Application of liver specific secretion factor FETUB in adjusting intramuscular fat content of chicken

Abstract

The invention discloses application of a liver specific secretion factor FETUB in adjusting the fat content in chicken muscle, and belongs to the technical field of poultry serum biochemical marking. The FETUB protein has significant difference in high intramuscular fat chicken and low intramuscular fat chicken, and can be used for identifying the intramuscular fat content of Gushi chicken and breeding Gushi chicken with high intramuscular fat. In addition, the invention further provides application and a screening method of the serum biochemical marker, and a new thought is provided for breeding work of Henan local chickens.

Description

Technical Field

[0001] The present invention relates to the technical field of avian serum biochemical markers, and particularly to the application of liver-specific secretory factor FETUB in regulating the intramuscular fat content of chickens. Background Art

[0002] Poultry meat is favored by consumers all over the world. However, the breeding for the weight gain of commercial broilers has led to a decrease in intramuscular fat content. Therefore, consumers prefer local chickens with better flavor. Intramuscular fat (IMF) refers to the fat deposited between muscle fibers and muscle bundles, and its content directly affects meat quality indicators such as meat color, flavor, juiciness, and tenderness, and is a key indicator for evaluating meat quality.

[0003] Gushi chickens originate from Gushi County, Henan Province and are a national geographical indication product in China. As a dual-purpose chicken for meat and eggs, Gushi chickens have excellent production performance and unique breed characteristics, and are one of the precious poultry breed resources in China. So far, there has been no research on using FETUB as a serum marker to detect the intramuscular fat content rate of Gushi chickens. To accelerate the improvement process of local chicken breeds in Henan Province, it is necessary to explore a new method for quickly breeding Gushi chickens with high intramuscular fat content, and to contribute to the breeding process of Gushi chickens in Henan by establishing serum biochemical markers.

[0004] The IMF content has a low to medium heritability, about 0.11 - 0.16, but selective breeding still significantly increases the IMF content, indicating that genetic methods are an effective way to improve IMF deposition. Therefore, analyzing the genetic basis and regulatory mechanism of chicken intramuscular fat deposition and developing molecular markers that can be used for breeding selection are of great significance for accelerating the genetic improvement of IMF content in chickens. Currently, the breeding for chicken intramuscular fat mainly relies on traditional methods. Researchers need to slaughter chickens after feeding them until the market time, and then use the Soxhlet extraction method to measure the intramuscular fat content in the muscle. Based on this, the generational breeding process is slow. Therefore, a more convenient and rapid breeding method is needed, which can not only avoid a long breeding cycle but also increase the intramuscular fat content, greatly improving the breeding process.

[0005] Poultry is different from mammals. The liver is the main site of lipid metabolism in chickens and is responsible for more than 90% of de novo fatty acid synthesis. As a bridge for signal communication between tissues, endocrine factors play important roles in biological processes such as maintaining the homeostasis of the energy, metabolism, and reproductive endocrine systems. As the main site of lipid synthesis in chickens, liver-secreted factors play important roles in regulating intramuscular fat deposition in chickens. Studies on mammals have shown that the liver, as an important endocrine organ of the body, can regulate lipid metabolism by producing and releasing endocrine factors that target adipose tissue. Fibroblast growth factor 21 (FGF21) is a member of the FGF superfamily of endocrine factors and is mainly produced in the liver. Hui et al. (2016) found that FGF21 exerts its regulatory effect on glucose and lipid metabolism in adipose tissue by binding to the complex receptor formed by the FGF receptor and β-klotho, promoting glucose uptake by adipocytes, enhancing mitochondrial oxidative capacity, enhancing PPARγ activity, and regulating lipolysis and thermogenic functions. Ghorpade et al. (2018) found that dipeptidyl peptidase-4 (DPP4) produced and secreted by hepatocytes acts on macrophages in adipose tissue through the bloodstream, activating the CAV1-IRAK1-TAK1 pathway, thereby promoting visceral adipose tissue inflammation and insulin resistance.

[0006] Fetuin B (FETUB) belongs to the cystatin superfamily of cysteine protease inhibitors. It is mainly synthesized and secreted by the liver and is involved in regulating physiological or pathological processes. It has been confirmed that it may be used as a serum biomarker for disease diagnosis. FETUB is involved in regulating insulin resistance, glucose metabolism, and hepatic steatosis. The research by Diao et al. (2016) showed that the expression level of FETUB in the serum of patients with chronic obstructive pulmonary disease was significantly higher than that in the healthy control group, and there was a significant correlation with the phenotypic features related to lung function, revealing that FETUB could be used as a serum biomarker for the diagnosis of chronic obstructive pulmonary disease. Zhan et al. (2020) confirmed through a gene gain-of-function strategy that the FETUB gene inhibits the activity of the PI3K / AKT signaling pathway, induces apoptosis, and inhibits the proliferation, migration, and invasion of prostate cancer cells, thereby inhibiting tumor growth. The research by Gao et al. (2022) found that the concentration of FETUB protein in the follicular fluid of patients with polycystic ovary syndrome was 2.978 times higher than that in the healthy population. FETUB can interact with transforming growth factor β receptor 2 (TGFR2) and restore the inhibitory effect of testosterone propionate on SMAD3 phosphorylation, thereby alleviating the oxidative stress and mitochondrial dysfunction induced by testosterone propionate in human ovarian granulosa cells, and ultimately inhibiting the secretion of estrogen and progesterone hormones, apoptosis, and oxidative stress ability. Meex et al. (2015) identified 168 hepatokines by proteomic methods, among which 32 were differentially expressed in steatotic and non-steatotic hepatocytes. They proposed that the protein signals of steatotic hepatocytes communicate with other cells to regulate the metabolic phenotype and identified FETUB as a hepatokine regulated by human steatosis. The research by Choi et al. (2012) found that the concentration of FETUB protein in the serum of obese mice induced by a high-fat diet was significantly higher than that in mice fed a normal diet. The research by Zhou et al. (2019) confirmed that the treatment with recombinant FETUB protein could increase the lipid accumulation in the liver caused by free fatty acids or a high-fat diet, reduce the phosphorylation level of AMPK, activate the LXR-SREBP1c pathway, reduce fatty acid oxidation, promote lipogenesis, and exacerbate hepatic steatosis; knocking down the FETUB gene produced the opposite results. These results indicate that FETUB, as a liver endocrine factor, can regulate various physiological processes of the body and has the potential to be used as a serum biomarker.

[0007] There is currently no report on whether FETUB can be used as a serum biomarker to predict the intramuscular fat content in chickens. Summary of the Invention

[0008] The purpose of the present invention is to provide the application of the liver-specific secreted factor FETUB in regulating the intramuscular fat content in chickens to solve the problems existing in the above-mentioned prior art.

[0009] To achieve the above purpose, the present invention provides the following solutions:

[0010] One of the technical solutions of the present invention is the application of the liver-specific secretion factor FETUB in regulating the intramuscular fat content of chickens.

[0011] Another technical solution of the present invention is a method for regulating the intramuscular fat content of chickens. Overexpressing the FETUB gene can increase the content of FETUB protein in the serum and thus increase the intramuscular fat content; reducing the expression of the FETUB gene can decrease the content of FETUB protein in the serum and thereby reduce the intramuscular fat content.

[0012] A further technical solution of the present invention is the application of the liver-specific secretion factor FETUB as a biomarker in identifying the intramuscular fat content of chickens.

[0013] Another technical solution of the present invention is the application of the liver-specific secretion factor FETUB as a biomarker in the preparation of products for identifying the intramuscular fat content of chickens.

[0014] Yet another technical solution of the present invention is the application of the liver-specific secretion factor FETUB in breeding chicken strains with low intramuscular fat content.

[0015] Another technical solution of the present invention is a method for breeding chicken strains with low intramuscular fat content, which includes the following steps: detecting the content of FETUB protein in the serum of individual chickens and selecting the individuals with low FETUB content in the chicken serum.

[0016] Based on the above technical solutions, the present invention has the following technical effects:

[0017] The serum biochemical marker provided by the present invention is the level of fetuin B (Fetuin B Protein, FETUB) in chicken serum. The specific steps are as follows: 1) Using an adeno-associated viral vector (AAV) to overexpress the FETUB gene in Gushi chickens and allowing them to freely eat and drink under the same feeding and management environment; 2) Measuring the serum FETUB concentration of the two groups of Gushi chickens; 3) Using the Soxhlet extraction method to measure the intramuscular fat content in the breast muscle and leg muscle of Gushi chickens. This index shows significant differences between chickens with high intramuscular fat content and those with low intramuscular fat content, and can be used for the identification of the intramuscular fat content of Gushi chickens and the breeding of Gushi chickens with high intramuscular fat content. In addition, the present invention also provides the application and screening method of this serum biochemical marker, providing new ideas for the breeding work of local chickens in Henan. Detailed Embodiments

[0018] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation embodiments of the present invention.

[0019] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0021] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are merely exemplary.

[0022] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0023] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are all purchased from commercial channels or have been made public.

[0024] The embodiments of the present invention provide the application of the liver-specific secretory factor FETUB in regulating the intramuscular fat content of chickens.

[0025] In some specific embodiments, overexpressing the FETUB gene increases the content of FETUB protein in the serum and increases the intramuscular fat content; reducing the expression of the FETUB gene reduces the content of FETUB protein in the serum and reduces the intramuscular fat content.

[0026] The embodiments of the present invention also provide a method for regulating the intramuscular fat content of chickens. Overexpressing the FETUB gene increases the content of FETUB protein in the serum and increases the intramuscular fat content; reducing the expression of the FETUB gene reduces the content of FETUB protein in the serum and reduces the intramuscular fat content.

[0027] The embodiments of the present invention also provide the application of the liver-specific secreted factor FETUB as a biomarker in identifying the intramuscular fat content of chickens.

[0028] In some specific embodiments, the content of FETUB protein in the serum of individual chickens is detected. The individuals with a low FETUB protein content have a lower intramuscular fat content than those with a high FETUB protein content.

[0029] The embodiments of the present invention also provide the application of the liver-specific secreted factor FETUB as a biomarker in the preparation of products for identifying the intramuscular fat content of chickens.

[0030] The embodiments of the present invention also provide the application of the liver-specific secreted factor FETUB in cultivating chicken lines with low intramuscular fat content.

[0031] The embodiments of the present invention also provide a method for cultivating chicken lines with low intramuscular fat content, including the following steps: detecting the content of FETUB protein in the serum of individual chickens, and selecting and retaining the individuals with a low FETUB content in the chicken serum.

[0032] Example 1

[0033] 1 Experimental materials

[0034] The experimental subjects (8-week-old Gushi chickens) were from the Yuanyang Germplasm Resource Farm of Henan Agricultural University and were allowed to freely eat and drink under the same feeding and management environment.

[0035] AAV-NC (liver-specific recombinant adeno-associated virus empty vector), AAV-FETUB (liver-specific recombinant adeno-associated virus vector of FETUB gene) were purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.; TG enzyme-linked immunosorbent assay kit, FETUB enzyme-linked immunosorbent assay kit were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

[0036] Main instruments: centrifuge, electronic scale, blood collection needle, vacuum blood collection tube.

[0037] 2 Experimental methods

[0038] 2.1 Selection of experimental individuals

[0039] Randomly select 12 8-week-old Gushi chickens, and randomly divide them into an experimental group and a control group (6 in each group), and perform in-situ liver injection of AAV-FETUB (experimental group) and AAV-EGFP (control group) respectively.

[0040] 2.2 Feeding method

[0041] The chicken flocks were all raised in the experimental chicken houses of Henan Agricultural University and were allowed to freely eat and drink under the same conditions. The nutritional levels during the raising period of the chicken flocks were as follows: the energy level was 12.40 MJ / kg, the protein level was 20.0%, the crude fiber level was 6.0%, and the crude ash level was 8.0%.

[0042] 2.3 Slaughter standards for experimental individuals

[0043] The slaughter experiment was completed in the laboratory of Henan Agricultural University. The body weights of the chicken flocks were measured weekly. After significant differences in body weights between the experimental group and the control group were observed, the chickens were slaughtered. After slaughter, the intramuscular fat contents of the breast muscles and leg muscles were determined using the Soxhlet extraction method.

[0044] 2.4 Determination of serum biochemical indexes

[0045] The operation was carried out according to the instructions of the chicken FETUB enzyme-linked immunosorbent assay (ELISA) kit. Then, the absorbance (OD) value was measured using an enzyme-labeled instrument at a wavelength of 450 nm. After drawing the standard curve, the FETUB content was calculated.

[0046] 2.5 Result statistics

[0047] Table 1 Comparison of FETUB contents in serum

[0048]

[0049] Table 1 shows the comparison of FETUB contents in the serum of the experimental group and the control group after slaughter. As can be seen from Table 1, after the in-situ injection of AAV-FETUB / AAV-EGFP into the chicken liver, the FETUB content in the serum of the experimental group increased significantly. Therefore, FETUB was successfully overexpressed in the chicken liver and the secretion amount of its encoded protein increased significantly.

[0050] Table 2 Statistics of body weight changes after injecting adeno-associated virus

[0051]

[0052] Table 2 shows the body weight measurement of 12 chickens in the experimental group and the control group after slaughter. As can be seen from Table 2, the average body weight of the experimental group was significantly higher than that of the control group. Under the conditions of the same feeding environment, feed, and drinking water, the experimental group showed higher feed conversion rates and body weight gains, indicating that FETUB promotes the body weight growth of chickens.

[0053] Table 3 Comparison of body fat traits of Gushi chickens after slaughter

[0054]

[0055] Table 3 shows the comparison of body fat traits between the experimental group and the control group of 12 Gushi chickens after slaughter. As can be seen from Table 3, the experimental group showed a lower intramuscular fat content (P < 0.01). It indicates that a high level of FETUB in the blood circulation of Gushi chickens is significantly correlated with a low intramuscular fat content.

[0056] The inventors also constructed an FETUB gene silencing vector. Twelve 8-week-old Gushi roosters were randomly selected and the FETUB gene silencing vector was injected in situ into the liver. After feeding according to the above method, the experimental individuals were slaughtered according to the standard and the serum biochemical indexes were measured. The results showed that after the in situ injection of the FETUB gene silencing vector into the chicken liver, the content of FETUB in the serum of the experimental individuals decreased significantly, the body weight decreased significantly, and the intramuscular fat content increased significantly. It shows that a low level of FETUB in the blood circulation of Gushi chickens is significantly correlated with a high intramuscular fat content.

[0057] Example 2

[0058] Fifteen sera each of Gushi hens with high and low intramuscular fat content at 43 weeks of age were selected, and the FETUB content in the two groups of sera was measured using an FETUB enzyme-linked immunosorbent assay kit. The results are shown in Table 4.

[0059] Table 4 Comparison of FETUB content in the Gushi chicken population

[0060]

[0061] Table 5 Comparison of the liver specific gravity of Gushi chickens

[0062]

[0063] As can be seen from Table 4, the FETUB content was detected in the sera of the 43-week-old Gushi hen population. The results showed that there were significant differences in FETUB between the chicken populations with high and low intramuscular fat content and there were no differences in the liver specific gravity between the two populations, indicating that there is a negative correlation between the FETUB content and the intramuscular fat content of chickens.

[0064] In summary, FETUB can be used as a serum biomarker for predicting the intramuscular fat content of chickens. According to the FETUB serum content, the individual chickens to be tested can be selected and bred to screen out excellent varieties. Specifically, individuals with a low FETUB content in the chicken serum are selected to increase the intramuscular fat content of chickens and improve the economic performance; individuals with a high FETUB content in the serum are eliminated.

[0065] Obviously, the above embodiments of the present invention are merely examples given to clearly illustrate the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims

1. Application of liver-specific secretory factor FETUB in regulating intramuscular fat content of chickens.

2. The application according to claim 1, characterized in that, Overexpressing the FETUB gene increases the content of FETUB protein in serum and increases intramuscular fat content; reducing the expression of the FETUB gene decreases the content of FETUB protein in serum and decreases intramuscular fat content.

3. A method for regulating intramuscular fat content in chickens, characterized in that, Overexpressing the FETUB gene increases the content of FETUB protein in serum and increases intramuscular fat content; reducing the expression of the FETUB gene decreases the content of FETUB protein in serum and decreases intramuscular fat content.

4. Application of liver-specific secretory factor FETUB as a biomarker in identifying intramuscular fat content of chickens.

5. The application according to claim 4, wherein Detecting the content of FETUB protein in the serum of individual chickens, the intramuscular fat content of individuals with low FETUB protein content is lower than that of individuals with high FETUB protein content.

6. Application of liver-specific secretory factor FETUB as a biomarker in the preparation of products for identifying intramuscular fat content of chickens.

7. Application of liver-specific secretory factor FETUB in breeding chicken lines with low intramuscular fat content.

8. A method for cultivating a chicken strain with low intramuscular fat content, characterized in that, It includes the following steps: detecting the content of FETUB protein in the serum of individual chickens and selecting individuals with low FETUB content in chicken serum.

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