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

Abstract

The present invention discloses a skin mucus protein molecular marker for accurately quantifying the alkalinity stress level of fish and a detection method thereof, belonging to the technical field of fish alkali tolerance. The present invention aims to solve the problem that there is currently a lack of protein molecular markers for the alkalinity stress level of fish, which in turn affects the efficiency of fish alkali tolerance breeding and selection. The present invention provides a marker for identifying carp fish with alkali tolerance, and the amino acid sequence of the marker is shown in SEQ ID NO.1. It will provide important support for the genetic breeding and selection of alkali-tolerant fish such as grass carp, crucian carp, and Leuciscus waleckii, and further promote the fishery development and utilization of medium and high-saline-alkali waters.

Description

Technical Field

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

[0002] Alkaline salts (NaHCO 3 and Na 2 CO 3 ) are more destructive to aquatic organisms than neutral salts (NaCl and Na 2 SO 4 ). High alkalinity can induce respiratory and metabolic alkalosis in freshwater fish, seriously affecting the growth, survival and reproduction of fish. Therefore, among the approximately 690 million mu of low-lying saline-alkali water area resources in China, currently only less than 2% of the low-saline-alkali water areas with an alkalinity below 10 mmol / L have been developed and utilized by transplanting and domesticating conventional freshwater and marine aquaculture economic animals. Most of the medium- and high-saline-alkali water areas with an alkalinity > 10 mmol / L are still in a deserted and idle state, and the development and utilization are imminent. To solve the above problems, improving the saline-alkali tolerance of aquaculture varieties is the key to realizing the efficient utilization of these resources. And accurately evaluating the alkalinity tolerance traits, especially by excavating biomarkers to indicate the response process of fish to alkaline environments, will provide important support for the genetic breeding of alkalinity-tolerant fish such as grass carp, crucian carp, and Leuciscus waleckii, and thus promote the fishery development and utilization of medium- and high-saline-alkali water areas.

[0003] To achieve this goal, accurately evaluating the alkalinity tolerance traits has become an important basis for genetic breeding. Alkalinity tolerance traits are usually regarded as typical threshold traits with discontinuous distribution in the field of quantitative genetics. Traditionally, lethal experiments are commonly used to evaluate the alkalinity tolerance degree, but lethal experiments cannot judge the long-term adaptation process of fish to alkaline environments during the breeding process, nor can they be directly used as an index for breeding alkalinity-tolerant fish varieties. Therefore, excavating biomarkers that can indicate the response process of alkaline environments is of great significance for more accurately evaluating the alkalinity tolerance traits. Summary of the Invention

[0004] The purpose of the present invention is to provide an α-globin fragment as a marker for assisting in the identification of the alkalinity stress level of fish and a detection method therefor, so as to solve the problem that there is currently a lack of protein molecular markers for the alkalinity stress level of fish, which in turn affects the breeding efficiency of fish alkalinity tolerance breeding.

[0005] The present invention provides an application of α-globin as a marker for identifying the alkalinity tolerance of Cyprinidae fish.

[0006] Further defined, the Cyprinidae fish are Carassius auratus dariensis from Lake Dali in Inner Mongolia ( Carassius auratus ) and Carassius auratus var. pingxiangensis from Pingxiang, Jiangxi ( Carassius auratus ); the sample to be detected is the mucus on the fish body surface.

[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 is the Inner Mongolia Dali Lake Crucian Carp ( Carassius auratus ) and Jiangxi Pingxiang colorful crucian carp ( Carassius auratus ), the sample to be tested is mucus on the surface of fish.

[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 50 mmol / 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 NaHCO 3 Solution.

[0019] Beneficial effects: The present invention uses an ultra-high performance liquid chromatography system combined with mass spectrometry to perform label-free quantification (LFQ) in the data-independent acquisition (DIA) mode. Mass spectrometry data is collected using a data-independent acquisition (DIA) scanning mode over a wide dynamic range to qualitatively and precisely quantify the components of α-globin for the following peptide segments. The α-globin expression level is used as a protein molecular marker to measure the alkalinity stress level of fish. Under the same intensity of alkalinity stress, a higher α-globin expression level indicates stronger alkali tolerance ability.

[0020] During the breeding process of alkali-tolerant fish varieties, under the condition of alkaline water stress, the α-globin expression level can be measured by sampling the skin mucus of breeding materials, and the alkali tolerance ability of fish can be accurately evaluated. Applied to breeding practice, it will greatly improve the selection efficiency of new alkali-tolerant fish varieties during the breeding process and significantly shorten the breeding cycle of new alkali-tolerant fish varieties. Brief description of the drawings

[0021] Figure 1 It is a cumulative distribution function diagram of the α-globin expression level in the skin mucus of fish with different alkali tolerance abilities. Detailed implementation manners

[0022] I. Reagents

[0023] NaHCO 3(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 freezer (Zhongke Meiling), liquid phase determination Vanquish™ Neo UHPLC (ThermoFisher), mass spectrometer Orbitrap Astral mass spectrometer (Thermo Fisher), low-temperature centrifuge (Scilogex / D3024R), freeze dryer (Labogene / Scan Speed ​​40), electrophoresis instrument (Bio-Rad), electrophoresis tank (purchased from Bio-Rad), electronic balance (Sartorius / BSA124S), vortex mixer (Photosynthesis / HY-6B), microplate reader (thermo / Multiskan FC), ice maker (Xueke), 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 coding gene sequence, SEQ ID NO.2:

[0030] AGACACACTTCTGTTTTCGCCAACATCTTTTTCTGAAGACAATCTAACTTGAGAAAAAGAAGACGCAGCAATGAGTCTCTCTGATAAGGACAAGGCTGTTGTGAAAGCCCTATGGGCTAAGATCGGCTCCAGAGCCGATGAGATCGGCGCTGAAGCCCTCGGCAGGTAATGTTGAATCACTCTCTTCCTAACTAAGGCTATGTAGAACTTTGTCAAATAACTTTGCAAAATAACATAAAGAAGTTAAAGTTTTGATTTATTCGAGTACCTCAAATGATCACCGAAATGAACAATGTTATTGCTTTCTTTCAGAATGCTGACCGTCTACCCTCAGACCAAGACCTACTTCTCTCACTGGTCTGACCTGAGCCCTGGGTCTGGTCCTGTGAAGAAGCATGGCAAGACTATCATGGGTGCTGTCGGTGATGCCGTTTCAAAGATAGACGACCTTGTGGGAGCTCTGTCCTCCCTGAGCGAACTTCATGCTTTCAAGCTGCGTATTGACCCGGCCAACTTCAAGGTACACCATGACAAAACTTACCTAAGAGTGTAATTTTAGACTTCTCATCATGTACATAATCGTTATAATGTGTTTCTGTGTTCCTTTTCTAGATCCTcgcacacaatgtgatcgtggtCATCGGCATGCTCTTCCCTGGAGACTTCACCCCAGAGGTTCACATGTCAGTTGACAAGTTTTTTCAGAACTTGGCTCTGGCTCTCTCTGAGAAATACCGCTAAACTCCCGGTGGGCATCCAAACGACACGGTGCGGCACCCGTGACCAACTCAAGTGTGATGTCTGAATAACATTTCTCAATAAAAGGCAGAGAATAAGGAA。

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

[0032] For Carassius auratus from Inner Mongolia's Dalihu Lake living in an extreme alkali-salt environment (pH 9.6) Carassius auratus and Carassius auratus from Pingxiang, Jiangxi living in a freshwater environment Carassius auratus)(such as fish with different alkali tolerance. It is known that Carassius auratus in Lake Dali, Inner Mongolia is the most alkali-tolerant, Carassius auratus in Pingxiang, Jiangxi is not alkali-tolerant, and the medium alkali-tolerant one is an outlier among the two. Among them, the medium alkali-tolerant individuals from Carassius auratus in Pingxiang, Jiangxi can be used as the selected parents to breed and improve the alkali tolerance of freshwater Carassius auratus.) Conduct a long-term stress experiment at an alkalinity of 50 mmol / L NaHCO 3 for 7 days, and then use an ultra-high performance liquid chromatography system combined with mass spectrometry to quantitatively determine the expression level of α-globin.

[0033] A total of 1 freshwater control group and 1 (50 mmol / L) NaHCO 3 alkalinity stress group were set up. NaHCO 3 (Analytical pure, Tianjin Kaitong Chemical Reagent Co., Ltd.) was used to prepare the corresponding alkalinity.

[0034] 30 experimental fish were stocked in the control group and the stress group respectively, and a 7-day alkalinity stress experiment was carried out in a 400 L indoor circulating aquarium (177 cm×57 cm×40 cm). No bait was fed during the experiment, oxygen was kept on, 1 / 2 of the water body was changed daily, and various indicators of the water body were monitored. NaHCO 3 The alkalinity was determined by titration method, the pH value was measured by a pH meter (pH 400 portable pH meter, Shanghai Peirui Instrument Co., Ltd.), the dissolved oxygen was measured by a dissolved oxygen meter (AZ 8371, Hengxin Industrial Co., Ltd.), and the temperature was measured by an ordinary thermometer.

[0035] During the experiment, the measured NaHCO 3 alkalinity of the control group and the experimental group was (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) °C. After the 7-day alkalinity stress experiment, 5 fish were randomly selected from the control group and the stress group respectively, and 1 ml of mucus on the fish body surface was scraped, pre-cooled with liquid nitrogen and stored in a -80 °C 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 100 mM Nacl) and DTT corresponding to 1 / 100 of the volume for dissolution, shake and mix well, and ultrasonically lyse in an ice-water bath for 5 min to fully lyse. React at 95°C for 8 - 15 min, ice-bath for 2 min, centrifuge at 4°C and 12,000 g for 15 min, take the supernatant, add a sufficient amount of IAM solution, and react in the dark for 1 h. Add 4 volumes of acetone pre-cooled to -20°C and precipitate at -20°C for at least 2 h, centrifuge at 4°C and 12,000 g for 15 min, and collect the precipitate. Then add 1 mL of acetone pre-cooled to -20°C to resuspend and wash the precipitate, collect the precipitate, air-dry it, and the precipitate is the total protein. Add an appropriate amount of Dissolved Buffer (DB buffer) to completely dissolve the protein precipitate.

[0037] Take the protein sample, add DB protein dissolution solution (8 M urea, 100 mM TEAB, pH = 8.5) to make up the volume to 100 μL, add trypsin and 100 mM TEAB buffer, mix well and digest at 37°C for 4 h, then add trypsin and CaCl2 and digest overnight. Add formic acid to adjust the pH to less than 3, mix well and centrifuge at room temperature and 12,000 g for 5 min, take the supernatant and slowly pass it through a C18 desalting column, then continuously wash it 3 times with the washing solution (0.1% formic acid, 3% acetonitrile), and then add an appropriate amount of elution solution (0.1% formic acid, 70% acetonitrile), collect the filtrate, and freeze-dry it.

[0038] Prepare mobile phase A solution (100% water, 0.1% formic acid) and B solution (80% acetonitrile, 0.1% formic acid). Dissolve the lyophilized powder with 10 µL of solution A, centrifuge at 14000 g for 20 min at 4 °C, take 200 ng of the supernatant as the sample for injection, and perform liquid chromatography - mass spectrometry detection. Use a Vanquish Neo nano - scale UHPLC system. The C18 pre - column is 174500 (5 mm × 300 µm, 5 µm, thermo), heat the column oven at 50 °C, and the C18 analytical column is ES906 (PepMap TM Neo UHPLC 150 µm x 15 cm, 2 µm, thermo). The liquid chromatography elution conditions are as shown above. Use a Thermo orbitrap astral mass spectrometer, an Easy - spray (ESI) ion source, set the ion spray voltage to 1.9 kV, the ion transfer tube temperature to 290 °C. The mass spectrometry adopts a data - dependent acquisition mode. The full - scan range of the first - stage mass spectrometry is m / z 380 - 980, the resolution of the first - stage mass spectrometry is set to 240000 (200 m / z), the AGC is set to 500%, the precursor ion window size is set to 2 - Th, the number of DIA windows is 300, the NCE is set to 25%, the second - stage m / z acquisition range is 150 to 2000, the resolution of the product ions Astral is set to 80000, and the maximum injection time is 3 ms. Generate the original mass spectrometry detection data (.raw). Use the database search software DIA - NN to search and analyze the raw file. The database search parameters are set as follows: the mass tolerance of the precursor ions is 10 ppm, and the mass tolerance of the fragment ions is 0.02 Da. The immobilized modification is the alkylation modification of cysteine, the variable modification is the oxidation modification of methionine, the N - terminal modification is acetylation modification, methionine loss, and methionine loss + acetylation, allowing a maximum of 1 missed cleavage site. To improve the quality of the analysis results, the DIA - NN software further filters the retrieval results: the peptide spectrum matches (PSMs) with a confidence level above 99% are considered reliable PSMs. Only retain the reliable peptide spectra and proteins, and perform FDR verification to remove the peptide segments and proteins with an FDR greater than 1%.

[0039] Then, compare the reliable peptide segments and proteins with the following peptide segments to qualitatively and precisely quantify the components of α - globin.

[0040] After the 7 - day alkalinity stress experiment, the results are as Figure 1The results showed that, based on the actual sequencing results of mass spectrometry of α-globin expression levels and according to the health and survival status of the test fish, different levels of alkali tolerance ability were exhibited, which could be clearly divided into high, medium, and low alkali tolerance levels. Among them, the α-globin expression level in the low alkali tolerance group was <5000, the α-globin expression level in the medium tolerance group was 53568.39 ± 9888.30, and the α-globin expression level in the high tolerance group was 72914.42 ± 22301.86. The multiple comparison of least square means reached an extremely significant level ( p <0.01). α-Globin can be directly used as a protein molecular marker for accurately quantifying the alkalinity stress level of fish.

[0041] Note: A and B belong to the low alkali tolerance group (A cannot induce the expression of a-globin under alkali stress, and B can produce a low level of 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 show the cumulative distribution of each value in the α-globin expression level data set, that is, for each value, it shows the percentage of data less than or equal to that value in the entire data set. The alkali tolerance ability levels of stressed fish can be distinguished by α-globin expression levels 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 50 mmol / 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 by the expression level of α-globin.

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

3. The use of α-globin as a marker for identifying the alkali resistance of carp fish, characterized in that: The amino acid sequence of the α-globin is shown in SEQ ID NO.

1.

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

5. Use of α-globin as a marker species for identifying alkaline-resistant carp fish, characterized in that: The amino acid sequence of the α-globin is shown in SEQ ID NO.

1.

6. The use according to claim 5, characterized in that: The carp fish are the Dali Lake crucian carp from Inner Mongolia and the colorful crucian carp from Pingxiang, Jiangxi, and the sample to be tested is mucus on the surface of the fish body.

7. Use of a gene encoding an α-globin fragment as a marker for identifying alkali resistance of carp fish, characterized in that: The gene encoding α-globin is shown as SEQ ID NO.2.

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