CN116284288A

CN116284288A - Chlamydomonas reinhardtii secretion peptide, transgenic Chlamydomonas reinhardtii, construction method and application thereof

Info

Publication number: CN116284288A
Application number: CN202310050318.XA
Authority: CN
Inventors: 庄惠琳; 欧耀辉; 陈若瑜; 王潮岗
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2023-02-01
Filing date: 2023-02-01
Publication date: 2023-06-23

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to chlamydomonas reinhardtii secretion peptide, a transgenic chlamydomonas reinhardtii, and a construction method and application thereof. The amino acid sequence of the chlamydomonas reinhardtii secretory leader peptide is selected from the amino acid sequences shown in SEQ ID NO: 1-3. When the secretory leader peptide with the amino acid sequence provided by the invention is used for expressing target proteins such as the antibacterial peptide, the antibacterial peptide expressed in chlamydomonas reinhardtii cells can be guided to be secreted outside cells, so that the efficient secretory expression of the antibacterial peptide is realized, the expression quantity is more than 40ng/mL, the isolated antibacterial peptide has higher biological activity, the growth and propagation of a plurality of aquaculture pathogenic bacteria such as vibrio alginolyticus, vibrio harveyi and vibrio parahaemolyticus can be effectively inhibited, the antibacterial rate reaches more than 90%, and the use of antibiotics can be effectively reduced, so that the antibacterial peptide obtained by secretory expression of the secretory leader peptide has important development potential in the aquaculture industry, especially in the aquaculture industry.

Description

Chlamydomonas reinhardtii secretion peptide, transgenic Chlamydomonas reinhardtii, construction method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to chlamydomonas reinhardtii secretion peptide, transgenic chlamydomonas reinhardtii, and a construction method and application thereof.

Background

The discovery and application of antibiotics lead people to be benefited, but the frequent use of antibiotics is equivalent to the directional screening of pathogenic bacteria in nature, so that microorganisms are easy to generate multiple drug resistance, and the application in the breeding industry can also cause environmental pollution and drug residues so as to cause food safety problems. AMPs are a generic name of small molecular polypeptides and small molecular proteins with broad-spectrum antibacterial activity which are induced in organisms, and are important components of the innate immune system of the organisms. Among them, anti-lipopolysaccharide factor (Anti-lipopolysaccharide factors, ALFs) is an antibacterial peptide in crustaceans, generally consisting of 98-123 amino acids, comprising a signal peptide sequence and a lipopolysaccharide binding domain as a functional region of ALFs, and inhibits or kills pathogenic microorganisms by binding lipopolysaccharide to destroy cell walls or cell membranes of bacteria. AMPs take cell membranes as action targets rather than special proteins, so microorganisms are not easy to generate drug resistance, are peptides, are easy to degrade in nature, cannot be accumulated in nature and cannot be remained in vivo, have no food safety problem, and have wide application prospects. However, the natural antibacterial peptide has very small content in organisms, has the problems of difficult extraction, low yield, long time, complex process, low in-vitro activity and the like, and cannot realize large-scale production, so that the method becomes the biggest obstacle for restricting AMPs from entering practical application. Therefore, it is of great importance to develop AMPs genetic engineering research, and designing or modifying natural antibacterial peptides by genetic engineering technology is a viable solution to the aforementioned problems.

Currently, heterologous expression of AMPs is achieved primarily by escherichia coli expression systems and yeast expression systems. The E.coli expression system is used as an expression system for expressing heterologous genes, has the advantages of high expression quantity, simple operation, low cost and the like, but because of the killing effect of AMPs on bacteria, the E.coli expression system cannot be used for directly expressing the antibacterial peptide with biological activity, and if the E.coli expression system is used for expression in the form of fusion protein, inclusion bodies are easy to form to reduce the activity of the expressed protein, and the folding, modification and processing biological processes of eukaryotic proteins are lacked, so that the obtained recombinant AMPs are low in activity and have cytotoxicity. Yeast belongs to eukaryotic microorganisms, can be processed and modified after translation, has a more complete gene expression regulation mechanism, can make up for the deficiency of an escherichia coli expression system, but also easily generates the problem of excessive glycosylation of expressed proteins, thereby influencing the activity of the expressed proteins. Moreover, both E.coli and yeast expression systems require heterotrophic fermentation, which consumes large amounts of materials and energy and is prone to environmental pollution.

Aiming at the defects of the expression system, in recent years, researchers develop a chlamydomonas reinhardtii expression system, namely, chlamydomonas reinhardtii which is taken as a single-cell eukaryotic green alga, combines the dual advantages of bacteria and higher plants, has the advantages of rapid growth, shorter life cycle, low culture cost, capability of post-translational modification and the like, does not contain endotoxin, has clear genetic background, and has a nucleus, chloroplast and mitochondria 3-set genetic transformation system, wherein the nuclear genome expression system is most used and the technology is most mature, and in addition, the chlamydomonas reinhardtii enters a human edible algae list and can be directly taken as the bait of aquatic organisms such as fishes, shrimps and the like, so the chlamydomonas reinhardtii expression system has unique advantages and can make up the related defects of escherichia coli and yeast expression systems. The latest research shows that the chlamydomonas reinhardtii can realize the intracellular and extracellular expression of the target protein, and the extracellular secretory expression has the advantages of convenient separation and purification, high product activity, convenient use and the like. The efficient recovery of AMPs in vitro can be realized by utilizing the secretion expression AMPs of chlamydomonas reinhardtii, the preparation of antibacterial peptide feed is simplified, and the efficient recovery of AMPs can be directly used as an antibacterial agent in aquaculture, so that the use of antibiotics is reduced, and a good foundation is laid for the practical application of antibacterial peptides. Therefore, the chlamydomonas formulation for secretory expression of the antibacterial peptide has important application potential in the aquaculture industry.

However, there are few studies on secretory expression of Chlamydomonas reinhardtii at present, and there are problems such as low secretion efficiency and even non-secretion of target proteins. Lauersen et al secrete expression luciferase with carbonic anhydrase 1 (Carbonic anhydrase, CAH 1) as leader peptide, the secretion expression amount of which is obviously smaller than that of E.coli or yeast expression system; in addition, the research results also show that CAH1 can interfere with the generation of recombinant proteins so as to further reduce the expression secretion amount. In addition, mayfield et al use the succinate dehydrogenase module as a secretory leader, and studies have shown that it interferes with the function of self-cleaving 2A peptide, resulting in the inability of mCherry proteins to be expressed and secreted. Therefore, perfecting or improving the secretion expression system of the chlamydomonas reinhardtii to solve the problem that the expression level of the antibacterial peptide in the chlamydomonas reinhardtii is low or even not expressed is an urgent need to be solved at present.

Disclosure of Invention

Aiming at the technical problems that the efficient secretory expression of the antibacterial peptide and the low or even non-expression of the antibacterial peptide in the chlamydomonas reinhardtii are difficult to realize by a chlamydomonas reinhardtii expression system in the prior art, the invention provides a chlamydomonas reinhardtii secretory leader peptide, provides a transgenic chlamydomonas reinhardtii containing the chlamydomonas reinhardtii secretory leader peptide, a construction method thereof, and application of the chlamydomonas reinhardtii secretory leader peptide or the transgenic chlamydomonas reinhardtii in the breeding industry.

In order to achieve the above purpose, the present invention is specifically realized by the following technical scheme:

the first aspect of the invention provides a chlamydomonas reinhardtii secretion leader peptide, the chlamydomonas reinhardtii secretion leader peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

Further, the amino acid sequence of the chlamydomonas reinhardtii secretory leader peptide is shown in SEQ ID NO: 2.

In a second aspect the present invention provides a polynucleotide for encoding a chlamydomonas reinhardtii secretory leader peptide as described above.

In a third aspect the present invention provides a transgenic chlamydomonas reinhardtii comprising an expression vector comprising a DNA sequence encoding a chlamydomonas reinhardtii secretory leader peptide as described above and a DNA sequence encoding an antibacterial peptide.

Further, the DNA sequence encoding the chlamydomonas reinhardtii secretory leader peptide is selected from the group consisting of SEQ ID NO: 4-6.

Further, the antibacterial peptide is anti-lipopolysaccharide factor 3 of the penaeus monodon, and the amino acid sequence of the antibacterial peptide is shown in SEQ ID NO:7, the DNA sequence is shown as SEQ ID NO: shown at 8.

Further, the expression vector further comprises a photoinduced promoter, and a DNA sequence encoding the chlamydomonas reinhardtii secretory leader peptide and a DNA sequence encoding the antibacterial peptide are sequentially positioned downstream of the photoinduced promoter.

Further, the starting vector for constructing the expression vector is pESVH.

The fourth aspect of the present invention provides a method for constructing transgenic Chlamydomonas reinhardtii as described above, comprising the steps of:

s1, connecting a DNA sequence for encoding the chlamydomonas reinhardtii secretory leader peptide and a DNA sequence for encoding the antibacterial peptide to a starting vector to obtain an expression vector;

s2, transforming the expression vector into a chlamydomonas reinhardtii receptor strain to obtain transgenic chlamydomonas reinhardtii.

Further, in step S1, a DNA sequence encoding the secretory leader peptide of Chlamydomonas reinhardtii and a DNA sequence encoding the antibacterial peptide are fused, a secretory leader peptide-antibacterial peptide fusion sequence is obtained through a gene synthesis technology, and then the secretory leader peptide-antibacterial peptide fusion sequence is inserted into the downstream of a promoter of an enzyme-cut linearized starting vector to obtain the expression vector.

Further, in step S2, the expression vector is transformed into the chlamydomonas reinhardtii receptor strain, which is a cell wall deficient strain JUV, by a "bead milling method".

In a fifth aspect the present invention provides the use of transgenic Chlamydomonas reinhardtii as described above in the aquaculture industry.

The invention has the advantages and positive effects that:

when the secretory leader peptide with the amino acid sequence provided by the invention is used for expressing target proteins such as the antibacterial peptide, the antibacterial peptide expressed in chlamydomonas reinhardtii cells can be guided to be secreted outside cells, so that the efficient secretory expression of the antibacterial peptide is realized, the expression quantity is more than 40ng/mL, the isolated antibacterial peptide has higher biological activity, the growth and propagation of a plurality of aquaculture pathogenic bacteria such as vibrio alginolyticus, vibrio harveyi and vibrio parahaemolyticus can be effectively inhibited, the antibacterial rate reaches more than 90%, and the use of antibiotics can be effectively reduced, so that the antibacterial peptide obtained by secretory expression of the secretory leader peptide has important development potential in the aquaculture industry, especially in the aquaculture industry.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing the expression vector pESVH-ALF3 of secretory leader-antibacterial peptide constructed in example 1 of the present invention;

FIG. 2 is a genomic PCR detection electrophoresis of transgenic Chlamydomonas reinhardtii of example 1 of the present invention; wherein, the liquid crystal display device comprises a liquid crystal display device,lanes 1-4, from left to right, are T-GH-3, T-IBD-1, T-PY-1 and T-CAH-5 genetic transformants, lane 5 is the positive control expression vector pESVH-ALF3, lane 6 is the untransformed Chlamydomonas reinhardtii receptor strain JUV, and lane 7 is the negative control H ₂ O；

FIG. 3 is an electrophoresis chart of RT-PCR detection of transgenic Chlamydomonas reinhardtii according to example 1 of the present invention; wherein, lane M is DL2000 DNAMmarker, 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 are positive control expression vector pESVH-ALF3;

FIG. 4 is a diagram of immunoblot analysis of transgenic Chlamydomonas reinhardtii according to example 1 of the present invention; wherein, lane N is the untransformed Chlamydomonas reinhardtii receptor 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 protein of interest, and Anti-alpha-Tubulin is the reference protein;

FIG. 5 is a graph showing the expression level of extracellular antimicrobial peptides of transgenic Chlamydomonas reinhardtii obtained by constructing different secretory leader peptides according to example 2 of the present invention; wherein 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;

FIG. 6 is a graph showing the antibacterial effect of the secretory expression of antibacterial peptides of different transgenic Chlamydomonas reinhardtii on Vibrio parahaemolyticus 12h according to example 3 of the present invention;

FIG. 7 is a graph showing the antibacterial effect of the secretory expression of antibacterial peptides from different transgenic Chlamydomonas reinhardtii of example 3 on Vibrio alginolyticus for 12 h;

FIG. 8 is a graph showing the antibacterial effect of the secretory expression of the antibacterial peptide of different transgenic Chlamydomonas reinhardtii of example 3 on Vibrio harveyi 12 h.

Detailed Description

The present invention will be described in further detail with reference to the following examples, in which the apparatus and reagents used in the respective examples and test examples are commercially available unless otherwise specified, in order to make the objects, technical schemes and advantages of the present invention more apparent. The specific embodiments described herein are to be considered in an illustrative sense only and are not intended to limit the invention.

Various modifications to the precise description of the invention will be readily apparent to those skilled in the art from the information contained herein without departing from the spirit or scope of the appended claims. It is to be understood that the scope of the invention is not limited to the defined processes, properties or components, as these embodiments, as well as other descriptions, are merely illustrative of specific aspects of the invention. Indeed, various modifications of the embodiments of the invention which are obvious to those skilled in the art or related fields are intended to be within the scope of the following claims.

For a better understanding of the present invention, and not to limit its scope, all numbers expressing quantities, percentages, and other values used in the present application are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated otherwise, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.

The invention screens and obtains the secretion peptide which can secrete target protein-antibacterial peptide to extracellular, further compares the activity and the expression efficiency of the antibacterial peptide secreted and expressed by the secretion peptide to obtain the secretion peptide far superior to the prior reported Arylsulfatase 2 (ARS 2) and Carbonic anhydrase (CAH 1), realizes that the antibacterial peptide is secreted to extracellular with high efficiency by taking the chlamydomonas reinhardtii as a host, and provides a solution for the extracellular expression of the antibacterial peptide with high activity in the chlamydomonas reinhardtii and the convenient application. The present invention has been completed on the basis of this finding.

In the context of the present invention, "secretory leader peptide", "leader peptide" and "signal peptide" are used interchangeably to direct secretory expression of a protein of interest. "secretion" refers to the transport of a protein or peptide molecule outside the cell after intracellular synthesis.

The embodiment of the invention provides a chlamydomonas reinhardtii secretion leader peptide, which is selected from at least one of IBD-1, GH-like and P-4-Y, wherein the amino acid sequences of the IBD-1, GH-like and P-4-Y are respectively shown as SEQ ID NO:1-3, specifically as follows:

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

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

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

According to published chlamydomonas reinhardtii genome sequence (GenBank accession number: NC-005353), signal peptide prediction software such as SignalP 4.0 is adopted to predict signal peptide to obtain candidate molecules, then screening is carried out according to the distribution condition of the candidate signal peptide in and out of cells, the activity and the expression efficiency of the antibacterial peptide secreted and expressed by different candidate signal peptides are further compared, the secretory leader peptides IBD-1, GH-like and P-4-Y shown by the amino acid sequences are screened to obtain, and when the IBD-1, GH-like and P-4-Y are respectively used for expressing the antibacterial peptide, the antibacterial peptide expressed in chlamydomonas reinhardtii cells can be led to be secreted out of cells, so that the efficient secretory expression of the antibacterial peptide is realized, the expression quantity is more than 40ng/mL, the purification of the extracellular antibacterial peptide is facilitated, and the separation and purification cost is greatly reduced. In addition, the separated antibacterial peptide has higher biological activity, can effectively inhibit the growth and reproduction of various aquaculture pathogenic bacteria such as vibrio alginolyticus, vibrio harveyi and vibrio parahaemolyticus, and has a bacteriostasis rate of more than 90%, which indicates that the antibacterial peptide secreted by the secretory leader peptide has important development potential in aquaculture, and meanwhile, the application of the antibacterial peptide in aquaculture is provided by adopting a Chlamydomonas reinhardtii expression system, such as a feed additive.

Preferably, the chlamydomonas reinhardtii secretory leader peptide is selected from GH-like, and the amino acid sequence is shown in SEQ ID NO: 2. The experiment shows that the GH-like secretory leader peptide expresses the highest amount of antibacterial peptide and has the best activity.

Another embodiment of the invention provides a polynucleotide for encoding a Chlamydomonas reinhardtii secretory leader peptide as described above.

The advantages of the polynucleotides over the prior art are the same as those of the chlamydomonas reinhardtii secretory leader peptide described above over the prior art and are not described in detail herein.

Illustratively, the nucleotide sequences encoding IBD-1, GH-like and P-4-Y are set forth in SEQ ID NO: 4-6.

It should be noted that the sequence of the polynucleotide is derived by conventional means such as codon encoding rules according to the amino acid sequence. It will be appreciated by those skilled in the art that the polynucleotides provided herein for encoding IBD-1, GH-like and P-4-Y are not intended to limit the scope of the invention.

Yet another embodiment of the present invention provides a transgenic chlamydomonas reinhardtii comprising an expression vector for expressing a chlamydomonas reinhardtii secretion leader peptide and an antibacterial peptide, the expression vector comprising a DNA sequence encoding the chlamydomonas reinhardtii secretion leader peptide and a DNA sequence encoding the antibacterial peptide as described above.

According to the invention, a Chlamydomonas reinhardtii expression system is adopted, and target proteins such as antibacterial peptides can be expressed outside cells of Chlamydomonas reinhardtii by utilizing IBD-1, GH-like and P-4-Y secretion guide peptides, so that product inhibition is effectively avoided, and the expression quantity of the target proteins is improved. The transgenic chlamydomonas reinhardtii of the present invention is cultured by the conventional method, the crude extract containing high concentration antibacterial peptide can be obtained by centrifugal separation of algal cells and freeze drying, and the high purity extracellular antibacterial peptide can be obtained by further separation and purification by the conventional method. The extracellular antibacterial peptide can effectively inhibit the growth of aquaculture microorganisms and reduce the use of antibiotics. In addition, the invention adopts chlamydomonas reinhardtii as host cells to produce the antibacterial peptide, which has the following advantages: 1) The genetic transformation operation is convenient, and various mutants are obtained for molecular biology research; 2) The method has the advantages that the method has the enzyme and organelle capable of modifying the antibacterial peptide, and can secrete the antibacterial peptide into a culture medium, so that the separation and purification steps are simplified; 3) The chlamydomonas reinhardtii is convenient to culture, can be used for photosynthesis autotrophy and energy heterotrophy, and has low cost and easy acquisition of culture medium; 4) The transgenic chlamydomonas reinhardtii expressed by the antibacterial peptide can be directly used as an antibacterial preparation in an aquaculture link, and has the characteristics of environmental protection and convenience.

Illustratively, the antibacterial peptide is anti-lipopolysaccharide factor 3 (ALFPm 3) of the penaeus monodon, and the amino acid sequence of the antibacterial peptide is shown in SEQ ID NO:7, the DNA sequence is shown as SEQ ID NO: shown at 8.

Optionally, the expression vector further comprises a light-induced promoter, and a DNA sequence encoding a Chlamydomonas reinhardtii secretory leader peptide as described above and a DNA sequence encoding an antibacterial peptide are located downstream of the light-induced promoter in sequence. Namely, from the 5'-3' end, a light-induced promoter, a DNA sequence encoding a Chlamydomonas reinhardtii secretory leader peptide and a DNA sequence encoding an antibacterial peptide are sequentially provided. The light-induced promoter can be used for improving the expression quantity of the target protein expressed by the chlamydomonas reinhardtii.

Alternatively, the starting vector from which the expression vector is constructed is pESVH.

The embodiment of the invention also provides a construction method of the transgenic chlamydomonas reinhardtii, which comprises the following steps:

s1, constructing an expression vector: ligating a DNA sequence encoding a Chlamydomonas reinhardtii secretory leader peptide as described above (e.g., the DNA sequence shown in SEQ ID NO: 4-6) and a DNA sequence encoding an antibacterial peptide (e.g., the DNA sequence shown in SEQ ID NO: 7) to a starting vector to obtain an expression vector;

s2, genetic transformation: and (3) transforming the expression vector into a chlamydomonas reinhardtii receptor strain to obtain the transgenic chlamydomonas reinhardtii.

The advantages of the construction method of the transgenic chlamydomonas reinhardtii with respect to the prior art are the same as those of the transgenic chlamydomonas reinhardtii described above with respect to the prior art, and will not be described again.

In the step S1, a DNA sequence for encoding the secretory leader peptide of Chlamydomonas reinhardtii and a DNA sequence for encoding the antibacterial peptide are fused, a secretory leader peptide-antibacterial peptide fusion sequence is obtained through a gene synthesis technology, and then the fusion sequence is inserted into the downstream of a promoter of an enzyme-cut linearization starting vector, so that an expression vector is obtained. In order to obtain a high expression level, the promoter is preferably a light-induced promoter, and the expression of the antibacterial peptide is induced by a strong light induction method.

In the step S2, an expression vector containing a secretory leader peptide-antibacterial peptide fusion sequence is transformed into a chlamydomonas reinhardtii receptor strain by a bead milling method for expression, so as to obtain a genetic transformant, genomic DNA, RNA and protein of the genetic transformant are detected, and transcriptional activity of the antibacterial peptide is analyzed, so that the transgenic chlamydomonas reinhardtii capable of being inherited stably is obtained.

Alternatively, the Chlamydomonas reinhardtii receptor strain is a cell wall deficient strain JUV, purchased from the C.america center.

Alternatively, the method of detecting genomic DNA of the genetic transformant comprises a PCR amplification method, and the secretion leader peptide-antibacterial peptide fusion sequence is detected whether to be successfully inserted into the starting vector by amplification of specific primers.

Alternatively, the method for detecting RNA of the genetic transformant includes a real-time PCR (RT-PCR) method. Transcript levels of the antibacterial peptide RNA were detected by RT-PCR.

Alternatively, the method of detecting the protein of the genetic transformant comprises Western immunoblotting. And identifying the expression quantity of the antibacterial peptide by a western immunoblotting method.

A further embodiment of the invention provides the use of a transgenic chlamydomonas reinhardtii as described above in aquaculture, in particular in aquaculture. Specifically, the high-purity and high-concentration antibacterial peptide can be obtained by culturing the transgenic chlamydomonas reinhardtii, has good biological activity, has obvious inhibition effect on growth and reproduction of various aquaculture pathogenic bacteria such as vibrio alginolyticus, vibrio harveyi and vibrio parahaemolyticus, can be used as a feed additive, and improves organism disease resistance and immunity, thereby promoting growth. Or the transgenic chlamydomonas reinhardtii is directly used as a feed additive to be applied to animal or aquatic feeds.

The invention will be further illustrated with reference to specific examples. The experimental methods in which specific conditions are not specified in the following examples are generally conducted under conventional conditions, for example, those described in the molecular cloning Experimental guidelines (fourth edition) published in Cold spring harbor laboratory, or are generally conducted under conditions recommended by the manufacturer.

EXAMPLE 1 construction of transgenic Chlamydomonas reinhardtii

1. Culture of Chlamydomonas reinhardtii

Cell wall-deficient chlamydomonas reinhardtii JUV was selected as the transgenic recipient strain (purchased from chlamydomonas americana center, st. Paul, MN 55108, usa).

The TAP medium was used as the medium for Chlamydomonas reinhardtii, and the formulation included: 2.42g Tris, 25mL 4 XBeijeinck salts, 1mL 1M (K) PO ₄ 1mL Trace element mixture, 975mL water, and adjusting pH to 6.95-7.05 with glacial acetic acid, and fixing volume to 1L. Wherein, the formula of 4×Beijerinck samples comprises: 16g NH ₄ Cl、2g CaCl ₂ ·2H ₂ O、4g MgSO ₄ ·7H ₂ O is dissolved in water, and the volume is fixed to 1L; the Trace element mixed solution comprises the following components: 11.4. 11.4g H ₃ BO ₃ 、5.6g MnCl ₂ ·4H ₂ O、22g ZnSO ₄ ·7H ₂ O、4.99g FeSO ₄ ·7H ₂ O、1.61g CoCl ₂ ·6H ₂ O、1.57g CuSO ₄ ·5H ₂ O、1.1g(NH ₄ ) ₆ Mo ₇ O ₂₄ ·4H ₂ O、50g Na ₂ EDTA was dissolved in water and pH adjusted to 6.5-6.8 with 20% KOH to 1L.

Culture conditions of Chlamydomonas reinhardtii: at 22-25deg.C, 90 μE/m ² Continuous culture under s-illumination, algae cells with 1×10 logarithmic growth phase concentration ⁶ -2×10 ⁶ cells/mL。

2. Construction of expression vectors

According to published Chlamydomonas reinhardtii genome sequence (GenBank accession number: NC_ 005353), the secretion signal peptide is predicted by adopting SignalP 4.0 software, and the obtained candidate molecules are screened according to the distribution condition of the protein in the cell and the cell, so that 5 extracellular secretion expressed signal peptides are finally obtained, and names, amino acid sequences and gene sequences in Chlamydomonas reinhardtii, which relate to the 5 extracellular secretion expressed signal peptides, are shown as follows:

TABLE 1 sequence information of Chlamydomonas reinhardtii secretory leader peptide candidate molecules

The antibacterial peptide is exemplified by Penaeus monodon antibacterial peptide-anti-lipopolysaccharide factor 3 (ALFPm 3), and other antibacterial peptides such as anti-lipopolysaccharide factor 11, crustin, lysozyme, etc. can be expressed according to practical requirements.

The amino acid sequence of ALFPm3 is:

QGWEAVAAAVASKIVGLWRNEKTELLGHECKFTVKPYLKRFQVYYKGRMWCPGWTAIRGEASTRSQSGVAGKTAKDFVRKAFQKGLISQQEANQWLSS (see SEQ ID NO: 7).

The gene expression sequence (5 '-3') of ALFPm3 in chlamydomonas reinhardtii is:

caagggtgggaggctgtggcagcggccgtcgccagcaagatcgtggggttgtggaggaacgaaaaaactgaacttctcggccacgagtgcaagttcaccgtcaagccttatttgaagagattccaggtgtactacaaggggaggatgtggtgcccaggctggacggccatcagaggagaagccagcacacgcagtcagtccggggtagctggaaagacagccaaagacttcgttcggaaagctttccagaaaggtctcatctctcaacaggaggccaaccagtggctcagctcatag (see SEQ ID NO: 8).

The signal peptide-antibacterial peptide fusion sequence is obtained through a gene synthesis technology, specifically, 5 secretory leader peptides and ALFPm3 in the table 1 are directly connected, and a 3 XHA tag is added, so that subsequent protein detection and purification are facilitated, and the obtained fusion sequence is sent to a gene synthesis company for synthesis, so that the secretory leader peptide-antibacterial peptide fusion sequence is obtained.

Then the synthesized secretory leader peptide-antibacterial peptide fusion sequence is connected between EcoRI and Pml I cleavage sites of the pESVH vector to obtain an expression vector named pESVH-ALF3, and the expression vector is transferred into escherichia coli for cloning by taking CAH signal peptide-antibacterial peptide fusion sequence (CAH-ALFPm 3) as an example, wherein the map of the expression vector is shown in figure 1.

The construction method of the pESVH vector comprises the following steps: the pESVH vector was obtained by selecting the Chlamydomonas reinhardtii PSaD promoter (PSaD pro) and the Chlamydomonas reinhardtii pasD terminator (PSaD ter) in place of the HSP70A-RBCS2 promoter and RBCS2 terminator of pH124 on the basis of the Chlamydomonas expression vector pH124 (see: wu Jixia, hu Zhangli, wang Chaogang, eficient expression of Green Fluorescent Protein (GFP) mediated by achimeric promoter in Chlamydomonas reinhardti, chinese Joumal of Oceanology and Limnolog,2008,26 (3): 242-247.).

3. Genetic transformation of expression vectors

Specifically, the genetic transformation is carried out by adopting a bead milling method, and an expression vector with a signal peptide-antibacterial peptide fusion sequence is introduced into a chlamydomonas reinhardtii genome for expression, and the method comprises the following steps:

preparing a transformation plasmid: coli containing the signal peptide-antibacterial peptide fusion sequence was inoculated into 15mL of LB liquid medium containing 100. Mu.g/mL of Amp, and cultured at 37℃and 200rpm for 12-16 hours. The culture broth was centrifuged at 8000rpm for 10min at room temperature and the supernatant was discarded. Plasmid DNA was extracted using TRANS EasyPure Plasmid MiniPrep kit kit: adding 500 mu L of Solution I added with RNase A, fully swirling thalli, adding 500 mu L of Solution II, gently mixing, not exceeding 5min, adding 700 mu L of Solution III, gently mixing for 5-10 times, centrifuging at room temperature for 10min at 12000 Xg, taking supernatant, adding the supernatant onto a centrifugal column sleeved with a 2mL collecting tube, centrifuging for 1min at 10000 Xg, discarding filtrate, adding 500 mu L of HBC Solution added with isopropanol, centrifuging for 1min at 10000 Xg, discarding filtrate, finally adding 700 mu LW Buffer, centrifuging for 1min at 10000 Xg, discarding filtrate, repeating the operation once, centrifuging for 2min at room temperature at 12000 Xg, removing the collecting tube, sleeving an EP tube of 1.5mL, dripping 80-100 mu L of sterile water preheated at 65 ℃, standing at room temperature for 2min, centrifuging for 1min at 12000 Xg, and obtaining plasmid DNA eluting with no protein, wherein the concentration of DNA is 901.4-1325.7 mu L.

Restriction enzyme digestion of plasmid DNA: the configuration of the cleavage reaction system was performed using FastDiget KpnI restriction enzyme (Thermo Fisher Scientific, mass., U.S.) as follows: mu.L of plasmid DNA, 1. Mu.L of KpnI, 3. Mu.L of 10 XBuffer, and 30. Mu.L of deionized water were used. The reaction was carried out at 37℃for 2 hours and heat-inactivated at 65℃for 10s. mu.L of the reaction solution was taken and 1. Mu.L of 5 Xloading Buffer was added thereto, and electrophoresis was performed in 1% agarose gel, whereby it was revealed that the plasmid DNA was completely digested and the band size was in accordance with the expectation, and the mixture was used as the next experiment.

Obtaining genetic transformants: inoculating Chlamydomonas reinhardtii cell wall deficient strain JUV into TAP liquid culture medium at 22deg.C, 90 μE×m ^-2 ×s ^-1 And (5) culturing under illumination. Up to OD ₇₅₀ To 1.0, the algal cell density in the culture solution at this time was about 1X 10 ⁶ cell/mL. The algae cells were collected by centrifugation at 5000rpm for 5min at room temperature. On a super clean bench, the algae cells were resuspended in 250. Mu.L of fresh TAP medium, the algae solution was added to a sterilized 1.5mL EP tube containing 300mg glass beads, and 20. Mu.L of plasmid DNA linearized with restriction enzymes was added and vortexed at 2500rpm for 25-30s on a vortexing shaker. The supernatant was carefully aspirated, transferred to a 50mL centrifuge tube containing 10mL TAP medium, wrapped with tin foil at 25℃and shake incubated at 100rpm for 22 hours in the dark. The algae cells were collected by centrifugation at 3000rpm for 5min at room temperature, leaving about 100. Mu.L of liquid to resuspend the algae cells. It was spread onto TAP solid medium containing 0.1% ampicillin and 0.001% bleomycin. 22 ℃ and 90 mu E m ^-2 ×s ^-1 Culturing for 1-2 weeks in reverse until monoclonal appears. The monoclonal algae colony is transferred onto a new square TAP plate containing 0.1% ampicillin and 0.001% bleomycin, and a genetic transformant capable of stably inheriting is obtained. Genetic transformants transformed with IBD-1-ALFPm3, GH-like-ALFPm3, P-4-Y-ALFPm3, CAH-ALFPm3 and ARS2-ALFPm3 fusion sequences were designated as T-IBD-1, T-GH-3, T-PY-1, T-CAH-5 and T-ARS-8, respectively.

4. Detection of expression of genetic transformants (transgenic Chlamydomonas reinhardtii)

1) PCR detection of genomic DNA

Extracting genomic DNA of genetic transformant: each genetic transformant was picked up in 20mL TAP liquid medium at 22℃and 90. Mu.E.times.m ^-2 ×s ^-1 Culturing at 100rpm for 5d. Subpackaging into 50mL centrifuge tubes, centrifuging at 5000rpm for 10min, discarding supernatant, and extracting genomic DNA of genetic transformant according to kit (Bebei Biotechnology Co., ltd. Ultra DNA lsolation, zhengzhou, china).

PCR detection of genomic DNA: a PCR reaction system was configured with 2×m5 HiPer plus Taq HiFi PCR mix (beijing polymeric biotechnology limited, beijing, china), and included: genomic DNA 2. Mu.L, upstream primer PSaD-P0.4. Mu.L, downstream primer PSaD-T0.4. Mu.L, 2 XM 5 HiPer plus Taq HiFi PCR mix. Mu. L, ddH ₂ O10. Mu.L. The PCR reaction flow is as follows: the reaction was terminated by 3min of pre-denaturation at 95 ℃,30 s denaturation at 95 ℃,20 s annealing at 56 ℃, 40s extension at 72 ℃, 35 cycles, 5min extension at 72 ℃ and 4 ℃.

Identifying 5 μL PCR reaction solution by 2% agarose gel electrophoresis, wherein the electrophoresis result is shown in FIG. 2, lane M is DL2000 marker, lanes 1-4 are genetic transformants, lane 5 is positive control (i.e. the expression vector pESVH-ALF3 of the invention), lane 6 is Chlamydomonas reinhardtii receptor strain JUV (non-transformed signal peptide-antibacterial peptide fusion sequence), and lane 7 is negative control (i.e. H) ₂ O). The 800bp band appears in the lanes of the genetic transformant, and the 700bp band appears in the genetic transformant and the chlamydomonas reinhardtii receptor strain JUV of the untransformed fusion sequence, because the genome itself also has a primer amplification site to obtain a 700bp fragment when the primer is designed, thus, one 700bp band is obtained in the chlamydomonas reinhardtii receptor strain JUV of the untransformed fusion sequence, and two 700bp bands exist in the genetic transformant at the same time. And (5) confirming according to the size of the gene design sequence, and primarily confirming to obtain positive transformants. The DNA sequences of the primers used are as follows:

PSaD-P (upstream primer): gggaattggaggtacgaccgagat (see SEQ ID NO: 13);

PSaD-T (downstream primer): agctccgatcccgtatcaatcagc (see SEQ ID NO: 14).

2) Transcriptional Activity assay of antibacterial peptides and ALFPm3

The genetic transformant monoclonal algae is picked up and inoculated into 10mL TAP liquid culture medium, 22 ℃,90 mu E multiplied by m ^-2 ×s ^-1 Culturing at 100rpm to OD ₇₅₀ 0.5-0.8. RNA was extracted using RNAfast200 kit (Fei Jie Shen, shanghai, china), and specific procedures included: 2mL of the algae solution was centrifuged at 6000rpm at 4℃for 10min to collect algae cells, and 100. Mu.L of RNase-free deionized water was used for resuspension. Subsequently 500. Mu.L RA2 was added, gently mixed 5 to 10 times, allowed to stand for 1min, and then added to a centrifuge column containing a 2mL collection tube, centrifuged at 12000 Xg for 1min, and the filtrate was discarded. 500. Mu.L Wash Buffer was added to the column, and the column was centrifuged at 12000 Xg for 1min, the filtrate is discarded. After repeating the above operation once, the column centrifugation (12000 Xg, 1 min) was performed, and the collection tube was discarded. Subsequently, the column was set in a 1.5mLEP tube, dried until no liquid remained in the column, and 50. Mu.L of RNase-free water was added to the column, followed by centrifugation at 12000 Xg for 1min. After mixing 4. Mu.L of the sample and 1. Mu.L of 5×loading Buffer, the mixture was separated by electrophoresis in a 1% agarose gel (voltage 160V, time 3 min), the RNA extraction effect was checked, the RNA integrity was analyzed, and 1. Mu.L of the sample was further taken and the concentration and purity of RNA were measured under Nanodrop.

By Yeasen

III 1st Strand cDNA Synthesis SuperMix for qRCR (gDNA digester plus) reverse transcription of the extracted RNA was performed. Mu.g of total RNA was taken, 3. Mu.L of 5X gDNA digester Mix, 15. Mu.L of RNase-free water was supplemented, and incubated at 42℃for 2min. Then add 5. Mu.L +.>

III Supermix plus, 20. Mu.L reverse transcription program system was prepared, and after gentle mixing with a pipetting gun, cDNA was obtained by reaction incubation at 55deg.C for 15min and stored at-20deg.C. 20. Mu.L of real-time PCR (RT-PCR) system was prepared: mu.L of cDNA (4. Mu.L of cDNA was diluted 5-fold after storage at-20 ℃), 0.4. Mu.L of each of the upstream and downstream primers (see Table 2), 10. Mu.L of 2 XTaq Mix, and 5.2. Mu.L of deionized water were taken.

TABLE 2 secretion leader peptide and primer information for ALFPm3 transcriptional Activity analysis

The RT-PCR reaction flow of the 5 signal peptides is respectively as follows:

ARS2 and IBD-1 secretory leader: pre-denaturation at 95℃for 3min, denaturation at 95℃for 30s, annealing at 58℃for 20s, extension at 72℃for 40s, cycling 35 times, extension at 72℃for 5min, and stabilization of the product at 12 ℃.

CAH secretory leader peptide: pre-denaturation at 95℃for 3min, denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 40s, cycling 35 times, extension at 72℃for 5min, and stabilization of the product at 12 ℃.

GH-like secretion leader peptide: pre-denaturation at 95℃for 3min, denaturation at 95℃for 30s, annealing at 58℃for 30s, extension at 72℃for 40s, cycling 35 times, extension at 72℃for 5min, and stabilization of the product at 12 ℃.

P-4-Y secretory leader: pre-denaturation at 95℃for 3min, denaturation at 95℃for 30s, annealing at 61℃for 30s, extension at 72℃for 40s, cycling 35 times, extension at 72℃for 5min, and stabilization of the product at 12 ℃.

mu.L of RT-PCR reaction solution was identified by 2% agarose gel electrophoresis (voltage 160V, time 15 min), the electrophoresis result is shown in FIG. 3, wherein lane M is DL2000 DNA Marker, lanes 1-11 are genetic transformants, and lane 12 is a positive control (CAH signal peptide expression vector). A band of about 250bp was obtained after electrophoresis, which was in agreement with the expectation, demonstrating that the introduced signal peptide-ALFPm 3 had transcriptional activity in transgenic algae.

3) Immunoblot analysis of antibacterial peptides

Crude isolation of the antibacterial peptide ALFPm 3: monoclonal algae of genetic transformant is picked up and inoculated into 10mL TAP liquid culture medium, 22 ℃,90 mu E multiplied by m ^-2 ×s ^-1 Culturing at 100rpm to OD ₇₅₀ 0.5-0.8; the cultures were transferred to new 50mL TAP medium at 22℃and 90. Mu.E.times.m ^-2 ×s ^-1 Culturing at 100rpm to OD ₇₅₀ 1.5-2.0. Adjusting the temperature of the shaking table to 40 ℃, and culturing the algae cells for 20min under the condition that the illumination condition is unchanged. Then the algae cells are restored to the normal culture condition, and are cultured for 20min at 40 ℃ again after being cultured for 5h, and then are restored to the normal culture condition and are cultured for 40min. Finally, the mixture is centrifuged at 5000rpm for 5min at 4 ℃, and the obtained supernatant is a crude extract containing the antibacterial peptide ALFPm 3.

Immunoblot (Western Blot) analysis of antibacterial peptides: 400mL of the crude ALFPm3 extract was measured, packaged into 10 50mL centrifuge tubes, sealed with sealing film, and frozen at-80 ℃. The next day, the frozen samples were removed and lyophilized in a freeze dryer for 2 days. Finally, the solution was dissolved in 4mL of PBS and stored at-20 ℃. Taking 40 mu L of sample, adding 10 mu L of 5 Xprotein loading buffer solution, fully and uniformly mixing, treating in a boiling water bath for 5-10min, and cooling to room temperature. Samples were taken at 5. Mu.L each and 180. Mu. 180kDa Prestained Protein Marker were subjected to SDS-PAGE to separate proteins. In the electrophoresis process, a PVDF membrane with a proper size is cut, soaked in absolute methanol for 1min, and soaked in 1 XPVDF membrane equilibrium solution in GenScrip's eBlot kit (GenScrip, USA) for 1min for later use. And after the electrophoresis is finished, taking out the gel, and soaking the gel in deionized water for 1min. The membrane clips were stacked according to GenScrip's eBlot kit instructions, and the membrane was automatically transferred for 6min from negative to positive as a foam-gel-PVDF membrane-foam. After the transfer, the gel and PVDF membrane were separated in deionized water, and the membrane was immersed in a 3% bsa solution and blocked at room temperature for 2 hours.

An anti-incubation with 1 XTBS solution at 1:1500 and 1:2000 dilution of mouse anti-alpha-tublin anti-ibody (Sigma, B30271) and mouse anti-HA Tag anti-ibody (Biolegend, 901513); placing an NC membrane where a target protein is located in an incubation box, selecting a mouse anti-HA Tag anti-ibody for the protein containing a 3 XHA Tag, and selecting a mouse anti-alpha-tublin anti-ibody for the alpha-tubulin; placing on a slow shaker at 4 ℃ for incubation overnight; after the incubation, the primary antibody was recovered, the membrane was immersed in a1 XTBST solution, and the membrane was washed with a horizontal shaker for 10min and repeated 3 times.

Secondary antibody incubation with 1×tbs solution at 1:2000 (Beijing Boolone, BF 03001); adding the secondary antibody into an incubation box after the primary antibody is incubated and the membrane is washed for 3 times, and incubating for 2 hours by a horizontal shaking table at room temperature; after incubation, the secondary antibody was discarded, and the membrane was immersed in 1×tbst solution, washed for 10min with a horizontal shaker, and repeated 3 times.

Development (hypersensitive ECL chemiluminescence method), following developer a: the solution B is 1:1, preparing a developing solution according to the volume ratio; pre-cooling the developing instrument when the temperature inside the instrument is minus 30 ℃; dropping a proper amount of developing solution on a plate in a developing instrument, taking out an NC film to be developed in an incubation box by using tweezers, sucking 1 XTBE solution on the film by using water absorbing paper, placing the film on the plate with the developing solution dropped thereon, and turning over the NC film to enable the front side and the back side of the film to fully contact the developing solution; the results of photographing after development by using a developing instrument are shown in FIG. 4, wherein lane N is Chlamydomonas reinhardtii receptor strain JUV (untransformed signal peptide-antimicrobial peptide fusion sequence), lanes 1-11 are genetic transformants, anti-HA is the target protein, and Anti-alpha-Tubulin is the reference protein. The results showed that the protein samples of Chlamydomonas reinhardtii receptor strain JUV and all genetic transformants were able to detect specific bands of alpha-tubulin, indicating successful protein immunoblot analysis experiments. Detection with anti-3 XHA tag antibody, specific bands can be detected at 16kD in the transformant, indicating that the antibacterial peptide of the present invention is an extracellular secreted antibacterial peptide.

The above shows that the invention successfully constructs the transgenic chlamydomonas reinhardtii capable of exocrine expression of the antibacterial peptide ALFPm3, and successfully realizes the secretory expression of the antibacterial peptide.

EXAMPLE 2 antibacterial peptide expression efficiency study of transgenic Chlamydomonas reinhardtii

On an ultra-clean workbench, sucking 5mL of TAP liquid culture medium, subpackaging into 10mL centrifuge tubes, picking a little algae with a small gun head, dropping into the culture medium, and controlling the temperature at 22deg.C and 90 μE×m ^-2 ×s ^-1 Culturing for 7d under continuous illumination; the culture was used as seed solution, and the culture was inoculated into a new 1000mL TAP liquid medium to give a cell density of 1X 10 ⁴ cell/mL，100μE×m ^-2 ×s ^-1 Culturing under continuous light for 7d to cell density of 1×10 ⁷ cell/mL; centrifuging at 5000rpm for 10min at 4deg.C, and collecting supernatant and algae cell precipitate respectively. Filtering the supernatant with 0.22 μm water-based filter membrane to remove cell debris and insoluble impurities to obtain extracellular protein, and ultrafiltering and concentrating extracellular protein at 4deg.C for 30min by centrifugation at 8000 Xg with ultrafilter tube with molecular weight cut-off of 3 kD. The obtained purified protein was quantitatively detected using an anti-3×ha tag antibody, and the result is shown in fig. 5.

The results show that the extracellular expression amounts of ALFPm3 mediated by IBD-1, GH-like and P-4-Y secretory leader peptide are 52ng/mL, 65ng/mL and 48ng/mL respectively, and the expression amount is greatly improved compared with the prior art. ARS2 secretion leader fails to secrete ALFPm3 extracellular, and CAH secretion leader mediates ALFPm3 extracellular expression at 24ng/mL. Thus, the GH-like secreted antimicrobial peptide is 1.7-fold higher than CAH secreted leader peptide, IBD-1 is 1.2-fold higher than CAH, and P-4-Y is 1-fold higher than CAH.

Example 3 use of transgenic Chlamydomonas reinhardtii secretion expressed antibacterial peptides

The bacterial pathogenic bacteria include vibrio alginolyticus, vibrio harveyi and vibrio parahaemolyticus (commercially available). The culture dish is held by the left hand, a seam is opened near the flame, the LB culture medium is poured into the culture dish rapidly, the culture dish is gently shaken after the cover is added, the culture medium is uniformly distributed at the bottom of the culture dish, and then the culture dish is horizontally placed on a table top, and the culture dish is a flat plate after the culture dish is coagulated. At the position near the flame, holding the bottom of the dish with the left hand and holding the inoculating loop with the right hand, picking the fungus loop, carrying out partition scribing on a flat plate, and culturing for 12-16 hours at 37 ℃ in an inverted mode until monoclonal appears. On an ultra clean bench, several 15mL centrifuge tubes were taken, 3mL LB medium was added, and the single clone was picked from the plate into the medium and cultured overnight at 37℃at 200 rpm. The following day, the overnight cultures were removed and inoculated at 1% into 15mL centrifuge tubes containing 3mL LB medium, and incubated at 37℃for 1h at 200rpm to allow bacteria to grow to log phase.

The antibacterial peptide ALFPm3 expressed by the transgenic Chlamydomonas reinhardtii extracellular is subjected to centrifugation, precipitation removal, supernatant filtration and concentration in example 2, so as to obtain an antibacterial peptide concentrated solution which is used for antibacterial experiments. Taking a plurality of 15mL centrifuge tubes, adding 3mL of LB culture medium and 3 mu L of bacterial culture, diluting the bacterial liquid 1000 times, and taking the diluted bacterial liquid as a sample for bacteriostasis experiments. Then, 150. Mu.L of diluted bacterial solution and 50. Mu.L of the antibacterial peptide concentrate were sequentially added to a sterile 96-well plate, and 50. Mu.L of 1 XPBS solution was added to the control group so that the total volume became 200. Mu.L. After the sample is added, the absorbance (OD) of the bacterial liquid sample at 600nm is measured by an enzyme-labeled instrument ₆₀₀ ) And (5) making a light absorption value of 0 h. Finally, the OD of the culture for 12h was detected ₆₀₀ . Will take 12h OD ₆₀₀ With 0h OD ₆₀₀ Subtracting to obtain the growth rate delta OD of the strain ₆₀₀ . At DeltaOD ₆₀₀ The values are taken as ordinate, bacterial growth curves are drawn, the bacteriostasis effects of vibrio parahaemolyticus, vibrio alginolyticus and vibrio harveyi are respectively shown in figures 6-8, wherein T-GH-3 is expressed by GH-like secretory leader peptide, T-IBD-1 is expressed by IBD-1 secretory leader peptide, T-PY-1 is expressed by P-4-Y secretory leader peptide, T-CAH-5 is expressed by CAH secretory leader peptide, T-ARS-8 is expressed by ARS2 secretory leader peptide, and the negative control is Chlamydomonas reinhardtii which does not express ALFPm 3. Bacteriostasis% = (absorbance value of blank bacterial liquid-absorbance value of sample bacterial liquid)/absorbance value of blank bacterial liquid.

From FIGS. 6 to 8, it can be seen that GH-like secretory leader peptide expression ALFPm3 shows the strongest antibacterial rate reaching 96.9% after co-culture of Chlamydomonas extract with bacteria such as Vibrio parahaemolyticus. The worst is that ARS2 secretory leader expresses ALFPm3 with substantially no antibacterial activity. The bacteriostasis rate of each genetic transformant on vibrio parahaemolyticus 12h is T-GH-3>T-IBD-1>T-PY-1>T-CAH-5>T-ARS-8 sequentially, and experimental results on vibrio alginolyticus and vibrio harveyi are approximately the same, so that the activity of the AlFPm3 expressed by GH-like secretory leader peptide is highest, and the antibacterial activity is strongest.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. A chlamydomonas reinhardtii secretory leader peptide, wherein the chlamydomonas reinhardtii secretory leader peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3.

2. The chlamydomonas reinhardtii secretory leader peptide according to claim 1, wherein the chlamydomonas reinhardtii secretory leader peptide has an amino acid sequence as shown in SEQ ID NO: 2.

3. A polynucleotide encoding the chlamydomonas reinhardtii secretory leader peptide of claim 1 or 2.

4. A transgenic chlamydomonas reinhardtii comprising an expression vector comprising a DNA sequence encoding a chlamydomonas reinhardtii secretory leader peptide according to claim 1 or 2 and a DNA sequence encoding an antimicrobial peptide.

5. The transgenic chlamydomonas reinhardtii according to claim 4, wherein the DNA sequence encoding the chlamydomonas reinhardtii secretory leader peptide is selected from the group consisting of SEQ ID NO: 4-6.

6. The transgenic chlamydomonas reinhardtii of claim 4, wherein said antimicrobial peptide is a krill anti-lipopolysaccharide factor 3 having an amino acid sequence as set forth in SEQ ID NO:7, the DNA sequence is shown as SEQ ID NO: shown at 8.

7. The transgenic chlamydomonas reinhardtii of claim 4, wherein said expression vector further comprises a light-induced promoter, and wherein the DNA sequence encoding said chlamydomonas reinhardtii secretory leader peptide and the DNA sequence encoding said antimicrobial peptide are positioned downstream of said light-induced promoter in sequence.

8. A method of constructing a transgenic chlamydomonas reinhardtii, for the preparation of a transgenic chlamydomonas reinhardtii according to any one of claims 4 to 7, comprising the steps of:

s1, connecting a DNA sequence for encoding chlamydomonas reinhardtii secretory leader peptide and a DNA sequence for encoding antibacterial peptide to a starting vector to obtain an expression vector;

9. The method for constructing transgenic chlamydomonas reinhardtii according to claim 8, wherein in step S1, a DNA sequence encoding a secretory leader peptide of chlamydomonas reinhardtii and a DNA sequence encoding the antibacterial peptide are fused, a secretory leader peptide-antibacterial peptide fusion sequence is obtained by a genetic synthesis technique, and then the secretory leader peptide-antibacterial peptide fusion sequence is inserted downstream of a promoter of a restriction enzyme linearized starting vector, which is pESVH, to obtain the expression vector;

in step S2, the expression vector is transformed into the chlamydomonas reinhardtii receptor strain, which is a cell wall defect type strain JUV, by a "bead milling method".

10. Use of the transgenic chlamydomonas reinhardtii according to any one of claims 4 to 7 in the aquaculture industry.