Acid-resistant microalgae construction method based on bicarbonate ion transporter protein

Abstract

The invention discloses an acid-resistant microalgae construction method based on bicarbonate ion transporter protein, and belongs to the field of biology and molecular biology. The method specifically comprises the following steps: taking Phatr3J33543 with a sequence structure as shown in SEQ ID No.1, Phatr3J50516 with a sequence structure as shown in SEQ ID No.2 or Phatr3Jdraft1806 with a sequence structure as shown in SEQ ID No.3 as a target gene, removing a termination codon of the target gene, and connecting a fluorescent protein gene with an initiation codon removed through a DNA (Deoxyribose Nucleic Acid) sequence for coding glycine to obtain a recombinant gene; inserting the recombinant gene into multiple cloning sites of a pPhaNR plasmid to obtain a vector plasmid; introducing the carrier plasmids into diatom cells through electroporation, and inoculating the diatom cells to an ESAW solid selective culture medium containing bleomycin to grow brown algal colonies; selecting the algal colonies with fluorescent protein signals from the algal colonies, namely the acid-resistant engineering algae. Compared with wild algae, the acid resistance of the engineering algae obtained by the invention is obviously improved. The method has important reference significance on acid-resistant diatom cultivation.

Description

[0001] This application is a divisional application of the invention patent with application number 202311052310.3, application date August 21, 2023, and invention name “A method for constructing acid-resistant engineered algae”. Technical Field

[0002] The invention belongs to the fields of biology and molecular biology, and particularly relates to a method for constructing acid-resistant microalgae based on bicarbonate ion transporter. Background Art

[0003] Microalgae, as single-celled microorganisms capable of photosynthesis, have biomass production and carbon fixation efficiencies far exceeding those of terrestrial plants. Microalgae production is considered an efficient way to reduce carbon dioxide emissions while also being convertible into high-value-added products. Therefore, methods for producing microalgae have received widespread attention. The current main research direction is to combine microalgae production with waste gas and wastewater treatment, thereby generating 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 hinders the growth of microalgae, resulting in low yields. Therefore, it is necessary to improve algae species to increase the yield of microalgae in acidic environments.

[0004] Major methods for improving microbial breeding include random mutagenesis, adaptive laboratory evolution, and genetic engineering. Adaptive laboratory evolution (ALE) utilizes artificially controlled stress environments to screen for beneficial spontaneous mutations, thereby accumulating variants within a population that are better able to withstand these stressful conditions. This simple and effective method can systematically optimize microbial fitness in stressful environments, thereby increasing biomass yield under adverse conditions. Based on the principle of screening for beneficial random spontaneous mutations within a population, ALE can cultivate algal strains with strong stress tolerance. However, different populations could theoretically follow different evolutionary pathways. If a single, constant selective pressure leads to the selection of variants with similar stress-resistance phenotypes across different populations, then the presence of identical gene mutations and differential regulation across these populations is more likely to contribute to their similar phenotypes. We use transcriptomes to reveal identical gene regulation across different populations to guide genetic engineering.

[0005] 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), making it a promising candidate for applications. However, marine algae thrive best in weakly alkaline conditions (typically seawater has a pH of around 8.0), while acidic conditions inhibit diatom growth, limiting their potential for producing high-value algal biomass directly from industrial waste gas, carbon dioxide. Therefore, the microalgae field urgently needs to develop methods for producing acid-tolerant engineered algae. Summary of the Invention

[0006] The present invention aims to address the problem of diatom growth being inhibited by acidic environments in industrial production and to provide a method for constructing acid-tolerant engineered algae. This method allows for the generation of acid-tolerant domesticated species through adaptive evolution, and the construction of acid-tolerant engineered algae through genetic engineering.

[0007] The specific technical solutions adopted in the present invention are as follows:

[0008] The present invention provides a method for constructing acid-resistant microalgae based on bicarbonate ion transporter, and the specific steps are as follows:

[0009] S1: A gene with annotated functions related to ion channels and pH homeostasis is selected as a target gene; the stop codon of the target gene is removed, and a fluorescent protein gene with the start codon removed is connected to a DNA sequence encoding glycine to obtain a recombinant gene; the target gene has a sequence structure as shown in SEQ ID No. 3, Phatr3_Jdraft1806;

[0010] S2: inserting the recombinant gene into the multiple cloning site of the pPhaNR plasmid having an antibiotic resistance gene to obtain a vector plasmid;

[0011] S3: mixing the vector plasmid and the salmon sperm DNA solution to obtain a mixed solution; placing the mixed solution in an ice bath, and then introducing the mixed solution into diatom cells that have undergone desalination pretreatment by electroporation; then placing the diatom cells in a dark place for a first culture; inoculating the diatom cells after the first culture onto an ESAW solid medium containing bleomycin, and performing a second culture until algal colonies grow on the solid medium;

[0012] S4: selecting algae colonies with fluorescent protein signals from the algae colonies obtained in step S3, which are acid-resistant engineered algae.

[0013] 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).

[0014] Preferably, the vector plasmid described in step S2 is amplified by introducing into Escherichia coli.

[0015] Preferably, the electroporation in step S3 is performed using an electroporator with the following parameters: field strength of 0.5 kV, capacitance of 25 μF, and resistance of 400 Ohm.

[0016] Preferably, the mass ratio of the vector plasmid to the salmon sperm DNA in the mixed solution in step S3 is 1:10.

[0017] Preferably, the diatom cells in step S3 are wild-type Phaeodactylum tricornutum cells.

[0018] Preferably, the desalination pretreatment step in step S3 is as follows: after collecting the Phaeodactylum tricornutum cells in the logarithmic growth phase by centrifugation, the cells are repeatedly centrifuged and resuspended, and the precipitated Phaeodactylum tricornutum cells are washed with a sorbitol solution to remove salts and then resuspended in a sorbitol solution.

[0019] Furthermore, the concentration of the sorbitol solution is 375 mM.

[0020] Preferably, the first culture is carried out in the dark for 24 hours, and the second culture is carried out for 2 to 3 weeks.

[0021] Preferably, the concentration of bleomycin in the ESAW solid selection medium in step S3 is 100 μg / L.

[0022] Preferably, the intensity of the fluorescent protein signal in step S4 is 6 to 10 times that of the green fluorescence in wild-type algal cells.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention provides a method for constructing acid-tolerant engineered algae, which can enhance the acid resistance of marine diatoms and improve their growth in low-pH stress environments. The engineered algae were constructed by inserting three target genes into the wild-type Phaeodactylum tricornutum. Experimental results showed that all three groups of engineered algae exhibited significantly improved tolerance to acidic stimulation, with the maximum growth rate of the engineered algae being 1.7 to 1.8 times that of the wild-type. This method has important implications for the cultivation of acid-tolerant diatoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the adaptive evolution curve of the wild-type Phaeodactylum tricornutum under low pH environmental pressure in Example 1;

[0026] Figure 2is the specific growth rate of the wild type and domesticated P. tricornutum under different acidic conditions in Example 1 (**, p<0.01; *, p<0.05);

[0027] Figure 3 Schematic diagram of the construction of acid-resistant engineered algae in Examples 2 to 4 (a) and a comparison diagram of the relative intensity of green fluorescence signals (b);

[0028] Figure 4 Figure 2 shows the growth performance of the acid-tolerant engineered algae and wild-type algae constructed in Examples 2 to 4 under pH 5.0 stimulation, 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 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 DESCRIPTION

[0029] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention may be combined accordingly without conflict.

[0030] Example 1 Breeding acid-resistant diatoms and obtaining acid-resistant target genes

[0031] (1) Carry out long-term subculture under artificially controlled constant environmental pressure to domesticate acid-resistant algae species.

[0032] The pH of the artificial seawater culture medium was controlled to be weakly acidic using 40 mM MES buffer, and the pH of the artificial seawater culture medium was 6.0. Wild-type Phaeodactylum tricornutum cells were subcultured in this culture medium under a continuous low pH pressure. The culture conditions were: light intensity 100 μmol / m 2 / s, the temperature was 22±1℃, and the subculture cycle was 5 days. At the end of each cycle, appropriate cells were collected by centrifugation and resuspended in the same volume (75mL) of fresh culture medium to maintain the same initial cell concentration in each cycle. After 16 subcultures, the average growth rate of the three groups of algae solutions increased significantly and reached a stable level at five days. Figure 1 As shown, the triangular algae populations ALE1, ALE2 and ALE3 with higher growth rates under acidic conditions were obtained.

[0033] To test the stability of the three groups of acclimated P. tricornutum, ALE1, ALE2 and ALE3 were restored to the optimal growth conditions and cultured for two weeks. The culture conditions were: light intensity 100 μmol / m 2 / s, the temperature was 22±1℃, 40mM Tris-HCl buffer was added to maintain the pH of the artificial seawater culture medium at 8.0 during the culture process, and then an appropriate amount of acclimated triangular shaped algae cells were collected by centrifugation. Each group of acclimated triangular shaped algae cells was divided into three parts, washed once with acidic culture medium of pH 6.5, pH 6.0 and pH 5.5, and then centrifuged to collect the cells and inoculate them into culture medium of corresponding pH value for stability test. After 5 days of culture, the growth performance of acclimated triangular shaped algae and wild type triangular shaped algae under acidic stimulation was compared. The results are as follows Figure 2 As shown, the growth rate of the domesticated P. tricornutum was increased compared with the wild type under all test conditions, with the growth rate increased by 27.5-110.4%.

[0034] (2) Transcriptome sequencing was used to identify the acid-resistant target genes common to the domesticated P. tricornutum.

[0035] The domesticated and wild algae strains were transferred from the optimal growth condition of pH = 8.0 to a low pH forced environment of pH = 6.0. The culture conditions were: light intensity 100 μmol / m 2 / s, the temperature was 22±1°C, and 40mM MES buffer was added to maintain a pH of 6.0 during the culture process. On day 5, cells were harvested, and total RNA was extracted for next-generation transcriptome sequencing. The transcriptome of the wild-type P. tricornutum stimulated by acid served as the control group, and differential expression analysis was performed on the transcriptomes of the three acclimated P. tricornutum groups under the same conditions.

[0036] Among genes with significant differential expression (fold change > 1) in two or more groups of domesticated 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, whose sequence structure is shown in SEQ ID No. 1; Phatr3_J50516, whose sequence structure is shown in SEQ ID No. 2; and Phatr3_Jdraft1806, whose sequence structure is shown in SEQ ID No. 3.

[0037] Example 2 Construction of engineered algae HI3310

[0038] This embodiment provides a method for constructing acid-resistant engineered algae using the sequence structure of Phatr3_J33543 as shown in SEQ ID No. 1. The specific steps are as follows:

[0039] (1) The stop codon at the 3' end of the Phatr3_J33543 gene, as shown in SEQ ID No. 1, was removed, and the start codon of the fluorescent protein (GFP) gene was removed by ligating a DNA sequence encoding five glycine residues to obtain a recombinant gene. Since the recombinant protein encoded by the recombinant gene contains the fluorescent protein sequence, subsequent screening by fluorescence microscopy is convenient.

[0040] The recombinant gene was inserted into the multiple cloning site of the pPhaNR plasmid (NCBI No. JN180663.1) to generate a vector plasmid. The pPhaNR plasmid is an amplification-capable plasmid that contains a gene that confers resistance to bleomycin, an antibiotic that can kill wild-type P. tricornutum cells.

[0041] The constructed vector plasmid is introduced into E. coli and amplified in the E. coli. After amplification in the E. coli, the vector plasmid is purified and used for later use.

[0042] (2) The expression vector plasmid obtained in step (1) is introduced into wild-type Phaeodactylum tricornutum cells using an electroporator as follows:

[0043] First, centrifuge at 1000 x g for 10 minutes to collect approximately 2 × 10 8 CFU triangular algae cells in the logarithmic growth phase. The centrifuged precipitate was washed three times with a 375mM sorbitol solution, repeatedly centrifuged and resuspended, and pretreated for desalting. Finally, the pretreated cells were resuspended in 100μL of a 375mM sorbitol solution, and 4μg of the vector plasmid obtained in step (1) and 40μg 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 transferred to a 2mm electroporation cuvette. Salmon sperm DNA helps the vector plasmid enter the algae cells.

[0044] The mixture was introduced into wild-type Phaeodactylum tricornutum cells using a Bio-Rad porator, with the following parameters: field strength 0.5 kV, capacitance 25 μF, and resistance 400 Ohm. The wild-type Phaeodactylum tricornutum cells containing the vector plasmid were allowed to stand in the dark for 24 hours and then inoculated into ESAW solid culture medium containing 100 μg / mL bleomycin. After 2-3 weeks, brown algal colonies that appeared on the culture medium were selected and transferred to a test tube containing artificial seawater culture medium. Culture conditions were: light intensity 100 μmol / m 2 / s, the temperature was 22±1°C, and 40 mM Tris-HCl buffer was added to maintain the pH at 8.0 during the culture process.

[0045] ESAW solid culture medium was prepared by preparing ESAW artificial seawater culture medium 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 ingredients of ESAW artificial seawater culture medium are shown in Table 1.

[0046] Table 1 ESAW artificial seawater culture medium composition

[0047]

[0048]

[0049] To avoid false positives in the brown algae selected from the bleomycin-containing ESAW solid medium, the algae cells were observed under a fluorescence microscope using an excitation wavelength of 488 nm and a detection wavelength of 525 nm. Algae expressing a fluorescent protein (GFP) signal were selected, indicating the acid-tolerant engineered algae, designated HI3310.

[0050] Example 3 Construction of engineered algae HI5021

[0051] This embodiment provides a method for constructing acid-resistant engineered algae using the sequence structure of Phatr3_J50516 as shown in SEQ ID No. 2. The specific steps are as follows:

[0052] (1) The stop codon at the 3' end of the Phatr3_J50516 gene, as shown in SEQ ID No. 2, was removed, and a fluorescent protein (GFP) gene, with the start codon removed, was ligated to a DNA sequence encoding five glycine residues to obtain a recombinant gene. Because the recombinant protein encoded by this recombinant gene contains a fluorescent protein sequence, subsequent screening using a fluorescence microscope is facilitated.

[0053] The recombinant gene was inserted into the multiple cloning site of the pPhaNR plasmid (NCBI No. JN180663.1) to generate a vector plasmid. The pPhaNR plasmid is an amplification-capable plasmid that contains a gene that confers resistance to bleomycin, an antibiotic that can kill wild-type P. tricornutum cells.

[0054] The constructed vector plasmid is introduced into E. coli and amplified in the E. coli. After amplification in the E. coli, the vector plasmid is purified and used for later use.

[0055] (2) The expression vector plasmid obtained in step (1) is introduced into wild-type Phaeodactylum tricornutum cells using an electroporator as follows:

[0056] First, centrifuge at 1000 x g for 10 minutes to collect approximately 2 × 10 8CFU triangular algae cells in the logarithmic growth phase. The centrifuged precipitate was washed three times with a 375mM sorbitol solution, repeatedly centrifuged and resuspended, and pretreated for desalting. Finally, the pretreated cells were resuspended in 100μL of a 375mM sorbitol solution, and 4μg of the vector plasmid obtained in step (1) and 40μg 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 transferred to a 2mm electroporation cuvette. Salmon sperm DNA helps the vector plasmid enter the algae cells.

[0057] The mixture was introduced into wild-type Phaeodactylum tricornutum cells using a Bio-Rad porator, with the following parameters: field strength 0.5 kV, capacitance 25 μF, and resistance 400 Ohm. The wild-type Phaeodactylum tricornutum cells containing the vector plasmid were allowed to stand in the dark for 24 hours and then inoculated into ESAW solid culture medium containing 100 μg / mL bleomycin. After 2-3 weeks, brown algal colonies that appeared on the culture medium were selected and transferred to a test tube containing artificial seawater culture medium. Culture conditions were: light intensity 100 μmol / m 2 / s, the temperature was 22±1°C, and 40 mM Tris-HCl buffer was added to maintain the pH at 8.0 during the culture process.

[0058] ESAW solid culture medium was prepared by preparing ESAW artificial seawater culture medium 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 ingredients of ESAW artificial seawater culture medium are shown in Table 1.

[0059] To avoid false positives in the brown algae selected from the bleomycin-containing ESAW solid medium, the algae cells were observed under a fluorescence microscope using an excitation wavelength of 488 nm and a detection wavelength of 525 nm. Algae expressing a fluorescent protein (GFP) signal were selected, indicating the acid-tolerant engineered algae, designated HI5021.

[0060] Example 4 Construction of engineered algae HI1831

[0061] This example provides a method for constructing acid-tolerant engineered algae using the sequence structure of Phatr3_Jdraft1806 as shown in SEQ ID No. 3. The protein encoded by the Phatr3_Jdraft1806 gene is primarily responsible for mediating transmembrane transport of bicarbonate and participating in maintaining cellular pH homeostasis. The specific steps are as follows:

[0062] (1) The stop codon at the 3' end of the Phatr3_Jdraft1806 gene, as shown in SEQ ID No. 3, was removed, and a fluorescent protein (GFP) gene, with the start codon removed, was ligated to a DNA sequence encoding five glycine residues to obtain a recombinant plasmid. Because the recombinant protein encoded by this recombinant gene contains a fluorescent protein sequence, subsequent screening using a fluorescence microscope is facilitated.

[0063] The recombinant gene was inserted into the multiple cloning site of the pPhaNR plasmid (NCBI No. JN180663.1) to generate a vector plasmid. The pPhaNR plasmid is an amplification-capable plasmid that contains a gene that confers resistance to bleomycin, an antibiotic that can kill wild-type P. tricornutum cells.

[0064] The constructed vector plasmid is introduced into E. coli and amplified in the E. coli. After amplification in the E. coli, the vector plasmid is purified and used for later use.

[0065] (2) The expression vector plasmid obtained in step (1) is introduced into wild-type Phaeodactylum tricornutum cells using an electroporator as follows:

[0066] First, centrifuge at 1000 x g for 10 minutes to collect approximately 2 × 10 8 CFU triangular algae cells in the logarithmic growth phase. The centrifuged precipitate was washed three times with a 375mM sorbitol solution, repeatedly centrifuged and resuspended, and pretreated for desalting. Finally, the pretreated cells were resuspended in 100μL of a 375mM sorbitol solution, and 4μg of the vector plasmid obtained in step (1) and 40μg 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 transferred to a 2mm electroporation cuvette. Salmon sperm DNA helps the vector plasmid enter the algae cells.

[0067] The mixture was introduced into wild-type Phaeodactylum tricornutum cells using a Bio-Rad porator, with the following parameters: field strength 0.5 kV, capacitance 25 μF, and resistance 400 Ohm. The wild-type Phaeodactylum tricornutum cells containing the vector plasmid were allowed to stand in the dark for 24 hours and then inoculated into ESAW solid culture medium containing 100 μg / mL bleomycin. After 2-3 weeks, brown algal colonies that appeared on the culture medium were selected and transferred to a test tube containing artificial seawater culture medium. Culture conditions were: light intensity 100 μmol / m 2 / s, the temperature was 22±1°C, and 40 mM Tris-HCl buffer was added to maintain the pH at 8.0 during the culture process.

[0068] ESAW solid culture medium was prepared by preparing ESAW artificial seawater culture medium 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 ingredients of ESAW artificial seawater culture medium are shown in Table 1.

[0069] To avoid false positives in the brown algae selected from the bleomycin-containing ESAW solid medium, the algae cells were observed under a fluorescence microscope using an excitation wavelength of 488 nm and a detection wavelength of 525 nm. Algae expressing a fluorescent protein (GFP) signal were selected, indicating the acid-tolerant engineered algae, designated HI1831.

[0070] The engineered algae constructed in Examples 2 to 4 were tested for acid stimulation. First, the growth performance of the three groups of engineered algae and wild-type P. tricornutum under optimal growth conditions was compared. The culture conditions were: light intensity 100 μmol / m 2 / s, the temperature was 22±1°C, and the pH of artificial seawater containing 40 mM Tris-HCl buffer was maintained at 8.0 during the culture process.

[0071] Then, an appropriate amount of cells were collected by centrifugation and washed once with an acidic culture medium at pH 5.0. The cells were collected by centrifugation again and inoculated into an artificial seawater culture medium at pH 5. After culturing for 5 days, the growth performance of the three groups of engineered algae and wild-type P. tricornutum under acidic stimulation was compared. Figure 4 The experimental results showed that wild-type P. tricornutum was strongly inhibited in an environment with a pH of 5.0, while the tolerance of the three groups of engineered algae to acidic stimulation was significantly improved, and the maximum growth rate of the engineered algae was 1.7 to 1.8 times that of the wild-type.

[0072] The embodiment described above is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

Claims

1. A method for constructing acid-resistant microalgae based on bicarbonate ion transporter, characterized in that: The specific steps are as follows: S1: A gene with annotated functions related to ion channels and pH homeostasis is selected as a target gene; the stop codon of the target gene is removed, and a fluorescent protein gene with the start codon removed is connected to a DNA sequence encoding glycine to obtain a recombinant gene; the target gene has a sequence structure as shown in SEQ ID No. 3, Phatr3_Jdraft1806; S2: inserting the recombinant gene into the multiple cloning site of the pPhaNR plasmid having an antibiotic resistance gene to obtain a vector plasmid; S3: mixing the vector plasmid and the salmon sperm DNA solution to obtain a mixed solution; ice-bathing the mixed solution and then introducing it into diatom cells that have undergone desalination pretreatment by electroporation; then placing the diatom cells in ESAW culture medium in the dark for a first culture; inoculating the diatom cells after the first culture onto ESAW solid selective medium containing bleomycin and performing a second culture until algal colonies appear on the solid selective medium; the diatom cells are wild-type Phaeodactylum tricornutum cells; S4: selecting algae colonies with fluorescent protein signals from the algae colonies obtained in step S3, which are acid-resistant engineered algae.

2. The method for constructing acid-resistant microalgae based on bicarbonate ion transporter according to claim 1, characterized in that: The pPhaNR plasmid described in step S2 contains the 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 microalgae based on bicarbonate ion transporter according to claim 1, characterized in that: The electroporation described in step S3 was performed using an electroporator with the following parameters set: field strength of 0.5 kV, capacitance of 25 μF, and resistance of 400 Ohm.

4. The method for constructing acid-resistant microalgae based on bicarbonate ion transporter according to claim 1, characterized in that: The mass ratio of the vector plasmid to the salmon sperm DNA in the mixed solution described in step S3 is 1:

10.

5. The method for constructing acid-resistant microalgae based on bicarbonate ion transporter according to claim 1, characterized in that: The desalination pretreatment step in step S3 is as follows: after collecting the P. tricornutum cells in the logarithmic growth phase by centrifugation, the cells are repeatedly centrifuged and resuspended, and the precipitated P. tricornutum cells are washed with a sorbitol solution to remove salt and then resuspended in a sorbitol solution.

6. The method for constructing acid-resistant microalgae based on bicarbonate ion transporter according to claim 5, characterized in that: The concentration of the sorbitol solution was 375 mM.

7. The method for constructing acid-resistant microalgae based on bicarbonate ion transporter according to claim 1, characterized in that: The first culture is carried out in the dark for 24 hours; the second culture is carried out for 2 to 3 weeks.

8. The method for constructing acid-resistant microalgae based on bicarbonate ion transporter according to claim 1, characterized in that: The concentration of bleomycin in the ESAW solid selection medium described in step S3 is 100 μg / L.

9. The method for constructing acid-resistant microalgae based on bicarbonate ion transporter according to claim 1, characterized in that: The intensity of the fluorescent protein signal in step S4 is 6 to 10 times that of the green fluorescence in wild-type algal cells.

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