Eurytopic chlamydomonas reinhardtii expression vector based on PSAD promoter and 3*Flag tag as well as construction method and application of eurytopic chlamydomonas reinhardtii expression vector

Abstract

The invention discloses an eurytopic chlamydomonas reinhardtii expression vector based on a PSAD promoter and a 3 * Flag tag as well as a construction method and application of the eurytopic chlamydomonas reinhardtii expression vector. The nucleotide sequence of the eurytopic chlamydomonas reinhardtii expression vector is shown as SEQ ID No.1; the vector comprises a PSAD promoter, a target gene insertion site EcoR V, a 3 * Flag tag at the 3'terminal, an rbcS2 terminator, a TUB2 promoter, a hygromycin B screening gene and an rbcS2 terminator. The construction method comprises the following steps: constructing a skeleton of a pHyg-PSAD-3Flag vector; constructing a sequence fragment fused with a 3 * Flag tag; and connecting the sequence fragment fused with the 3 * Flag tag with a plasmid skeleton fragment through T4 DNA (deoxyribonucleic acid) enzyme so as to construct the pHyg-PSAD-3Flag vector. According to the invention, the expression plasmid of the chlamydomonas pHyg-PSAD-3Flag can be used for efficiently screening algae strains capable of stably expressing target protein.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and 3×Flag tag, its construction method, and its application. Background Technology

[0002] As a model organism, the single-celled eukaryotic Chlamydomonas reinhardtii, besides being known as photosynthetic yeast, has shown great potential in various applications for the production of proliferative metabolites and recombinant proteins, based on in-depth research over the past few decades. The following characteristics make Chlamydomonas reinhardtii an excellent system for producing recombinant proteins: easy growth, rapid reproduction, and low cost; simple and efficient transformation; complete genomic information; the ability to accurately fold and assemble complex proteins; and well-developed molecular biology techniques. Modification of Chlamydomonas genes using various promoters and markers has become a new direction for the continuous development of genetic engineering tools.

[0003] FLAG-tagged proteins are small, hydrophilic polypeptides (DYKDDDDK) encoding eight amino acids, and were the first protein tags designed for fusion proteins. The Kozak sequence constructed in the vector allows for higher expression efficiency of FLAG-tagged fusion proteins in eukaryotic expression systems. As a fusion expression tag, FLAG does not mask other epitopes and domains in the fusion protein, is less likely to alter its function, secretion, or transport, and can be recognized by anti-FLAG antibodies. This facilitates the detection and identification of FLAG-containing fusion proteins using methods such as Western blotting. Designing three Flag sequences together (3×Flag) further enhances expression efficiency, antibody binding, and detection sensitivity. Currently, Flag tags are widely used in protein expression, purification, identification, functional studies, and protein-protein interactions.

[0004] In conclusion, constructing a diverse, modular, standardized, and rapidly interchangeable universal expression plasmid vector is a necessary infrastructure for Chlamydomonas reinhardtii functional genomics, cell biology, and synthetic biology research to move towards a high-throughput and systematic stage. Therefore, seeking a plasmid vector with a 3×Flag tag and its construction method is particularly important. Summary of the Invention

[0005] This invention provides a broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and 3×Flag tag, along with its construction method and applications. This method, using the resistance gene Hyg+ and the PSAD promoter, enables simultaneous expression of the resistance selection gene and the target gene on a single vector during target gene protein expression in Chlamydomonas reinhardtii. This couples the expression of the resistance selection gene with the target gene, increasing the yield of positive transformants. Furthermore, the strategy of using a tandem PSAD promoter and an endogenous promoter allows for efficient expression of the target gene, resulting in stable and high expression of the target protein. The construction of this expression vector enriches the existing Chlamydomonas reinhardtii gene expression vector library and expands the genetic manipulation platform for Chlamydomonas cell factories.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and a 3×Flag tag, comprising a PSAD promoter, a target gene insertion site EcoR V, a 3' end 3×Flag tag, an rbcS2 terminator, a TUB2 promoter, a hygromycin B selection gene, and an rbcS2 terminator; wherein, the nucleotide sequence of the PSAD promoter is shown in SEQ ID No. 2, the nucleotide sequence of the target gene insertion site EcoR V is shown in SEQ ID No. 3, the nucleotide sequence of the 3' end 3×Flag tag is shown in SEQ ID No. 4, the nucleotide sequence of the TUB2 promoter is shown in SEQ ID No. 5, the nucleotide sequence of the hygromycin B selection gene is shown in SEQ ID No. 6, the nucleotide sequence of the rbcS2 terminator is shown in SEQ ID No. 7, and the nucleotide sequence of the broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and the 3×Flag tag, namely the pHyg-PSAD-3Flag vector, is shown in SEQ ID No. 7. As shown in IDNo.1.

[0007] This invention also provides a method for constructing a broadly applicable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and a 3×Flag tag, comprising the following steps: (1) Construction of the backbone of the pHyg-PSAD-3Flag vector: The plasmid pHyg-PSAD-3HA was digested with EcoR V and Bgl II, and its nucleotide sequence is shown in SEQ ID No.8. Two double-stranded linear DNA fragments with lengths of 147 bp and 5427 bp were obtained. The digestion products were analyzed by electrophoresis. The 5427 bp fragment was purified and recovered and the DNA concentration was determined for later use. (2) Constructing a sequence fragment fused with a 3×Flag tag: Using plasmid psAD-IFT46 Flag-strep Hyg+ as a template, the nucleotide sequence of plasmid psAD-IFT46 Flag-strep Hyg+ is shown in SEQ ID No. 9. The PCR amplification of the fragment with the nucleotide start and end positions of 5432-5547 bp was performed. The upstream primer 3Flag-F and the downstream primer 3Flag-R were used. The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID No. 10 and SEQ ID No. 11, respectively. The 5' end of the primer pair contains the EcoR V site. At the same time, the primer was designed with a Bgl II restriction site sequence. The DNA amplification product with a length of 137 bp was purified and recovered. Then, EcoR V and Bgl II were used for restriction modification to obtain a sequence fragment fused with a 3×Flag tag with a length of 112 bp. (3) The PCR product of the sequence fragment fused with the 3×Flag tag obtained in step (2) was purified, the concentration of the fragment was determined, and the fragment fused with the 3×Flag tag was ligated with the backbone fragment 5427 bp obtained in step (1) by T4 DNA ligation method to form a widely adaptable Chlamydomonas reinhardtii expression plasmid, namely pHyg-PSAD-3Flag vector, with a full length of 5539 bp.

[0008] Preferably, the PCR preparation system in step (2) is as follows: ; The PCR procedure is as follows:

[0009] Preferably, in step (1), the electrophoretic analysis is performed by separating the target fragment using 1% agarose gel electrophoresis; in steps (1) and (2), the purification and recovery are performed using a gel recovery kit and a PCR column purification kit.

[0010] Preferably, in step (3), the T4 DNA ligation method involves ligating the sequence fragment fused with the 3×Flag tag to the plasmid backbone structure fragment 5427 bp using T4 DNA ligase at 22 °C for 2 h; the T4 ligation system is as follows: This invention also provides an application of a broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and a 3×Flag tag in screening positive transformed algal strains. The specific application process is as follows: pHyg-PSAD-3Flag is digested with EcoR V site enzymes, and the target gene is directionally integrated into the pHyg-PSAD-3Flag expression vector through homologous recombination via the left and right homologous arms of the cleavage or by enzyme digestion-ligation method to obtain the final target gene expression plasmid; the final target gene expression plasmid is linearized with restriction endonuclease and then electroporated into Chlamydomonas reinhardtii cells to be tested; positive transformed algal strains stably expressing the target protein are obtained by screening with Hygromycin B plates and Western blotting.

[0011] Compared with the prior art, the present invention has the following advantages: (1) This invention successfully constructed a broadly adaptable Chlamydomonas reinhardtii expression vector pHyg-PSAD-3Flag. The DNA fragment to be expressed was inserted into pHyg-PSAD-3Flag, and the expression plasmid of the novel target gene was successfully constructed through steps such as plasmid transformation, colony identification, plasmid extraction, enzyme digestion identification and DNA sequencing. Subsequently, after DNA linearization and electroporation transformation, it was integrated into the Chlamydomonas cell genome to realize the expression of the target gene and subsequent research. This method, through the resistance gene Hyg+ and PSAD promoter, enables the simultaneous expression of the resistance selection gene and the target gene on a single vector when expressing the target gene protein in Chlamydomonas reinhardtii, so that the expression of the resistance selection gene and the target gene are coupled, improving the positive transformant acquisition rate. Furthermore, the strategy of tandem PSAD promoter and endogenous promoter can be used to efficiently express the target gene, thereby obtaining stable and high expression of the target protein. The construction of this expression vector enriches the existing Chlamydomonas reinhardtii gene expression vector library and expands the genetic operation platform of Chlamydomonas cell factory.

[0012] (2) This invention employs the "strong constitutive" PSAD promoter strategy of Chlamydomonas reinhardtii, which can more effectively drive the expression of exogenous genes while reducing the risk of non-expression by other expression vectors. This promoter contains a light-responsive enhancer element and has high activity under photoautotrophic or mixed nutrient conditions. This modular approach can be directly used for routine overexpression and can also be upgraded through enhancer, intron, UTR optimization, and other strategies. The protein tag 3×Flag does not cover other protein epitopes and domains in the fusion protein in Chlamydomonas cells, which not only enhances expression efficiency and detection sensitivity but also provides more options and methods for expressing one or more different target genes in Chlamydomonas cells. This is beneficial for studying the expression, purification, identification, structure, localization, and function of target proteins, and further facilitates the revelation of the related molecular mechanisms between different target proteins. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of 1% agarose gel electrophoresis of the backbone vector digested with EcoR V and Bgl II enzymes. Figure 2 This is a schematic diagram of the final sequence structure of the widely adaptable Chlamydomonas reinhardtii expression vector pHyg-PSAD-3Flag. Detailed Implementation

[0014] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. Unless otherwise specified, the methods described below are conventional methods.

[0015] A broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and a 3×Flag tag includes a PSAD promoter, a target gene insertion site EcoR V, a 3' end 3×Flag tag, an rbcS2 terminator, a TUB2 promoter, a hygromycin B selection gene, and an rbcS2 terminator. The nucleotide sequence of the PSAD promoter is shown in SEQ ID No. 2, the nucleotide sequence of the target gene insertion site EcoR V is shown in SEQ ID No. 3, the nucleotide sequence of the 3' end 3×Flag tag is shown in SEQ ID No. 4, the nucleotide sequence of the TUB2 promoter is shown in SEQ ID No. 5, the nucleotide sequence of the hygromycin B selection gene is shown in SEQ ID No. 6, the nucleotide sequence of the rbcS2 terminator is shown in SEQ ID No. 7, and the nucleotide sequence of the broadly adaptable Chlamydomonas reinhardtii expression vector, namely the pHyg-PSAD-3Flag vector, is shown in SEQ ID No. 1.

[0016] A method for constructing a broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and 3×Flag tag, comprising the following steps: (1) Construction of the backbone of the pHyg-PSAD-3Flag vector: The plasmid pHyg-PSAD-3HA was digested with EcoR V and Bgl II, and its nucleotide sequence is shown in SEQ ID No. 8, resulting in two double-stranded linear DNA segments with lengths of 147 bp and 5427 bp, respectively. Figure 1 As shown; the 5427 bp fragment was recovered by 1% agarose gel electrophoresis and the DNA concentration was determined for later use; (2) Constructing a sequence fragment fused with a 3×Flag tag: Using plasmid psAD-IFT46 Flag-strep Hyg+ as a template, the nucleotide sequence of plasmid psAD-IFT46 Flag-strep Hyg+ is shown in SEQ ID No. 9. The PCR amplification of the fragment with the nucleotide start and end positions of 5432-5547 bp was performed. The upstream primer 3Flag-F and the downstream primer 3Flag-R were used. The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID No. 10 and SEQ ID No. 11, respectively. The 5' end of the primer pair contains the EcoR V site. At the same time, the primer was designed with a Bgl II restriction site sequence. The DNA amplification product with a length of 137 bp was gel recovered and then modified by EcoR V and Bgl II restriction enzyme digestion to obtain a sequence fragment fused with a 3×Flag tag with a length of 112 bp. The PCR preparation system is as follows: The PCR procedure is as follows: (3) The PCR product of the 112 bp sequence fragment fused with the 3×Flag tag obtained in step (2) was purified, the fragment concentration was determined, and the 3×Flag tag fused fragment was ligated with the 5427 bp backbone fragment obtained in step (1) using the T4 DNA ligation method to form the broadly adaptable expression plasmid Chlamydomonas reinhardtii expression vector, namely the pHyg-PSAD-3Flag vector, with a full length of 5539 bp, as shown below. Figure 2 As shown; the T4 DNA ligation method involves ligating a sequence fragment fused with a 3×Flag tag to a 5427 bp plasmid backbone structure fragment using T4 DNA ligase at 22 °C for 2 h; the T4 ligation system is as follows: An application of a broadly adaptable *Chlamydomonas reinhardtii* expression vector based on the PSAD promoter and 3×Flag tag in screening positive transformed algal strains is described. The specific process is as follows: After digestion with the EcoR V site, the target gene can be directionally integrated into the pHyg-PSAD-3Flag expression vector via homologous recombination through the left and right homologous arms of the digestion site or by enzyme digestion-ligation method, obtaining the final target gene expression plasmid. The final target gene expression plasmid is linearized with restriction endonucleases and then electroporated into *Chlamydomonas reinhardtii* cells for testing. Positive transformed algal strains stably expressing the target protein are obtained through screening using Hygromycin B plates and Western blotting. The screening process includes preparing TAP liquid medium; preparing linearized fragments of the recombinant plasmid; preparing Hyg+ resistance selection plates; electroporation transformation; and screening and identification of transformants. The specific process is shown below: a. Preparation of TAP liquid culture medium: Prepare TAP medium according to the following formula, dispense it into aeration bottles after preparation, and autoclave at 121 °C for 20 min.

[0017] TAP liquid culture medium formulation (for 1 L): b. Preparation of linearized fragments of recombinant plasmids: The recombinant plasmid was digested with a suitable restriction endonuclease. After digestion, the enzyme was inactivated according to the instructions of the different restriction endonucleases and stored at -20 °C for later use. This is the linearized recombinant plasmid to be transformed.

[0018] c. Preparation of Hyg+ resistance screening plates: After preparing TAP liquid medium as described above, add 15 g / L of agar powder and autoclave at 121 °C for 20 min. After the medium cools to a temperature that is not too hot to handle, add hygromycin B (Hyg+, 50 mg / mL stock solution, 2500× dilution) in a clean bench to make the final concentration of Hyg+ in the medium 20 μg / mL. Shake well and pour in about 15 mL of medium. Gently rotate the petri dish to distribute the medium evenly at the bottom of the plate. Cover the dish and store at 4 °C after solidification for later use. d. Electroconvulsive transformation: ①Culturing Chlamydomonas cells: In a clean bench, a suitable amount of Chlamydomonas cells were picked from a plate and inoculated into an aeration bottle containing sterilized TAP medium. The cells were cultured continuously under light and aeration at 23±0.5 °C and 8000 Lx for 3-4 days until the cell concentration reached 1-2 × 10⁷ cells / mL. The growth status of the Chlamydomonas cells was observed under a microscope. If the cells were in good condition, they were transferred to a fresh, sterilized TAP liquid aeration bottle to achieve a final transfer concentration of 1 × 10⁶ cells / mL. Continuous light and aeration culture was then maintained for 20 h until the final concentration reached 4 × 10⁶ cells / mL. The growth status of the Chlamydomonas cells was observed under a microscope again. If the cells were in good condition, they could be used for electroporation transformation.

[0019] ② Counting of Chlamydomonas cells: In a clean bench, take 50 µL of Chlamydomonas cells from an aeration bottle, place them in an EP tube, and add 50 µL of 5% glutaraldehyde fixative to fix the Chlamydomonas cells. Mix well. Add 10 µL of the above solution to one side of a hemocytometer, gently cover with a coverslip, and count the cells under a regular optical microscope. First, locate the large squares, then locate the four corner squares, count them (count the top ones but not the bottom ones, count the left ones but not the right ones), and record the counts. Estimate the cell concentration using the formula: number of cells in the four corner squares × 2 × 4 × 10⁴ cells / mL.

[0020] ③ Cleaning and pre-cooling of the electrocution cup: Inside the clean bench, each electrocution cup is first washed with 1 mL of anhydrous ethanol and then air-dried. It is then washed with a sterile 1×TAP + 60 mM sorbitol solution and air-dried. Finally, it is pre-cooled in a -20 °C refrigerator.

[0021] ④ Collection of Chlamydomonas cells: In a clean bench, take a small amount of cultured cells and count them using a hemocytometer. Calculate the required cell volume for each transformation, based on the requirement of 250 μL of cells at a concentration of 1 × 10⁸ cells / mL. Transfer an appropriate amount of cells to a 50 mL centrifuge tube, centrifuge at 2500 rpm for 3 min, discard the supernatant, and wash once with pre-chilled sterile 1×TAP + 60 mM sorbitol (resuspend in 1×TAP + 60 mM sorbitol, centrifuge at 2500 rpm for 3 min, discard the supernatant). Finally, resuspend the cells in pre-chilled sterile 1×TAP + 60 mM sorbitol to the required transformation volume and incubate on ice for 10 min.

[0022] ⑤ Square wave electric shock conversion: In a clean bench, add 250 μL of 1×10⁸ algae / mL solution and 500-1000 ng of linearized recombinant plasmid fragments to a pre-cooled electroporation cup. Gently tap to mix and then quickly transfer to ice. Wipe the sides of the electroporation cup dry with absorbent paper. Use a BTX ECM830 electroporator with the parameters set to 500 V, 4 ms, 6 bands, and an electroporation interval of 100 ms. After electroporation, place on ice for 5-10 min.

[0023] ⑥ Cell overnight recovery: After electroporation, the cells were transferred to 50 mL sterile centrifuge tubes containing 10 mL of sterile 1×TAP + 60 mM sorbitol solution and allowed to recover overnight on a shaker under low light conditions.

[0024] ⑦ Prepare a 20% starch solution: Weigh an appropriate amount of starch and prepare a 20% starch solution. Wash once with anhydrous ethanol, then twice with distilled water, discarding the supernatant after each wash. Centrifuge at 1000 rpm for 2 min, and bring to a final volume with 70% ethanol. Place at room temperature. In a clean bench, mix the 20% starch solution thoroughly. Measure the required volume (2 mL) for each conversion. Wash twice with TAP, then once with sterile 1×TAP + 60 mM sorbitol solution, discarding the supernatant after each wash. Centrifuge at 1000 rpm for 2 min, then resuspend in sterile 1×TAP + 60 mM sorbitol to the appropriate volume. Finally, add sterile 20% PEG 6000 to achieve a final concentration of 0.4%.

[0025] ⑧ Coating plate: Centrifuge the recovered cells at 2500 rpm for 3 min, remove the supernatant, add 2 mL of the prepared 20% starch solution to each tube to resuspend and mix well, add 1 mL of the mixture to each antibiotic plate, spread it evenly, air dry, seal with sealing film, invert in a light incubator, and culture for 4-7 days under normal conditions.

[0026] e. Screening and identification of transformants Transformants grown on Hyg+ resistance selection plates were picked in a clean bench, spread onto TAP plates, and labeled. They were cultured under standard conditions for 3-4 days. Once transformant colonies had formed, 1.2 mL of TAP medium was added to each well of a 24-well plate in a clean bench, labeled, and colonies were picked and transferred to the corresponding wells. The plates were sealed and placed in a light incubator for 2-3 days under standard conditions with a shaker. Transformed algal cells were collected, with 1 × 10⁷ cells collected in each 1.5 mL EP tube and frozen at -80 °C. Western blotting was used to identify the expression of the target gene based on the tag protein.

[0027] Following the above screening process, monoclonal antibodies with a positivity rate of ≥95% can be obtained within 2 weeks. Subsequently, through multidimensional experiments such as Western blotting and fluorescence colocalization, the subcellular spatiotemporal distribution and biological function of the target protein can be analyzed.

Claims

1. A broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and a 3×Flag tag, characterized in that, The expression vector includes a PSAD promoter, a target gene insertion site EcoR V, a 3×Flag tag at the 3' end, an rbcS2 terminator, a TUB2 promoter, a hygromycin B selection gene, and an rbcS2 terminator. The nucleotide sequence of the PSAD promoter is shown in SEQ ID No. 2, the nucleotide sequence of the target gene insertion site EcoR V is shown in SEQ ID No. 3, the nucleotide sequence of the 3×Flag tag at the 3' end is shown in SEQ ID No. 4, the nucleotide sequence of the TUB2 promoter is shown in SEQ ID No. 5, the nucleotide sequence of the hygromycin B selection gene is shown in SEQ ID No. 6, the nucleotide sequence of the rbcS2 terminator is shown in SEQ ID No. 7, and the nucleotide sequence of the broadly adaptable Chlamydomonas reinhardtii expression vector, namely the pHyg-PSAD-3Flag vector, is shown in SEQ ID No.

1.

2. A method for constructing a broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and 3×Flag tag as described in claim 1, characterized in that, Includes the following steps: (1) Construction of the backbone of the pHyg-PSAD-3Flag vector: The nucleotide sequence of the plasmid pHyg-PSAD-3HA, as shown in SEQ ID No. 8, was obtained by double digestion with EcoR V and Bgl II to obtain two double-stranded linear DNA segments with lengths of 147 bp and 5427 bp, respectively. The enzyme digestion products were analyzed by electrophoresis. The 5427 bp fragment was purified and recovered, and the DNA concentration was determined for later use. (2) Constructing a sequence fragment fused with a 3×Flag tag: Using plasmid psAD-IFT46 Flag-strep Hyg+ as a template, the nucleotide sequence of plasmid psAD-IFT46 Flag-strep Hyg+ is shown in SEQ ID No.

9. The PCR amplification of the fragment with the nucleotide start and end positions of 5432-5547 bp was performed. The upstream primer 3Flag-F and the downstream primer 3Flag-R were used. The nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID No. 10 and SEQ ID No. 11, respectively. The 5' end of the primer pair contains the EcoR V site. At the same time, the primer was designed with a Bgl II restriction site sequence. The DNA amplification product with a length of 137 bp was purified and recovered. Then, EcoR V and Bgl II were used for restriction modification to obtain a sequence fragment fused with a 3×Flag tag with a length of 112 bp. (3) The PCR product of the sequence fragment fused with the 3×Flag tag obtained in step (2) was purified, the concentration of the fragment was determined, and the fragment fused with the 3×Flag tag was ligated with the backbone fragment 5427 bp obtained in step (1) by T4 DNA ligation method to form a widely adaptable Chlamydomonas reinhardtii expression vector, namely pHyg-PSAD-3Flag vector, with a full length of 5539 bp.

3. The method for constructing a broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and 3×Flag tag according to claim 2, characterized in that, In step (2), the PCR preparation system is as follows: ； The PCR procedure is as follows: 。 4. The method for constructing a broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and 3×Flag tag according to claim 2, characterized in that, In step (1), the electrophoresis analysis is performed by separating the target fragment using 1% agarose gel electrophoresis; in steps (1) and (2), the purification and recovery are performed using a gel recovery kit and a PCR column purification kit.

5. The method for constructing a broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and 3×Flag tag according to claim 2, characterized in that, In step (3), the T4 DNA ligation method involves ligating a sequence fragment fused with a 3×Flag tag to a 5427 bp plasmid backbone structure fragment at 22 °C for 2 h using T4 DNA ligase; the T4 ligation system is as follows:

6. The application of the broadly adaptable Chlamydomonas reinhardtii expression vector based on the PSAD promoter and 3×Flag tag as described in claim 1 in screening positive transformed algal strains, the specific application process is as follows: pHyg-PSAD-3Flag is digested with EcoR V site enzyme, and homologous recombination is performed through the left and right homologous arms of the cleavage or the target gene is directionally integrated into the pHyg-PSAD-3Flag expression vector by enzyme digestion-ligation method to obtain the final target gene expression plasmid; the final target gene expression plasmid is linearized with restriction endonuclease and then electroporated into Chlamydomonas reinhardtii cells to be tested; positive transformed algal strains stably expressing the target protein are obtained by screening by Hygromycin B plate and immunoblotting method.

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