阅读说明：本技术 一种曼氏无针乌贼衰老过程中的代谢生物标志物 (Metabolism biomarker in sepiella maindroni aging process ) 是由 郭宝英 徐鸣 祁鹏志 许中天 于 2019-09-26 设计创作，主要内容包括：本发明提供一种曼氏无针乌贼衰老过程中的代谢生物标志物,属于基因工程技术领域,该代谢生物标志物包含尿酸,其筛选方法包括：采集不同生殖阶段个体的血清样本；将血清样本进行预处理；对血清样本依次进行液相色谱-质谱联用分析；对LC-MS数据处理进行筛选,筛选出代谢生物标志物；对代谢生物标志物进行对比和指认；构建生物标志物的代谢通路并进行分析。本发明通过高效液相色谱-串联质谱技术对不同生殖阶段曼氏无针乌贼的血清进行微量代谢组学分析,经过筛选,相比产卵前,尿酸的含量在产卵后有大幅的下降,产卵前含量/产卵后含量＝2.16,VIP＝1.5385,是一种潜在的代谢生物标志物。(The invention provides a metabolic biomarker in the process of Sepiella maindroni aging, which belongs to the technical field of genetic engineering, the metabolic biomarker comprises uric acid, and the screening method comprises the following steps: collecting serum samples of individuals in different reproductive stages; pretreating a serum sample; sequentially carrying out liquid chromatography-mass spectrometry combined analysis on the serum sample; screening LC-MS data processing to screen out metabolic biomarkers; comparing and assigning metabolic biomarkers; metabolic pathways for biomarkers were constructed and analyzed. According to the invention, the serum of Sepiella maindroni at different reproductive stages is subjected to micro-metabonomic analysis by a high performance liquid chromatography-tandem mass spectrometry technology, and the content of uric acid is greatly reduced after egg laying compared with that before egg laying after screening, wherein the content before egg laying/after egg laying is 2.16, VIP is 1.5385, and the sepiella maindroni is a potential metabolic biomarker.)

1. A metabolic biomarker in the aging process of Sepiella maindroni, which is characterized in that: which comprises uric acid.

2. A metabolic biomarker according to claim 1, characterised in that: the pre-egg concentration/post-egg concentration of uric acid in the aging process of Sepiella maindroni is 2.16.

3. A metabolic biomarker according to claim 1, characterised in that: VIP ═ 1.5385 of said uric acid.

4. A method of screening for metabolic biomarkers according to claim 1 or 2 or 3, characterized by: the method comprises the following steps:

collecting serum samples of Sepiella maindroni individuals at different reproductive stages in the aging process;

pre-treating the serum sample;

sequentially carrying out liquid chromatography-mass spectrometry combined analysis on the serum sample;

screening the liquid chromatography-mass spectrometry data processing to screen out metabolic biomarkers;

comparing and assigning the metabolic biomarkers;

and constructing a metabolic pathway of the biomarker and analyzing.

5. The screening method according to claim 4, wherein: the Sepiella maindroni individuals are selected from the group consisting of before sexual maturity and when spawning, and when dying after spawning.

6. The screening method according to claim 4, wherein: the pretreatment comprises the following specific steps: adding 2-4 times volume of acetonitrile into the serum sample, mixing for 1-3min, standing at 4 deg.C for 5-15min, centrifuging at 10000-.

7. The screening method according to claim 4, wherein: the liquid chromatography conditions are as follows: adopting an ACQUITYUPLC liquid chromatography system of Waters company; a chromatographic column: WatersACQUITYTTMUPLCHSST 3 column, 2.1mm × 100mm, 1.8 μm, 40 deg.C; the mobile phase A is 0.1% formic acid water solution, and the mobile phase B is 0.1% formic acid-acetonitrile solution.

8. The screening method according to claim 4, wherein: the mass spectrum conditions are as follows: a Waters SYNAPT G2-S mass spectrometer was used; collection quality range: 100-1500 Da; an acquisition mode: measuring positive and negative ions; capillary voltage: 3(ESI +); 2.5 (ESI-); taper hole voltage: 100V; collision energy: 6-45 eV; temperature of ionization source: 120 ℃; temperature of the liquid removing agent: 500 ℃; taper hole air flow rate: 60L/h.

9. The screening method according to claim 4, wherein: the relative concentration of the uric acid is 4333.277 before sexual maturity and spawning; 2188.035 when spawning; 2001.929 before and after spawning.

10. Use of the metabolic biomarker of claim 1, 2 or 3 for predicting and/or assessing sepiella maindroni aging.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a metabolic biomarker in the process of Sepiella maindroni aging.

Background

Sepiella maindroni (Sepiella japonica) belongs to the phylum mollusca, class cephalopoda, order decacarpales, family Sepiellae and genus Sepiella, is an important economic species in the east China sea, and has high edible, medicinal and economic values. The life cycle of Sepiella maindroni is generally only one year, and the resource amount of the Sepiella maindroni is composed of the supplement population in the current year. However, since the middle of the 70's of the 20 th century, the resources of sepiella maindroni in our country have been greatly destroyed or even have tended to be exhausted due to the deterioration of environmental conditions and the overfishing of fishermen. In order to quickly recover the Sepiella maindroni resources in China and carry out resource repair work, the strength of artificially culturing the Sepiella maindroni is increased, and the propagation and releasing of seeds are carried out, so that the valuable natural resources are recovered to a certain extent and become an important task at present. Sepiella maindroni can mate and lay eggs after gonads develop and mature, female bodies after laying eggs are thin and weak, and die in succession soon, and some dead female ovaries still have immature ova with different sizes. In the egg laying period of cultured Sepiella maindroni, the female Sepiella maindroni is found to have great egg laying energy consumption and high death speed, and a large amount of eggs are left in the abdomen during death and are not produced. The Sepiella maindroni has lower fecundity than fishes and shrimps, the average fecundity is only 1800 grains, the egg laying amount is only 40%, a large amount of eggs are remained in the ovary when the parents die and are not produced, the huge waste of the eggs is caused, the egg laying amount is seriously reduced, the propagation of the Sepiella maindroni resources is seriously hindered, and the Sepiella maindroni population can not be effectively supplemented. If the aging of the cuttlefish can be delayed, the smooth hatching of fertilized eggs of the cuttlefish is ensured, and the fertilized eggs of the cuttlefish can die at least after the eggs are discharged, so that the aims of improving the culture yield of the cuttlefish and maintaining the cuttlefish resources can be fulfilled. Under the condition of the current Sepiella maindroni resource failure, the protection and repair of the Sepiella maindroni become more and more gradual, and the problem how to improve the oviposition amount of the Sepiella maindroni, delay the aging and prolong the service life of the Sepiella maindroni is the most urgent problem to be solved. However, no relevant research report of Sepiella maindroni exists at home and abroad at present. If the method can be used for researching the improvement of the egg laying amount and the aging mechanism of the Sepiella maindroni, great influence is inevitably generated in the Sepiella maindroni culture industry, and the method has great significance for the development of artificial breeding and proliferation and release industries. During the different stages of Sepiella maindroni aging, the content of certain metabolites in the serum can change significantly, and the change indicates the aging degree, and the metabolites can also be used as potential biomarkers in the aging process.

Disclosure of Invention

An object of the present invention is to provide a metabolic biomarker in the aging process of sepiella maindroni.

The technical scheme adopted by the invention for realizing the purpose is as follows: a metabolic biomarker in the aging process of sepiella maindroni, comprising uric acid.

Preferably, the pre-egg content/post-egg content of uric acid in the aging process of sepiella maindroni is 2.16.

Preferably, the VIP (weight variation importance ranking) of uric acid is 1.5385. The weight variation importance ranking (VIP) value is a variable importance factor. The higher the VIP value, the higher the contribution rate of the metabolites, and generally the VIP > 1 is considered to have statistical significance, which indicates that uric acid is a metabolic biomarker in the aging process of Sepiella maindroni.

Still another object of the present invention is to provide a method for screening the metabolic biomarkers, comprising:

collecting serum samples of Sepiella maindroni individuals at different reproductive stages in the aging process;

pre-treating the serum sample;

sequentially carrying out liquid chromatography-mass spectrometry combined analysis on the serum sample;

screening the liquid chromatography-mass spectrometry data processing to screen out metabolic biomarkers;

comparing and assigning metabolic biomarkers;

metabolic pathways for biomarkers were constructed and analyzed.

Preferably, the individual Sepiella maindroni is selected from the group consisting of pre-oviposition, and dying after oviposition in sexual maturity.

Preferably, the pretreatment comprises the following specific steps: adding 2-4 times volume of acetonitrile into the serum sample, mixing for 1-3min, standing at 4 deg.C for 5-15min, centrifuging at 10000-.

Preferably, the liquid chromatography conditions are: adopting an ACQUITYUPLC liquid chromatography system of Waters company; a chromatographic column: WatersACQUITYTTMUPLCHSST 3 column, 2.1mm × 100mm, 1.8 μm, 40 deg.C; the mobile phase A is 0.1% formic acid water solution, and the mobile phase B is 0.1% formic acid-acetonitrile solution.

Preferably, the mass spectrometry conditions are: a Waters SYNAPT G2-S mass spectrometer was used; collection quality range: 100-1500 Da; an acquisition mode: measuring positive and negative ions; capillary voltage: 3(ESI +); 2.5 (ESI-); taper hole voltage: 100V; collision energy: 6-45 eV; temperature of ionization source: 120 ℃; temperature of the liquid removing agent: 500 ℃; taper hole air flow rate: 60L/h.

Preferably, the relative concentration of uric acid is 4333.277 before sexual maturation and oviposition; 2188.035 when spawning; 2001.929 before and after spawning.

Still another object of the present invention is to provide a use of the above metabolic biomarker for predicting and/or evaluating sepiella maindroni aging.

Compared with the prior art, the invention has the beneficial effects that: the serum of the sepiella maindroni at different reproductive stages is subjected to micro metabonomics analysis by a high performance liquid chromatography-tandem mass spectrometry technology, a metabolic biomarker in the aging process of the sepiella maindroni is searched, and compared with the serum before spawning, the content of Uric acid (Uric acid) is greatly reduced after spawning, the content before spawning/the content after spawning is 2.16, and VIP (weight variation importance ranking) is 1.5385, so that the serum is a potential metabolic biomarker and can be used for predicting the aging of the sepiella maindroni.

The metabolic biomarker in the sepiella maindroni aging process is provided by adopting the technical scheme, so that the defects of the prior art are overcome, the design is reasonable, and the operation is convenient.

Drawings

FIG. 1 shows the results of the LC-MS data normalization process in example 1 of the present invention;

FIG. 2 is a result of one-way analysis of variance in example 1 of the present invention;

FIG. 3 is a dendrogram of cluster analysis in example 1 of the present invention;

FIG. 4 is a heat map of cluster analysis in example 1 of the present invention;

FIG. 5 is a score plot of the PCA analysis in example 1 of the present invention;

FIG. 6 is a score chart of PLS-DA analysis in example 1 of the present invention;

FIG. 7 is a diagram of metabolic pathways in example 1 of the present invention.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

The following further describes embodiments of the present invention with reference to specific examples.