CN111454917B - A kind of methionine sulfoxide reductase and its encoding gene, preparation method and use - Google Patents

Abstract

The invention discloses a methionine sulfoxide reductase which is selected from any one of the following (a1) - (a 3): (a1) the amino acid sequence is shown as SEQ ID NO. 1; (a2) is an amino acid sequence with at least 85 percent of homology with the amino acid sequence shown in SEQ ID NO. 1; (a3) homologues, derivatives, fragments or mutants having the amino acid sequence shown in (a) or (b) without loss of biological activity. The invention provides the sequence of the methionine sulfoxide reductase of haematococcus pluvialis for the first time, lays a foundation for the genetic engineering expression of the selenoprotein MsrA, and is favorable for realizing the large-scale industrial production of the methionine sulfoxide reductase with high catalytic activity. The invention also discloses a gene, a recombinant expression vector, a recombinant cell line or a recombinant strain for coding the methionine sulfoxide reductase, which is favorable for realizing the in-vitro genetic engineering expression of MsrA and reduces the acquisition cost of the MsrA.

Description

A kind of methionine sulfoxide reductase and its encoding gene, preparation method and use

technical field

The invention relates to the technical field of genetic engineering, in particular to a methionine sulfoxide reductase, a methionine sulfoxide reductase gene, a recombinant expression vector, a recombinant cell line or a recombinant strain and use thereof, and a cDNA sequence of the methionine sulfoxide reductase gene. Preparation method, preparation method of methionine sulfoxide reductase, detection primer and detection probe of methionine sulfoxide reductase, chip or kit.

Background technique

Oxygen in the organism can generate reactive oxygen compounds (ROS, Reactive Oxygen Species) after incomplete reduction during normal metabolism, mainly including superoxide anion free radicals, hydrogen peroxide, peroxynitrite anion, hypochlorous acid, singlet oxygen and hydroxyl radicals, etc. ROS can interact with a series of biomolecules, causing oxidative damage or affecting many signal transduction processes, regulating cell proliferation, differentiation and apoptosis. The occurrence, development and treatment of diseases such as ALS and ALS are closely related.

The main targets of ROS formation after cells are stressed are two sulfur-containing amino acids, cysteine (Cys) and methionine (Met). Cys and Met are very sensitive to reactive oxygen species, and their sulfur atoms are easily oxidized. The oxidation of Met leads to the formation of methionine sulfoxide (MetO) in proteins. Met is a common starting amino acid in protein synthesis. The production of MetO may lead to changes in protein structure and abnormal catalytic function, affecting the normal function of cells. To counteract ROS damage, organisms have evolved a variety of ROS scavenging and repair mechanisms, including low molecular weight compounds and antioxidant enzymes that combat oxidative stress. Methionine sulfoxide reductase (Msrs) is currently the only identified reductase characteristic of methionine sulfoxide residues in proteins, which can reduce methionine sulfoxide (MetO) to methionine (Met), regulate protein functions, and regulate related signaling pathways. , Repair oxidative damage protein, prevent oxidative stress and so on. At present, studies have shown that Msrs play a protective role in the physiological and pathological processes related to aging, neurodegenerative diseases, and cataracts.

Methionine sulfoxide reductase (Msrs) widely exists in most prokaryotic and eukaryotic organisms such as bacteria and humans. The most studied Msrs that have been found in organisms mainly include methionine sulfoxide reductase A (MsrA) and methionine sulfoxide. Reductase B (MsrB) two. MsrA can specifically reduce proteins and free methionine-S-sulfoxide, while MsrB can only reduce methionine-R-sulfoxide in proteins. The conserved catalytic site of MsrA contains a selenocysteine residue (Sec or U) is a selenoprotein MsrA. Compared with a conserved site of cysteine (Cys), the selenoprotein MsrA does not contain selenium. The catalytic activity of 10-50 times was improved, indicating that selenocysteine residues (Sec or U) improved the catalytic activity of redox. Currently, selenium-containing forms of MsrA are found in bacteria, algae, and invertebrates.

Obtaining the selenoprotein MsrA with high catalytic activity is of great significance for the development and utilization of antioxidants and Msrs-related drugs. Due to the extremely low content of selenoprotein MsrA in animals and plants, a large amount of MsrA extracted from animals and plants is not only costly, but also destroys ecological resources. Therefore, how to obtain a large amount of expressed selenoprotein MsrA is an important problem to be solved at this stage.

SUMMARY OF THE INVENTION

Therefore, the technical problem to be solved by the present invention is to overcome the high extraction cost of the selenoprotein MsrA in the prior art and the inability to realize its mass production and expression, thereby providing a methionine sulfoxide reductase that can be used for genetic engineering expression. .

For this reason, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a methionine sulfoxide reductase, the methionine sulfoxide reductase is selected from any one of the following (a1)-(a3):

(a1) Its amino acid sequence is shown in SEQ ID NO.1;

(a2) is an amino acid sequence with at least 85% homology with the amino acid sequence shown in SEQ ID NO.1;

(a3) A homolog, derivative, fragment or mutant having the amino acid sequence shown in (a) or (b) that does not lose biological activity.

In a second aspect, the present invention provides a methionine sulfoxide reductase gene, the methionine sulfoxide reductase gene encoding the methionine sulfoxide reductase according to claim 1.

Optionally, the above-mentioned methionine sulfoxide reductase gene, the methionine sulfoxide reductase gene is selected from any one shown in the following (b1)-(b2):

(b1) Its cDNA sequence is shown in SEQ ID NO.2;

(b2) is a nucleotide sequence having at least 85% homology with the nucleotide sequence shown in SEQ ID NO. 2.

In a third aspect, the present invention provides a recombinant expression vector, a recombinant cell line or a recombinant strain comprising the above-mentioned methionine sulfoxide reductase gene.

In a fourth aspect, the present invention provides a medicine comprising the above-mentioned methionine sulfoxide reductase or the above-mentioned methionine sulfoxide reductase gene.

In a fifth aspect, the present invention provides the above-mentioned methionine sulfoxide reductase, methionine sulfoxide reductase gene, or the recombinant expression vector, recombinant cell line or recombinant strain of the methionine sulfoxide reductase gene in the following (C1)-(C3) Use of at least one of:

(C1) reducing methionine sulfoxide, or preparing a product for reducing methionine sulfoxide;

(C2) as an antioxidant, or to prepare a product for anti-oxidation;

(C3) preparing a drug for treating Msrs-related diseases.

In a sixth aspect, the present invention provides a method for preparing the above-mentioned methionine sulfoxide reductase, comprising:

S1, construct and express the recombinant expression vector of methionine sulfoxide reductase as claimed in claim 1;

S2, transforming the target strain with the recombinant expression vector to obtain a recombinant strain;

S3, fermenting and culturing the recombinant strain to induce protein expression;

S4, separate and purify the protein expressed by the recombinant strain to obtain methionine sulfoxide reductase.

In a seventh aspect, the present invention provides a method for preparing the cDNA sequence of the above-mentioned methionine sulfoxide reductase gene, characterized in that it comprises the following steps:

(1) Extract the total RNA of Haematococcus pluvialis and reverse-transcribe it into cDNA;

(2) Amplify the cDNA of Haematococcus pluvialis as a template to obtain a cDNA fragment of the methionine sulfoxide reductase gene;

(3) performing 5'-RACE amplification and 3'-RACE amplification on the cDNA fragment of the methionine sulfoxide reductase gene to obtain a 5'-RACE amplified fragment and a 3'-RACE amplified fragment;

(4) After sequencing the 5'-RACE amplified fragment and the 3'-RACE amplified fragment, sequence splicing is performed to obtain the cDNA sequence of the methionine sulfoxide reductase gene;

Preferably, the amplification primers for the cDNA fragment of the methionine sulfoxide reductase gene include: HpMsrA-F, whose nucleotide sequence is shown in SEQ ID NO. 3; HpMsrA-R, whose nucleotide sequence is shown in SEQ ID NO.4 shows;

Preferably, the amplification primers of the 5'-RACE amplified fragment include: GSP5-1, whose nucleotide sequence is shown in SEQ ID NO.5; GSP5-2, whose nucleotide sequence is shown in SEQ ID NO.6 shown; GSP5-3, its nucleotide sequence is shown in SEQ ID NO.7;

Preferably, the amplification primers of the 3'-RACE amplified fragment include: GSP3-1, whose nucleotide sequence is shown in SEQ ID NO.8; GSP3-2, whose nucleotide sequence is shown in SEQ ID NO.9 shown; GSP3-3, the nucleotide sequence of which is shown in SEQ ID NO.10.

In an eighth aspect, the present invention provides a detection primer for methionine sulfoxide reductase, the detection primer is designed based on the methionine sulfoxide reductase gene of claim 2 or 3;

Preferably, the detection primers include: HpMsrA-qF, whose nucleotide sequence is shown in SEQ ID NO.11; HpMsrA-qR, whose nucleotide sequence is shown in SEQ ID NO.12.

In a ninth aspect, the present invention provides a probe, chip or kit for detecting the above-mentioned methionine sulfoxide reductase or methionine sulfoxide reductase gene.

The technical scheme of the present invention has the following advantages:

1. The methionine sulfoxide reductase provided by the present invention is selected from any one of the following (a1)-(a3): (a1) its amino acid sequence is shown in SEQ ID NO.1; (a2) is the same as SEQ ID NO.1; The amino acid sequence shown in NO.1 has at least 85% homology; (a3) The amino acid sequence shown in (a) or (b) has a homologue, derivative, fragment or mutation that does not lose biological activity body.

The methionine sulfoxide reductase with the amino acid sequence shown in SEQ ID NO.1 is derived from Haematococcus pluvialis, which is a single-cell freshwater microalgae, and is a single-celled algae with strong resistance to stress. biology. Haematococcus pluvialis accumulates a high content of astaxanthin in the cells of Haematococcus pluvialis under the stress of high temperature, high light and other environmental conditions. Aging and so on are important. The present invention provides the sequence of the methionine sulfoxide reductase of Haematococcus pluvialis for the first time, and the research finds that there is a highly conserved GUFW catalytic center in the amino acid sequence of the methionine sulfoxide reductase, which can significantly improve the redox catalytic activity. The selenocysteine residue (U) shows that the methionine sulfoxide reductase with the amino acid sequence shown in SEQ ID NO.1 is a selenoprotein MsrA with high catalytic activity. By providing the amino acid sequence of the methionine sulfoxide reductase derived from Haematococcus pluvialis, the present invention lays a foundation for the genetic engineering expression of the selenoprotein MsrA, and is beneficial to realizing large-scale industrial production of the methionine sulfoxide reductase with high catalytic activity. Production reduces the cost of obtaining methionine sulfoxide reductase, thereby providing conditions for the development and utilization of therapeutic drugs for Msrs-related diseases such as high-efficiency antioxidants, neurodegenerative diseases, and cataracts.

2. The methionine sulfoxide reductase gene provided by the present invention encodes the above-mentioned methionine sulfoxide reductase, and the methionine sulfoxide reductase gene is selected from any of the following (b1)-(b2): (b1) Its cDNA The sequence is shown in SEQ ID NO.2; (b2) is a nucleotide sequence with at least 85% homology with the nucleotide sequence shown in SEQ ID NO.2. The use of the above nucleotide sequence is beneficial to construct an in vitro expression vector of methionine sulfoxide reductase, and to screen recombinant strains that highly express methionine sulfoxide reductase, so as to achieve high in vitro expression of methionine sulfoxide reductase and reduce methionine sulfoxide reductase. production cost.

3. The recombinant expression vector comprising the methionine sulfoxide reductase gene provided by the present invention is suitable for screening recombinant cell lines or recombinant strains that stably express high methionine sulfoxide reductase, and can achieve methionine sulfoxide reduction through the fermentation culture of the recombinant strains Large-scale in vitro expression and production of enzymes.

4. The methionine sulfoxide reductase provided by the present invention can achieve antioxidative effect by reducing methionine sulfoxide and repairing oxidatively damaged proteins. Therefore, methionine sulfoxide reductase can act as an antioxidant or prepare a product for antioxidant use. In addition, studies have shown that Msrs protein plays a role in the occurrence and development of neurodegenerative diseases and other related diseases, and the methionine sulfoxide reductase provided by the present invention provides conditions for the development of therapeutic drugs for Msrs related diseases.

5. The preparation method of methionine sulfoxide reductase provided by the present invention adopts genetic engineering method to realize the in vitro expression of methionine sulfoxide reductase, reduces the production cost of methionine sulfoxide reductase, and is beneficial to the realization of methionine sulfoxide reductase Large-scale industrial mass production.

6. The detection primers, probes, chips or kits for methionine sulfoxide reductase provided by the present invention can be used to detect the expression level of methionine sulfoxide reductase. , heavy metal cadmium, selenium, etc.), the expression of methionine sulfoxide reductase in Haematococcus pluvialis changes significantly. Therefore, by detecting the expression of methionine sulfoxide reductase in Haematococcus pluvialis in water, it is possible to achieve The monitoring and control of unfavorable factors in the environment is conducive to the management and control of unfavorable factors in the environment.

Description of drawings

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

Fig. 1 is the cDNA sequence of Haematococcus pluvialis methionine sulfoxide reductase gene and its corresponding amino acid sequence diagram provided in Example 1 of the present invention;

Fig. 2 is the SECIS element structure diagram of Haematococcus pluvialis methionine sulfoxide reductase gene provided in Example 1 of the present invention;

Fig. 3 is the sequence alignment result of the MsrA sequence of Haematococcus pluvialis provided by the embodiment of the present invention and the methionine sulfoxide reductase of multispecies;

Fig. 4 is the expression change situation of the methionine sulfoxide reductase gene of Haematococcus pluvialis provided by Experimental Example 1 of the present invention under the influence of different environmental stress factors;

Figure 5 shows the changes in expression and enzyme activity of the methionine sulfoxide reductase gene of Haematococcus pluvialis provided in Experimental Example 2 of the present invention under the influence of different concentrations of selenite.

Detailed ways

The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

The experimental reagents used in the following examples are all conventional experimental reagents, which can be directly obtained from the market;

Example 1

The present embodiment provides a method for cloning Haematococcus pluvialis methionine sulfoxide reductase gene, which specifically includes the following steps:

(1) Extract the total RNA of Haematococcus pluvialis, and reverse-transcribe the total RNA into cDNA using the SMARTer® PCR cDNA Synthesis Kit (Clontech, USA).

(2) The cDNA of Haematococcus pluvialis was used as the template, and the amplification primers HpMsrA-F and HpMsrA-R were used for amplification to obtain the cDNA amplified fragment of the methionine sulfoxide reductase gene in Haematococcus pluvialis. The nucleotide sequence of HpMsrA-F is shown in SEQ ID NO.3, and the nucleotide sequence of HpMsrA-R is shown in SEQ ID NO.4. PCR amplification conditions were: 95°C for 2 min; (95°C for 30 s, 58°C for 45 s, 72°C for 1 min, 35 cycles); 72°C for 10 min.

(3) 5'-RACE amplification and 3'-RACE amplification are performed on the cDNA fragment in step (2), wherein the primers used for 5'-RACE amplification include: GSP5-1, the nucleotide sequence of which is as follows: SEQ ID NO.5; GSP5-2, whose nucleotide sequence is shown in SEQ ID NO.6; GSP5-3, whose nucleotide sequence is shown in SEQ ID NO.7; 3'-RACE amplification using The primers include: GSP3-1, whose nucleotide sequence is shown in SEQ ID NO.8; GSP3-2, whose nucleotide sequence is shown in SEQ ID NO.9; GSP3-3, whose nucleotide sequence is shown in SEQ ID NO.9 Shown in SEQ ID NO.10. After 5'-RACE amplification and 3'-RACE amplification, 5'-RACE amplified fragments and 3'-RACE amplified fragments were obtained. PCR amplification conditions were: 94°C for 30 s, 68°C for 30 s, 72°C for 3 min, 25 cycles.

(4) After sequencing the 5'-RACE amplified fragment and the 3'-RACE amplified fragment in step (3), perform sequence splicing to obtain the cDNA sequence of the methionine sulfoxide reductase gene, whose cDNA sequence is as shown in SEQ ID NO. .2, the corresponding amino acid sequence of methionine sulfoxide reductase is shown in SEQ ID NO.1.

Figure 1 shows the cDNA sequence of Haematococcus pluvialis methionine sulfoxide reductase gene and its corresponding amino acid sequence, the codon U in the figure represents the coding site of selenocysteine, and the amino acid sequence can be found in the methionine sulfoxide Reductase has a conserved catalytic center of GUFW. The underlined open reading frame region in the amino acid sequence corresponds to the peptide methionine sulfoxide reductase (PSMR) family, and the underlined in the cDNA sequence is the SElenoCysteine Insertion Sequence (SECIS). Figure 2 shows the secondary structure of the SECIS element. It can be seen from the figure that the SECIS element of the methionine sulfoxide reductase gene has conserved structures such as the iconic top loop and AG/GA quartet. In the presence of the SECIS element, the methionine sulfoxide UGA in the mRNA of the reductase gene can be used as the codon of selenocysteine to encode selenocysteine, which makes the methionine sulfoxide reductase of Haematococcus pluvialis as a selenium with high catalytic activity. Protein MsrA. The identity of MsrA of Haematococcus pluvialis with the homologous gene protein sequence of Volvox algae is 65.5%, and it has a significant selenoprotein gene structure.

Figure 3 shows the results of the sequence alignment of the MsrA sequence of Haematococcus pluvialis with the methionine sulfoxide reductases of the following species: B.floridae, N.vectensis_b, T.adhaerens, D.vulgaris, H.pluvialis, M.commoda , D.rerio, H.sapiens, E.coli, S.cerevisiae, A.thaliana . Dark shading indicates the complete consensus sequence, light shading indicates the conserved substitution sequence, and the highly conserved GUFW motif is the catalytic center of the MsrA gene with U selenocysteine in it.

In this example, the full-length cDNA sequence of the methionine sulfoxide reductase gene of Haematococcus pluvialis was obtained for the first time, and the sequence of the methionine sulfoxide reductase of Haematococcus pluvialis was provided. Through sequence analysis, it was found that there is a highly conserved catalytic center of GUFW in the amino acid sequence of methionine sulfoxide reductase, which has a selenocysteine residue (U) that can significantly improve the redox catalytic activity, indicating that it has SEQ ID NO. The methionine sulfoxide reductase with the amino acid sequence shown in .1 is a selenoprotein MsrA with high catalytic activity. By providing the amino acid sequence of the methionine sulfoxide reductase derived from Haematococcus pluvialis, the present invention lays a foundation for the genetic engineering expression of the selenoprotein MsrA, and is beneficial to realizing large-scale industrial production of the methionine sulfoxide reductase with high catalytic activity. Production reduces the cost of obtaining methionine sulfoxide reductase, thereby providing conditions for the development and utilization of therapeutic drugs for Msrs-related diseases such as high-efficiency antioxidants, neurodegenerative diseases, and cataracts.

Example 2

The present embodiment provides a method for preparing methionine sulfoxide reductase, which specifically includes the following steps:

S1, construct a recombinant expression vector expressing the methionine sulfoxide reductase of the amino acid sequence shown in SEQ ID NO.1, specifically:

The restriction enzymes of XbaI and XhoI were introduced into both ends of the cDNA fragment of the methionine sulfoxide reductase gene, and pET-28a (purchased from Solarbio) was used as the vector. The gene fragment of MsrA was digested with enzyme, and the digested fragment was recovered by gel to obtain the linear plasmid fragment of 5'XbaI-pET-28a-3'XhoI and the gene fragment of 5'XbaI-MsrA-3'XhoI.

5'XbaI-pET-28a-3'XhoI and 5'XbaI-MsrA-3'XhoI recovered by digestion were added to 10 μL of T4 ligase system, and ligated overnight at 16°C. The DH5a competent cells were transformed with the ligation product, screened after plating, and positive clones were screened by bacterial liquid PCR to obtain the recombinant expression vector of MsrA, which was named pET-28a-MsrA.

S2, transforming the target strain with the recombinant expression vector to obtain a recombinant strain; specifically: transforming BL21 competent cells with the recombinant expression vector pET-28a-MsrA, and screening the recombinant transformants to obtain a recombinant strain containing the MsrA gene.

S3, fermenting and culturing the recombinant strain to induce protein expression; specifically: inoculating the transformed BL21 competent cells into LB medium, shaking at 37°C, fermenting and culturing the recombinant strain, and using IPTG to induce The recombinant strain expresses the protein, and determines the suitable IPTG addition amount and protein induction conditions with high protein expression.

S4, separate and purify the protein expressed by the recombinant strain to obtain methionine sulfoxide reductase. Specifically: crushing the recombinant strain after inducing protein expression in step S3, isolating the protein expressed in the strain, and purifying the methionine sulfoxide reductase expressed in the recombinant strain by column chromatography affinity chromatography to obtain the target protein.

Example 3

This embodiment provides a recombinant expression vector and a recombinant strain comprising a methionine sulfoxide reductase gene, wherein the methionine sulfoxide reductase gene is specifically a cDNA fragment having the nucleotide sequence shown in SEQ ID NO. 2.

Specifically, the recombinant expression vector is the recombinant expression vector pET-28a-MsrA constructed in Example 2 with pET-28a as the vector and XbaI and XhoI as the cloning sites; the recombinant strain is the recombinant expression vector pET-28a- The recombinant transformants obtained by transforming MsrA into BL21 competent cells.

The recombinant expression vector pET-28a-MsrA and the recombinant strain provided in this example can be used for in vitro induced expression of methionine sulfoxide reductase, so as to reduce the production cost of methionine sulfoxide reductase and realize large-scale industrial production.

Example 4

This example provides an antioxidant, comprising the methionine sulfoxide reductase purified in Example 2. In Example 2, methionine sulfoxide reductase has the amino acid sequence shown in SEQ ID NO. 1, and is a selenoprotein MsrA with GUFW as the active center. Oxidative, can act as an efficient antioxidant.

Experimental example 1

1. Experimental purpose: To detect and monitor the effect of environmental stress factors on the expression level of methionine sulfoxide reductase gene in Haematococcus pluvialis.

2. Experimental method:

1) Haematococcus cells were treated with H ₂ O ₂ , strong light, cadmium nitrite and glyphosate-based herbicides as representatives. Three concentrations of treatments were designed for each factor, including: hydrogen peroxide H ₂ O ₂ (20 , 40, 80 mg/L), high light (80, 150, 350 mol photons m ^-2 s ^-1 ), glyphosate (2, 4, 8 mg/L) and cadmium nitrite (3, 6, 12 mg /L) is designed to handle groups. Each treatment was replicated three times, and expression levels were sampled and recorded 3, 6, and 12 hours after exposure treatment.

2) Design qRT-PCR primers according to Haematococcus pluvialis MsrA gene sequence, and obtain the following detection primers: HpMsrA-qF, whose nucleotide sequence is shown in SEQ ID NO.11; HpMsrA-qR, whose nucleotide sequence is shown in Shown in SEQ ID NO.12. Using the above-mentioned detection primers, the samples from the treatment group and the control group of Haematococcus pluvialis cells in exponential growth phase (with a density of about 3 × 10 ⁵ cells per ml) were analyzed by fluorescence quantitative PCR. The protein was actin, and the primers for detecting the expression of actin protein were actin-qF: 5'-AGCGGGAGATAGTGCGGGACA-3', actin-qR: 5'-ATGCCCACCGCCTCAATGC-3'. The qRT-PCR amplification conditions were: 95°C for 30 s; 95°C for 10 s, 60°C for 30 s, 40 cycles.

3. Experimental results

The experimental results are shown in Figure 4, and the details are as follows:

Figure 4A shows the detection results of the expression level of MsrA after exposure to H ₂ O ₂ for different time periods. It can be seen from Figure 4A that after exposure to H ₂ O ₂ for 3 hours, there was no significant change in all treatment groups and the control group, and with the prolongation of treatment time , compared with the corresponding control group, all 40 and 80 mg/L treatment groups reached significant levels, while the expression of HpMsrA increased to the highest level after 12 hours of treatment (1.8-fold and 3.4-fold higher than that of the control group, respectively, P < 0.01 ), while the 20 mg/LH ₂ O ₂ group was consistent with the control group.

Figure 4B shows the detection results of ^MsrA expression levels as a function of time under different light intensities. The ^algal cells are sensitive to the increased light intensities, because the high light treatment may have Induced intracellular ROS accumulation, HpMsrA transcript levels were significantly up-regulated under light intensities ranging from 80 to 350 mol photons m ^-2 s ^-1 in the range of 3 h to 6 h. Return to normal by 12h, and all highlight groups maintain the same level.

Figure 4C shows the detection results of the expression level of MsrA as a function of time after exposure to different concentrations of glyphosate. As a broad-spectrum systemic herbicide, glyphosate may interfere with the synthesis of aromatic amino acids. The expression of HpMsrA showed an increasing pattern with time and was not affected by the dose change. The transcript increased slightly compared with the control 3 hours after treatment, but there was no significant difference. Glyphosate continued to promote the expression of MsrA from 6 hours to 12 hours. The expression of HpMsrA reached a peak at 12 hours after 8 mg/L glyphosate treatment, which was more than 9 times that of the control.

Figure 4D shows the detection results of the expression level of MsrA over time after exposure to different concentrations of Cd ²⁺ . Cadmium (Cd), as a harmful metal that pollutes the aquatic environment, has a dose-dependent effect on the expression of HpMsrA. In each treatment group, transcripts were stably expressed over different time periods. There was no significant difference between the control group and the 3 mg/L cadmium nitrate-treated group, and the transcript levels at 6 and 12 hours were twice that of the control group. 6 mg/L cadmium nitrate treatment could rapidly increase the transcript expression and reached the highest level at 6 h, but the 12 mg/L group was always lower than the 6 mg/L group.

Experimental example 2

1. Experimental purpose: To detect the changes of methionine sulfoxide reductase gene expression level and enzyme activity in Haematococcus pluvialis under selenium treatment.

2. Experimental method

1) Haematococcus pluvialis was exposed to different concentrations of selenite (0, 3, 13, 23 mg/mL) and detected by real-time quantitative PCR after 3, 6 and 12 hours of selenite exposure mRNA transcript levels of MsrA gene in Haematococcus pluvialis.

2) A specific colorimetric method was used to monitor the activity of MsrA after sodium selenite treatment. In the MsrA reaction system, the oxidation of the dithiol compound occurs simultaneously with the reduction of methyl sulfoxide to methyl sulfide, so it can be obtained by The catalytic activity of MsrA was monitored by reducing the amount of dithiol substrate. The reaction is based on Ellman's reagent (dithiobisnitrobenzoic acid, DTNB) reacting with dithiothreitol (DTT) and producing a color that can be detected spectrophotometrically at the OD412 wavelength (Wu et al., 2013). Enzyme activity was studied at equal time intervals.

3. Experimental results

The test results are shown in Figure 5:

The left panel of Figure 5 shows the time-dependent results of Haematococcus pluvialis methionine sulfoxide reductase gene expression exposed to different concentrations of selenite. The detection results showed that Haematococcus cells were highly sensitive to selenite, and the expression of HpMsrA mRNA was significantly higher than that of the control (10-40 times). Expression levels in each treatment group increased over time, reaching a peak after 12 hours of treatment with 13 mg/L selenite. The results showed that no significant differences were observed in the control group.

The right panel of Figure 5 shows the time-dependent results of Haematococcus pluvialis methionine sulfoxide reductase activity exposed to different concentrations of selenite. In the presence of selenite, the enzymatic activity was significantly increased, with a more than 1.8-fold increase compared to the control group. Furthermore, it is worth noting that the HpMsrA enzyme activity level increased approximately 7-fold in the 23 mg/L group after 6 hours of exposure, followed by a decrease after 12 hours.

Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. And the obvious changes or changes derived from this are still within the protection scope of the present invention.

SEQUENCE LISTING

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<400> 5

cgtcattcgc gtttgcgg 18

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (GSP5-2)

<400> 6

cgagttgcgg tcctggcaca 20

<210> 7

<211> 19

<212> DNA

<213> Artificial sequences (GSP5-3)

<400> 7

acgaccgcag atgtggagc 19

<210> 8

<211> 18

<212> DNA

<213> Artificial sequence (GSP3-1)

<400> 8

ggaagagccg tgatgtgc 18

<210> 9

<211> 18

<212> DNA

<213> Artificial sequences (GSP3-2)

<400> 9

acagtgccat cacaagac 18

<210> 10

<211> 18

<212> DNA

<213> Artificial sequences (GSP3-3)

<400> 10

tttgccgttt ggctgctt 18

<210> 11

<211> 17

<212> DNA

<213> Artificial sequence (HpMsrA-qF)

<400> 11

gctcggctga ttttggc 17

<210> 12

<211> 21

<212> DNA

<213> Artificial sequence (HpMsrA-qR)

<400> 12

tgtaggtgac gacatctggg t 21

Claims (8)

1. A methionine sulfoxide reductase, wherein the amino acid sequence of the methionine sulfoxide reductase is shown in SEQ ID NO.1. 2 . A methionine sulfoxide reductase gene, wherein the methionine sulfoxide reductase gene encodes the methionine sulfoxide reductase according to claim 1 . 3 . The methionine sulfoxide reductase gene according to claim 2 , wherein the cDNA sequence of the methionine sulfoxide reductase gene is shown in SEQ ID NO. 2. 4 . 4. A recombinant expression vector, recombinant cell line or recombinant strain comprising the methionine sulfoxide reductase gene of claim 2 or 3. 5. The methionine sulfoxide reductase of claim 1, the methionine sulfoxide reductase gene of claim 2 or 3, or the recombinant expression vector and recombinant cell line of the methionine sulfoxide reductase gene of claim 4 Or use of recombinant strains in at least one of the following (C1)-(C2): (C1) preparing a product for reducing methionine sulfoxide; (C2) Preparation of products for anti-oxidation. 6. a preparation method of methionine sulfoxide reductase as claimed in claim 1, is characterized in that, comprising: S1, construct and express the recombinant expression vector of methionine sulfoxide reductase as claimed in claim 1; S2, transforming the target strain with the recombinant expression vector to obtain a recombinant strain; S3, fermenting and culturing the recombinant strain to induce protein expression; S4, separate and purify the protein expressed by the recombinant strain to obtain methionine sulfoxide reductase. 7. a method for preparing the cDNA sequence of the described methionine sulfoxide reductase gene of claim 3, is characterized in that, comprises the steps: (1) Extract the total RNA of Haematococcus pluvialis and reverse-transcribe it into cDNA; The amplification primers of the cDNA fragment of the methionine sulfoxide reductase gene include: HpMsrA-F, whose nucleotide sequence is shown in SEQ ID NO.3; HpMsrA-R, whose nucleotide sequence is shown in SEQ ID NO.4 shown; (2) Amplify the cDNA of Haematococcus pluvialis as a template to obtain a cDNA fragment of the methionine sulfoxide reductase gene; (3) performing 5'-RACE amplification and 3'-RACE amplification on the cDNA fragment of the methionine sulfoxide reductase gene to obtain a 5'-RACE amplified fragment and a 3'-RACE amplified fragment; The amplification primers of the 5'-RACE amplified fragment include: GSP5-1, whose nucleotide sequence is shown in SEQ ID NO.5; GSP5-2, whose nucleotide sequence is shown in SEQ ID NO.6 ; GSP5-3, its nucleotide sequence is as shown in SEQ ID NO.7; The amplification primers of the 3'-RACE amplified fragment include: GSP3-1, whose nucleotide sequence is shown in SEQ ID NO.8; GSP3-2, whose nucleotide sequence is shown in SEQ ID NO.9 ; GSP3-3, its nucleotide sequence is as shown in SEQ ID NO.10; (4) After sequencing the 5'-RACE amplified fragment and the 3'-RACE amplified fragment, sequence splicing is performed to obtain the cDNA sequence of the methionine sulfoxide reductase gene. 8. A detection primer for methionine sulfoxide reductase, wherein the detection primer is designed based on the methionine sulfoxide reductase gene of claim 2 or 3; The detection primers include: HpMsrA-qF, whose nucleotide sequence is shown in SEQ ID NO.11; HpMsrA-qR, whose nucleotide sequence is shown in SEQ ID NO.12.

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