Method for regulating and controlling key genes to improve high-temperature resistance and stress resistance of carbon sequestration microalgae

Abstract

The invention relates to a microalgae carbon sequestration technology, and aims to provide a method for regulating and controlling a key gene to improve high-temperature resistance and stress resistance of carbon sequestration microalgae. Comprising the following steps: regulating and controlling the expression quantity of four key genes in microalgae cells by performing temperature gradient domestication on the microalgae cells; the final temperature condition of the domestication culture is 42 DEG C, so that the expression quantity of each key gene is increased to several times of the original expression quantity; and (2) carrying out transfer culture on the domesticated microalgae strain, and verifying the genetic stability of the high temperature resistance of the microalgae strain. According to the invention, the adaptability of the microalgae to high-temperature stress can be enhanced, the biomass production efficiency of the microalgae in a high-temperature environment is improved, and the method has important application value in the fields of biological energy production, environmental restoration and the like; as the microalgae can well grow in high-temperature environments such as high-temperature regions and desert arid zones, the large-scale application of the microalgae carbon sequestration technology in the recovery and treatment of the carbon dioxide in the flue gas of the power plant in the high-temperature regions can be expanded.

Description

Technical Field

[0001] The present invention relates to microalgae carbon fixation technology, and in particular to a method for regulating key genes to improve the high temperature tolerance and stress resistance of carbon fixation microalgae. Background Art

[0002] With the increasing severity of global climate change and environmental pollution, the development of sustainable bioenergy and environmental protection technologies has become a top priority. Microalgae carbon fixation, as one of the most promising CO2 bio-sequestration technologies in recent years, can continuously and efficiently absorb and convert CO2 from power plant flue gas to achieve carbon reduction. Microalgae are known for their excellent photosynthetic ability, rapid growth rate, and tenacious survival ability under harsh environmental conditions. They are widely used in the absorption of carbon dioxide, purification of wastewater, and production of biodiesel and biogasoline. However, in high-temperature areas, desert arid areas, etc., the sensitivity of microalgae to high-temperature environments limits their large-scale application in fixing carbon dioxide from power plant flue gas in high-temperature areas.

[0003] High temperature can inhibit the growth of microalgae and can be regarded as a growth adversity. Existing research on improving the high temperature resistance of microalgae to enhance its ability to resist adverse growth mainly focuses on using mutagen regulation, ultraviolet mutagenesis breeding and other means to increase its growth rate and tolerance at specific temperatures. However, the disadvantage of mutagen regulation is that the number of favorable mutations produced is relatively small, and most mutagens are toxic and cause great harm to the environment. The disadvantage of ultraviolet mutagenesis is that the direction of mutation is uncertain and random, and it is impossible to obtain the desired target traits in a targeted manner, and there are few meaningful mutations.

[0004] Therefore, proposing a method that can accurately and effectively improve the high temperature resistance of microalgae is of great significance for improving the stability and economy of microalgae in industrial production. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a method for regulating key genes to improve the high temperature tolerance and stress resistance of carbon-fixing microalgae.

[0006] To solve the technical problem, the solution of the present invention is:

[0007] A method for regulating key genes to improve the high temperature tolerance and stress resistance of carbon-fixing microalgae is provided, comprising the following steps:

[0008] (1) By subjecting microalgae cells to temperature gradient acclimation, the expression levels of four key genes in the microalgae cells, namely, a gene encoding ribosomal protein (RP-S10), a gene encoding ubiquitin-small subunit ribosomal protein (UBA80), a gene encoding serine / threonine protein kinase (SNRK2), and a gene encoding photosystem IIP680 reaction center D1 protein (psbA) were regulated; wherein the final temperature condition of the acclimation culture was 42°C, which increased the expression levels of each key gene to 6.8-13.2 times, 5.5-10.5 times, 5.0-8.0 times, and 3.6-7.5 times of the original expression levels, respectively;

[0009] (2) The domesticated microalgae strains were cultured and the genetic stability of the strains' high temperature resistance was verified.

[0010] As a preferred embodiment of the present invention, the microalgae is Chlorella or Scenedesmus.

[0011] As a preferred embodiment of the present invention, during the temperature gradient culture and transfer culture verification, the lighting condition is maintained at 2000 lux, the air flow rate is 25 ml / min per liter of algae liquid, and BG-11 culture medium is used.

[0012] As a preferred embodiment of the present invention, the preparation method of BG-11 culture medium is as follows: add the following amounts of components to 1L of water: 1 mg of disodium ethylenediaminetetraacetate, 6 mg of ammonium ferric citrate, 6 mg of citric acid monohydrate, 36 mg of calcium chloride dihydrate, 75 mg of magnesium sulfate heptahydrate, 45 mg of dipotassium hydrogen phosphate trihydrate, and 20 mg of sodium carbonate; after mixing, sterilize at 121°C for 15 minutes; then add 1.5 g of sodium nitrate, mix, sterilize again at 121°C for 15 minutes, and cool for use.

[0013] As a preferred embodiment of the present invention, in step (1), the temperature gradient acclimation of the microalgae cells specifically includes:

[0014] (1.1) Inoculate microalgae cells into BG-11 medium and culture in a constant temperature water bath at 25°C until the biomass density reaches 1.0-1.2 g / L / d;

[0015] (1.2) Carry out four stages of temperature gradient acclimatization:

[0016] In the first stage, the new culture medium was inoculated with an initial biomass density of 0.25 g / L / d and cultured in a constant temperature water bath at 30 °C for three cycles, each cycle lasting 5 days;

[0017] In the second stage, the microalgae cells cultured in the first stage were taken and inoculated into the new culture medium at a biomass density of 0.25 g / L / d, and cultured in a constant temperature water bath at 35°C for three cycles, each cycle lasting 5 days;

[0018] In the third stage, the microalgae cells cultured in the second stage were taken and inoculated into the new culture medium at a biomass density of 0.25 g / L / d, and cultured in a constant temperature water bath at 40°C for three cycles, each cycle lasting 5 days;

[0019] In the fourth stage, the microalgae cells cultured in the third stage were taken and inoculated into new culture medium at a biomass density of 0.25 g / L / d, and cultured in a constant temperature water bath at 42°C for 3 cycles, each cycle lasting 5 days.

[0020] (1.3) Screening of algae strains after temperature gradient acclimation:

[0021] The microalgae cells after the fourth stage of culture were cultured on a new culture medium at 42°C for 15 days, the algae strain with the highest growth rate was selected, and re-inoculated into the new culture medium for a further 15 days. After 3 to 5 rounds of screening in this way, the final microalgae community was collected as the algae strain with the highest growth rate and good adaptability to high temperature for verification of genetic stability.

[0022] As a preferred embodiment of the present invention, in the step (1), several parallel groups are always set during the domestication process, and the expression levels of the four key genes in each group of microalgae are measured by transcriptomics after each transfer culture; all transcripts obtained by transcriptome sequencing are compared with the database, and differential gene analysis is performed between the domesticated strain and the original strain group; and the group with the best gene expression level is selected for the next transfer culture, and finally the expression levels of the four key genes are adjusted to the above range.

[0023] As a preferred embodiment of the present invention, in the step (2), the genetic stability of the high temperature resistance of the algae strain is verified according to the following method: the algae strain is inoculated into a new culture medium with an initial biomass density of 0.5 g / L / d, and is cultured continuously for 5 cycles at 42°C, each cycle being 5 days; the light is maintained at 2000 lux during the culture process, and an air flow rate of 25 ml / min is introduced into each liter of algae liquid; if the growth rate of the algae strain in the fifth cycle is not less than 90% of the maximum growth rate of the microalgae in the first cycle, the high temperature resistance of the algae strain is deemed to be genetically stable.

[0024] As a preferred embodiment of the present invention, the growth rate v of microalgae is calculated according to the following formula:

[0025] v=(n2-n1) / (t2-t1)

[0026] Where n1 is the biomass density at the previous sampling time t1, g / L; n2 is the biomass density at the next sampling time t2, g / L.

[0027] As a preferred embodiment of the present invention, samples are taken every day during the cultivation of the algae strain, and the biomass density n of the microalgae is obtained by a drying method:

[0028] Pre-weigh the mass m1 of the glass fiber filter that has been dried to a constant weight, and the mass m2 of a blank glass fiber filter of the same size; measure the algae solution with a volume of V = 20 mL and filter it using a vacuum filtration device; put the filtered microalgae biomass together with the glass fiber filter into an air drying oven at 80°C and dry it under blast conditions for 12 hours; take out the sample, and weigh its mass m3 after it is completely cooled to prevent the sample and the glass fiber filter from absorbing moisture; weigh the mass m4 of the blank glass fiber filter under the same temperature conditions, and calculate the biomass density n of the microalgae according to the following formula:

[0029] n=[(m3-m1)+(m2-m4)] / V.

[0030] As a preferred embodiment of the present invention, when the microalgae strains that have been verified to have genetic stability and high temperature resistance are used in industry, they need to be expanded for cultivation; the inoculation density during cultivation is 0.15-0.25 g / L, the temperature is maintained at 42°C, the light is 2000 lux, and the air flow rate per liter of algae liquid is 25 ml / min.

[0031] Description of the invention principle:

[0032] The present invention utilizes temperature gradient acclimation to increase the expression levels of four key genes in Scenedesmus or Chlorella cells: a gene encoding ribosomal protein (RP-S10), a gene encoding ubiquitin-small subunit ribosomal protein (UBA80), a gene encoding serine / threonine protein kinase (SNRK2), and a gene encoding photosystem IIP680 reaction center D1 protein (psbA). Among them, the upregulation of RP-S10 expression helps to improve the efficiency and accuracy of protein synthesis in microalgae under high temperature. Ribosomal proteins play a key role in maintaining the stability and function of the protein synthesis factory, the ribosome, which is essential for maintaining cell function under high temperature stress. UBA80 is related to the ubiquitination and degradation process of proteins. The increase in its expression helps to more effectively remove damaged or inactivated proteins at high temperatures, thereby maintaining intracellular protein homeostasis and function. SNRK2 is involved in regulating a variety of cellular stress responses, including heat stress. The upregulation of its expression helps to activate the heat stress signal transduction pathway and enhance the adaptability and tolerance of microalgae to high temperature. psbA encodes the reaction center D1 protein of photosystem II, which is a key component in photosynthesis. Increasing the expression of psbA helps to enhance the efficiency of photosynthesis in microalgae under high temperatures, thereby improving energy production and maintaining growth.

[0033] The above four genes are commonly found in Scenedesmus or Chlorella. At a high temperature of 42°C, microalgae cells with high temperature resistance have a survival advantage and can reproduce more efficiently.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The present invention can enhance the adaptability of microalgae to high temperature adversity and improve its efficiency in producing biomass in high temperature environment, and has important application value in the fields of bioenergy production and environmental remediation. According to experimental verification, microalgae can grow normally under high temperature adversity of 42°C, and the maximum growth rate increases from 0.12-0.22 g / L / d before improvement to 0.58-0.65 g / L / d.

[0036] 2. The high-temperature resistant microalgae cultivated by the method of the present invention can grow well in high-temperature environments such as high-temperature areas and desert arid areas, thus expanding the large-scale application of microalgae carbon fixation technology in the recovery and disposal of carbon dioxide from flue gas in power plants in high-temperature areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The present invention is a flow chart of the control method. DETAILED DESCRIPTION

[0038] The specific implementation scheme of the present invention is described in detail below.

[0039] Part I Implementation of the Invention

[0040] 1. Methods for regulating key genes to improve the ability of carbon-fixing microalgae to tolerate high temperatures and stresses:

[0041] (1) The microalgae used in the present invention is Chlorella or Scenedesmus. The microalgae cells are subjected to temperature gradient acclimation to regulate the expression of four key genes in the microalgae cells, namely, a gene encoding ribosomal protein (RP-S10), a gene encoding ubiquitin-small subunit ribosomal protein (UBA80), a gene encoding serine / threonine protein kinase (SNRK2), and a gene encoding photosystem IIP680 reaction center D1 protein (psbA); wherein the final temperature condition of the acclimation culture is 42°C, so that the expression of each key gene is increased to 6.8 to 13.2 times, 5.5 to 10.5 times, 5.0 to 8.0 times, and 3.6 to 7.5 times of the original expression, respectively; specifically including:

[0042] (1.1) Inoculate microalgae cells into BG-11 medium and culture in a constant temperature water bath at 25°C until the biomass density reaches 1.0-1.2 g / L / d;

[0043] (1.2) Carry out four stages of temperature gradient acclimatization:

[0044] In the first stage, the new culture medium was inoculated with an initial biomass density of 0.25 g / L / d and cultured in a constant temperature water bath at 30 °C for three cycles, each cycle lasting 5 days;

[0045] In the second stage, the microalgae cells cultured in the first stage were taken and inoculated into the new culture medium at a biomass density of 0.25 g / L / d, and cultured in a constant temperature water bath at 35°C for three cycles, each cycle lasting 5 days;

[0046] In the third stage, the microalgae cells cultured in the second stage were taken and inoculated into the new culture medium at a biomass density of 0.25 g / L / d, and cultured in a constant temperature water bath at 40°C for three cycles, each cycle lasting 5 days;

[0047] In the fourth stage, the microalgae cells cultured in the third stage were taken and inoculated into new culture medium at a biomass density of 0.25 g / L / d, and cultured in a constant temperature water bath at 42°C for 3 cycles, each cycle lasting 5 days.

[0048] (1.3) Screening of algae strains after temperature gradient acclimation:

[0049] The microalgae cells after the fourth stage of culture were cultured on a new culture medium at 42°C for 15 days, the algae strain with the highest growth rate was selected, and re-inoculated into the new culture medium for a further 15 days. After 3 to 5 rounds of screening in this way, the final microalgae community was collected as the algae strain with the highest growth rate and good adaptability to high temperature for verification of genetic stability.

[0050] 6. The method according to claim 1, characterized in that, in the step (1), several parallel groups are always set during the domestication process, and the expression levels of the four key genes in each group of microalgae are measured by transcriptomics after each transfer culture; all transcripts obtained by transcriptome sequencing are compared with the database, and differential gene analysis is performed between the domesticated strain and the original strain group; the group with the best gene expression level is selected for the next transfer culture, and finally the expression levels of the four key genes are adjusted to the said range.

[0051] The workflow of transcriptomics includes sequencing experiments and machine testing: eukaryotic mRNA sequencing is based on a high-throughput sequencing platform to sequence all mRNAs transcribed from specific tissues or cells of eukaryotic organisms at a certain period. Its operation process includes: extracting total RNA, enriching mRNA with Oligo dT, fragmenting mRNA, reverse synthesizing cDNA, connecting adapters, fragment screening and library enrichment; machine sequencing can be selected from NovaSeq X Plus platform machine sequencing and DNBSEQ-T7 platform machine sequencing. This part of the content belongs to the prior art. The applicant commissioned an external commercial sequencing service agency to perform sequencing. This operation step does not belong to the innovative content of the present invention, so it will not be repeated.

[0052] (2) The domesticated microalgae strains were cultured and the genetic stability of the strains' high temperature resistance was verified.

[0053] The algae strain was inoculated into a new culture medium at an initial biomass density of 0.5 g / L / d and cultured continuously at 42°C for 5 cycles, each cycle being 5 days. The light was maintained at 2000 lux during the culture process, and an air flow rate of 25 ml / min was introduced into each liter of algae liquid. If the growth rate of the algae strain in the fifth cycle was not less than 90% of the maximum growth rate of the microalgae in the first cycle, the high temperature resistance of the algae strain was deemed to be genetically stable.

[0054] In the present invention, the growth rate v of microalgae is calculated according to the following formula:

[0055] v=(n2-n1) / (t2-t1)

[0056] Where n1 is the biomass density at the previous sampling time t1, g / L; n2 is the biomass density at the next sampling time t2, g / L.

[0057] During the cultivation of algae strains, samples were taken every day, and the biomass density n of the microalgae was obtained by the drying method: the mass m1 of the glass fiber filter membrane that had been dried to a constant weight and the mass m2 of the blank glass fiber filter membrane of the same size were weighed in advance; the algae liquid with a volume of V = 20mL was measured and filtered using a vacuum filtration device; the filtered microalgae biomass and the glass fiber filter membrane were placed in an air drying oven at 80°C and dried under blast conditions for 12 hours; the sample was taken out and its mass was weighed as m3 after it was completely cooled to prevent the sample and the glass fiber filter membrane from absorbing moisture; the mass of the blank glass fiber filter membrane under the same temperature conditions was weighed as m4, and the biomass density n of the microalgae was calculated as follows:

[0058] n=[(m3-m1)+(m2-m4)] / V.

[0059] During the temperature gradient culture and transfer culture verification process of the present invention, the lighting condition of 2000 lux was maintained, an air flow rate of 25 ml / min was introduced into each liter of algae liquid, and BG-11 culture medium was used.

[0060] The preparation method of BG-11 culture medium is as follows: add the following amounts of components to 1L of water: 1 mg of disodium ethylenediaminetetraacetate, 6 mg of ammonium ferric citrate, 6 mg of citric acid monohydrate, 36 mg of calcium chloride dihydrate, 75 mg of magnesium sulfate heptahydrate, 45 mg of dipotassium hydrogen phosphate trihydrate, and 20 mg of sodium carbonate; after mixing, sterilize at 121°C for 15 minutes; then add 1.5 g of sodium nitrate, mix, sterilize again at 121°C for 15 minutes, and cool for use.

[0061] When the microalgae strain with genetic stability verified to be resistant to high temperature in the present invention is used in industry, it needs to be expanded and cultured; the inoculation density during culture is 0.15-0.25 g / L, the temperature is maintained at 42°C, the light is 2000 lux, and the air flow rate per liter of algae liquid is 25 ml / min.

[0062] 2. The significance of increasing the expression of four key genes in microalgae in the present invention:

[0063] In microalgae cells, genes encoding ribosomal proteins (RP-S10), genes encoding ubiquitin-small subunit ribosomal proteins (UBA80), genes encoding serine / threonine protein kinases (SNRK2), and genes encoding photosystem IIP680 reaction center D1 proteins (psbA) are all recorded in various public gene databases (such as KEGG databases) together with other genes. Through in-depth research by the inventor team of the applicant, it was found that there is a certain correlation between these four key genes, and they work together on the high temperature resistance mechanism of microalgae. SNRK2 kinase can regulate gene expression by phosphorylating other proteins or transcription factors, including the possible influence of RP-S10 and UBA80 expression. At the same time, the improvement of photosynthesis efficiency (by upregulating psbA) can provide more energy for microalgae cells and help microalgae cells cope with the metabolic pressure brought by high temperature. The synergistic effect of these enzymes helps microalgae maintain normal physiological functions under high temperature conditions and improves its high temperature resistance and stress resistance. In this way, microalgae can better adapt to high temperature environments, which is of great significance for improving the robustness and economy of microalgae in industrial production.

[0064] (1) The gene encoding ribosomal protein S10 (RP-S10) is an important component of the ribosome. As part of the 40S subunit, it plays a key role in the protein synthesis process. The function of RP-S10 is not limited to structural support, but also involves regulating the assembly and stability of ribosomes, thereby ensuring the efficiency and accuracy of protein synthesis. Under high temperature conditions, protein synthesis in microalgae cells may be affected by heat stress, resulting in protein misfolding or aggregation, thereby interfering with the normal function of the cells. Therefore, upregulating the expression of RP-S10 can enhance the function of ribosomes and help cells maintain normal protein synthesis rate and quality in high temperature environments, thereby maintaining the physiological stability and adaptability of cells. This regulatory mechanism is crucial for the survival of microalgae under high temperature conditions because it ensures that cells can still effectively synthesize essential proteins in adversity to support their growth and reproduction.

[0065] (2) The gene encoding ubiquitin-small subunit ribosomal protein S27Ae (UBA80, also known as RPS27A) is a transcription factor related to protein ubiquitination and degradation. Its main function in microalgae cells is to regulate protein stability and degradation through the ubiquitin-proteasome pathway. It can interact with proteins such as RNF168 and participate in DNA damage response and repair. Under high temperature conditions, proteins in microalgae cells may be affected by heat stress, resulting in protein misfolding or inactivation. Upregulating the expression of UBA80 can enhance the cell's ability to clear damaged or inactivated proteins, thereby maintaining the homeostasis of intracellular proteins and protecting cells from oxidative damage and other adverse effects caused by high temperature.

[0066] (3) Genes encoding serine / threonine-protein kinase (SNRK2) Kinases are a class of plant-specific serine / threonine kinases that are widely involved in the response of microalgae to abiotic stresses, including high temperature. SNRK2 kinase enhances the stress resistance of microalgae by regulating the expression of downstream genes. Specifically, SNRK2 can regulate gene expression by activating transcription factors related to stress response (such as bZIP and AREB / ABF), thereby improving the adaptability of microalgae to high temperature environments. Under high temperature conditions, the upregulation of SNRK2 kinase may promote the activation of intracellular signal transduction pathways and enhance the response of microalgae to heat stress. It can also help microalgae to remove reactive oxygen species produced under high temperature conditions by regulating the expression of antioxidant enzymes, thereby protecting cells from oxidative damage.

[0067] (4) The synthesis efficiency of the D1 protein in the photosystem II P680 reaction center directly affects the repair efficiency of the PSII complex, and thus affects the efficiency of photosynthesis. Under high temperature stress, increasing the expression level of the gene encoding the D1 protein (photosystem II P680reaction center D1 protein, psbA) helps to accelerate the replacement of damaged D1 proteins in the PSII complex and maintain the continuous progress of photosynthesis. In addition, the rapid turnover of D1 proteins is essential for maintaining the stability of the photosynthetic electron transport chain. Increasing the expression level of psbA helps to quickly replace damaged D1 proteins and maintain the normal operation of the photosynthetic electron transport chain. By enhancing the efficiency of photosynthesis and protecting PSII, microalgae with increased psbA expression can produce more energy under high temperature conditions to support their growth and metabolic activities, thereby improving their ability to withstand high temperatures.

[0068] Part II Specific implementation examples

[0069] Except for the differences specifically indicated in the embodiments, other undescribed operating steps or operating conditions shall be performed in accordance with the contents of the technical solution in the first part.

[0070] Embodiment 1:

[0071] The Scenedesmus used in this example was from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences (Wuhan), and was numbered Scenedesmus quadricauda FACHB-44.

[0072] (1) First, Scenedesmus was inoculated into BG-11 culture medium and cultured in a constant temperature water bath at 25°C until the biomass density reached 1.0 g / L / d.

[0073] (2) The temperature gradient acclimation at 30℃, 35℃, 40℃ and 42℃ was used to increase the expression levels of four key genes contained in Scenedesmus, among which: the gene encoding ribosomal protein (RP-S10) increased by 6.8 times, the gene encoding ubiquitin-small subunit ribosomal protein (UBA80) increased by 5.5 times, the gene encoding serine / threonine protein kinase (SNRK2) increased by 6.3 times, and the gene encoding photosystem IIP680 reaction center D1 protein (psbA) increased by 3.6 times.

[0074] The microalgae cells after the fourth stage of culture were cultured on a new culture medium at 42°C for 15 days, the fastest growing algae strain was selected, and re-inoculated into the new culture medium and cultured for another 15 days. After four rounds of screening in this way, the final microalgae community was collected as the algae strain with the highest growth rate and good adaptability to high temperatures.

[0075] (3) Verify the genetic stability of the obtained high-temperature resistant algae.

[0076] (4) Comparative test:

[0077] Thermotolerant Scenedesmus was cultured in a 500 ml conical flask in an artificial greenhouse, with 400 ml of BG-11 medium in the flask; the inoculation density was 0.25 g / L, cultured at 42°C, the light intensity was 2000 lux, and the continuous air flow rate was 25 ml / min. On the fourth day of culture, the maximum growth rate of the thermotolerant acclimated strain was 0.58 g / L / d; after five generations of continuous culture, the maximum growth rate was 0.55 g / L / d, which can be considered to have good genetic stability.

[0078] The original Scenedesmus that had not been domesticated was cultured under exactly the same conditions (starting time, inoculation concentration, culture medium, temperature, light, ventilation, etc.). On the third day after the start of the culture, the highest growth rate was measured to be 0.12 g / L / d; after continuing to culture until the fifth day, the Scenedesmus turned yellow and died, which indicates that the original Scenedesmus could not adapt to the high temperature environment and could not survive continuously.

[0079] Embodiment 2:

[0080] The Chlorella Pyrenoidosa used in this example was purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences (Wuhan) and was numbered FACHB-9.

[0081] (1) First, Chlorella was inoculated into BG-11 medium and cultured in a constant temperature water bath at 25°C until the biomass density reached 1.08 g / L / d.

[0082] (2) The temperature gradient acclimation at 30℃, 35℃, 40℃ and 42℃ increased the expression levels of four key genes contained in Chlorella. Among them, the gene encoding ribosomal protein (RP-S10) increased by 13.2 times, the gene encoding ubiquitin-small subunit ribosomal protein (UBA80) increased by 10.5 times, the gene encoding serine / threonine protein kinase (SNRK2) increased by 8 times, and the gene encoding photosystem IIP680 reaction center D1 protein (psbA) increased by 7.2 times.

[0083] The microalgae cells after the fourth stage of culture were cultured on a new culture medium at 42°C for 15 days, the fastest growing algae strain was selected, and re-inoculated into the new culture medium and cultured for another 15 days. After three rounds of screening in this way, the final microalgae community was collected as the algae strain with the highest growth rate and good adaptability to high temperature.

[0084] (3) Verify the genetic stability of the obtained high-temperature resistant Chlorella.

[0085] (4) Comparative test:

[0086] Thermotolerant Chlorella was cultured in a 500 ml conical flask in an artificial greenhouse, with 400 ml of BG-11 culture medium in the flask; the inoculation density was 0.25 g / L, cultured at 42°C, the light intensity was 2000 lux, and the continuous air flow rate was 25 ml / min. On the fourth day of culture, the maximum growth rate of the thermotolerant acclimated strain was 0.65 g / L / d; after five generations of continuous culture, the maximum growth rate was 0.61 g / L / d, which can be considered to have good genetic stability.

[0087] Untamed Chlorella vulgaris was cultured under exactly the same conditions (starting time, inoculation concentration, culture medium, temperature, light, ventilation, etc.). On the third day after the start of the culture, the highest growth rate was measured to be 0.22 g / L / d; after continuing to culture until the fifth day, the Chlorella vulgaris turned yellow and died, indicating that Chlorella vulgaris could not adapt to the high temperature environment and could not survive continuously.

[0088] Embodiment 3:

[0089] The Scenedesmus obliquus used in this example was from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences (Wuhan), with the serial number Scenedesmus sp. FACHB-489.

[0090] (1) First, Scenedesmus was inoculated into BG-11 culture medium and cultured in a constant temperature water bath at 25°C until the biomass density reached 1.2 g / L / d.

[0091] (2) The temperature gradient acclimation at 30°C, 35°C, 40°C and 42°C was used to increase the expression levels of four key genes contained in the scenedesmus, among which: the gene encoding ribosomal protein (RP-S10) increased by 7.2 times, the gene encoding ubiquitin-small subunit ribosomal protein (UBA80) increased by 8 times, the gene encoding serine / threonine protein kinase (SNRK2) increased by 5 times, and the gene encoding photosystem IIP680 reaction center D1 protein (psbA) increased by 7.5 times.

[0092] The microalgae cells after the fourth stage of culture were cultured on a new culture medium at 42°C for 15 days, and the fastest growing algae strain was selected and re-inoculated into the new culture medium for further culture for 15 days. After five rounds of screening in this way, the last microalgae community was collected as the algae strain with the highest growth rate and good adaptability to high temperature.

[0093] (3) Verify the genetic stability of the obtained high-temperature resistant algae.

[0094] (4) Comparative test:

[0095] Thermotolerant Scenedesmus was cultured in a 500 ml conical flask in an artificial greenhouse, with 400 ml of BG-11 medium in the flask; the inoculation density was 0.2 g / L, cultured at 42°C, the light intensity was 2000 lux, and the continuous air flow rate was 25 ml / min. On the fourth day of culture, the maximum growth rate of the thermotolerant acclimated strain was 0.61 g / L / d; after five generations of continuous culture, the maximum growth rate was 0.59 g / L / d, which can be considered to have good genetic stability.

[0096] The original Scenedesmus that had not been domesticated was cultured under exactly the same conditions (starting time, inoculation concentration, culture medium, temperature, light, ventilation, etc.). On the third day after the start of the culture, the highest growth rate was measured to be 0.16 g / L / d; after continuing to culture until the fifth day, the Scenedesmus turned yellow and died, indicating that the original Scenedesmus could not adapt to the high temperature environment and could not survive continuously.

[0097] It can be seen from the comparative test data of each embodiment that, compared with the original algae strain, the present invention can effectively improve the high temperature resistance and stress resistance of carbon-fixing microalgae, so that the high temperature resistant domesticated strain can maintain a good growth state at 42°C, and the growth rate is greatly improved. It can be seen that if the method of the present invention is applied industrially, it will greatly improve the use of microalgae in high temperature areas to absorb and convert power plant flue gas CO2 to achieve carbon reduction goals. Compared with the microalgae that are generally required in the prior art, the present invention can further save the refrigeration energy required in the temperature control process of microalgae growth.

Claims

1. A method for regulating key genes to improve the high temperature tolerance and stress resistance of carbon-fixing microalgae, characterized in that: The following steps are involved: (1) By subjecting microalgae cells to temperature gradient acclimation, the expression levels of four key genes in the microalgae cells, namely, RP-S10, a gene encoding ribosomal protein, UBA80, a gene encoding ubiquitin-small subunit ribosomal protein, SNRK2, a gene encoding serine / threonine protein kinase, and psbA, a gene encoding D1 protein of the photosystem IIP680 reaction center, were regulated; wherein the final temperature condition of the acclimation culture was 42°C, which increased the expression levels of the key genes to 6.8 to 13.2 times, 5.5 to 10.5 times, 5.0 to 8.0 times, and 3.6 to 7.5 times of the original expression levels, respectively; (2) The domesticated microalgae strains were cultured and the genetic stability of the strains' high temperature resistance was verified.

2. The method according to claim 1, characterized in that The microalgae is Chlorella or Scenedesmus.

3. The method according to claim 1, characterized in that During the temperature gradient culture and transfer culture verification, the lighting condition was maintained at 2000 lux, the air flow rate was 25 ml / min per liter of algae liquid, and BG-11 culture medium was used.

4. The method according to claim 3, characterized in that The preparation method of BG-11 culture medium is as follows: add the following amounts of components to 1L of water: 1 mg of disodium ethylenediaminetetraacetate, 6 mg of ammonium ferric citrate, 6 mg of citric acid monohydrate, 36 mg of calcium chloride dihydrate, 75 mg of magnesium sulfate heptahydrate, 45 mg of dipotassium hydrogen phosphate trihydrate, and 20 mg of sodium carbonate; after mixing, sterilize at 121°C for 15 minutes; then add 1.5 g of sodium nitrate, mix, sterilize again at 121°C for 15 minutes, and cool for use.

5. The method according to claim 1, characterized in that In the step (1), the temperature gradient acclimation of the microalgae cells specifically includes: (1.1) Inoculate microalgae cells into BG-11 medium and culture in a constant temperature water bath at 25°C until the biomass density reaches 1.0-1.2 g / L / d; (1.2) Carry out four stages of temperature gradient acclimatization: In the first stage, the new culture medium was inoculated with an initial biomass density of 0.25 g / L / d and cultured in a constant temperature water bath at 30 °C for three cycles, each cycle lasting 5 days; In the second stage, the microalgae cells cultured in the first stage were taken and inoculated into the new culture medium at a biomass density of 0.25 g / L / d, and cultured in a constant temperature water bath at 35°C for three cycles, each cycle lasting 5 days; In the third stage, the microalgae cells cultured in the second stage were taken and inoculated into the new culture medium at a biomass density of 0.25 g / L / d, and cultured in a constant temperature water bath at 40°C for three cycles, each cycle lasting 5 days; In the fourth stage, the microalgae cells cultured in the third stage were taken and inoculated into new culture medium at a biomass density of 0.25 g / L / d, and cultured in a constant temperature water bath at 42°C for 3 cycles, each cycle lasting 5 days. (1.3) Screening of algae strains after temperature gradient acclimation: The microalgae cells after the fourth stage of culture were cultured on a new culture medium at 42°C for 15 days, the algae strain with the highest growth rate was selected, and re-inoculated into the new culture medium for a further 15 days. After 3 to 5 rounds of screening in this way, the final microalgae community was collected as the algae strain with the highest growth rate and good adaptability to high temperature for verification of genetic stability.

6. The method according to claim 1, characterized in that In the step (1), several parallel groups are always set during the domestication process, and the expression levels of the four key genes in each group of microalgae are measured by transcriptomics after each transfer culture; all transcripts obtained by transcriptome sequencing are compared with the database, and differential gene analysis is performed between the domesticated strain and the original strain group; and the group with the best gene expression level is selected for the next transfer culture, and finally the expression levels of the four key genes are adjusted to the range.

7. The method according to claim 1, characterized in that In the step (2), the genetic stability of the high temperature resistance of the algae strain is verified according to the following method: the algae strain is inoculated into a new culture medium at an initial biomass density of 0.5 g / L / d, and is cultured continuously for 5 cycles at 42°C, each cycle being 5 days; the light intensity is maintained at 2000 lux during the culture process, and an air flow rate of 25 ml / min is introduced into each liter of algae liquid; if the growth rate of the algae strain in the fifth cycle is not less than 90% of the maximum growth rate of the microalgae in the first cycle, the high temperature resistance of the algae strain is deemed to be genetically stable.

8. The method according to claim 5 or 7, characterized in that: The growth rate v of microalgae is calculated as follows: v=(n2-n1) / (t2-t1) Where n1 is the biomass density at the previous sampling time t1, g / L; n2 is the biomass density at the next sampling time t2, g / L.

9. The method according to claim 8, characterized in that During the cultivation of the algae strain, samples were taken every day and the biomass density n of the microalgae was obtained by drying method: Pre-weigh the mass m1 of the glass fiber filter that has been dried to a constant weight, and the mass m2 of a blank glass fiber filter of the same size; measure the algae solution with a volume of V = 20 mL and filter it using a vacuum filtration device; put the filtered microalgae biomass together with the glass fiber filter into an air drying oven at 80°C and dry it under blast conditions for 12 hours; take out the sample, and weigh its mass m3 after it is completely cooled to prevent the sample and the glass fiber filter from absorbing moisture; weigh the mass m4 of the blank glass fiber filter under the same temperature conditions, and calculate the biomass density n of the microalgae according to the following formula: n=[(m3-m1)+(m2-m4)] / V.

10. The method according to claim 1, characterized in that When microalgae strains that have been proven to have genetic stability and high temperature resistance are used in industry, they need to be expanded for cultivation; the inoculation density during cultivation is 0.15-0.25g / L, the temperature is maintained at 42°C, the light is 2000lux, and the air flow rate per liter of algae liquid is 25ml / min.

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