High constitutive activity-based PSAD strong promoter driven exogenous gene expression vector system and construction method and application thereof

Abstract

The invention relates to a high constitutive activity-based PSAD strong promoter driven exogenous gene expression vector system and a construction method and application thereof. The nucleotide sequence of the system is shown as SEQ ID No.1; the main sequence of the vector system is sequentially composed of the following components: a PSAD promoter, an exogenous gene insertion site EcoR V, a 3 * HA tag at the 3'terminal, an rbcS2 terminator, a TUB2 promoter, a hygromycin B (Hyg +) screening gene and an rbcS2 terminator. The construction method comprises the following steps: constructing an outer edge skeleton sequence fragment of the vector system; synthesizing a DNA fragment of the PSAD promoter; and carrying out in-vitro homologous recombination splicing on the skeleton sequence fragment and a DNA fragment of a PSAD promoter, so as to finally obtain the pHyg-PSAD-3HA expression vector. The high-constitutive strong promoter PSAD is added, so that transcription and expression of an exogenous gene can be efficiently driven, and the gene expression is quicker and more stable.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a PSAD strong promoter-driven exogenous gene expression vector system with high constitutive activity, its construction method, and its application in Chlamydomonas reinhardtii. Background Technology

[0002] Chlamydomonas reinhardtii, belonging to the Chlorophyta phylum, is a single-celled eukaryotic aquatic organism that has long been used as a model organism to study various biological processes such as photosynthesis, cilia, and cell cycle control. As a biotechnology platform for algae, it boasts advantages such as simple growth conditions, ease of large-scale cultivation, and economic sustainability; moreover, the application of biotechnology on this platform has become increasingly mature, making Chlamydomonas an important "cell super factory." In recent years, the genetic engineering of Chlamydomonas to produce recombinant proteins and other high-value products, as well as the production of algal lipids as biofuels, has gradually gained widespread attention.

[0003] The PSAD promoter was first reported in spinach in 1994. The psad gene sequence of *Chlamydomonas reinhardtii* was cloned and sequenced in 2001, and in 2013 it was evaluated as one of the excellent promoters for transgene expression in *Chlamydomonas reinhardtii*. The psad gene is a nuclear gene encoding a highly expressed protein of subunit II of the photosynthetic system I reaction center in photosynthetic organisms. Generally, gene expression is positively correlated with promoter strength, and promoters encoding genes that express highly expressed proteins in vivo are usually considered strong promoters. The PSAD promoter is recognized as one of the endogenous strong promoters for stable gene expression in *Chlamydomonas*. Therefore, exploring PSAD strong promoter-driven exogenous gene expression vector systems with high constitutive activity is particularly important for exogenous gene expression in *Chlamydomonas reinhardtii* and its application as a "cell superfactory."

[0004] Currently, there are still shortcomings in the selection of promoters for *Chlamydomonas reinhardtii* gene expression: firstly, the number of available promoters is limited, and single-gene expression vectors cannot meet the expression needs of various genes, sometimes resulting in restricted expression of certain genes under study; secondly, co-expression systems have reached a bottleneck, and further research is needed to develop promoter tandem schemes. Therefore, exploring strong promoters and a rich and diverse library of expression vectors will be beneficial to the establishment of *Chlamydomonas reinhardtii*'s "cellular superfactory". Summary of the Invention

[0005] This invention provides a system for driving exogenous gene expression vectors based on a highly constitutively active PSAD strong promoter, along with its construction method and applications. This system pre-installs a C-terminal 3×HA tag within a universal vector. Through a fusion expression strategy, it enables subcellular localization analysis, protein purification, and functional studies of target exogenous proteins. By coupling dominant selection genes and exogenous genes within the same system, and employing a single-transformation rather than co-transformation strategy, this system improves the expression success rate of exogenous proteins in *Chlamydomonas reinhardtii* cells, greatly facilitating subsequent screening and detection. The addition of the highly constitutively active PSAD strong promoter enables efficient driving of target gene transcription and expression, resulting in faster and more stable exogenous target gene expression. Therefore, this invention provides researchers with a highly efficient and stable *Chlamydomonas reinhardtii* expression vector, enriching the genetic manipulation platform of this "chassis organism," and is expected to provide a powerful tool for basic biological research such as gene function studies within *Chlamydomonas reinhardtii* cells, as well as for biotechnology development such as the expression of exogenous bioactive substances.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a PSAD strong promoter-driven exogenous gene expression vector system based on high constitutive activity, wherein the main sequence of the vector system is composed of the following components in sequence: PSAD promoter, exogenous gene insertion site EcoR V, 3×HA tag at the 3' end, rbcS2 terminator, TUB2 promoter, hygromycin B (Hyg+) selection gene, and rbcS2 terminator; wherein, the nucleotide sequence of the PSAD promoter is shown in SEQ ID No. 2, the nucleotide sequence of the exogenous gene insertion site EcoR V is shown in SEQ ID No. 3, the nucleotide sequence of the 3×HA 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 (Hyg+) 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 PSAD strong promoter-driven exogenous gene expression vector system based on high constitutive activity is shown in SEQ ID No. 1.

[0007] This invention also provides a method for constructing a PSAD strong promoter-driven exogenous gene expression vector system with high constitutive activity, comprising the following steps: (1) Construction of the outer backbone sequence fragment of the vector system: using plasmid pHyg-3HA as a template, the nucleotide sequence of plasmid pHyg-3HA is shown in SEQ ID No.8. It is digested with EcoR V and analyzed by electrophoresis to obtain a linearized fragment of 4777 bp. The fragment is then recovered and its concentration is detected for later use. (2) Synthesis of PSAD promoter DNA fragment: 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. At the same time, PCR amplification of the target nucleotide position of the fragment at 705-1507 bp is performed using upstream primer SI-PSAD-F and downstream primer SI-PSAD-R. The nucleotide sequences of the upstream primer and downstream primer are shown in SEQ ID No.10 and SEQ ID No.11, respectively. After amplification, DNA with a length of 842 bp can be obtained. Its two ends are designed with homologous arms at both ends of the EcoRV cleavage site of the outer backbone sequence constructed in step (1) through primer design, thereby obtaining the PSAD promoter DNA fragment. The concentration is measured for later use. (3) The outer backbone sequence fragment obtained in step (1) and the PSAD promoter DNA fragment obtained in step (2) are spliced ​​together by in vitro homologous recombination to form the exogenous gene expression vector system pHyg-PSAD-3HA, with a full length of 5574 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, and the fragment is recovered by purifying and recovering it using a gel recovery kit.

[0010] Preferably, in step (3), the homologous recombination splicing involves reacting the PSAD promoter DNA fragment with the outer backbone sequence fragment obtained in step (1) at 50 °C for 1 h using a homologous recombination enzyme. The homologous recombination system is set as follows: This invention also provides an application of a PSAD strong promoter-driven exogenous gene expression vector system with high constitutive activity in Chlamydomonas reinhardtii. The specific application process is as follows: cloning or synthesizing an exogenous gene fragment, inserting it into the EcoR V site of the pHyg-PSAD-3HA vector in vitro via homologous recombination or enzyme ligation to construct the final expression plasmid; linearizing the final expression plasmid and transforming it into Chlamydomonas cells using electroporation; obtaining plasmid-transformed positive algal strains through preliminary screening with hygromycin B; and finally, using Western blotting and anti-HA antibody screening to select algal strains that stably express the target protein.

[0011] Compared with the prior art, the present invention has the following advantages: (1) This invention successfully constructed a PSAD strong promoter-driven exogenous gene expression vector system with high constitutive activity. In this system, the dominant selection gene Hyg+ and the exogenous gene are tandemly connected in the same expression vector system, which effectively avoids the shortcomings of the Chlamydomonas co-transformation strategy, namely, the selection gene is integrated into the genome while the exogenous target gene is not integrated into the cell genome. This achieves the coupling of expression between the resistance selection gene and the target gene, improves the positive transformant acquisition rate, and thus obtains stable expression of the target protein, which is beneficial for studying the molecular mechanisms related to the protein structure, location and function of the target protein.

[0012] (2) This invention develops a highly efficient and compatible Chlamydomonas reinhardtii nuclear expression platform, pHyg-PSAD-3HA, driven by the strong promoter PSAD, with the target gene accepted by the EcoR V single enzyme restriction site, a 3×HA tag fused to the C-terminus, and a double rbcS2 terminator to enhance mRNA stability. The vector can be constructed within 48 hours through a three-step method of "backbone assembly - PSAD fragment cloning - in vitro homologous recombination". After linearization, electroporation can be performed, and positive algal strains can be obtained within 7-10 days. The target protein accounts for ≥0.3% of the total soluble protein. Western blotting, immunofluorescence, and cell localization can be performed directly using anti-HA antibodies to detect the expression of the target gene without the need to prepare exogenous specific antibodies. This universal platform significantly shortens the research and development cycle of gene function research, metabolic pathway reconstruction, and high-value-added exogenous protein production, providing a plug-and-play efficient tool for Chlamydomonas reinhardtii basic biology and microalgal biomanufacturing. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of 1% agarose gel electrophoresis of the EcoR V digested pHyg-3HA vector (4777 bp); Figure 2 This is a schematic diagram of the sequence structure of a PSAD strong promoter-driven exogenous gene expression vector system with high constitutive activity. 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 highly constitutive PSAD strong promoter-driven exogenous gene expression vector system is disclosed. The main sequence of the vector system comprises the following components in sequence: a PSAD promoter, an exogenous gene insertion site EcoR V, a 3' terminating 3×HA tag, an rbcS2 terminator, a TUB2 promoter, a hygromycin B (Hyg+) selection gene, and the rbcS2 terminator. The nucleotide sequence of the PSAD promoter is shown in SEQ ID No. 2, the nucleotide sequence of the exogenous gene insertion site EcoR V is shown in SEQ ID No. 3, the nucleotide sequence of the 3' terminating 3×HA 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 (Hyg+) 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 highly constitutive PSAD strong promoter-driven exogenous gene expression vector system is shown in SEQ ID No. 1.

[0016] A method for constructing a PSAD strong promoter-driven exogenous gene expression vector system with high constitutive activity includes the following steps: (1) Construction of the outer backbone sequence fragment of the vector system: Using plasmid pHyg-3HA as a template, the nucleotide sequence of plasmid pHyg-3HA is shown in SEQ ID No. 8. The fragment was digested with EcoR V and analyzed by electrophoresis to obtain a linearized fragment of 4777 bp, as shown in SEQ ID No. 8. Figure 1 As shown, the glue was then recovered and its concentration was tested for future reference. (2) Synthesis of PSAD promoter DNA fragment: 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. At the same time, PCR amplification of the target nucleotide position of the fragment at 705-1507 bp is performed using upstream primer SI-PSAD-F and downstream primer SI-PSAD-R. The nucleotide sequences of the upstream primer and downstream primer are shown in SEQ ID No.10 and SEQ ID No.11, respectively. After amplification, a DNA fragment of 842 bp in length is obtained. The two ends of the fragment are designed with homologous arms at both ends of the EcoR V cleavage site of the outer backbone sequence constructed in step (1), thereby obtaining the PSAD promoter DNA fragment. The concentration is measured for later use. The PCR preparation system is as follows: The PCR instrument program is set as follows: (3) The outer backbone sequence fragment obtained in step (1) and the DNA fragment of the PSAD promoter obtained in step (2) are spliced ​​together by in vitro homologous recombination to form the exogenous gene expression vector system pHyg-PSAD-3HA, with a full length of 5574 bp. Figure 2 As shown; the homologous recombination splicing is performed by reacting the PSAD promoter DNA fragment with the outer backbone sequence fragment obtained in step (1) at 50 °C for 1 h for in vitro recombination using homologous recombination enzyme. The homologous recombination system is set as follows: The application of a PSAD strong promoter-driven exogenous gene expression vector system with high constitutive activity in *Chlamydomonas reinhardtii* is described. The specific application process is as follows: A foreign gene fragment is cloned or synthesized and inserted into the EcoR V site of the pHyg-PSAD-3HA vector in vitro via homologous recombination or enzyme ligation to construct the final expression plasmid; the final expression plasmid is linearized and transformed into *Chlamydomonas reinhardtii* cells using electroporation; plasmid-transformed positive algal strains are obtained through preliminary screening with hygromycin B; and algal strains stably expressing the target protein are screened again using Western blotting with an anti-HA antibody. The screening process includes preparing TAP liquid medium; culturing *Chlamydomonas reinhardtii* cells to be transformed; preparing the linearized recombinant plasmid fragment; preparing Hyg+ resistance selection plates; collecting cells and performing square wave electroporation transformation; and screening and identifying transformants. The specific process is shown below: a. Prepare TAP liquid culture medium: Prepare the TAP medium according to the TAP medium formula, pour it into an aeration bottle, and autoclave at 121 °C for 20 min. The TAP liquid medium formula is as follows: b. Culturing Chlamydomonas cells to be transformed: ①Preparation: Place the sterilized TAP culture medium, inoculation loop or sterilized toothpick into the laminar flow hood and sterilize with ultraviolet light for at least 20 minutes before use.

[0017] ②Culturing S0 generation cells: In a clean bench, use an inoculation loop or toothpick to pick up fresh experimental algal strains and inoculate them into sterilized TAP liquid culture medium aeration bottles. Culture them continuously under light and aeration at 23±0.5°C and 8000 Lx light intensity for 3-4 days until the cell concentration is 1-2×107 cells / mL.

[0018] ③ Transferring S1 generation cells: Transfer S0 generation cells into fresh, sterilized TAP liquid aeration bottles at a concentration of 1×106 cells / mL. Continue culturing under continuous light and aeration for 20 h until the final concentration reaches 4×106 cells / mL, at which point they can be used for electroporation transformation.

[0019] c. Preparation of linearized fragments of recombinant plasmids: The recombinant plasmid was digested with a suitable restriction endonuclease to obtain a linearized fragment, which was then detected by 1% agarose gel electrophoresis. After detection, the fragment was purified and recovered using a gel extraction kit, and the concentration was measured. This purified fragment is the linearized target plasmid to be transformed.

[0020] d. Preparation of Hyg+ resistance screening plates: ①Preparation of TAP solid medium: After preparing TAP liquid medium as described above, add 15 g / L of agar powder and then autoclave at 121°C for 20 min.

[0021] ② Preparation of Hyg+ resistance plates: After the above culture medium has cooled to a temperature that is not too hot to handle, add hygromycin B (Hyg+, 50 mg / mL stock solution, diluted 2500×) to the clean bench to make the final concentration of Hyg+ in the culture medium 20 μg / mL. Shake well and pour into plates. After solidification, the Hyg+ resistance screening plates are obtained.

[0022] e. Cell collection and square wave electroporation: ① Cell collection: Take a small amount of cultured cells in a clean bench and count them using a hemocytometer. Calculate the number of cells needed for each transformation based on the required concentration of 1×10⁸ cells / mL (250 μL). Centrifuge the collected cells at 2500 rpm for 3 min, discard the supernatant, and resuspend them in pre-cooled sterile 1×TAP + 60 mM sorbitol. Centrifuge again at 2500 rpm for 3 min, discard the supernatant, and finally resuspend the cells in pre-cooled sterile 1×TAP + 60 mM sorbitol to the required volume for transformation. Incubate on ice for 10 min.

[0023] ② Square wave electroporation conversion: First, pre-cool the 0.4 cm electroporation cups in a clean bench. Each electroporation cup is first washed with 1 mL of anhydrous ethanol and then air-dried, then washed with sterile 1×TAP + 60 mM sorbitol solution and air-dried, and finally pre-cooled at -20°C. Then, 250 μL of 1×10⁸ cells / mL algal solution and 500-1000 ng of linearized recombinant plasmid fragments are added to the pre-cooled electroporation cups. After gently tapping to mix, the cups are quickly transferred to ice. The cups are dried with absorbent paper. A BTX ECM830 electroporator is used with the parameters set to 500 V, 4 ms, 6 bands, and an electroporation interval of 100 ms. After electroporation, the cups are placed on ice for 5-10 min. Finally, after electroporation, the cells were transferred into 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] ③ Plate coating: Prepare a 20% starch solution, wash it once with water and ethanol, then twice with distilled water. Each washing condition is centrifugation at 1000 rpm for 2 min. After each washing, discard the supernatant, bring the solution to a fixed volume with 70% ethanol, and place it at room temperature. In a clean bench, mix the 20% starch solution thoroughly. Measure the required volume (2 mL) for each conversion. Take the corresponding volume of starch, wash it twice with TAP, then once with sterile 1×TAP + 60 mM sorbitol solution. Each washing condition is centrifugation at 1000 rpm for 2 min. After each washing, discard the supernatant, then resuspend the starch in sterile 1×TAP + 60 mM sorbitol to the required volume. Finally, add sterile 20% PEG 6000 to bring the final concentration to 0.4%. 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 at 23±0.5°C, 14 / 10 h light / dark cycle, and 8000 Lx.

[0025] f. Screening and identification of transformants ① Transformant selection: Select the transformants grown on the Hyg+ resistance selection plate in the clean bench, spread them on the TAP plate, mark them, and incubate them for 3-4 days at 23±0.5°C, 14 / 10 h light / dark cycle, and 8000 Lx.

[0026] ② Expanding the culture of transformants: Add 1.2 mL of TAP medium to each well of a 24-well plate in a clean bench, mark the wells, pick algae and place them into the corresponding wells, seal the plates and place them in a light incubator. Culture in a shaker for 2-3 days at 23±0.5°C, 14 / 10 h light / dark cycle, and 8000 Lx.

[0027] ③ Screening of transformants: 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.

[0028] Through the above steps, algal strains containing the target protein can be efficiently screened, thereby enabling further research on the structure, location, and function of the target protein.

Claims

1. A PSAD strong promoter-driven exogenous gene expression vector system with high constitutive activity, characterized in that, The main sequence of the vector system consists of the following components in sequence: PSAD promoter, exogenous gene insertion site EcoR V, 3×HA tag at the 3' end, rbcS2 terminator, TUB2 promoter, hygromycin B (Hyg+) selection gene, and rbcS2 terminator; wherein, the nucleotide sequence of PSAD promoter is shown in SEQ ID No. 2, the nucleotide sequence of exogenous gene insertion site EcoR V is shown in SEQ ID No. 3, the nucleotide sequence of 3×HA tag at the 3' end is shown in SEQ ID No. 4, the nucleotide sequence of TUB2 promoter is shown in SEQ ID No. 5, the nucleotide sequence of hygromycin B (Hyg+) selection gene is shown in SEQ ID No. 6, the nucleotide sequence of rbcS2 terminator is shown in SEQ ID No. 7, and the nucleotide sequence of the exogenous gene expression vector system based on the highly constitutive active PSAD strong promoter is shown in SEQ ID No.

1.

2. A method for constructing a PSAD strong promoter-driven exogenous gene expression vector system based on high constitutive activity as described in claim 1, characterized in that, Includes the following steps: (1) Construction of the outer backbone sequence fragment of the vector system: using plasmid pHyg-3HA as a template, the nucleotide sequence of plasmid pHyg-3HA is shown in SEQ ID No.

8. It is digested with EcoR V and analyzed by electrophoresis to obtain a linearized fragment of 4777 bp. The fragment is then recovered and its concentration is detected for later use. (2) Synthesis of PSAD promoter DNA fragment: 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. At the same time, PCR amplification of the target nucleotide position of the fragment at 705-1507 bp is performed using upstream primer SI-PSAD-F and downstream primer SI-PSAD-R. The nucleotide sequences of the upstream primer and downstream primer are shown in SEQ ID No.10 and SEQ ID No.11, respectively. After amplification, a DNA fragment of 842 bp in length is obtained. The two ends of the fragment are designed with homologous arms at both ends of the EcoR V cleavage site of the outer backbone sequence constructed in step (1), thereby obtaining the PSAD promoter DNA fragment. The concentration is measured for later use. (3) The outer backbone sequence fragment obtained in step (1) and the PSAD promoter DNA fragment obtained in step (2) are spliced ​​together by in vitro homologous recombination to form the exogenous gene expression vector system pHyg-PSAD-3HA, with a full length of 5574 bp.

3. The method for constructing a PSAD strong promoter-driven exogenous gene expression vector system based on high constitutive activity 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 PSAD strong promoter-driven exogenous gene expression vector system based on high constitutive activity according to claim 2, characterized in that, In step (1), the electrophoretic analysis is performed by separating the target fragment using 1% agarose gel electrophoresis, and the fragment is recovered by purifying and recovering it using a gel recovery kit.

5. The method for constructing a PSAD strong promoter-driven exogenous gene expression vector system based on high constitutive activity according to claim 2, characterized in that, In step (3), the homologous recombination splicing involves reacting the PSAD promoter DNA fragment with the outer backbone sequence fragment obtained in step (1) at 50 °C for 1 h using a homologous recombination enzyme. The homologous recombination system is set as follows:

6. The application of the PSAD strong promoter-driven exogenous gene expression vector system based on high constitutive activity as described in claim 1 in Chlamydomonas reinhardtii, the specific application process being as follows: cloning or synthesizing an exogenous gene fragment, inserting it into the EcoR V site of the pHyg-PSAD-3HA vector in vitro via homologous recombination or enzyme ligation to construct the final expression plasmid; linearizing the final expression plasmid and transforming it into Chlamydomonas cells using electroporation; obtaining plasmid-transformed positive algal strains through preliminary screening with hygromycin B; and further screening using Western blotting with anti-HA antibody to identify algal strains stably expressing the target protein.

Images