Skin mucus protein molecular marker for accurately quantifying alkalinity stress level of fish and detection method of skin mucus protein molecular marker

Abstract

The invention discloses a skin mucus protein molecular marker for accurately quantifying the fish alkalinity stress level and a detection method thereof, and belongs to the technical field of fish alkali resistance. The invention aims to solve the problem that the alkali-resistant breeding efficiency of fishes is influenced due to the lack of protein molecular markers for fish alkalinity stress level at present. The invention provides a marker for identifying alkali-resistant cyprinid fishes. The amino acid sequence of the marker is shown as SEQ ID NO. 1. Important support is provided for alkali-resistant genetic breeding of fishes such as grass carps, crucian carps and leuciscus waleckii, and fishery development and utilization in medium and high saline-alkali water areas are further promoted.

Description

Technical Field

[0001] The present invention belongs to the technical field of fish alkali resistance, and specifically relates to a skin mucus protein molecular marker for accurately quantifying the alkalinity stress level of fish and a detection method thereof. Background Art

[0002] Alkali salts (NaHCO3 and Na2CO3) are more destructive to aquatic organisms than neutral salts (NaCl and Na2SO4). High alkalinity can induce respiratory and metabolic alkalosis in freshwater fish, seriously affecting the growth, survival and reproduction of fish. Therefore, of the approximately 690 million mu of low-lying saline-alkali water resources in my country, less than 2% are currently used. Low-salinity-alkali waters with alkalinity below 10mmol / L are developed and utilized by transplanting and domesticating conventional freshwater and seawater aquatic economic animals. Most of the medium and high saline-alkali waters with alkalinity >10mmol / L are still in a state of wasteland and idleness, and development and utilization are imminent. To solve the above problems, improving the salt-alkali tolerance of aquaculture species is the key to achieving efficient utilization of these resources. Accurate evaluation of alkaline resistance traits, especially by mining biomarkers to indicate the response process of fish to alkaline environments, will provide important support for the genetic breeding of grass carp, crucian carp, and walleye fish, and thus promote the development and utilization of fisheries in medium and high saline-alkali waters.

[0003] In order to achieve this goal, accurate evaluation of alkali resistance traits has become an important basis for genetic breeding. In the field of quantitative genetics, alkali resistance traits are usually regarded as typical threshold traits with discontinuous distribution. Traditionally, lethal experiments are often used to evaluate the degree of alkali resistance. However, lethal experiments cannot determine the long-term adaptation process of fish to alkaline environment during breeding, nor can they be directly used as indicators for the selection of fish alkali-resistant varieties. Therefore, the discovery of biomarkers that can indicate the response process to alkaline environment is of great significance for more accurate evaluation of alkali resistance traits. Summary of the invention

[0004] The purpose of the present invention is to provide a method for assisting in the identification of fish alkalinity stress level and its detection using α-globin fragments as markers, so as to solve the problem of the current lack of protein molecular markers of fish alkalinity stress level, thereby affecting the efficiency of fish alkali-resistant breeding and selection.

[0005] The invention provides an application of alpha-globin as a marker for identifying the alkali resistance of carp fish.

[0006] It is further defined that the carp fish are Carassius auratus from Dali Lake, Inner Mongolia and Carassius auratus from Pingxiang, Jiangxi; and the sample to be tested is mucus on the surface of fish bodies.

[0007] The invention provides an application of alpha-globin as a marker species for identifying alkali-resistant carp fish.

[0008] It is further defined that the carp fish are Carassius auratus from Dali Lake, Inner Mongolia and Carassius auratus from Pingxiang, Jiangxi, and the sample to be tested is mucus on the surface of the fish body.

[0009] It is further defined that the amino acid sequence of the α-globin is as shown in SEQ ID NO.1.

[0010] Further defined, the alkalinity performance is divided into a non-alkali resistance level, a medium alkali resistance level and a high alkali resistance level.

[0011] The invention provides application of a gene encoding an alpha-globin fragment as a marker for identifying the alkali resistance of carp fish.

[0012] It is further defined that the gene encoding α-globin is as shown in SEQ ID NO.2.

[0013] The present invention provides a method for identifying the alkali resistance of carp fish, and the specific steps of the identification method are as follows:

[0014] Step 1: Prepare 50mmol / L carbonate solution;

[0015] Step 2: Stress the carp fish in a carbonate solution for several days and scrape the mucus on the surface of the fish body;

[0016] Step 3: Detect the level of α-globin in the fish surface mucus using mass spectrometry;

[0017] Step 4: Identify the level of alkali resistance of carp fish through the level of α-globin; the expression level of α-globin in low alkali resistance level fish is <5000, the expression level of α-globin in medium alkali resistance level fish is 53568.39±9888.30, and the expression level of α-globin in high alkali resistance level fish is 72914.42±22301.86.

[0018] It is further defined that the carbonate solution in step 1 is a NaHCO3 solution.

[0019] Beneficial effect: The present invention utilizes an ultra-high performance liquid chromatography system in combination with a mass spectrometer to use label free quantification (LFQ) in a data-independent acquisition (DIA) mode to collect mass spectrometry data in a wide dynamic range using a data-independent scanning mode (DIA) to compare the α-globin obtained by the following peptide segments for component qualitative and precise quantification. The expression level of α-globin is used as a protein molecular marker to measure the alkalinity stress level of fish. Under the same intensity of alkalinity stress, the higher the expression level of α-globin, the stronger its alkali resistance.

[0020] In the breeding process of alkali-resistant fish varieties, under alkaline water stress conditions, the expression of α-globin can be measured by sampling the skin mucus of breeding materials, which will enable accurate evaluation of the fish's alkali resistance. Applied in breeding practice, it will greatly improve the selection efficiency of new alkali-resistant fish varieties during the breeding process and significantly shorten the selection cycle of new alkali-resistant fish varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the cumulative distribution function diagram of α-globin expression in skin mucus of fish with different alkali resistance. DETAILED DESCRIPTION

[0022] 1. Reagents

[0023] NaHCO3 (analytical grade, Tianjin Kaitong Chemical Reagent Co., Ltd.), iRT kit (purchased from Biognosys), Bradford protein quantification kit (purchased from Beyotime), dithiothreitol (DTT, purchased from Sigma / D9163-25G), iodoacetamide (IAM, purchased from Sigma / I6125-25G), sodium dodecyl sulfate (SDS, purchased from Sinopharm), urea (purchased from Sinopharm / 10023218), mass spectrometry grade trypsin (purchased from Promega / V5280), ammonium bicarbonate (purchased from Sigma / 5330050050), LC-MS grade ultrapure water (purchased from Thermo Fisher Chemical / W6-4), triethylammonium bicarbonate buffer (TEAB, purchased from Sigma / T7408-500ML), LC-MS grade acetonitrile (purchased from Thermo Fisher Chemical / A955-4), LC-MS grade formic acid (purchased from Thermo Fisher Scientific / A117-50), acetone (purchased from Beijing Chemical Plant / 11241203810051), ammonia water (purchased from Sigma / 221228-500ML-A), low abundance protein enrichment kit [ProteoMiner low abundance protein enrichment kit (purchased from Bio-Rad / 1633007), NP], trifluoroacetic acid (TFA, purchased from Sigma / T6508-100ML).

[0024] 2. Instrument model and source

[0025] pH meter (pH 400 portable pH meter, Shanghai Peirui Instrument Co., Ltd.), dissolved oxygen meter (AZ 8371, Hengxin Industrial Co., Ltd.), -80℃ ultra-low temperature refrigerator (Zhongke Meiling), liquid phase Vanquish TM Neo UHPLC (Thermo Fisher), mass spectrometer Orbitrap Astral mass spectrometer (Thermo Fisher), low-temperature centrifuge (Scilogex / D3024R), freeze dryer (Labogene / Scan Speed ​​40), electrophoresis apparatus (Bio-Rad), electrophoresis tank (purchased from Bio-Rad), electronic balance (Sartorius / BSA124S), vortex mixer (Photosynthesis / HY-6B), microplate reader (thermo / Multiskan FC), ice maker (Xue Ke), tissue grinder (Shanghai Jingxin / 24 holes), ultrasonic cell disruptor (Ningbo Xinzhi / JY92-11N).

[0026] Example 1. A marker for identifying alkali-resistant carp

[0027] α-Globin sequence, SEQ ID NO.1:

[0028] MSLSDKDKAVVKALWAKIGSRADEIGAEALGRMLTVYPQTKTYFSHWSDLSPGSGPVKKHGKTIMGAVGDAVSKIDDLVGALSSLSELHAFKLRIDPANFKILAHNVIVVIGMLFPGDFTPEVHMSVDKFFQNLALALSEKYR.

[0029] α-globin encoding gene sequence, SEQ ID NO.2:

[0030] AGACACACTTCTGTTTTCGCCAACATCTTTTTCTGAAGACAATCTAACTTGAGAAAAAGAAGACGCAGCAATGAGTCTCTCTGATAAGGACAAGGCTGTTGTGAAAGCCCTATGGGCTAAGATCGGCTCCAGAGCCGATGAGATCGGCGCTGAAGCCCTCGGCAGGTAATGTTGAATCACTCTCTTCCTAACTAAGGCTATGTAGAACTTTGTCAAATAACTTTGCAAAATAACATAAAGAAGTTAAAGTTTTGATTTATTCGAGTACCTCAAATGATCACCGAAATGAACAATGTTATTGCTTTCTTTCAGAATGCTGACCGTCTACCCTCAGACCAAGACCTACTTCTCTCACTGGTCTGACCTGAGCCCTGGGTCTGGTCCTGTGAAGAAGCATGGCAAGACTATCATGGGTGCTGTCGGTGATGCCGTTTCAAAGATAGACGACCTTGTGGGAGCTCTGTCCTCCCTGAGCGAACTTCATGCTTTCAAGCTGCGTATTGACCCGGCCAACTTCAAGGTACACCATGACAAAACTTACCTAAGAGTGTAATTTTAGACTTCTCATCATGTACATAATCGTTATAATGTGTTTCTGTGTTCCTTTTCTAGATCCTcgcacacaatgtgatcgtggtCATCGGCATGCTCTTCCCTGGAGACTTCACCCCAGAGGTTCACATGTCAGTTGACAAGTTTTTTCAGAACTTGGCTCTGGCTCTCTCTGAGAAATACCGCTAAACTCCCGGTGGGCATCCAAACGACACGGTGCGGCACCCGTGACCAACTCAAGTGTGATGTCTGAATAACATTTCTCAATAAAAGGCAGAGAATAAGGAA。

[0031] Example 2. A method for identifying the alkali tolerance of cyprinid fish

[0032] A 7-day medium- and long-term stress experiment with 50mmol / LNaHCO3 alkalinity was conducted on fish with different alkali resistance, such as the Dari Lake crucian carp (Carassius auratus) from Inner Mongolia living in an extreme alkaline-salt environment (pH 9.6) and the colorful crucian carp (Carassius auratus) from Pingxiang, Jiangxi living in a freshwater environment (it is known that the Dari Lake crucian carp from Inner Mongolia is the most alkali-resistant, the colorful crucian carp from Pingxiang, Jiangxi is not alkali-resistant, and the moderate alkali resistance is the outlier between the two. Among them, the moderate alkali-resistant individuals from Pingxiang colorful crucian carp from Jiangxi can be used as selected parents to breed and improve the alkali resistance of freshwater crucian carp. Then an ultra-high performance liquid chromatography system combined with mass spectrometry was used to quantitatively determine the expression level of α-globin.

[0033] A freshwater control group and a (50mmol / L) NaHCO3 alkalinity stress group were set up. NaHCO3 (analytical grade, Tianjin Kaitong Chemical Reagent Co., Ltd.) was used to prepare the corresponding alkalinity.

[0034] Thirty experimental fish were placed in the control group and the stress group, respectively, and the alkalinity stress experiment was carried out for 7 days in a 400L indoor circulating aquarium (177cm×57cm×40cm). No feeding was given during the experiment, oxygenation was maintained, 1 / 2 of the water was replaced daily, and various water indicators were monitored. NaHCO3 alkalinity was determined by titration, pH value was determined by pH meter (pH 400 portable pH meter, Shanghai Peirui Instrument Co., Ltd.), dissolved oxygen was determined by dissolved oxygen meter (AZ 8371, Hengxin Industrial Co., Ltd.), and temperature was determined by ordinary thermometer.

[0035] During the experiment, the alkalinity of the control group and the experimental group was measured to be (0.51±0.04)mM and (50.65±1.35)mM, respectively, the pH was 7.79±0.19 and pH 9.12±0.35, respectively, and the salinity was (0.1±0.02)g / L and (3.6±0.52)g / L, respectively. The dissolved oxygen in the water was (7.51±0.65)mg / L, and the water temperature was (17.84±2.73)℃. After the 7-day alkalinity stress experiment, 5 fish in each control group and stress group were randomly selected to scrape 1ml of mucus from the surface of the fish, which was pre-cooled with liquid nitrogen and stored in a -80℃ ultra-low temperature refrigerator.

[0036] Take out the tissue sample, grind it into powder at low temperature, quickly transfer it to a centrifuge tube pre-cooled with liquid nitrogen, add an appropriate amount of SDT (containing 100mM Nacl) and 1 / 100 of the volume of DTT to dissolve, shake and mix, and fully lyse it in an ice water bath for 5 minutes. React at 95℃ for 8-15min, ice bath for 2min, centrifuge at 4℃ and 12000g for 15min, take the supernatant and add sufficient IAM solution, and place it in the dark to react for 1h. Add 4 times the volume of -20℃ pre-cooled acetone to precipitate at -20℃ for at least 2h, centrifuge at 4℃ and 12000g for 15min, and collect the precipitate. Then add 1mL of -20℃ pre-cooled acetone to resuspend and wash the precipitate, collect the precipitate, and air dry. The precipitate is the total protein. Add an appropriate amount of Dissolved Buffer (DB buffer) to thoroughly dissolve the protein precipitate.

[0037] Take a protein sample, add DB protein dissolving solution (8M urea, 100mM TEAB, pH = 8.5) to make up the volume to 100μL, add trypsin and 100mM TEAB buffer, mix well and digest at 37℃ for 4h, then add trypsin and CaCl2 for digestion overnight. Add formic acid to adjust the pH to less than 3, mix well and centrifuge at room temperature and 12000g for 5min, take the supernatant and slowly pass it through the C18 desalting column, then use the cleaning solution (0.1% formic acid, 3% acetonitrile) to wash continuously for 3 times, then add an appropriate amount of eluent (0.1% formic acid, 70% acetonitrile), collect the filtrate, and freeze-dry.

[0038] Prepare mobile phase A (100% water, 0.1% formic acid) and B (80% acetonitrile, 0.1% formic acid). Dissolve the freeze-dried powder in 10 μL of A solution, centrifuge at 14000 g for 20 min at 4°C, take 200 ng of the supernatant and inject it for liquid mass spectrometry. A VanquishNeo nanoliter UHPLC system was used, with a C18 precolumn of 174500 (5 mm × 300 μm, 5 μm, thermo), heated in a 50 °C column oven, and a C18 analytical column of ES906 (PepMap TM Neo UHPLC 150 μm x 15 cm, 2 μm, thermo). The elution conditions of the liquid chromatography are shown in Table 1. A Thermo orbitrap astral mass spectrometer was used, with an Easy-spray (ESI) ion source, the ion spray voltage was set to 1.9 kV, the ion transfer tube temperature was set to 290 °C, the mass spectrometer adopted a data-dependent acquisition mode, and the full scan range of the primary mass spectrometer was m / z 380-980, the primary mass spectrometry resolution was set to 240000 (200m / z), AGC was set to 500%, the parent ion window size was set to 2-Th, the number of DIA windows was 300, the NCE was set to 25%, the secondary m / z acquisition range was 150 to 2000, the daughter ion resolution Astral was set to 80000, and the maximum injection time was 3ms. The raw data (.raw) of mass spectrometry detection was generated. The raw files were searched and analyzed using the library search software DIA-NN. The library search parameters were set as follows: the mass tolerance of the precursor ion was 10ppm, and the mass tolerance of the fragment ion was 0.02Da. The fixed modification was alkylation modification of cysteine, the variable modification was methionine oxidation modification, the N-terminal modification was acetylation modification, methionine loss, and methionine loss + acetylation, and a maximum of 1 missed cleavage site was allowed. In order to improve the quality of analysis results, DIA-NN software further filters the search results: peptide spectrum matches (PSMs) with a credibility of more than 99% are credible PSMs, and only credible peptide spectrum matches and proteins are retained, and FDR verification is performed to remove peptides and proteins with FDR greater than 1%.

[0039] Then, the authentic peptides and proteins were compared with the following peptides to achieve the qualitative and accurate quantification of the components of α-globin.

[0040] After 7 days of alkalinity stress experiment, the results are as follows Figure 1The results showed that according to the actual sequencing results of α-globin expression mass spectrometry, different levels of alkali tolerance were shown according to the health and survival status of the tested fish, and they could be clearly divided into high, medium and low alkali tolerance. Among them, the α-globin expression level of the low alkali tolerance group was <5000, the α-globin expression level of the medium tolerance group was 53568.39±9888.30, and the α-globin expression level of the high tolerance group was 72914.42±22301.86. The difference of the least squares mean multiple comparison reached an extremely significant level (p<0.01). α-globin can be directly used as a protein molecular marker for accurate quantification of the alkalinity stress level of fish.

[0041] Note: A and B belong to the low alkali tolerance group (A cannot induce the expression of α-globin under alkali stress, and B can produce low-level expression, but both show low alkali tolerance), C is the high alkali tolerance group, and D is the medium alkali tolerance group. This figure is a graphical representation method used to describe the statistical distribution of a data set. Figure 1 It is used to display the cumulative distribution of each value in the α-globin expression data set, that is, for each value, it shows the percentage of data less than or equal to this value in the entire data set. The α-globin expression level can be used to distinguish the alkali resistance level of stressed fish in an intuitive and concise graphical method.

Claims

1. A method for identifying the alkali resistance of carp fish, characterized in that: The specific steps of the identification method are as follows: Step 1: Prepare 50mmol / L carbonate solution; Step 2: Stress the carp fish in a carbonate solution for several days and scrape the mucus on the surface of the fish body; Step 3: Detect the level of α-globin in the fish surface mucus using mass spectrometry; Step 4: Identify the level of alkali resistance of carp fish through the level of α-globin; the expression level of α-globin in low alkali resistance level fish is <5000, the expression level of α-globin in medium alkali resistance level fish is 53568.39±9888.30, and the expression level of α-globin in high alkali resistance level fish is 72914.42±22301.

86.

2. The method according to claim 1, characterized in that The carbonate solution in step 1 is a NaHCO3 solution.

3. Application of α-globin as a marker for identifying the alkali resistance of carp fish.

4. The use according to claim 3, characterized in that: The carp fish are Carassius auratus from Dali Lake, Inner Mongolia and Carassius auratus from Pingxiang, Jiangxi; the sample to be tested is mucus on the surface of the fish body.

5. Application of α-globin as a marker species for identifying alkaline-resistant carp fish.

6. The use according to claim 5, characterized in that: The carp fish are Carassius auratus from Dali Lake, Inner Mongolia and Carassius auratus from Pingxiang, Jiangxi; and the fish bodies have mucus on their surfaces.

7. The use according to any one of claims 3 to 6, characterized in that: The amino acid sequence of the α-globin is shown in SEQ ID NO.

1.

8. The use according to any one of claims 3 to 6, characterized in that: Alkali performance is divided into non-alkali resistance level, medium alkali resistance level and high alkali resistance level.

9. Application of genes encoding α-globin fragments as markers for identifying alkali resistance of carp fish.

10. The use according to claim 9, characterized in that: The gene encoding α-globin is shown as SEQ ID NO.2.

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