A method for constructing acid-resistant engineered algae

Abstract

The present invention discloses a method for constructing acid-resistant engineered algae, belonging to the fields of biology and molecular biology. The specific method is as follows: Using Phatr3_J33543 with the sequence structure shown in SEQ ID No.1, Phatr3_J50516 with the sequence structure shown in SEQ ID No.2, or Phatr3_Jdraft1806 with the sequence structure shown in SEQ ID No.3 as the target gene, removing the stop codon of the target gene, and connecting the fluorescent protein gene without the start codon through the DNA sequence encoding glycine to obtain a recombinant gene; inserting the recombinant gene into the multiple cloning site of the pPhaNR plasmid to obtain a vector plasmid; introducing the vector plasmid into diatom cells by electroporation and then inoculating them onto the ESAW solid selection medium containing bleomycin, and growing brown colonies; selecting the colonies with fluorescent protein signals in the colonies, which are the acid-resistant engineered algae. Compared with the wild algal species, the acid-resistant ability of the engineered algae obtained by the present invention is significantly improved. The present invention has important reference significance for the cultivation of acid-resistant diatoms.

Description

Technical Field

[0001] The present invention belongs to the fields of biology and molecular biology, and particularly relates to a method for constructing acid-tolerant engineered microalgae. Background Art

[0002] As single-celled microorganisms capable of photosynthesis, microalgae have biomass production efficiency and carbon fixation efficiency far exceeding those of terrestrial plants. The production of microalgae is considered an efficient way to reduce carbon dioxide emissions and can be converted into high-value products. Therefore, methods for producing microalgae have received extensive attention. The current main research direction is to combine microalgae production with waste gas and wastewater treatment to generate economic benefits while reducing carbon dioxide emissions and purifying wastewater. However, the technical bottleneck of this production strategy is that the acidic environment caused by greenhouse gases will hinder the growth of microalgae, resulting in low yields. Therefore, it is necessary to improve algal strains to increase the yield of microalgae in acidic environments.

[0003] The main methods for improving microbial breeding include random mutagenesis, adaptive laboratory evolution, and genetic engineering. Adaptive Laboratory Evolution (ALE) uses an artificially controlled stress environment to screen for beneficial spontaneous mutations, thereby accumulating variants in the population that are more capable of overcoming the stress environment. This method is simple and effective and can systematically optimize the fitness of microorganisms in a stress environment, thereby increasing biomass production under adverse conditions. Based on the principle of screening for spontaneous beneficial random mutations in the population, ALE can cultivate algal strains with strong stress resistance. However, different populations may theoretically have different evolutionary pathways. If a single constant selection pressure leads to the screening of variants with similar stress-resistant phenotypes in different populations, then the same gene mutations and differential regulations that occur in different populations are more likely to act on their similar phenotypes. We use transcriptomics to reveal the same gene regulation in different populations to guide genetic engineering modification.

[0004] Phaeodactylum tricornutum is a marine diatom. It grows rapidly and is rich in high-value bioactive components, including unsaturated fatty acids (such as EPA) and carotenoids (such as fucoxanthin), and has potential application value. However, marine algae are most suitable for growth under weakly alkaline conditions (the pH of seawater is generally about 8.0), and acidic conditions will inhibit the growth of diatoms, restricting their application in the production of high-value algal biomass using industrial waste gas carbon dioxide directly. Therefore, there is an urgent need in the field of microalgae to develop methods for obtaining acid-tolerant engineered microalgae. Summary of the Invention

[0005] The object of the present invention is to solve the problem that the growth of diatoms is inhibited by an acidic environment in industrial production, and to provide a method for constructing acid-resistant engineered algae. According to this method, acid-resistant domesticated species can be obtained through adaptive evolution, and acid-resistant engineered algal species can be constructed through genetic engineering.

[0006] The specific technical solution adopted by the present invention is as follows:

[0007] The present invention provides a method for constructing acid-resistant engineered algae, and the specific steps are as follows:

[0008] S1: Genes related to annotation function, ion channels, and pH homeostasis are used as target genes; the stop codons of the target genes are removed, and a fluorescent protein gene with the start codon removed is connected through a DNA sequence encoding glycine to obtain a recombinant gene; the target genes are Phatr3_J33543 with a sequence structure as shown in SEQ ID No.1, Phatr3_J50516 with a sequence structure as shown in SEQ ID No.2, or Phatr3_Jdraft1806 with a sequence structure as shown in SEQ ID No.3.

[0009] S2: The recombinant gene is inserted into the multiple cloning site of the pPhaNR plasmid with an antibiotic resistance gene to obtain a vector plasmid.

[0010] S3: The vector plasmid and salmon sperm DNA solution are mixed to obtain a mixed solution; after the mixed solution is ice-bathed, it is introduced into diatom cells that have completed desalting pretreatment by electroporation, and then the diatom cells are placed in the dark for the first culture; the diatom cells after the first culture are inoculated onto an ESAW solid medium containing bleomycin for the second culture until algal colonies appear on the solid medium.

[0011] S4: Select the algal colonies with fluorescent protein signals among the algal colonies obtained in step S3, which are the acid-resistant engineered algae.

[0012] Preferably, the pPhaNR plasmid in step S2 contains a bleomycin resistance gene (ZeoR / bleoR); the inserted recombinant gene is located between the endogenous nitrate reductase promoter and terminator, and its gene expression is regulated by the nitrate reductase promoter (pNR).

[0013] Preferably, the vector plasmid in step S2 is amplified by introducing it into Escherichia coli.

[0014] Preferably, the electroporation in step S3 is carried out using an electroporator, and the set parameters are as follows: the field strength is 0.5 kV, the capacitance is 25 μF, and the resistance is 400 Ohm.

[0015] Preferably, the mass ratio of the carrier plasmid to salmon sperm DNA in the mixed solution in step S3 is 1:10.

[0016] Preferably, the diatom cells in step S3 are wild-type Phaeodactylum tricornutum cells.

[0017] Preferably, the desalting pretreatment step in step S3 is as follows: After centrifuging to collect Phaeodactylum tricornutum cells in the logarithmic growth phase, the precipitated Phaeodactylum tricornutum cells are washed with a sorbitol solution to remove salt and then resuspended in the sorbitol solution by repeated centrifugation and resuspension.

[0018] Furthermore, the concentration of the above sorbitol solution is 375 mM.

[0019] Preferably, the first culture is carried out in the dark for 24 h, and the second culture is carried out for 2 - 3 weeks.

[0020] Preferably, the concentration of bleomycin in the ESAW solid selection medium described in step S3 is 100 μg / L.

[0021] Preferably, the intensity of the fluorescent protein signal in step S4 is 6 - 10 times that of the green fluorescence in wild-type algal cells.

[0022] The present invention has the following beneficial effects compared with the prior art:

[0023] The present invention provides a method for constructing acid-tolerant engineered algae, which can enhance the acid tolerance of marine diatoms and improve the growth of the constructed engineered algae in a low-pH stress environment. The present invention constructs engineered algae by inserting three target genes into wild-type Phaeodactylum tricornutum respectively. Through experiments, it can be obtained that the acid tolerance of the three groups of engineered algae is significantly improved, and the maximum growth rate of the engineered algae is 1.7 to 1.8 times that of the wild type. This method has important reference significance for the cultivation of acid-tolerant diatoms. Description of the Drawings

[0024] Figure 1 It is the adaptive evolution process curve of wild-type Phaeodactylum tricornutum under low-pH environmental stress in Example 1;

[0025] Figure 2 It is the specific growth rate of wild-type and domesticated Phaeodactylum tricornutum under different acidic conditions in Example 1 (**, p < 0.01; *, p < 0.05);

[0026] Figure 3 It is the schematic diagram of constructing acid-tolerant engineered algae (a) and the comparison diagram of the relative intensity of green fluorescence signals (b) in Examples 2 - 4;

[0027] Figure 4To implement the growth performance of the acid-tolerant engineered algae and wild-type algae stimulated by pH = 5.0 in Examples 2 to 4, where (a) is the growth curve, (b) is the fluorescence quantum yield, (c) is the maximum specific growth rate under optimal conditions and low pH stress conditions, and (d) is the increase multiple of the maximum specific growth rate of the transformant relative to the wild type (compared with the control group: NS, no significant difference, ***, p < 0.001; **, p < 0.01). Detailed implementation manners

[0028] The present invention will be further described and explained below in conjunction with the accompanying drawings and specific implementation manners. The technical features of each implementation manner in the present invention can be combined correspondingly without conflict.

[0029] Example 1 Breeding acid-tolerant diatoms and obtaining acid-tolerant target genes

[0030] (1) Subculture for a long time under artificially controlled constant environmental pressure to domesticate acid-tolerant algal species.

[0031] Use 40 mM MES buffer to control the pH in the artificial seawater culture medium to remain stable at weakly acidic, and the pH of the artificial seawater culture medium is 6.0. Use this culture medium to subculture wild-type Phaeodactylum tricornutum cells under continuous low pH stress. The culture conditions are: light intensity 100 μmol / m 2 / s, temperature 22 ± 1 °C, and the subculture cycle is 5 days. At the end of each cycle, appropriate cells are collected by centrifugation and then resuspended in the same volume (75 mL) of fresh culture medium to keep the initial cell concentration consistent for each cycle. After 16 subcultures, the average growth rates of the three groups of algal solutions at five days were significantly increased and reached stability, as Figure 1 shown, to obtain Phaeodactylum tricornutum populations ALE1, ALE2, and ALE3 with higher growth rates under acidic conditions.

[0032] To test the stability of the three domesticated Phaeodactylum tricornutum strains ALE1, ALE2, and ALE3, ALE1, ALE2, and ALE3 were all restored to the optimal growth conditions for two weeks. The culture conditions are: light intensity 100 μmol / m 2 / s, at a temperature of 22 ± 1 °C, 40 mM Tris-HCl buffer was added to maintain the pH of the artificial seawater culture medium at 8.0 during the culture process. Then, an appropriate amount of acclimated Phaeodactylum tricornutum cells was collected by centrifugation. Each group of acclimated Phaeodactylum tricornutum cells was divided into three parts, and the cells were washed once with acidic culture media at pH 6.5, pH 6.0, and pH 5.5 respectively, and then collected by centrifugation. The cells were inoculated into the culture media with corresponding pH values for stability testing. After 5 days of culture, the growth performances of the acclimated Phaeodactylum tricornutum and the wild-type Phaeodactylum tricornutum under acidic stimulation were compared. The results are as Figure 2 shown. The growth rate of the acclimated Phaeodactylum tricornutum was increased relative to the wild-type in all test conditions, and the growth rate was increased by 27.5 - 110.4%.

[0033] (2) Identify the acid tolerance target genes commonly present in the above-mentioned acclimated Phaeodactylum tricornutum through transcriptome sequencing.

[0034] The acclimated strain and the wild-type strain were transferred from the optimal growth condition of pH = 8.0 to the low-pH stress environment of pH = 6.0. The culture conditions were as follows: light intensity 100 μmol / m 2 / s, at a temperature of 22 ± 1 °C, 40 mM MES buffer was added to maintain the pH at 6.0 during the culture process. Cells were collected on the 5th day, and total RNA was extracted for second-generation transcriptome sequencing. Using the transcriptome of the wild-type Phaeodactylum tricornutum under acidic stimulation as the control group, differential expression analysis was performed on the transcriptomes of the three groups of acclimated Phaeodactylum tricornutum under the same conditions.

[0035] Among the genes with prominent differential expression (change multiple > 1) in two or more groups of acclimated Phaeodactylum tricornutum, several genes with annotated functions related to ion channels and pH homeostasis were selected as target genes. The selected target genes were Phatr3_J33543 with a sequence structure as shown in SEQ ID No.1, Phatr3_J50516 with a sequence structure as shown in SEQ ID No.2, and Phatr3_Jdraft1806 with a sequence structure as shown in SEQ ID No.3.

[0036] Example 2 Construction of engineered alga HI3310

[0037] This example provides a method for constructing an acid-tolerant engineered alga using Phatr3_J33543 with a sequence structure as shown in SEQ ID No.1. The specific steps are as follows:

[0038] (1) Remove the stop codon at the 3'-end of the Phatr3_J33543 gene with the sequence structure as shown in SEQ ID No.1, and remove the fluorescent protein (GFP) gene at the start codon by ligating a DNA sequence encoding 5 glycines to obtain a recombinant gene. Since the recombinant protein encoded by this recombinant gene contains a fluorescent protein sequence, it is convenient for subsequent screening by fluorescence microscopy.

[0039] Insert the above recombinant gene plasmid into the multiple cloning site of the pPhaNR plasmid (NCBI accession number: JN180663.1) to obtain a vector plasmid. The pPhaNR plasmid is a plasmid with amplification ability, and this plasmid contains a gene that can resist bleomycin. Bleomycin is an antibiotic that can kill wild-type Phaeodactylum tricornutum cells.

[0040] Introduce the constructed vector plasmid into Escherichia coli and allow the vector plasmid to amplify in Escherichia coli. After amplification from Escherichia coli, the vector plasmid is purified for standby.

[0041] (2) Introduce the expression vector plasmid obtained in step (1) into wild-type Phaeodactylum tricornutum cells using an electroporator, and the method is as follows:

[0042] First, centrifuge at 1000 x g for 10 minutes to collect approximately 2×10 8 CFU of Phaeodactylum tricornutum cells in the logarithmic growth phase. Wash the centrifuged precipitate three times with a 375 mM sorbitol solution, centrifuge and resuspend repeatedly for desalting pretreatment. Finally, resuspend the pretreated cells in 100 μL of a 375 mM sorbitol solution, add 4 μg of the vector plasmid obtained in step (1) and 40 μg of salmon sperm DNA to obtain a mixture. Incubate the mixture in an ice-water bath for 10 minutes and transfer it to a 2 mm electroporation cuvette. Salmon sperm DNA helps the vector plasmid enter the algal cells.

[0043] Introduce the above mixture into wild-type Phaeodactylum tricornutum cells using an electroporator (Bio-Rad), and set the parameters as follows: the field strength is 0.5 kV, the capacitance is 25 μF, and the resistance is 400 Ohm. After allowing the wild-type Phaeodactylum tricornutum cells transfected with the vector plasmid to stand in the dark for 24 hours, inoculate them onto an ESAW solid medium containing 100 μg / mL bleomycin. After 2 - 3 weeks, select the brown colonies that appear on the medium and pick them into a test tube containing artificial seawater culture solution. The culture conditions are: light intensity 100 μmol / m 2 / s, temperature 22 ± 1 °C, and add 40 mM Tris-HCl buffer to maintain the pH value at 8.0 during the culture process.

[0044] The ESAW solid medium was prepared by formulating the ESAW artificial seawater culture solution according to the following formula, adjusting the pH to 8.0, adding 1% (w / v) agar powder, and sterilizing at 121 °C for 30 minutes. The specific components of the ESAW artificial seawater culture solution are shown in Table 1.

[0045] Table 1 Composition of ESAW artificial seawater culture solution

[0046]

[0047] To avoid false positives in the brown algal colonies selected from the above ESAW solid medium containing bleomycin, the above algal cells also need to be observed by a fluorescence microscope. The excitation wavelength of the fluorescence microscope is 488 nm, the detection wavelength is 525 nm, and the algal species with a fluorescent protein (GFP) signal is selected, which is the constructed acid-resistant engineered alga, denoted as HI3310.

[0048] Example 3 Construction of engineered alga HI5021

[0049] This example provides a method for constructing an acid-resistant engineered alga using Phatr3_J50516 with a sequence structure as shown in SEQ ID No. 2. The specific steps are as follows:

[0050] (1) Remove the stop codon at the 3' end of the Phatr3_J50516 gene with a sequence structure as shown in SEQ ID No. 2, and ligate the fluorescent protein (GFP) gene without the start codon through a DNA sequence encoding 5 glycines to obtain a recombinant gene. Since the recombinant protein encoded by this recombinant gene contains a fluorescent protein sequence, it is convenient for subsequent screening by a fluorescence microscope.

[0051] Insert the above recombinant gene into the multiple cloning site of the pPhaNR plasmid (NCBI accession number: JN180663.1) to obtain a vector plasmid. The pPhaNR plasmid is a plasmid with amplification ability, and this plasmid contains a gene that can resist bleomycin. Bleomycin is an antibiotic that can kill wild-type Phaeodactylum tricornutum cells.

[0052] Introduce the constructed vector plasmid into Escherichia coli and allow the vector plasmid to amplify in Escherichia coli. The vector plasmid is purified for later use after amplification from Escherichia coli.

[0053] (2) Introduce the expression vector plasmid obtained in step (1) into wild-type Phaeodactylum tricornutum cells using an electroporator. The method is as follows:

[0054] First, centrifuge at 1000 x g for 10 minutes to collect approximately 2×10 8Cells of *Phaeodactylum tricornutum* in the logarithmic growth phase of CFU. The centrifuged precipitate was washed three times with 375 mM sorbitol solution, centrifuged and resuspended repeatedly for desalting pretreatment. Finally, the pretreated cells were resuspended in 100 μL of 375 mM sorbitol solution, and 4 μg of the vector plasmid obtained in step (1) and 40 μg of salmon sperm DNA were added to obtain a mixed solution. The mixed solution was placed in an ice-water bath for 10 minutes and then transferred to a 2-mm cuvette for electroporation. Salmon sperm DNA helps the vector plasmid enter algal cells.

[0055] The above mixed solution was introduced into wild-type *Phaeodactylum tricornutum* cells using a electroporator (Bio-Rad), and the parameters were set as follows: the field strength was 0.5 kV, the capacitance was 25 μF, and the resistance was 400 Ohm. After the wild-type *Phaeodactylum tricornutum* cells transfected with the vector plasmid were allowed to stand in the dark for 24 hours, they were inoculated onto an ESAW solid medium containing 100 μg / mL bleomycin. After 2-3 weeks, the brown colonies that appeared on the selective medium were picked and transferred to a test tube containing artificial seawater culture solution. The culture conditions were as follows: the light intensity was 100 μmol / m 2 / s, the temperature was 22 ± 1 °C, and 40 mM Tris-HCl buffer was added to maintain the pH value at 8.0 during the culture process.

[0056] The ESAW solid medium was prepared by formulating the ESAW artificial seawater culture solution, adjusting the pH to 8.0, adding 1% (w / v) agar powder, and autoclaving at 121 °C for 30 minutes. The specific components of the ESAW artificial seawater culture solution are shown in Table 1.

[0057] To avoid false positives in the brown colonies selected from the above ESAW solid medium containing bleomycin, the above algal cells also need to be observed by a fluorescence microscope. The excitation wavelength of the fluorescence microscope was 488 nm, and the detection wavelength was 525 nm. The algal species with fluorescence protein (GFP) signals were selected, which was the constructed acid-tolerant engineered alga, denoted as HI5021.

[0058] Example 4 Construction of engineered alga HI1831

[0059] This example provides a method for constructing an acid-tolerant engineered alga using Phatr3_Jdraft1806 with the sequence structure shown in SEQ ID No. 3. The specific steps are as follows:

[0060] (1) Remove the stop codon at the 3' end of the Phatr3_Jdraft1806 gene with the sequence structure as shown in SEQ ID No. 3, and connect the fluorescent protein (GFP) gene without the start codon through a DNA sequence encoding 5 glycines to obtain a recombinant plasmid. Since the recombinant protein encoded by this recombinant gene contains a fluorescent protein sequence, it is convenient for subsequent screening by fluorescence microscopy.

[0061] Insert the above recombinant gene into the multiple cloning site of the pPhaNR plasmid (NCBI accession number: JN180663.1) to obtain a vector plasmid. The pPhaNR plasmid is a plasmid with amplification ability, and this plasmid contains a gene that can resist bleomycin. Bleomycin is an antibiotic that can kill wild-type Phaeodactylum tricornutum cells.

[0062] Introduce the constructed vector plasmid into Escherichia coli and allow the vector plasmid to amplify in Escherichia coli. After amplification from Escherichia coli, the vector plasmid is purified for standby.

[0063] (2) Introduce the vector plasmid expressed in step (1) into wild-type Phaeodactylum tricornutum cells using an electroporator, and the method is as follows:

[0064] First, centrifuge at 1000 x g for 10 minutes to collect approximately 2×10 8 CFU of Phaeodactylum tricornutum cells in the logarithmic growth phase. Wash the centrifuged precipitate three times with a 375 mM sorbitol solution, centrifuge and resuspend repeatedly for desalting pretreatment. Finally, resuspend the pretreated cells in 100 μL of a 375 mM sorbitol solution, add 4 μg of the vector plasmid obtained in step (1) and 40 μg of salmon sperm DNA to obtain a mixture. Incubate the mixture in an ice-water bath for 10 minutes and transfer it to a 2 mm special cuvette for electroporation. Salmon sperm DNA helps the vector plasmid enter algal cells.

[0065] Introduce the above mixture into wild-type Phaeodactylum tricornutum cells using an electroporator (Bio-Rad), and set the parameters as follows: the field strength is 0.5 kV, the capacitance is 25 μF, and the resistance is 400 Ohm. After allowing the wild-type Phaeodactylum tricornutum cells introduced with the vector plasmid to stand in the dark for 24 hours, inoculate them into an ESAW solid medium containing 100 μg / mL bleomycin. After 2 - 3 weeks, select the brown colonies that appear on the culture medium and pick them into a test tube containing artificial seawater culture solution. The culture conditions are: light intensity 100 μmol / m 2 / s, temperature 22 ± 1 °C, and add 40 mM Tris-HCl buffer to maintain the pH value at 8.0 during the culture process.

[0066] The ESAW solid medium was prepared by formulating the ESAW artificial seawater culture solution according to the following formula, adjusting the pH to 8.0, adding 1% (w / v) agar powder, and sterilizing at 121 °C for 30 minutes. The specific components of the ESAW artificial seawater culture solution are shown in Table 1.

[0067] To avoid false positives in the brown colonies selected from the above ESAW solid medium containing bleomycin, the above algal cells also need to be observed under a fluorescence microscope. The excitation wavelength of the fluorescence microscope is 488 nm, and the detection wavelength is 525 nm. The algal species with fluorescent protein (GFP) signals are selected, which is the constructed acid-tolerant engineered alga, denoted as HI1831.

[0068] An acid stimulation experiment was conducted to verify the engineered algae constructed in Examples 2-4. First, the growth performance of three groups of engineered algae and wild-type Phaeodactylum tricornutum was compared under optimal growth conditions. The culture conditions were as follows: light intensity 100 μmol / m 2 / s, temperature 22 ± 1 °C, and the pH of the artificial seawater containing 40 mM Tris-HCl buffer was maintained at 8.0 during the culture process.

[0069] Then, an appropriate amount of cells was collected by centrifugation and washed once with an acidic culture solution with pH = 5.0. The cells were collected by centrifugation again and inoculated into the artificial seawater culture solution with pH = 5. After 5 days of culture, the growth performance of the three groups of engineered algae and wild-type Phaeodactylum tricornutum under acid stimulation was compared, as specifically Figure 4 shown. The experimental results showed that the growth of wild-type Phaeodactylum tricornutum was strongly inhibited in an environment with pH = 5.0, while the tolerance of the three groups of engineered algae to acid stimulation was significantly improved. The maximum growth rate of the engineered algae was 1.7 to 1.8 times that of the wild type.

[0070] The above-described embodiments are only a preferred solution of the present invention, but they are not intended to limit the present invention. Those of ordinary skill in the relevant technical field can still make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for constructing acid-resistant engineered algae, characterized in that, The specific steps are as follows: S1: Use the genes related to the annotation function, ion channels, and pH homeostasis as the target genes; remove the stop codons of the target genes, and connect the fluorescent protein genes with the start codons removed through the DNA sequence encoding glycine to obtain recombinant genes; The target gene is Phatr3_J50516 with the sequence structure shown in SEQ ID No.2; S2: Insert the recombinant gene into the multiple cloning site of the pPhaNR plasmid with an antibiotic resistance gene to obtain a vector plasmid; S3: Mix the vector plasmid and the salmon sperm DNA solution to obtain a mixture; after ice-bathing the mixture, introduce it into the diatom cells that have completed desalting pretreatment by electroporation, and then place the diatom cells in an ESAW culture medium and place them in the dark for the first culture; inoculate the diatom cells after the first culture onto an ESAW solid selection medium containing bleomycin for the second culture until algal colonies appear on the solid selection medium; the diatom cells are wild-type Phaeodactylum tricornutum ( Phaeodactylum tricornutum ) cells; S4: Select the colonies with fluorescent protein signals from the colonies obtained in step S3, which are the acid-resistant engineered algae.

2. The method for constructing acid-resistant engineered algae according to claim 1, characterized in that, The pPhaNR plasmid described in step S2 contains a bleomycin resistance gene ZeoR / bleoR ; the inserted recombinant gene is located between the endogenous nitrate reductase promoter and terminator, and its gene expression is regulated by the nitrate reductase promoter pNR.

3. The method for constructing acid-resistant engineered algae according to claim 1, characterized in that, In step S3, the electroporation introduction uses an electroporator, and the set parameters are as follows: the field strength is 0.5 kV, the capacitance is 25 μF, and the resistance is 400 Ohm.

4. The method for constructing acid-resistant engineered algae according to claim 1, characterized in that, In the mixed solution in step S3, the mass ratio of the vector plasmid to salmon sperm DNA is 1:

10.

5. The method for constructing acid-resistant engineered algae according to claim 1, characterized in that, The desalting pretreatment step in step S3 is as follows: After centrifuging to collect Phaeodactylum tricornutum cells in the logarithmic growth phase, through repeated centrifugation and resuspension, the precipitated Phaeodactylum tricornutum cells are washed with a sorbitol solution to remove salt and then resuspended in the sorbitol solution.

6. The method for constructing acid-resistant engineered algae according to claim 5, characterized in that, The concentration of the sorbitol solution is 375 mM.

7. The method for constructing acid-resistant engineered algae according to claim 1, characterized in that, The first culture is carried out in the dark for 24 h; the second culture lasts for 2 - 3 weeks.

8. The method for constructing acid-resistant engineered algae according to claim 1, characterized in that, In the ESAW solid selection medium in step S3, the concentration of bleomycin is 100 μg / L.

9. The method for constructing acid-resistant engineered algae according to claim 1, characterized in that, In step S4, the intensity of the fluorescent protein signal is 6 - 10 times that of the green fluorescence in wild-type algal cells.

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