Chlamydomonas reinhardtii secreting signal peptide and transgenic chlamydomonas reinhardtii, and construction method and application thereof

Abstract

The present application belongs to the technical field of genetic engineering, and particularly relates to Chlamydomonas reinhardtii secretion leader peptide and transgenic Chlamydomonas reinhardtii as well as a construction method and application thereof. The amino acid sequence of the Chlamydomonas reinhardtii secretion leader peptide is selected from at least one of SEQ ID NO: 1-3. The secretion leader peptide with the amino acid sequence as described above is used for expressing a target protein such as an antibacterial peptide, can guide the antibacterial peptide expressed in Chlamydomonas reinhardtii cells to be secreted to the extracellular, realize efficient secretion expression of the antibacterial peptide, and the expression amount is more than 40 ng / mL. Moreover, the separated antibacterial peptide has high biological activity, can effectively inhibit the growth and reproduction of various water aquaculture pathogenic bacteria such as Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus, the antibacterial rate is more than 90%, and the use of antibiotics can be effectively reduced. Therefore, the antibacterial peptide obtained by the foregoing secretion leader peptide secretion expression has important development potential in the aquaculture industry, especially in the water aquaculture industry.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to Chlamydomonas reinhardtii secretory peptides and transgenic Chlamydomonas reinhardtii, their construction methods and applications. Background Technology

[0002] The discovery and application of antibiotics have greatly benefited humanity. However, frequent use of antibiotics is equivalent to targeted screening of pathogens in nature, easily leading to multidrug resistance in microorganisms. In the aquaculture industry, their application can also cause environmental pollution and drug residues, thus triggering food safety issues. Because antimicrobial peptides (AMPs) have different mechanisms of action than traditional antibiotics and offer advantages such as environmental friendliness, safety, and low resistance rates, they are considered ideal alternatives to antibiotics and have broad application prospects in the pharmaceutical industry, feed, and food additives. AMPs are a collective term for small molecule polypeptides and proteins with broad-spectrum antibacterial activity induced in organisms and are important components of the organism's innate immune system. Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides found in crustaceans, typically composed of 98-123 amino acids. They include a signal peptide sequence and a lipopolysaccharide-binding domain. The lipopolysaccharide-binding domain serves as the functional region of ALFs, binding to lipopolysaccharides to disrupt bacterial cell walls or membranes, thereby inhibiting or killing pathogenic microorganisms. Since AMPs target the cell membrane rather than specific proteins, microorganisms are less likely to develop resistance. Furthermore, as peptides, they are easily degraded in nature, do not accumulate in the environment, and do not remain in the body, thus posing no food safety concerns and possessing broad application prospects. However, the content of natural antimicrobial peptides in organisms is extremely low, and extraction is difficult, yields are low, time-consuming, processes are complex, and in vitro activity is low, hindering large-scale production. This is the biggest obstacle restricting the practical application of AMPs. Therefore, conducting AMP genetic engineering research is of great significance, and designing or modifying natural antimicrobial peptides through genetic engineering technology is a feasible way to solve the aforementioned problems.

[0003] Currently, heterologous expression of AMPs is mainly achieved through E. coli and yeast expression systems. E. coli expression systems, as the earliest established systems for expressing heterologous genes, have advantages such as high expression levels, simple operation, and low cost. However, due to the bactericidal effect of AMPs, biologically active antimicrobial peptides cannot be directly expressed using E. coli expression systems. If fusion proteins are used, inclusion bodies are easily formed, reducing the activity of the expressed protein. Furthermore, the lack of biological processes for folding, modifying, and processing eukaryotic proteins often results in recombinant AMPs with low activity and cytotoxicity. Yeast, being a eukaryotic microorganism, can perform post-translational processing and modification, possessing a more complete gene expression regulation mechanism, which can compensate for the shortcomings of E. coli expression systems. However, yeast is also prone to excessive glycosylation of expressed proteins, thus affecting their activity. Moreover, both E. coli and yeast expression systems require heterotrophic fermentation, a process that consumes large amounts of resources and energy and easily causes environmental pollution.

[0004] To address the shortcomings of the aforementioned expression systems, researchers have recently developed the *Chlamydomonas reinhardtii* expression system. As a single-celled eukaryotic green algae, *Chlamydomonas reinhardtii* combines the advantages of bacteria and higher plants, exhibiting rapid growth, a short life cycle, low culture costs, and the ability to undergo post-translational modification. Furthermore, it is endotoxin-free, has a clear genetic background, and possesses three genetic transformation systems: nuclear, chloroplast, and mitochondrial. Among these, the nuclear genome expression system is the most widely used and technologically mature. In addition, *Chlamydomonas reinhardtii* is already on the list of edible algae for humans and can be directly used as food for aquatic organisms such as fish and shrimp. Therefore, the *Chlamydomonas reinhardtii* expression system has unique advantages and can compensate for the deficiencies of expression systems such as *E. coli* and yeast. Recent research has found that *Chlamydomonas reinhardtii* can achieve intracellular and extracellular expression of target proteins. Extracellular secretion expression offers advantages such as convenient isolation and purification, high product activity, and ease of use. By utilizing Chlamydomonas reinhardtii to express AMPs, efficient in vitro recovery of AMPs can be achieved, simplifying the production of antimicrobial peptide feeds. Furthermore, it can be directly used as an antimicrobial agent in aquaculture, reducing antibiotic use. This lays a solid foundation for the practical application of antimicrobial peptides. Therefore, Chlamydomonas preparations expressing antimicrobial peptides have significant application potential in the aquaculture industry.

[0005] However, current research on the secretory expression of *Chlamydomonas reinhardtii* is limited, and problems such as low secretion efficiency and even failure to secrete target proteins remain. Lauersen et al. used carbonic anhydrase 1 (CAH1) as a guide peptide for the secretory expression of luciferase, but the secretion level was significantly lower than that of *E. coli* or yeast expression systems. Furthermore, their results showed that CAH1 interferes with the production of recombinant proteins, further reducing the expression and secretion levels. Mayfield et al. used a succinate dehydrogenase component as a secretory guide peptide, but their research showed that it interfered with the function of the self-cleaved 2A peptide, resulting in the inability to express and secrete mCherry proteins. Therefore, improving or refining the *Chlamydomonas reinhardtii* secretory expression system to address the low or non-expression levels of antimicrobial peptides in *Chlamydomonas reinhardtii* is an urgent problem that needs to be solved. Summary of the Invention

[0006] To address the technical problems of existing Chlamydomonas reinhardtii expression systems failing to achieve efficient secretory expression of antimicrobial peptides, and low or even non-existent expression levels of antimicrobial peptides in Chlamydomonas reinhardtii, this invention provides a Chlamydomonas reinhardtii secretory peptide, a transgenic Chlamydomonas reinhardtii containing the secretory peptide, a method for constructing the transgenic Chlamydomonas reinhardtii, and the application of the aforementioned Chlamydomonas reinhardtii secretory peptide or transgenic Chlamydomonas reinhardtii in aquaculture.

[0007] To achieve the above objectives, the present invention is specifically implemented through the following technical solutions:

[0008] The first aspect of the present invention provides a Chlamydomonas reinhardtii secretory peptide, wherein the amino acid sequence of the Chlamydomonas reinhardtii secretory peptide is selected from at least one of the following as shown in SEQ ID NO: 1-3.

[0009] Furthermore, the amino acid sequence of the Chlamydomonas reinhardtii secretory peptide is shown in SEQ ID NO: 2.

[0010] A second aspect of the present invention provides a polynucleotide for encoding the Chlamydomonas reinhardtii secretory peptide as described above.

[0011] A third aspect of the present invention provides a transgenic Chlamydomonas reinhardtii containing an expression vector, the expression vector comprising a DNA sequence encoding a Chlamydomonas reinhardtii secretory peptide as described above and a DNA sequence encoding an antimicrobial peptide.

[0012] Furthermore, the DNA sequence encoding the Chlamydomonas reinhardtii secretory peptide is selected from at least one of the following: SEQ ID NO: 4-6.

[0013] Furthermore, the antimicrobial peptide is Penaeus monodon antilipopolysaccharide factor 3, whose amino acid sequence is shown in SEQ ID NO: 7 and DNA sequence is shown in SEQ ID NO: 8.

[0014] Furthermore, the expression vector also includes a photoinducible promoter, with the DNA sequence encoding the Chlamydomonas reinhardtii secretory peptide and the DNA sequence encoding the antimicrobial peptide located sequentially downstream of the photoinducible promoter.

[0015] Furthermore, the starting vector for constructing the expression vector is pESVH.

[0016] The fourth aspect of this invention provides a method for constructing the transgenic Chlamydomonas reinhardtii as described above, comprising the following steps:

[0017] S1. The DNA sequence encoding the secretory peptide of Chlamydomonas reinhardtii and the DNA sequence encoding the antimicrobial peptide are ligated into the starting vector to obtain the expression vector;

[0018] S2. The expression vector is transformed into the recipient algal strain of Chlamydomonas reinhardtii to obtain transgenic Chlamydomonas reinhardtii.

[0019] Further, in step S1, the DNA sequence encoding the Chlamydomonas reinhardtii secretory peptide and the DNA sequence encoding the antimicrobial peptide are fused together to obtain the secretory peptide-antimicrobial peptide fusion sequence through gene synthesis technology. Then, the secretory peptide-antimicrobial peptide fusion sequence is inserted downstream of the promoter of the enzyme-cleaved linearized starting vector to obtain the expression vector.

[0020] Further, in step S2, the expression vector is transformed into the Chlamydomonas reinhardtii recipient algal strain by bead milling, wherein the Chlamydomonas reinhardtii recipient algal strain is a cell wall-deficient algal strain JUV.

[0021] The fifth aspect of this invention provides the application of the genetically modified Chlamydomonas reinhardtii as described above in aquaculture.

[0022] The advantages and positive effects of this invention are as follows:

[0023] The secretory peptide with the amino acid sequence described above provided by this invention can guide the secretion of antimicrobial peptides expressed in Chlamydomonas reinhardtii cells to the extracellular space when used to express target proteins such as antimicrobial peptides, achieving highly efficient secretory expression of antimicrobial peptides with an expression level of over 40 ng / mL. Moreover, the isolated antimicrobial peptides have high biological activity and can effectively inhibit the growth and reproduction of various aquaculture pathogens such as Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahaemolyticus, with an inhibition rate of over 90%, which can effectively reduce the use of antibiotics. Therefore, the antimicrobial peptides obtained by secretory expression of the aforementioned secretory peptides have significant development potential in aquaculture, especially in the aquaculture industry. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The image shows the spectrum of the secretory peptide-antimicrobial peptide expression vector pESVH-ALF3 constructed in Example 1 of this invention;

[0026] Figure 2 This is an electrophoresis diagram of the genome PCR detection of transgenic Chlamydomonas reinhardtii in Example 1 of the present invention; wherein, lanes 1-4 from left to right are the genetic transformants T-GH-3, T-IBD-1, T-PY-1 and T-CAH-5, lane 5 is the positive control expression vector pESVH-ALF3, lane 6 is the untransformed Chlamydomonas reinhardtii recipient strain JUV, and lane 7 is the negative control H2O;

[0027] Figure 3 This is an electrophoresis diagram of the RT-PCR detection of transgenic Chlamydomonas reinhardtii from Example 1 of the present invention; wherein, lane M is DL2000 DNA Marker, lanes 1-11 are genetic transformants (lanes 1-2 are T-GH-3, lanes 3-4 are T-IBD-1, lanes 5-6 are T-PY-1, lanes 7-8 are T-CAH-5, lanes 9-11 are T-ARS-8), and lane 12 is the positive control expression vector pESVH-ALF3;

[0028] Figure 4 This is an immunoblot analysis diagram of the transgenic Chlamydomonas reinhardtii from Example 1 of the present invention; wherein, lane N is the untransformed Chlamydomonas reinhardtii recipient strain JUV, lanes 1-11 are genetic transformants (lanes 1-2 are T-GH-3, lanes 3-4 are T-IBD-1, lanes 5-6 are T-PY-1, lanes 7-8 are T-CAH-5, lanes 9-11 are T-ARS-8), Anti-HA is the target protein, and Anti-α-Tubulin is the internal control protein;

[0029] Figure 5 This is a graph showing the expression levels of extracellular antimicrobial peptides in transgenic Chlamydomonas reinhardtii obtained from different secretion-guided peptides constructed in Example 2 of the present invention; where IBD-1, GH-like, P-4-Y, CAH and ARS2 are T-IBD-1, T-GH-3, T-PY-1, T-CAH-5 and T-ARS-8 genetic transformants, respectively;

[0030] Figure 6This is a graph showing the antibacterial effect of different transgenic Chlamydomonas reinhardtii secreted and expressed antimicrobial peptides on Vibrio parahaemolyticus after 12 hours in Example 3 of the present invention.

[0031] Figure 7 This is a graph showing the antimicrobial effect of different transgenic Chlamydomonas reinhardtii secreted and expressed antimicrobial peptides on Vibrio alginolyticus after 12 hours in Example 3 of the present invention.

[0032] Figure 8 This is a graph showing the antimicrobial effect of different transgenic Chlamydomonas reinhardtii secreted and expressed antimicrobial peptides on Vibrio harveyi over 12 hours in Example 3 of the present invention. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0034] Based on the information contained in this application, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0035] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0036] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0037] This invention screened secretory peptides that theoretically can secrete the target protein—antimicrobial peptide—out of the cell. Further comparison of the activity and expression efficiency of the antimicrobial peptide expressed by these secreted peptides revealed secretory peptides that are far superior to those currently reported, such as Arylsulfatase 2 (ARS2) and Carbonic anhydrase (CAH1). This enables efficient extracellular secretion of antimicrobial peptides using *Chlamydomonas reinhardtii* as a host, providing a solution for the highly active extracellular expression and convenient application of antimicrobial peptides in *Chlamydomonas reinhardtii*. Based on this, the invention was completed.

[0038] In the context of this invention, the terms "secretion-guided peptide," "guide peptide," and "signal peptide" are used interchangeably to guide the secretory expression of a target protein. "Secretion" refers to the transport of a protein or peptide molecule from its intracellular synthesis to the extracellular space.

[0039] This invention provides a Chlamydomonas reinhardtii secretion-guiding peptide, wherein the Chlamydomonas reinhardtii secretion-guiding peptide is selected from at least one of IBD-1, GH-like, and P-4-Y, and the amino acid sequences of IBD-1, GH-like, and P-4-Y are shown in SEQ ID NO: 1-3, respectively, as follows:

[0040] IBD-1: MPSSSMKLFAALLIACMAQTSMA (see SEQ ID NO: 1);

[0041] GH-like: MRRAIALGVGLALLGLLLPGSLA (see SEQ ID NO: 2);

[0042] P-4-Y: MARRLLLALALAAVLGLAHA (see SEQ ID NO: 3).

[0043] Based on the published genome sequence of *Chlamydomonas reinhardtii* (GenBank accession number: NC_005353), this invention uses signal peptide prediction software such as SignalP 4.0 to predict signal peptides and obtain candidate molecules. Then, the distribution of candidate signal peptides intracellularly and extracellularly is used for screening. Furthermore, the activity and expression efficiency of antimicrobial peptides secreted by different candidate signal peptides are compared. The screening yielded secretion-guided peptides IBD-1, GH-like, and P-4-Y, as shown in the above amino acid sequences. When IBD-1, GH-like, and P-4-Y are used to express antimicrobial peptides, they can guide the secretion of antimicrobial peptides expressed intracellularly in *Chlamydomonas reinhardtii* cells to the extracellular space, achieving highly efficient secretory expression of antimicrobial peptides with expression levels exceeding 40 ng / mL. This facilitates the purification of extracellular antimicrobial peptides and significantly reduces the cost of separation and purification. Moreover, the isolated antimicrobial peptides have high biological activity and can effectively inhibit the growth and reproduction of various aquatic pathogens such as Vibrio alginolyticus, Vibrio harveyi, and Vibrio parahaemolyticus, with an inhibition rate of over 90%. This indicates that the antimicrobial peptides obtained by the secretion of the aforementioned secretory peptides of this invention have significant development potential in aquaculture. At the same time, the use of the Chlamydomonas reinhardtii expression system provides a safe environment for the application of antimicrobial peptides, such as as feed additives in aquaculture.

[0044] Preferably, the Chlamydomonas reinhardtii secretory peptide is selected from GH-like peptides, and its amino acid sequence is shown in SEQ ID NO: 2. Experiments have shown that GH-like secretory peptides express the highest amount of antimicrobial peptides and exhibit the best activity.

[0045] Another embodiment of the present invention provides a polynucleotide for encoding the Chlamydomonas reinhardtii secretory peptide as described above.

[0046] The advantages of the polynucleotide over the prior art are the same as those of the Chlamydomonas reinhardtii secretory peptide over the prior art as described above, and will not be repeated here.

[0047] For example, the nucleotide sequences encoding IBD-1, GH-like, and P-4-Y are shown in SEQ ID NO: 4-6, respectively.

[0048] It should be noted that the sequences of the polynucleotides can be derived from the amino acid sequences using conventional methods such as codon encoding rules. Those skilled in the art should understand that the polynucleotides provided by this invention for encoding IBD-1, GH-like, and P-4-Y are not intended to limit the scope of protection of this invention.

[0049] In another embodiment of the present invention, a transgenic Chlamydomonas reinhardtii is provided, the transgenic Chlamydomonas reinhardtii containing an expression vector for expressing Chlamydomonas reinhardtii secretory peptides and antimicrobial peptides, the expression vector comprising a DNA sequence encoding the Chlamydomonas reinhardtii secretory peptide as described above and a DNA sequence encoding the antimicrobial peptide.

[0050] This invention employs a Chlamydomonas reinhardtii expression system, utilizing IBD-1, GH-like, and P-4-Y secretory peptides to express target proteins, such as antimicrobial peptides, extracellularly into Chlamydomonas reinhardtii cells, effectively avoiding product inhibition and increasing the expression level of the target protein. Using conventional methods to cultivate the transgenic Chlamydomonas reinhardtii of this invention, centrifugation to separate algal cells, and freeze-drying, a crude extract containing a high concentration of antimicrobial peptides can be obtained. Further purification using conventional methods yields high-purity extracellular antimicrobial peptides. These extracellular antimicrobial peptides can effectively inhibit the growth of aquaculture microorganisms, reducing the use of antibiotics. In addition, the present invention has the following advantages in using Chlamydomonas reinhardtii as a host cell to produce antimicrobial peptides: 1) Genetic transformation is convenient, and various mutant strains have been obtained for molecular biology research; 2) It has enzymes and organelles that can modify antimicrobial peptides and can also secrete antimicrobial peptides into the culture medium, simplifying the separation and purification steps; 3) Chlamydomonas reinhardtii is easy to cultivate, as it can be both photosynthetically autotrophic and chemoheterotrophic, and the culture medium is inexpensive and readily available; 4) Transgenic Chlamydomonas reinhardtii expressing antimicrobial peptides can be directly used as an antimicrobial agent in aquaculture, which is environmentally friendly and convenient.

[0051] For example, the antimicrobial peptide is the anti-lipopolysaccharide factor 3 (ALFPm3) of Penaeus monodon, whose amino acid sequence is shown in SEQ ID NO: 7 and DNA sequence is shown in SEQ ID NO: 8.

[0052] Optionally, the expression vector further includes a photoinducible promoter, with the DNA sequence encoding the Chlamydomonas reinhardtii secretory peptide and the DNA sequence encoding the antimicrobial peptide located sequentially downstream of the photoinducible promoter. That is, from the 5'-3' end, the sequence is the photoinducible promoter, the DNA sequence encoding the Chlamydomonas reinhardtii secretory peptide, and the DNA sequence encoding the antimicrobial peptide. Using a photoinducible promoter can increase the expression level of the target protein in Chlamydomonas reinhardtii.

[0053] Optionally, the starting vector for constructing the expression vector is pESVH.

[0054] This invention also provides a method for constructing the transgenic Chlamydomonas reinhardtii as described above, comprising the following steps:

[0055] S1. Constructing the expression vector: The DNA sequence encoding the secretory peptide of Chlamydomonas reinhardtii as described above (e.g., the DNA sequence shown in SEQ ID NO: 4-6) and the DNA sequence encoding the antimicrobial peptide (e.g., the DNA sequence shown in SEQ ID NO: 7) are ligated into the starting vector to obtain the expression vector;

[0056] S2. Genetic transformation: The expression vector is transformed into the recipient algal strain of Chlamydomonas reinhardtii to obtain transgenic Chlamydomonas reinhardtii.

[0057] The advantages of the method for constructing the transgenic Chlamydomonas reinhardtii over the prior art are the same as those of the transgenic Chlamydomonas reinhardtii over the prior art as described above, and will not be repeated here.

[0058] In step S1, the DNA sequence encoding the secretory peptide of *Chlamydomonas reinhardtii* and the DNA sequence encoding the antimicrobial peptide are fused to obtain the secretory peptide-antimicrobial peptide fusion sequence using gene synthesis technology. This fusion sequence is then inserted downstream of the promoter of a linearized starting vector to obtain the expression vector. To achieve high expression levels, a photoinducible promoter is preferred, and the expression of the antimicrobial peptide is induced using strong light induction.

[0059] In step S2, the expression vector containing the secretory peptide-antimicrobial peptide fusion sequence is transformed into the Chlamydomonas reinhardtii recipient algal strain for expression using the "bead milling method" to obtain genetic transformants. The genomic DNA, RNA, and protein of the genetic transformants are detected, and the transcriptional activity of the antimicrobial peptide is analyzed to obtain transgenic Chlamydomonas reinhardtii that can be stably inherited.

[0060] Optionally, the Chlamydomonas reinhardtii receptor strain is a cell wall-deficient strain JUV, purchased from the Chlamydomonas Research Center in the United States.

[0061] Optionally, the method for detecting the genomic DNA of the genetic transformant includes PCR amplification, which uses specific primers to amplify and detect whether the secretory peptide-antimicrobial peptide fusion sequence has been successfully inserted into the starting vector.

[0062] Optionally, the method for detecting the RNA of the genetic transformants includes real-time PCR (RT-PCR). The transcriptional level of the antimicrobial peptide RNA is detected by RT-PCR.

[0063] Optionally, the method for detecting proteins in the genetic transformants includes Western blotting. Western blotting is used to identify the expression levels of antimicrobial peptides.

[0064] Another embodiment of the present invention provides the application of the transgenic *Chlamydomonas reinhardtii* described above in aquaculture, particularly in the aquaculture industry. Specifically, by culturing the transgenic *Chlamydomonas reinhardtii* of the present invention, high-purity and high-concentration antimicrobial peptides can be obtained. These antimicrobial peptides have good biological activity and significantly inhibit the growth and reproduction of various aquatic pathogens such as *Vibrio alginolyticus*, *Vibrio harveyi*, and *Vibrio parahaemolyticus*. They can be used as feed additives to improve the disease resistance and immunity of organisms, thereby promoting growth. Alternatively, the transgenic *Chlamydomonas reinhardtii* of the present invention can be directly applied as a feed additive in animal or aquatic feed.

[0065] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.

[0066] Example 1: Construction of transgenic Chlamydomonas reinhardtii

[0067] 1. Cultivation of Chlamydomonas reinhardtii

[0068] The cell wall-deficient Chlamydomonas reinhardtii JUV was selected as the recipient strain for transgenesis (purchased from the Chlamydomonas Research Center, St. Paul, MN 55108, USA).

[0069] TAP medium was used as the culture medium for *Chlamydomonas reinhardtii*. The formulation included: 2.42 g Tris, 25 mL 4×Beijerinck salts, 1 mL 1 M(K)PO4, 1 mL Trace trace element solution, and 975 mL water. The pH was adjusted to 6.95-7.05 using glacial acetic acid, and the volume was brought to 1 L. The 4×Beijerinck salts formulation consisted of: 16 g NH4Cl, 2 g CaCl2·2H2O, and 4 g MgSO4·7H2O dissolved in water and brought to a final volume of 1 L. The Trace trace element solution formulation included: 11.4 g H3BO3, 5.6 g MnCl2·4H2O, 22 g ZnSO4·7H2O, 4.99 g FeSO4·7H2O, 1.61 g CoCl2·6H2O, 1.57 g CuSO4·5H2O, and 1.1 g (NH4)6Mo7O 24 Dissolve 4H2O and 50g Na2EDTA in water, adjust the pH to 6.5-6.8 with 20% KOH, and bring the volume to 1L.

[0070] Culture conditions for Chlamydomonas reinhardtii: 22-25℃, 90 μE / m 2 Under continuous irradiation conditions, the concentration of algal cells in the logarithmic growth phase was 1×10⁻⁶. 6 -2×10 6 cells / mL.

[0071] 2. Construction of the expression carrier

[0072] Based on the published genome sequence of *Chlamydomonas reinhardtii* (GenBank accession number: NC_005353), SignalP4.0 software was used to predict secretory signal peptides. Candidate molecules were screened based on their intracellular and extracellular protein distribution, ultimately resulting in five extracellularly secreted signal peptides. The names, amino acid sequences, and gene sequences of these five extracellularly secreted signal peptides in *Chlamydomonas reinhardtii* are shown below:

[0073] Table 1. Sequence information of candidate molecules for secretory peptides from Chlamydomonas reinhardtii.

[0074]

[0075] The antimicrobial peptide is exemplary selected from the antimicrobial peptide of Penaeus monodon—anti-lipopolysaccharide factor 3 (ALFPm3). In other embodiments, other antimicrobial peptides, such as anti-lipopolysaccharide factor 11, Crusin, and lysozyme, may also be selected to express according to actual needs.

[0076] The amino acid sequence of ALFPm3 is as follows:

[0077] QGWEAVAAAVASKIVGLWRNEKTELLGHECKFTVKPYLKRFQVYYKGRMWCPGWTAIRGEASTRSQSGVAGKTAKDFVRKAFQKGLISQQEANQWLSS (see SEQ ID NO: 7).

[0078] The gene expression sequence (5'-3') of ALFPm3 in Chlamydomonas reinhardtii is as follows:

[0079] caagggtgggaggctgtggcagcggccgtcgccagcaagatcgtggggttgtggaggaacgaaaaaactgaacttctcggccacgagtgcaagttcaccgtcaagccttatttgaagagattccaggtgtactacaaggggaggatgtggt gcccaggctggacggccatcagaggagaagccagcacacgcagtcagtccggggtagctggaaagacagccaaagacttcgttcggaaagctttccagaaaggtctcatctctcaacaggaggccaaccagtggctcagctcatag (see SEQ ID NO: 8).

[0080] The signal peptide-antimicrobial peptide fusion sequence was obtained through gene synthesis technology. Specifically, the five secretory peptides in Table 1 were directly linked to ALFPm3 and tagged with 3×HA to facilitate subsequent protein detection and purification. The obtained fusion sequence was sent to a gene synthesis company for synthesis to obtain the secretory peptide-antimicrobial peptide fusion sequence.

[0081] The synthesized secretory peptide-antimicrobial peptide fusion sequence was then ligated between the EcoRI and PmlI restriction sites of the pESVH vector to obtain the expression vector, named pESVH-ALF3. Taking the CAH signal peptide-antimicrobial peptide fusion sequence (CAH-ALFPm3) as an example, its expression vector map can be found in [reference needed]. Figure 1 The expression vector was transferred into E. coli for cloning.

[0082] The pESVH vector was constructed by replacing the HSP70A-RBCS2 promoter and RBCS2 terminator of the Chlamydomonas reinhardti expression vector pH124 (see literature: Wu Jinxia, ​​Hu Zhangli, Wang Chaogang, Eficient expression of green fluorescent protein (GFP) mediated by achimeric promoter in Chlamydomonas reinhardti, Chinese Journal of Oceanology and Limnolog, 2008, 26(3):242-247.) with the Chlamydomonas reinhardti psaD promoter (psaD pro) and Chlamydomonas reinhardti psaD terminator (psaD ter) to obtain the pESVH vector.

[0083] 3. Genetic transformation of expression vectors

[0084] Specifically, the genetic transformation was carried out using the "bead milling method," in which an expression vector carrying a signal peptide-antimicrobial peptide fusion sequence was introduced into the genome of Chlamydomonas reinhardtii for expression, including the following steps:

[0085] Preparation of transformation plasmid: E. coli containing the signal peptide-antimicrobial peptide fusion sequence were inoculated into 15 mL of LB liquid medium containing 100 μg / mL Amp and cultured at 37°C and 200 rpm for 12-16 h. At room temperature, the culture broth was centrifuged at 8000 rpm for 10 min, and the supernatant was discarded. Plasmid DNA was extracted using the TRANS EasyPure Plasmid MiniPrep kit: 500 μL of Solution I (containing RNase A) was added, and the bacterial cells were thoroughly vortexed. Then, 500 μL of Solution II was added, and the mixture was gently mixed for no more than 5 minutes. Next, 700 μL of Solution III was added, and the mixture was gently mixed 5-10 times. The column was centrifuged at 12000×g for 10 minutes at room temperature. The supernatant was collected and transferred to a centrifuge column fitted with a 2 mL collection tube. The column was centrifuged at 10000×g for 1 minute, and the filtrate was discarded. Then, 500 μL of HBC Solution (containing isopropanol) was added, and the column was centrifuged at 10000×g for 1 minute, and the filtrate was discarded. Finally, 700 μL of Washing Buffer was added, and the column was centrifuged at 10000×g for 1 minute, and the filtrate was discarded. This process was repeated once. The column was then centrifuged at 12000×g for 2 minutes at room temperature. The collection tube was removed, and a 1.5 mL EP tube was attached. 80-100 μL of the EP tube was added to the center of the membrane in the centrifuge column. Preheated sterile water at 65℃ was left to stand at room temperature for 2 minutes, and then centrifuged at 12000×g for 1 minute to elute the plasmid DNA, yielding 100 μL of protein-free plasmid DNA with a concentration between 901.4 and 1325.7 ng / μL.

[0086] Plasmid DNA digestion: The digestion reaction was prepared using FastDigest KpnI restriction endonuclease (Thermo Fisher Scientific, Massachusetts, USA). The reaction mixture consisted of 2 μL plasmid DNA, 1 μL KpnI, and 3 μL 10× Buffer, with the final volume made up to 30 μL with deionized water. The reaction was incubated at 37°C for 2 hours, followed by heat inactivation at 65°C for 10 seconds. 5 μL of the reaction mixture was then added to 1 μL of 5× Loading Buffer and electrophoresed on a 1% agarose gel. The results showed that the plasmid DNA was completely digested, and the band size was as expected, indicating that the DNA could be used for further experiments.

[0087] Obtaining genetic transformants: The cell wall-deficient Chlamydomonas reinhardtii strain JUV was inoculated into TAP liquid medium and incubated at 22°C for 90 μE × m -2 ×s -1 Cultured under light. Until OD 750 At a concentration of 1.0, the algal cell density in the culture medium is approximately 1 × 10⁻⁶. 6cell / mL. At room temperature, centrifuge at 5000 rpm for 5 min to collect algal cells. Resuspend the algal cells in 250 μL of fresh TAP medium on a clean bench. Add the algal solution to a sterile 1.5 mL EP tube containing 300 mg glass beads, then add 20 μL of restriction endonuclease-linearized plasmid DNA. Vortex at 2500 rpm for 25-30 s. Carefully aspirate the supernatant and transfer it to a 50 mL centrifuge tube containing 10 mL of TAP medium. Incubate at 25°C, wrapped in aluminum foil to protect from light, and shake at 100 rpm for 22 hours. At room temperature, centrifuge at 3000 rpm for 5 min to collect algal cells, resuspending the cells in approximately 100 μL of liquid. Spread the resuspended cells onto TAP solid medium containing 0.1% ampicillin and 0.001% bleomycin. Incubate at 22°C, 90 μE × m -2 ×s -1 The algal colonies were inverted and cultured for 1-2 weeks until single colonies appeared. The single-clone colonies were then transferred to new square TAP plates containing 0.1% ampicillin and 0.001% bleomycin to obtain genetically transformed individuals capable of stable inheritance. The genetically transformed individuals containing the fusion sequences IBD-1-ALFPm3, GH-like-ALFPm3, P-4-Y-ALFPm3, CAH-ALFPm3, and ARS2-ALFPm3 were named T-IBD-1, T-GH-3, T-PY-1, T-CAH-5, and T-ARS-8, respectively.

[0088] 4. Expression detection of genetically transformed strains (transgenic Chlamydomonas reinhardtii)

[0089] 1) PCR detection of genomic DNA

[0090] Genomic DNA extraction from transformants: Pick each transformant into 20 mL of TAP liquid medium and incubate at 22°C at 90 μE × m -2 ×s -1 Cultured at 100 rpm for 5 days. Aliquoted into 50 mL centrifuge tubes, centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. Then, genomic DNA was extracted from the genetic transformants according to the kit (Bebebio Biotechnology Co., Ltd. Ultra DNA Isolation, Zhengzhou, China).

[0091] PCR detection of genomic DNA: The PCR reaction system was prepared using 2×M5 HiPer plus Taq HiFi PCR mix (Beijing Polymer Biotechnology Co., Ltd., Beijing, China). The PCR reaction system included: 2 μL genomic DNA, 0.4 μL upstream primer psaD-P, 0.4 μL downstream primer psaD-T, 10 μL 2×M5 HiPer plus Taq HiFi PCR mix, and 10 μL ddH2O. The PCR reaction procedure was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 56℃ annealing for 20 s, 72℃ extension for 40 s, 35 cycles, 72℃ extension for 5 min, and termination of the reaction at 4℃.

[0092] 5 μL of PCR reaction solution was analyzed by 2% agarose gel electrophoresis. The electrophoresis results are shown in the figure. Figure 2 Lane M was the DL2000 marker, lanes 1-4 were genetic transformants, lane 5 was the positive control (i.e., the expression vector pESVH-ALF3 of this invention), lane 6 was the recipient strain JUV of *Chlamydomonas reinhardtii* (unconverted signal peptide-antimicrobial peptide fusion sequence), and lane 7 was the negative control (i.e., H2O). An 800bp band appeared in all lanes containing the genetic transformants, and a 700bp band appeared in both the genetic transformants and the unconverted *Chlamydomonas reinhardtii* recipient strain JUV. This is because the genome itself also has primer amplification sites during primer design, resulting in a 700bp fragment. Therefore, the unconverted *Chlamydomonas reinhardtii* recipient strain JUV showed a 700bp band, while the genetic transformants showed both 700bp and 800bp bands. Based on the gene design sequence size, positive transformants were preliminarily confirmed. The DNA sequences of the primers used are shown below:

[0093] psaD-P (upstream primer): gggaattggaggtacgaccgagat (see SEQ ID NO: 13);

[0094] psaD-T (downstream primer): agctccgatcccgtatcaatcagc (see SEQ ID NO: 14).

[0095] 2) Transcriptional activity analysis of antimicrobial peptides and ALFPm3

[0096] Select single-clone algal colonies from the genetically transformed individuals and inoculate them into 10 mL of TAP liquid medium. Incubate at 22°C and 90 μE × m -2 ×s -1 Incubate at 100 rpm until OD 750The concentration was 0.5-0.8. RNA was extracted using the RNAfast200 kit (Feijie Biotechnology, Shanghai, China). The specific steps included: collecting algal cells by centrifuging 2 mL of algal culture at 6000 rpm for 10 min at 4°C, and resuspending in 100 μL of RNase-free deionized water. Then, 500 μL of RA2 was added, gently mixed 5-10 times, allowed to stand for 1 min, and then transferred to a centrifuge column containing a 2 mL collection tube. The column was centrifuged at 12000 × g for 1 min, and the filtrate was discarded. 500 μL of Wash Buffer was added to the column, and the column was centrifuged at 12000 × g for 1 min, and the filtrate was discarded. This process was repeated once, followed by empty column centrifugation (12000 × g, 1 min), and the collection tube was discarded. The column was then placed in a 1.5 mL EP tube and allowed to air dry until no liquid remained. 50 μL of RNase-free water was added to the column, and the column was centrifuged at 12000 × g for 1 min. Take 4 μL of sample and mix with 1 μL of 5× Loading Buffer, then separate by electrophoresis in 1% agarose gel (voltage 160V, time 3 min) to check the RNA extraction effect and analyze the RNA integrity. Then take 1 μL and determine the concentration and purity of RNA under Nanodrop.

[0097] Using Yeasen III. The extracted RNA was reverse transcribed using the first-strand cDNA Synthesis SuperMix for qRCR (gDNA digester plus). 1 μg of total RNA was added to 3 μL of 5×gDNA digester mix, and the volume was increased to 15 μL with RNase-free water. The mixture was incubated at 42°C for 2 min. Then, 5 μL of... III. Using SuperMix plus, a 20 μL reverse transcription program was prepared. After gently mixing with a pipette, the mixture was incubated at 55°C for 15 min to obtain cDNA, which was then stored at -20°C. A 20 μL real-time PCR (RT-PCR) system was prepared: 4 μL of cDNA (the original sample stored at -20°C was diluted 5-fold and then 4 μL was taken), 0.4 μL each of forward and reverse primers (see Table 2), 10 μL of 2×Taq Mix, and 5.2 μL of deionized water.

[0098] Table 2 Primer information used for analysis of secretory peptide and ALFPm3 transcriptional activity.

[0099]

[0100] The RT-PCR reaction procedures for the five signal peptides are as follows:

[0101] ARS2 and IBD-1 secretion-guided peptides: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 30 s, annealing at 58℃ for 20 s, extension at 72℃ for 40 s, repeated 35 times, extension at 72℃ for 5 min, and product stabilization at 12℃.

[0102] CAH secretion-guided peptide: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 30 s, annealing at 60℃ for 30 s, extension at 72℃ for 40 s, cycled 35 times, extension at 72℃ for 5 min, and stabilized at 12℃.

[0103] GH-like secretion-guided peptide: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 40 s, cycled 35 times, extension at 72℃ for 5 min, and stabilized at 12℃.

[0104] P-4-Y secretion-guided peptide: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 30 s, annealing at 61℃ for 30 s, extension at 72℃ for 40 s, cycled 35 times, extension at 72℃ for 5 min, and stabilized at 12℃.

[0105] 5 μL of RT-PCR reaction solution was analyzed by 2% agarose gel electrophoresis (160 V, 15 min). The electrophoresis results are shown below. Figure 3 In the study, lane M was the DL2000 DNA Marker, lanes 1-11 were genetic transformants, and lane 12 was the positive control (expression vector of CAH signal peptide). Electrophoresis yielded a band of approximately 250 bp, consistent with expectations, demonstrating that the introduced signal peptide-ALFPm3 has transcriptional activity in the transgenic algae.

[0106] 3) Immunoblotting analysis of antimicrobial peptides

[0107] Crude isolation of the antimicrobial peptide ALFPm3: Single-clonal algal colonies of genetically transformed individuals were picked and inoculated into 10 mL of TAP liquid medium, incubated at 22°C and 90 μE×m. -2 ×s -1 Incubate at 100 rpm until OD 750 The concentration was 0.5-0.8; the culture was then transferred to a fresh 50mL LTAP medium and incubated at 22°C at 90μE×m. -2 ×s -1 Incubate at 100 rpm until OD 750 The concentration was 1.5-2.0. The shaker temperature was adjusted to 40℃, while the light conditions remained unchanged. The algal cells were cultured under these conditions for 20 minutes. Then, the algal cells were returned to normal culture conditions and cultured for 5 hours. Afterward, they were cultured again at 40℃ for 20 minutes, and then returned to normal culture conditions for 40 minutes. Finally, the cells were centrifuged at 4℃ and 5000 rpm for 5 minutes. The supernatant obtained was the crude extract containing the antimicrobial peptide ALFPm3.

[0108] Western blotting analysis of antimicrobial peptides: 400 mL of crude ALFPm3 extract was aliquoted into ten 50 mL centrifuge tubes, sealed tightly with sealing film, and frozen at -80°C. The next day, the frozen samples were removed and freeze-dried for 2 days. Finally, the samples were dissolved in 4 mL of PBS and stored at -20°C. 40 μL of sample was added to 10 μL of 5× protein loading buffer, mixed thoroughly, and incubated in a boiling water bath for 5-10 min, then cooled to room temperature. 5 μL of each sample was taken, along with 5 μL of 180 kDa Prestained Protein Marker, for SDS-PAGE to separate the proteins. During electrophoresis, a suitable PVDF membrane was cut, soaked in anhydrous methanol for 1 min, and then soaked in 1× PVDF membrane equilibration buffer from the GenScrip eBlot kit (Genscrip, USA) for 1 min. After electrophoresis, the gel was removed and soaked in deionized water for 1 min. Stack the transfer clamps according to the GenScrip eBlot kit instructions, from negative to positive electrode, sponge pad-gel-PVDF membrane-sponge pad, and perform automatic transfer for 6 minutes. After transfer, separate the gel and PVDF membrane in deionized water, and then immerse the membrane in 3% BSA solution for 2 hours at room temperature.

[0109] Primary antibody incubation was performed by diluting mouse anti-α-tublin antibody (Sigma, B30271) and mouse anti-HA Tag antibody (Biolegend, 901513) with 1×TBS solution at ratios of 1:1500 and 1:2000, respectively. The NC membrane containing the target protein was placed in an incubation chamber. For proteins containing 3×HA tags, mouse anti-HA Tag antibody was used, and for α-tubulin, mouse anti-α-tublin antibody was used. The membrane was incubated overnight on a slow shaker at 4°C. After incubation, the primary antibody was recovered, and the membrane was immersed in 1×TBST solution and washed on a horizontal shaker for 10 min. This process was repeated 3 times.

[0110] Secondary antibody incubation: Dilute Goat anti-mouse IgG antibody (Beijing Bio-Long, BF03001) at a ratio of 1:2000 with 1×TBS solution; add the secondary antibody to the incubation chamber after primary antibody incubation and washing the membrane 3 times, and incubate on a horizontal shaker at room temperature for 2 hours; after incubation, discard the secondary antibody, immerse the membrane in 1×TBST solution, and wash on a horizontal shaker for 10 minutes, repeating 3 times.

[0111] Development (Ultrasensitive ECL chemiluminescence): Prepare the developing solution according to a 1:1 volume ratio of developing solution A to developing solution B; pre-cool the developing instrument until the internal temperature reaches -30℃; add an appropriate amount of developing solution to the plate in the developing instrument; remove the NC membrane to be developed from the incubation box with tweezers, absorb the 1×TBST solution on the membrane with absorbent paper, place it on the plate with the developing solution added, and flip the NC membrane so that both sides of the membrane are fully in contact with the developing solution; expose using the developing instrument, and take pictures after development. The results are shown in [Figure number missing]. Figure 4 In this study, lane N represents the *Chlamydomonas reinhardtii* recipient strain JUV (unconverted signal peptide-antimicrobial peptide fusion sequence), lanes 1-11 represent genetic transformants, Anti-HA represents the target protein, and Anti-α-Tubulin represents the internal control protein. Results showed that a specific band of α-tubulin was detected in the protein samples of both the *Chlamydomonas reinhardtii* recipient strain JUV and all genetic transformants, indicating the success of the protein immunoblotting analysis. Using an anti-3×HA tag antibody, a specific band at 16 kDa was detected in all transformants, demonstrating that the antimicrobial peptide of this invention is an extracellularly secreted antimicrobial peptide.

[0112] The above demonstrates that the present invention has successfully constructed a transgenic Chlamydomonas reinhardtii capable of extracellularly expressing the antimicrobial peptide ALFPm3, and has successfully achieved the secretory expression of the antimicrobial peptide.

[0113] Example 2: Study on the expression efficiency of antimicrobial peptides in transgenic Chlamydomonas reinhardtii

[0114] On the clean bench, take 5 mL of TAP liquid culture medium and aliquot it into 10 mL centrifuge tubes. Use a pipette tip to pick up a small amount of algae and add it to the culture medium. Incubate at 22°C and 90 μE×m. -2 ×s -1 The cells were cultured under continuous light for 7 days. Using this culture as a seed culture, the cells were inoculated into fresh 1000 mL of TAP liquid medium to achieve a cell density of 1 × 10⁻⁶ cells / mL. 4 cell / mL, 100μE×m -2 ×s -1 Under continuous light, the cells were cultured for 7 days until the cell density reached 1×10⁻⁶. 7 The sample was centrifuged at 4℃ and 5000 rpm for 10 min, and the supernatant and algal cell precipitate were collected separately. The supernatant was filtered through a 0.22 μm aqueous filter membrane to remove cell debris and insoluble impurities, yielding extracellular proteins. These proteins were then concentrated by ultrafiltration using a 3 kDa ultrafiltration tube, centrifuged at 4℃ and 8000 × g for 30 min. The purified proteins were quantified using an anti-3×HA tag antibody, and the results are shown in [Figure number missing]. Figure 5 .

[0115] The results showed that the extracellular expression levels of ALFPm3 mediated by IBD-1, GH-like, and P-4-Y secretory peptides were 52 ng / mL, 65 ng / mL, and 48 ng / mL, respectively, representing a significant increase compared to existing technologies. The ARS2 secretory peptide failed to secrete ALFPm3 extracellularly, while the CAH secretory peptide mediated an extracellular expression level of 24 ng / mL. Therefore, the antimicrobial peptide secreted by GH-like peptides was 1.7 times higher than that secreted by CAH peptides, IBD-1 was 1.2 times higher than CAH, and P-4-Y was 1 time higher than CAH.

[0116] Example 3: Application of antimicrobial peptides secreted and expressed by transgenic Chlamydomonas reinhardtii

[0117] The pathogenic bacteria involved include *Vibrio alginolyticus*, *Vibrio harveyi*, and *Vibrio parahaemolyticus* (commercially available). Hold the petri dish in your left hand and slightly open the lid near a flame. Quickly pour in approximately 15 mL of LB medium. After capping, gently shake the dish to distribute the medium evenly at the bottom. Then place it flat on a table and allow it to solidify to form an agar plate. Holding the dish near a flame, use your left hand to hold the bottom and your right hand to hold an inoculation loop. Pour a loopful of bacteria onto the agar plate and streak it in sections. Incubate at 37°C inverted for 12-16 hours until single colonies appear. On a clean bench, take several 15 mL centrifuge tubes, add 3 mL of LB medium, and pick single colonies from the agar plate into the medium. Incubate overnight at 37°C and 200 rpm. The next day, remove the overnight culture and inoculate it at a ratio of 1% into 15 mL centrifuge tubes containing 3 mL of LB medium. Incubate at 37°C and 200 rpm for 1 hour to allow the bacteria to reach the logarithmic growth phase.

[0118] The antimicrobial peptide ALFPm3, expressed extracellularly in transgenic Chlamydomonas reinhardtii, was centrifuged to remove the precipitate, and the supernatant was filtered and concentrated as described in Example 2 to obtain a concentrated antimicrobial peptide solution, which was used for antibacterial experiments. Several 15mL centrifuge tubes were taken, and 3mL of LB medium and 3μL of bacterial culture were added to dilute the bacterial solution 1000-fold. This diluted bacterial solution was used as the sample for the antibacterial experiment. Then, sterile 96-well plates were taken, and 150μL of diluted bacterial solution and 50μL of concentrated antimicrobial peptide solution were added sequentially. For the control group, 50μL of 1×PBS solution was added to bring the total volume to 200μL. After adding the samples, the absorbance (OD) of the bacterial solution sample at 600nm was measured using a microplate reader. 600 ), calculate the absorbance at 0 h. Finally, measure the OD after 12 h of culture. 600 12h OD 600 With 0h OD 600 Subtraction yields the growth rate ΔOD of the bacterium. 600 With ΔOD 600 The vertical axis was used to plot bacterial growth curves. The antibacterial effects of Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio harveyi are shown in the figure below. Figure 6-8Among them, T-GH-3 expresses ALFPm3 via GH-like secretory peptide, T-IBD-1 expresses ALFPm3 via IBD-1 secretory peptide, T-PY-1 expresses ALFPm3 via P-4-Y secretory peptide, T-CAH-5 expresses ALFPm3 via CAH secretory peptide, and T-ARS-8 expresses ALFPm3 via ARS2 secretory peptide. The negative control is Chlamydomonas reinhardtii, which does not express ALFPm3. The inhibition rate % = (absorbance value of blank bacterial solution - absorbance value of sample bacterial solution) / absorbance value of blank bacterial solution.

[0119] from Figure 6-8 It can be seen that after co-culturing Chlamydomonas extract with bacteria such as Vibrio parahaemolyticus, ALFPm3 expressed with GH-like secretory peptide showed the strongest antibacterial rate, reaching 96.9%. The worst was ALFPm3 expressed with ARS2 secretory peptide, showing virtually no antibacterial activity. The 12-hour inhibition rates of each genetic transformant against Vibrio parahaemolyticus were, in descending order: T-GH-3 > T-IBD-1 > T-PY-1 > T-CAH-5 > T-ARS-8. The experimental results against Vibrio alginolyticus and Vibrio harveyi were roughly the same, indicating that ALFPm3 expressed with GH-like secretory peptide exhibited the highest activity and the strongest antibacterial activity.

[0120] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A genetically modified Chlamydomonas reinhardtii, characterized in that, The transgenic Chlamydomonas reinhardtii contains an expression vector, which includes a DNA sequence encoding a secretory peptide of Chlamydomonas reinhardtii, a DNA sequence encoding an antimicrobial peptide, and a photoinducible promoter. The DNA sequence encoding the secretory peptide and the DNA sequence encoding the antimicrobial peptide are located downstream of the photoinducible promoter. The amino acid sequence of the Chlamydomonas reinhardtii secretory peptide is selected from one of the following as shown in SEQ ID NO: 1-3, and the antimicrobial peptide is Penaeus monodon anti-lipopolysaccharide factor 3, the amino acid sequence of which is shown in SEQ ID NO:

7.

2. The transgenic Chlamydomonas reinhardtii as described in claim 1, characterized in that, The DNA sequence encoding the Chlamydomonas reinhardtii secretory peptide is selected from one of the sequences shown in SEQ ID NO: 4-6.

3. The transgenic Chlamydomonas reinhardtii as described in claim 1, characterized in that, The DNA sequence of the anti-lipopolysaccharide factor 3 of the tiger prawn is shown in SEQ ID NO:

8.

4. A method for constructing a transgenic Chlamydomonas reinhardtii, characterized in that, The method for preparing the transgenic Chlamydomonas reinhardtii as described in any one of claims 1-3 comprises the following steps: S1. The DNA sequence encoding the secretory peptide of Chlamydomonas reinhardtii and the DNA sequence encoding the antimicrobial peptide are ligated into a starting vector to obtain an expression vector, wherein the starting vector is pESVH; S2. The expression vector is transformed into the recipient algal strain of Chlamydomonas reinhardtii to obtain transgenic Chlamydomonas reinhardtii.

5. The method for constructing transgenic Chlamydomonas reinhardtii as described in claim 4, characterized in that, In step S1, the DNA sequence encoding the secretory peptide of Chlamydomonas reinhardtii and the DNA sequence encoding the antimicrobial peptide are fused together to obtain the secretory peptide-antimicrobial peptide fusion sequence through gene synthesis technology. Then, the secretory peptide-antimicrobial peptide fusion sequence is inserted downstream of the promoter of the enzyme-cleaved linearized starting vector to obtain the expression vector. In step S2, the expression vector is transformed into the Chlamydomonas reinhardtii recipient algal strain using the "bead milling method". The Chlamydomonas reinhardtii recipient algal strain is a cell wall-deficient algal strain JUV.

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