CN112684064A - Serum metabolic marker for screening stress-resistant mutton sheep and application thereof - Google Patents
Serum metabolic marker for screening stress-resistant mutton sheep and application thereof Download PDFInfo
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Abstract
本发明涉及一种用于抗应激胁迫肉羊的血清代谢标志物及其应用，属于畜牧生产和应用生物技术领域，本发明通过收集抗应激胁迫肉羊和非抗应激胁迫肉羊外周血样本，采用超高效液相色谱‑质谱技术，结合系统的生物信息学分析方法，从而对抗热应激和非抗应激肉羊血清样本中差异代谢物进行筛选。所述标志物为吲哚‑3‑乙酸甲酯，通过所述代谢物标志物以及检测该代谢标志物的试剂，可以筛选耐热肉羊，结果准确客观可靠，且无需提供其他组织样品，为耐热肉羊的筛选奠定基础，具有较好的推广价值。
The invention relates to a serum metabolic marker for anti-stress stress mutton sheep and its application, belonging to the field of animal husbandry production and applied biotechnology. Using ultra-performance liquid chromatography-mass spectrometry technology combined with a systematic bioinformatics analysis method to screen differential metabolites in serum samples of heat-stressed and non-stressed mutton sheep. The marker is indole-3-methyl acetate, and the metabolite marker and the reagent for detecting the metabolite marker can be used to screen heat-resistant mutton sheep, and the results are accurate, objective and reliable, and there is no need to provide other tissue samples. The screening of hot mutton sheep lays the foundation and has good promotion value.
Description
Technical Field
The invention belongs to the technical field of livestock production and applied biology, and particularly relates to a serum metabolic marker for screening stress-resistant mutton sheep, a screening method and application.
Background
The mutton has delicious meat quality, has the effects of warming the sex, tonifying qi, nourishing yin, warming the middle warmer and tonifying the stomach, and the nutritive value of the mutton is increasingly valued by consumers, so that the demand of the market on the mutton is continuously increased.
With the increasing demand of mutton market and the increasing emphasis on ecological environment protection, mutton sheep breeding has been gradually changed from traditional household-based scatter breeding into intensive breeding. In tropical and subtropical regions in south China, the temperature can reach over 38 ℃ in summer, the duration is long, intensive cultivation is carried out under a high-temperature condition, and the heat stress of mutton sheep is easily caused. The heat stress can reduce the dry matter feed intake of the mutton sheep, increase the respiratory rate and the heart rate, reduce the abundance of rumen microbial flora, finally reduce the digestibility of nutrient substances, influence the production performance and bring great loss to the mutton sheep breeding industry in south China.
Metabolites are the end products of gene expression. Metabonomics is a newly developed subject after genomics, transcriptomics and proteomics, directly uses animal body fluid as a sample for detection, quantitatively detects endogenous metabolites by means of a modern analysis technology with high flux, high sensitivity and high precision, excavates the change rule of the whole metabolites and metabolites, finds the relative relationship between biological change and metabolite change of an organism and reflects the essence of the organism change. Therefore, the method has the characteristics of high speed, accuracy, high sensitivity and the like. At present, no relevant report for screening stress resistance mutton sheep biomarkers exists, and serum detection is noninvasive and non-invasive, so that how to screen and apply the stress resistance mutton sheep biomarkers by researching mutton sheep serum metabolites has important significance for solving stress of mutton sheep and breeding new stress resistance mutton sheep varieties.
Disclosure of Invention
The invention aims to provide a serum metabolic marker for screening stress-resistant mutton sheep, a screening method and application, and provides a method for non-invasively screening stress-resistant mutton sheep by using serum.
The invention realizes the purpose through the following technical scheme:
the invention provides a serum metabolic marker for screening stress resistant mutton sheep, wherein the serum metabolic marker is indole-3-methyl acetate.
The invention also provides application of the serum metabolic marker in screening stress resistant mutton sheep, which specifically comprises the following steps: judging the heat stress resistance of the mutton sheep according to the concentration of the serum metabolic marker in the mutton sheep serum sample, wherein the lower the concentration, the higher the stress resistance of the mutton sheep, and the smaller the stress resistance of the mutton sheep.
As a further optimization of the present invention, the stress resistance refers to heat stress resistance.
The invention also provides a method for screening stress resistant mutton sheep by using the serum metabolic marker, which comprises the following steps:
(1) taking the known stress-resistant mutton sheep blood or non-stress-resistant mutton sheep blood as a standard test sample, taking the mutton sheep blood to be detected as a detection sample, and centrifuging to obtain a serum sample;
(2) detecting the concentration of indole-3-methyl acetate in a serum sample;
(3) and calculating the average concentration value of the indole-3-methyl acetate of the standard test sample as a reference level, wherein if the detection sample is lower than the reference level, the detection sample has higher stress resistance than the standard test sample, and otherwise, the detection sample has lower stress resistance.
As a further optimization scheme of the invention, in the step (2), an ultra performance liquid chromatography-mass spectrometry combined analysis method is adopted to detect the concentration of indole-3-methyl acetate in the serum sample, and the steps comprise:
the method comprises the following steps: extraction of serum metabolites
Adding 3 times volume of the extract and an internal standard into a serum sample, uniformly mixing, standing at-20 ℃ for 2h, centrifuging at 4000 rpm at 4 ℃ for 20min, and taking the supernatant to obtain a serum metabolite;
step two: preparation of indole-3-methyl acetate standard
Dissolving an indole-3-methyl acetate standard substance to prepare a standard substance stock solution of 10nmol/L, preparing a mixed standard solution from the stock solution, and performing gradient dilution to obtain a calibration solution;
step three: mass spectrometric analysis of samples
Detecting serum metabolites and a calibration solution by using an ultra performance liquid chromatography-mass spectrometry combined analysis method;
liquid chromatography conditions: separating and detecting metabolites by using a Waters 2D UPLC series Q active high resolution mass spectrometer, wherein the used chromatographic column is a BEH C18 chromatographic column, positive ion mode mobile phases are aqueous solution containing 0.1 volume percent of formic acid as A liquid and 100 percent methanol containing 0.1 volume percent of formic acid as B liquid, and negative ion mode mobile phases are aqueous solution containing 10mM of ammonia formate as A liquid and 95 percent methanol containing 10mM of ammonia formate as B liquid, and the elution is carried out by adopting the following gradient in percentage by volume: 0-1 min, 2% of solution B; 1-9 min, 2% -98% of liquid B; 9-12 min, and 98% of liquid B; 12-12.1 min, 98% of liquid B to 2% of liquid B; 12.1-15 min, 2% of solution B. The flow rate was 0.35mL/min, the column temperature was 45 ℃ and the amount of sample was 5. mu.L.
Mass spectrum conditions: and (3) performing primary and secondary mass spectrum data acquisition by using a Q active mass spectrometer, wherein the mass-nuclear ratio range of mass spectrum scanning is 70-1050, the primary resolution is 70,000, the AGC is 3e6, and the maximum injection time is 100 ms. According to the intensity of the parent ions, Top3 is selected for fragmentation, secondary information is collected, the secondary resolution is 17,500, AGC is 1e5, the maximum injection time is 50ms, and the fragmentation energy is set as: 20, 40, 60eV, ion source parameter settings: the flow rate of the sheath gas is 40, the flow rate of the auxiliary gas is 10, the spray voltage positive ion mode is 3.80, the negative ion mode is 3.20, the temperature of an ion transmission tube is 320 ℃, and the heating temperature of the auxiliary gas is 350 ℃;
step four: and drawing a standard curve chart according to the detection result of the ultra performance liquid chromatography tandem mass spectrometry of the calibration solution, and calculating to obtain the content data of the indole-3-methyl acetate in the sample to be detected according to the standard curve chart.
As a further optimization scheme of the invention, the extracting solution is composed of methanol and CAN in a volume ratio of 2: 1.
As a further optimization of the invention, the internal standard is indole-3-acetic acid Methyl ester (Methyl indole-3-acetate).
The invention has the beneficial effects that:
the metabolic marker of the stress-resistant mutton sheep is indole-3-methyl acetate, and the indole-3-methyl acetate is used as the marker for detection, so that the method has the characteristics of high accuracy and high sensitivity (AUC is more than 0.800), and can be directly used as an evaluation index of the stress-resistant mutton sheep.
Drawings
FIG. 1 is a PCA classification scatter diagram showing the metabolic profiles in serum of the heat stress resistant group (HS) and the non-heat stress resistant group (DS), wherein A is a PCA diagram in a negative ion mode and B is a PCA diagram in a positive ion mode.
FIG. 2 is a bar graph showing the relative expression of indole-3-acetic acid methyl ester as a serum metabolite in Hu Sheep (HS) and Du Po sheep (DS).
FIG. 3 is an ultra performance liquid chromatography tandem mass spectrometry detection chart of indole-3-methyl acetate.
Detailed Description
The present application will now be described in further detail with reference to the drawings, it should be noted that the following detailed description is given for illustrative purposes only and is not to be construed as limiting the scope of the present application, as those skilled in the art will be able to make numerous insubstantial modifications and adaptations to the present application based on the above disclosure.
Example 1
1. Material
The methods used in this example are conventional methods known to those skilled in the art unless otherwise specified, and the reagents and other materials used therein are commercially available products unless otherwise specified.
2. Method of producing a composite material
2.1 screening of serum for Heat stress related metabolites
(1) Collecting a serum sample:
collecting blood of 6 stress-resistant mutton sheep (HS, Hu sheep) and 6 non-stress-resistant mutton sheep (DS, Dorper sheep) under heat stress condition, placing in a negative pressure tube without heparin, centrifuging at 1500g for 15-20min, and collecting supernatant, wherein the supernatant is the required 12 serum samples; serum samples were stored in a-80 ℃ freezer for use.
(2) Extraction of serum metabolites
Adding 3 times volume of extract (methanol: acetonitrile: 2:1, v: v, precooling at-20 ℃) and indole-3-methyl acetate as an internal standard into the 12 serum samples, uniformly mixing, standing at-20 ℃ for 2h, centrifuging at 4000 rpm at 4 ℃ for 20min, taking the supernatant and placing in a sample loading bottle to obtain the serum metabolite.
(3) Mass spectrometric analysis of samples
Performing ultra performance liquid chromatography-mass spectrometry analysis on 12 serum samples in sequence, wherein the experimental conditions are as follows:
liquid chromatography conditions: separation and detection of metabolites was performed using Waters 2D UPLC (Waters, USA) tandem Q active high resolution Mass spectrometer (Thermo Fisher Scientific, USA). The column used was a BEH C18 column (1.7 μm 2.1 × 100mm, Waters, USA). The positive ion mode mobile phase was an aqueous solution (solution A) containing 0.1% by volume of formic acid and 100% methanol (solution B) containing 0.1% by volume of formic acid, and the negative ion mode mobile phase was an aqueous solution (solution A) containing 10mM ammonia formate and 95% methanol (solution B) containing 10mM ammonia formate. Elution was performed with the following gradient, in volume percent: 0-1 min, 2% of solution B; 1-9 min, 2% -98% of liquid B; 9-12 min, and 98% of liquid B; 12-12.1 min, 98% of liquid B to 2% of liquid B; 12.1-15 min, 2% of solution B. The flow rate was 0.35mL/min, the column temperature was 45 ℃ and the amount of sample was 5. mu.L.
Mass spectrum conditions: first and second mass spectral data acquisition was performed using a Q exact mass spectrometer (Thermo Fisher Scientific, USA). The mass-spectrometric scanning nucleus ratio range is 70-1050, the primary resolution is 70,000, the AGC is 3e6, and the maximum Injection Time (IT) is 100 ms. According to the parent ion intensity, Top3 was selected for fragmentation, secondary information was collected, secondary resolution was 17,500, AGC was 1e5, maximum Injection Time (IT) was 50ms, fragmentation energy (fragmented) was set as: 20, 40 and 60 eV. Ion source (ESI) parameter settings: a Sheath gas flow rate (Sheath gas flow rate) of 40, an auxiliary gas flow rate (Aux gas flow rate) of 10, a Spray voltage (KV)) of 3.80 in a positive ion mode, a negative ion mode of 3.20, an ion transfer tube temperature (Capillary temp.) of 320 ℃, and an auxiliary gas heating temperature (Aux gas heater temp.) of 350 ℃; finally, a 12 sample total ion flow graph is obtained, namely the serum metabolism fingerprint.
(4) Multivariate statistical analysis, screening biomarkers:
the sample serum metabolism fingerprint data is subjected to peak extraction, retention time correction within and among groups, combined ion combination, deletion value filling, background peak marking and metabolite identification by using Compound discover 3.0(Thermo Fisher Scientific, USA), and finally, information such as Compound molecular weight, retention time, peak area and identification result is derived. Based on the difference comparison of the non-heat stress resistant animal spectrogram and the heat stress resistant animal spectrogram, SPSS statistical software is used for screening different metabolites among groups in a mode of combining multivariate statistical analysis (PCA) and univariate analysis (Fold-Change (FC) and T test (Student's T test)), and the metabolites can be considered as biomarkers which can possibly influence the metabolism of the heat stress resistant animals.
As a result, it was found that the anti-heat stress group (HS) and the non-anti-heat stress group (DS) had a distinct degree of discrimination, mainly along the horizontal axis, the anti-heat stress group (HS) samples mainly gathered on the right side, and the non-anti-heat stress group (DS) mainly gathered on the left side (fig. 1). Compared with a non-heat-resistant stress group, the content of indole-3-methyl acetate (Propylparaben) in the serum metabolite is remarkably reduced, indole-3-methyl acetate is predicted to be a serum metabolite related to heat stress resistance of the mutton sheep, and the indole-3-methyl acetate is used as a serum metabolic marker for screening the stress-resistant mutton sheep to realize screening of the stress-resistant mutton sheep.
The change of the markers in stress-resistant mutton sheep and the corresponding predicted area under stress (AUC) are shown in the following table 1:
table 1: information on serum metabolic markers
The detailed change histogram of the serum metabolism markers in the serum of meat sheep without heat stress resistance and stress resistance is shown in figure 2, and the metabolite concentration of indole-3-methyl acetate in the stress resistance meat sheep is obviously reduced.
Example 2
The embodiment provides a method for screening stress-resistant mutton sheep by using indole-3-methyl acetate as a marker, which comprises the following steps:
(1) extracting the blood of the known stress-resistant mutton sheep or non-stress-resistant mutton sheep as a standard test sample and the blood of the mutton sheep to be detected as a detection sample, centrifuging to obtain a serum sample, and storing at-80 ℃.
(2) Detecting the concentration of indole-3-methyl acetate in a serum sample; in this example, an expression level of indole-3-methyl acetate is obtained by a quantitative analysis method of ultra performance liquid chromatography-mass spectrometry, and the detailed steps are as follows:
a) extraction of serum metabolites: adding 3 times volume of extractive solution (methanol: ACN 2:1, v: v-20 deg.C precooled) and 2 kinds of internal standard into the above serum sample, mixing, standing at-20 deg.C for 2h, centrifuging at 4 deg.C and 4000g for 20min, and collecting supernatant and placing in a sample bottle.
b) Dissolving indole-3-methyl acetate standard to prepare 10nmol/L standard stock solution, preparing mixed standard solution from the stock solution, performing gradient dilution to obtain calibration solution, diluting indole-3-methyl acetate calibration solution by 2 times, and performing ultra performance liquid chromatography-mass spectrometry monitoring analysis.
c) And (3) performing ultra performance liquid chromatography-mass spectrometry analysis, wherein specific liquid chromatography conditions and mass spectrometry conditions refer to the step (3) in the example 1, and the concentration information of indole-3-methyl acetate in the metabolites of the sample is obtained.
d) And drawing a standard curve chart according to the detection result of the ultra performance liquid chromatography tandem mass spectrometry of the calibration solution, and calculating to obtain the content data of the indole-3-methyl acetate in the sample to be detected according to the standard curve chart.
FIG. 2 shows a graph of the detection result of indole-3-methyl acetate standard, in which the abscissa represents the peak time, the ordinate represents the peak height, and the position indicated by the arrow is the indole-3-methyl acetate peak position.
(3) And taking the average concentration value of the standard test sample as a reference level, comparing the concentration of the indole-3-methyl acetate of the mutton sheep to be tested with the reference level, if the concentration of the indole-3-methyl acetate is lower than the reference level, indicating that the mutton sheep to be tested has higher heat stress resistance than the standard test sample, otherwise indicating that the mutton sheep to be tested has lower heat stress resistance than the standard test sample.
As in this example 2, 3 individuals of the 6 non-stress-resistant mutton sheep of example 1 were randomly selected as test a, test b and test c, and their relative serum concentrations were: 90779.3, 81955.8, 89008.1. The mean metabolite concentration for all subjects was calculated as 87247.7 as the reference level of indole-3-acetic acid methyl ester in this group of test sheep. For one of the tested sheep, such as the tested sheep b, the concentration of the indole-3-methyl acetate is 45937.8, which is lower than 87247.7, namely the concentration of the metabolite in the serum of the stress resistant mutton sheep is lower, and the tested sheep b is presumed to have higher heat stress resistance.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.
Claims (9)
1. The serum metabolic marker of the stress-resistant mutton sheep is characterized by being indole-3-methyl acetate.
2. Use of the serum metabolic marker of claim 1 in screening stress-resistant mutton sheep.
3. The use of the serum metabolism marker in screening stress resistance mutton sheep according to claim 2, wherein the heat stress resistance of the mutton sheep is judged according to the concentration of the serum metabolism marker in a mutton sheep serum sample, and the lower the concentration, the greater the stress resistance of the mutton sheep, and the smaller the stress resistance of the mutton sheep.
4. The use of a serum metabolic marker in screening stress-resistant mutton sheep according to claim 2, wherein the stress resistance refers to heat stress resistance.
5. A method for screening stress resistant mutton sheep is characterized by comprising the following steps:
(1) taking the known stress-resistant mutton sheep blood or non-stress-resistant mutton sheep blood as a standard test sample, taking the mutton sheep blood to be detected as a detection sample, and centrifuging to obtain a serum sample;
(2) detecting the concentration of indole-3-methyl acetate in a serum sample;
(3) and calculating the average concentration value of the indole-3-methyl acetate of the standard test sample as a reference level, wherein if the detection sample is lower than the reference level, the detection sample has higher stress resistance than the standard test sample, and otherwise, the detection sample has lower stress resistance.
6. The method for screening mutton sheep resisting stress according to claim 5, wherein in the step (2), the concentration of indole-3-methyl acetate in the serum sample is detected by using an ultra performance liquid chromatography-mass spectrometry combined analysis method.
7. The method for screening stress-resistant mutton sheep according to claim 6, wherein the step of the ultra-high performance liquid chromatography-mass spectrometry analysis method comprises the following steps:
the method comprises the following steps: extraction of serum metabolites
Adding 3 times volume of the extract and an internal standard into a serum sample, uniformly mixing, standing at-20 ℃ for 2h, centrifuging at 4000 rpm at 4 ℃ for 20min, and taking the supernatant to obtain a serum metabolite;
step two: preparation of indole-3-methyl acetate standard
Dissolving an indole-3-methyl acetate standard substance to prepare a standard substance stock solution of 10nmol/L, preparing a mixed standard solution from the stock solution, and performing gradient dilution to obtain a calibration solution;
step three: mass spectrometric analysis of samples
Detecting serum metabolites and a calibration solution by using an ultra performance liquid chromatography-mass spectrometry combined analysis method;
liquid chromatography conditions: separating and detecting metabolites by using a Waters 2D UPLC series Q active high resolution mass spectrometer, wherein the used chromatographic column is a BEH C18 chromatographic column, positive ion mode mobile phases are aqueous solution containing 0.1 volume percent of formic acid as A liquid and 100 percent methanol containing 0.1 volume percent of formic acid as B liquid, and negative ion mode mobile phases are aqueous solution containing 10mM of ammonia formate as A liquid and 95 percent methanol containing 10mM of ammonia formate as B liquid, and the elution is carried out by adopting the following gradient in percentage by volume: 0-1 min, 2% of solution B; 1-9 min, 2% -98% of liquid B; 9-12 min, and 98% of liquid B; 12-12.1 min, 98% of liquid B to 2% of liquid B; 12.1-15 min, 2% of solution B. The flow rate was 0.35mL/min, the column temperature was 45 ℃ and the amount of sample was 5. mu.L.
Mass spectrum conditions: and (3) performing primary and secondary mass spectrum data acquisition by using a Q active mass spectrometer, wherein the mass-nuclear ratio range of mass spectrum scanning is 70-1050, the primary resolution is 70,000, the AGC is 3e6, and the maximum injection time is 100 ms. According to the intensity of the parent ions, Top3 is selected for fragmentation, secondary information is collected, the secondary resolution is 17,500, AGC is 1e5, the maximum injection time is 50ms, and the fragmentation energy is set as: 20, 40, 60eV, ion source parameter settings: the flow rate of the sheath gas is 40, the flow rate of the auxiliary gas is 10, the spray voltage positive ion mode is 3.80, the negative ion mode is 3.20, the temperature of an ion transmission tube is 320 ℃, and the heating temperature of the auxiliary gas is 350 ℃;
step four: and drawing a standard curve chart according to the detection result of the ultra performance liquid chromatography tandem mass spectrometry of the calibration solution, and calculating to obtain the content data of the indole-3-methyl acetate in the sample to be detected according to the standard curve chart.
8. The method for screening mutton sheep resisting stress according to claim 7, wherein the extract is composed of methanol to CAN in a volume ratio of 2: 1.
9. The method for screening stress-resistant mutton sheep according to claim 7, wherein the internal standard is indole-3-methyl acetate.
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