
FIG. 2 is a schematic diagram of screening for high-lipid mutants;
FIG. 3 shows the genetic stability of the high-lipid mutant (1-AT60-5 represents the first generation of the fifth strain under 60s conditions, similar to the other mutants);
FIG. 4 shows the expression patterns of the genes accD and dgat7566 in oil and fat.
Detailed Description
The invention is further described with reference to the following figures and examples,
the invention provides a method for mutagenizing and screening microalgae with high oil yield by using ARTP, and establishes a high-efficiency screening method for microalgae with high oil yield by using ARTP. The method comprises the following steps of firstly carrying out ARTP mutagenesis on algae liquid to obtain various character mutants, and then carrying out an efficient screening method to obtain an algae strain with target characters and high oil yield, wherein the method comprises the following steps:
(1) selecting proper concentration of algae liquid for mutagenesis, namely culturing the algae liquid to logarithmic phase, and keeping the concentration at 106About one/mL; the method for pretreating the algae liquid in the step comprises the following steps: the optimum mutagenesis phase of the microalgae is logarithmic phase, the cell activity is strong, and the cell concentration is 106About one per milliliter, 0.1mL of algae liquid at the current period is taken for mutagenesis; 5mL of the medium was mixed with 5mL of 10% glycerol to a final concentration of 5% as a protective solution after mutagenesis.
(2) Determining basic parameters of the plasma and leading the device to be ventilated and electrified stably before mutagenesis; in the step, the mutagen instrument controls the input voltage of a power supply to be 120V and the current to be 1A, the introduced air flow is 5L/min, and the mutagenesis distance is adjusted to be 2 mm. The instrument was preheated and aerated for 10 minutes prior to mutagenesis.
(3) The time is taken as the variable of the mutagenesis process, and the algae are also mutagenized; in the step, the specific operation of carrying out mutagenesis on the algae liquid is as follows: the time for mutagenizing the algal solution was 0s, 10s, 20s, 30s, 40s, 50s, 60s, 65s, and 70 s. Before mutagenesis, an objective table below the plasma is wiped by alcohol, algae liquid to be mutagenized is stored on the objective table by utilizing a plastic cover which is sterilized in advance and has the diameter of 1cm, and the distance between the cover and an emergent port of the plasma device is adjusted to be 2 mm. Setting the plasma voltage to be 120V and the current to be 1A, leading in air flow to be 5L/min and stabilizing the pressure 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.
(4) Protecting mutants generated by mutagenesis by using a culture medium (BG-11) containing 5% of glycerol and stabilizing for 3 hours; in the step, the mutagenized algae liquid is required to be transferred into a BG-11 culture medium mixed solution containing 5% of glycerol, and the glycerol at a high concentration can protect the generated mutant from keeping the current mutation character.
(5) Diluting the mutagenized algae liquid and the original strain without mutagenesis 103After doubling, uniformly coating the solid culture medium in a sterile environment; the operation of the dilution coating flat plate in the step is as follows: transferring mutagenized 100 μ L algae solution together with sterilized cover to 10mL mixed nutrient solution (BG-11 medium containing 5% glycerol) and placing in light incubator for stabilization for 3h, adding stabilized 1mL mutagenized algae solution into 9mL mixed solution (BG-11 medium containing 5% glycerol) for dilution, and diluting to 103And (4) doubling. And finally, adding 200 mu L of diluted mutant algae liquid to a 90mm solid plate containing BG-11 culture medium, uniformly coating the mixture by using a sterilized triangular coating rod, immediately covering the cover, and inversely placing the cover 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.
(6) Culturing the coated solid, performing inverted culture observation on a constant-temperature illumination incubator, respectively counting algae colonies grown from a mutagenesis group and a control group after culturing for 10d, and calculating the lethality at each mutagenesis time;
(7) selecting algae strains with larger diameter and darker color from the grown mutagenic algae colonies by using a sterilized inoculating loop, inoculating the algae strains to a 6-hole plate, and culturing and screening the algae strains by using a liquid culture medium; in the steps (6) and (7), both solid culture and 6-hole plate culture are required to be cultured in a constant-temperature illumination incubator, the illumination period is 12 hours: 12 hours, and the culture temperature is 25 +/-1 ℃. After solid culture for 10 days, forming more obvious algae colonies, counting the algae colonies of the mutagenesis group and the control group, calculating the lethality at each mutagenesis time, drawing a lethality curve, and calculating the lethality according to the following formula:
lethality rate ═ number of colonies of control group algae-colonies of mutagenized group algae)/number of colonies of non-mutagenized group algae × 100%
The determination index of the optimal mutagenesis time is more than 95 percent of the lethality. And selecting mutant algae with larger diameter and darker color from the grown mutant algae by using a sterilized inoculating loop, and carrying out liquid culture on the mutant algae in a 6-well plate.
(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 screening method of the 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; in the steps (8) and (9), the specific growth rate and the relative fluorescence value of the algae cells are respectively measured by an ultraviolet spectrophotometer at 680nm under a fluorescence spectrophotometer, namely, the fluorescence value (the excitation wavelength is 480, and the emission wavelength is 500-650nm) under the emission wavelength of 570nm through the oil fluorescence before and after the nile red is used for dyeing the mutant algae liquid.
(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; and (3) mainly performing leak-filling screening on the mutants with high specific growth rate or the mutants with high relative fluorescence value according to a second screening principle that the product value of the specific growth rate and the relative fluorescence value is greater than 35% of that of the control strain. The first screening principle considered in the step can screen mutation of dominant algae strains with rapid growth speed and grease accumulation capacity, namely the dominant algae strains have two dominant traits; and acquiring the single dominant character of the dominant mutant with high growth rate or high grease accumulation through a second screening principle. All high oil mutants can be obtained by two screening principles.
(11) And (4) subculturing the screened dominant algae according to the combined screening principle in the step (10), 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, so that the genetic stable expression of the high-fat gene is met. The genetic stability considered in this step is based on the requirement whether the specific growth rate and relative fluorescence value differences between generations are significant and the screening conditions can be maintained.
(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. The mutant strain with high oil yield obtained in the step is subjected to amplification culture, and after the periodic culture is finished, the algae liquid is collected by a centrifugal method and stored at the temperature of minus 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.
Examples
In this example, Scenedesmus was purchased from the fresh water algae bank of Wuhan institute of aquatic organisms, academy of sciences, China. The ARTP mutagenesis system used was purchased from Soumann plasma technology Co., Ltd, Nanjing.
1. Culturing Scenedesmus by BG11 culture medium, inoculating 200mL Scenedesmus solution into 500mL triangular conical flask, sealing the flask with sterile membrane, placing in constant temperature illumination incubator, and culturing by semi-continuous culture mode. Average light intensity: 80 μmol. m-1·s-1And the illumination period is as follows: 12L:12D, culture temperature: (25. + -. 1). degree.C. The position of the flask was randomly changed and shaken 3-4 times per day.
2. The cell concentration was 10 at the logarithmic phase6About one/mL, the cells are more viable at this time, and the mutagenesis is more suitable, and 0.1mL of algae solution is taken in a sterilized plastic cover to be subjected to mutagenesis.
3. Preparing 5mL of sterilized BG-11 culture medium and 5mL of sterilized glycerol solution with the volume fraction of 10%, adding the sterilized glycerol solution into a 50mL plastic centrifuge tube, and enabling the final volume fraction of glycerol to be 5% to serve as mutant protection solution and nutrient solution after mutagenesis;
4. the mutagenesis operation comprises the following specific steps:
(1) the mutagenic instrument controls the input voltage of the power supply to be 120V, the current is 1A, the flow of the introduced air is 5L/min, and the voltage is stabilized for 10 min.
(2) In the embodiment, taking the adjustment of mutagenesis time as an example, the mutagenesis time is respectively 0s, 10s, 20s, 30s, 40s, 50s, 60s, 65s and 70s, before mutagenesis, an objective table below the plasma is wiped by alcohol, an algae liquid to be mutagenized is stored and placed on the objective table by utilizing a plastic cover which is sterilized in advance and has the diameter of 1cm, and the distance between the cover and an emergent port of the plasma device is adjusted to be 2 mm. 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.
(3) The mutagenized algal solution was transferred to BG11 medium containing 5% glycerol (5mL BG11 medium +5mL 10% glycerol solution) and resuspended and stabilized in a light incubator for 3 h.
(4) 1mL of the suspended algal solution (a mixed solution containing 5% glycerol and BG-11 medium) was added to 9mL of a mixed solution (BG-11 medium containing 5% glycerol) and diluted. Adding 200 μ L diluted algae solution into 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. The culture conditions are that the illumination period is 12h:12h, and the culture temperature is 25 +/-1 ℃. Three plates were coated as replicates for each mutagenic condition and a blank was made with non-mutagenized algal solution.
(5) After 10d of culture, algae colonies of the mutagenesis group and the control group are counted, the lethality rate is calculated and drawn up, and the lethality rate is calculated according to the following formula:
lethality rate ═ number of non-mutagenized group algae colonies)/number of non-mutagenized group algae colonies × 100%
According to the theory of mutation breeding, positive mutation is more easily obtained when the fatality rate is above 95%. Selecting the mutant algae colony with larger diameter and darker color by using an inoculating loop, transferring the mutant algae colony into a 6-pore plate for liquid culture, wherein the culture conditions are as follows: average light intensity: 80 μmol. m-1·s-1And the illumination period is as follows: 12L:12D, culture temperature: (25. + -. 1). degree.C. To prevent the occurrence of phenomena such as adherence, static settlement, etc., the six-well plate was gently shaken 2 times a day, each time for 1 minute.
Finally, 60s and 65s are determined as the optimal mutagenesis time (the lethality reaches more than 95 percent and the lethality reaches 100 percent after 70 s).
(5) Monitoring the absorbance OD680 and the fluorescence value of the oil dyed by Nile Red on the third day and the eighth day, wherein OD680nm is the absorbance value measured by an ultraviolet spectrophotometer at the wavelength of 680nm, and the relative fluorescence value measurement method comprises the steps of taking 2mL of algae liquid, adding 1mL of 25% dimethyl sulfoxide, simultaneously taking two groups (one group is blank), carrying out ultrasonic treatment for 3min by using ultrasonic waves (500KW), adding 50 mu L of Nile Red reagent (100mg/L) into one group, and after keeping out of the sun for 15min, measuring the fluorescence value (the light wavelength is 480, and the emission wavelength range is 500 plus 650nm) under the emission light wavelength of 570nm by using a fluorescence spectrophotometer. Obtaining the specific growth rate and the relative fluorescence value of each mutant strain, and evaluating the cell proliferation speed and the oil accumulation condition of each mutant strain;
(6) the specific screening principle takes the specific growth rate of more than 10% of the normal plants and the relative fluorescence value of more than 20% of the normal plants as a first screening principle (priority); and (3) taking the product value of the specific growth rate and the relative fluorescence value which is more than 35% of that of the normal strain as a leak-stopping screening principle, wherein the condition is mainly used for leak-stopping screening of the high specific growth rate mutant or the high relative fluorescence value mutant. During the screening process, AT is named after each mutant, and the mutants are distinguished by the mutagenesis time, for example, AT60-1 represents the first strain selected AT60 s. The mutagenic strain screened by the above principle is inoculated into a 6-well plate and subcultured under the same conditions.
(7) And (3) measuring the change of the specific growth rate and the relative fluorescence value of scenedesmus in the 6-hole plate, and analyzing by using SPSS data statistical software to determine whether the difference of subculture is obvious (based on the requirement of screening conditions), wherein the situation that the difference is not obvious is that the subculture character is stable, and the genetic stable expression of the high-fat gene is met. As shown in fig. 3:
(8) after the oil production stability of each mutant strain is determined, carrying out amplification culture on each dominant mutant strain (culturing in a 250mL conical flask under constant culture conditions), and after periodic culture is finished, collecting algae liquid by a centrifugal method and storing in a refrigerator at minus 80 ℃. Referring to the sequences of the oil genes accD and dgat7566 for regulating oil anabolism in gen bank (https:// www.ncbi.nlm.nih.gov /), the expression of each oil gene is determined by using a q-PCR technology. As shown in fig. 4:
through the measurement of the expression of the grease gene, the grease gene expression level of the mutant strain is found to be far higher than that of a normal strain, and the high-yield oil mutant can be obtained by utilizing the screening.
The inventor screens out a dominant mutant strain with stable genetic property and oil yield reaching 343mg/L by the method, which is improved by 117 percent compared with a normal strain, and greatly improves the possibility of preparing biodiesel by microalgae biomass. As shown in table 1:
TABLE 1 comparison of biomass and fat accumulation of the 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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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292838A (en) * | 2021-12-31 | 2022-04-08 | 西安建筑科技大学 | Mutagenesis and screening method for improving sugar yield of microalgae |
| CN114317517A (en) * | 2021-12-31 | 2022-04-12 | 西安建筑科技大学 | Mutagenesis and screening method for improving yield of microalgae grease |
| CN114457065A (en) * | 2022-03-09 | 2022-05-10 | 宿州学院 | Normal-pressure room-temperature plasma microorganism mutagenesis method |
| CN114672421A (en) * | 2022-03-02 | 2022-06-28 | 陕西海斯夫生物工程有限公司 | Method for cultivating and screening microalgae with high tocopherol content |
| CN115044474A (en) * | 2022-06-17 | 2022-09-13 | 陕西海斯夫生物工程有限公司 | Microalgae mutant strain with high tocopherol content, screening method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104711249A (en) * | 2013-12-13 | 2015-06-17 | 中国科学院大连化学物理研究所 | Artificial microalgae strain breeding method |
| CN106754862A (en) * | 2016-12-22 | 2017-05-31 | 昆明藻能生物科技有限公司 | A kind of selection of the hidden dinoflagellate mutation algae strain of Seedling height ability |
-
2019
- 2019-12-04 CN CN201911229869.2A patent/CN110885816A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104711249A (en) * | 2013-12-13 | 2015-06-17 | 中国科学院大连化学物理研究所 | Artificial microalgae strain breeding method |
| CN106754862A (en) * | 2016-12-22 | 2017-05-31 | 昆明藻能生物科技有限公司 | A kind of selection of the hidden dinoflagellate mutation algae strain of Seedling height ability |
Non-Patent Citations (5)
| Title |
|---|
| BIN LIU等: "Mutation Breeding of Extracellular Polysaccharide-Producing Microalga Crypthecodinium cohnii by a Novel Mutagenesis with Atmospheric and Room Temperature Plasma", 《INT. J. MOL. SCI.》 * |
| SONG CAO等: "Improving of lipid productivity of the oleaginous microalgae Chlorella pyrenoidosa via atmospheric and room temperature plasma (ARTP)", 《BIORESOURCE TECHNOLOGY》 * |
| XIN SUN等: "Strategies for enhanced lipid production of Desmodesmus sp. mutated by atmospheric and room temperature plasma with a new efficient screening method", 《JOURNAL OF CLEANER PRODUCTION》 * |
| 曹旭鹏等: "基于常压室温等离子体技术的金藻诱变筛选方法", 《中国生物工程杂志》 * |
| 艾江宁等: "生长速度快且油脂产率高的湛江等鞭金藻诱变株的筛选", 《微生物学通报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292838A (en) * | 2021-12-31 | 2022-04-08 | 西安建筑科技大学 | Mutagenesis and screening method for improving sugar yield of microalgae |
| CN114317517A (en) * | 2021-12-31 | 2022-04-12 | 西安建筑科技大学 | Mutagenesis and screening method for improving yield of microalgae grease |
| CN114672421A (en) * | 2022-03-02 | 2022-06-28 | 陕西海斯夫生物工程有限公司 | Method for cultivating and screening microalgae with high tocopherol content |
| CN114457065A (en) * | 2022-03-09 | 2022-05-10 | 宿州学院 | Normal-pressure room-temperature plasma microorganism mutagenesis method |
| CN114457065B (en) * | 2022-03-09 | 2024-10-01 | 宿州学院 | Normal-pressure room-temperature plasma microorganism mutagenesis method |
| CN115044474A (en) * | 2022-06-17 | 2022-09-13 | 陕西海斯夫生物工程有限公司 | Microalgae mutant strain with high tocopherol content, screening method and application thereof |
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