Application of liver specific secretion factor FETUB in regulation of abdominal fat percentage of chicken

Abstract

The invention discloses application of a liver specific secretion factor FETUB in regulation of chicken abdominal fat percentage, and belongs to the technical field of poultry serum biochemical marking. The liver specific secretion factor FETUB has significant difference between high abdominal fat rate chickens and low abdominal fat rate chickens, and can be used for identifying the abdominal fat amount of Gushi chickens and breeding low abdominal fat series Gushi chickens. 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 secretion factor FETUB in regulating the abdominal fat rate of chickens. Background Art

[0002] Poultry meat is the second largest consumed meat in China after pork. In recent decades, commercial chickens bred through genetic selection for high growth rate and carcass yield have made great progress in meat production rate and shortening the slaughter cycle. However, at the same time, this has led to a significant increase in the abdominal fat rate of commercial chickens. Excessive abdominal fat can cause an increase in bone diseases, metabolic diseases, and mortality, thus having an adverse impact on carcass weight, feed utilization rate, age at first laying, egg production, meat quality, etc.

[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 abdominal fat 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 a low abdominal fat type, and contribute to the breeding process of Gushi chickens in Henan by establishing serum biochemical markers.

[0004] The abdominal fat weight and abdominal fat rate of chickens are direct indicators for evaluating the degree of abdominal fat deposition, with heritability of approximately 0.62 and 0.24 respectively, belonging to high heritability traits, indicating that genetic selection is an effective way to improve excessive abdominal fat deposition in chickens. Currently, the breeding direction for improving the abdominal fat rate of chickens mainly relies on traditional phenotypic selection. People need to measure the body weight and abdominal fat weight after slaughtering the chickens after feeding for a certain period to calculate the abdominal fat rate of the chickens, and the breeding process of generations is slow. Therefore, a more convenient and fast breeding method is needed, which can not only shorten the breeding generation interval but also reduce the abdominal fat rate, 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. Fatty acids are converted into lipid substances such as triglycerides in the liver. Then, the lipid substances are assembled by apolipoproteins to form very low density lipoprotein particles (VLDL) and secreted into the blood, transported to the abdomen through the peripheral vascular system. After being decomposed by lipoprotein lipase (LPL) in tissues to generate fatty acids, they are taken up by adipocytes and re-esterified to form triglyceride (TG), and stored and accumulated in the form of lipid droplets to form abdominal fat. Multiple serum substances are involved in the regulation and participation in this process. Among them, endocrine factors released from tissue cells into the blood circulation are the core mediators of inter-tissue communication. As a bridge for signal communication between tissues, endocrine factors play an important role in biological processes such as maintaining the homeostasis of the energy, metabolism, and reproductive endocrine systems. The liver is the main site of lipid synthesis in chickens, and liver-secreted factors play an important role in regulating abdominal fat deposition in chickens.

[0006] Fetuin B (FETUB) belongs to the cystatin superfamily of cysteine protease inhibitors, is mainly synthesized and secreted by the liver, and is related to the regulation of 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. Meex et al. (2015) identified 168 liver factors by proteomic methods, among which 32 were differentially expressed in steatotic and non-steatotic hepatocytes, suggesting that the protein signals of steatotic hepatocytes communicate with other cells to regulate the metabolic phenotype, and identified FETUB as a hepatocyte factor regulated by human steatosis. The level of FETUB is positively correlated with the content of TG in the human body, and the level of FETUB in obese mice induced by a high-fat diet, patients with non-alcoholic fatty liver, and diabetic patients is significantly higher than that in mice and humans with normal body types. The serum FETUB protein concentration in patients with hepatic steatosis and type II diabetes is significantly higher than that in healthy people, and is positively correlated with insulin resistance in rodents. FETUB can impair the insulin activity of myotubes and hepatocytes and cause glucose intolerance in mice; knocking down the FETUB gene improves glucose metabolism in obese mice. These results indicate that FETUB, as a liver endocrine factor, can regulate various physiological and pathological processes in humans.

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

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

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

[0010] One of the technical solutions of the present invention is the application of the liver-specific secretory factor FETUB in regulating the abdominal fat rate of chickens.

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

[0012] A third technical solution of the present invention is the application of the liver-specific secretory factor FETUB as a biomarker in identifying the abdominal fat rate of chickens.

[0013] A fourth technical solution of the present invention is the application of the liver-specific secretory factor FETUB as a biomarker in the preparation of products for identifying the abdominal fat rate of chickens.

[0014] A fifth technical solution of the present invention is the application of the liver-specific secretory factor FETUB in breeding chicken strains with a low abdominal fat rate.

[0015] A sixth technical solution of the present invention is a method for breeding chicken strains with a low abdominal fat rate, which includes the following steps: detecting the content of FETUB protein in the serum of individual chickens and selecting individuals with a 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) Overexpressing the FETUB gene in Gushi chickens using an adeno-associated viral vector (AAV) and allowing free feeding and drinking under the same feeding management environment; 2) Measuring the concentrations of FETUB and triglyceride (TG) in the serum of the two groups of Gushi chickens; 3) Measuring the body weight and abdominal fat weight of Gushi chickens and calculating the abdominal fat rate. This index shows significant differences between chickens with a high abdominal fat rate and those with a low abdominal fat rate, and can be used for the identification of abdominal fat in Gushi chickens and the breeding of Gushi chickens with a low abdominal fat line. In addition, the present invention also provides the application and screening method of this serum biochemical marker, providing a new idea for the breeding work of local chickens in Henan. Detailed Embodiments

[0018] The various exemplary embodiments of the present invention will be described in detail. In the embodiments, unless otherwise specified, the methods are all conventional methods, and the reagents, unless otherwise specified, are all conventional commercially available reagents or reagents prepared by conventional methods. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0019] It should be understood that the terms used in the present invention are only for describing particular 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. Any 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 can be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation 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 embodiments of the present invention 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 publicly disclosed.

[0024] The embodiments of the present invention provide the application of the liver-specific secretion factor FETUB in regulating the abdominal fat rate of chickens.

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

[0026] An embodiment of the present invention also provides a method for regulating the abdominal fat rate of chickens. Overexpressing the FETUB gene can increase the content of FETUB protein in the serum and increase the abdominal fat rate of chickens; reducing the expression of the FETUB gene can reduce the content of FETUB protein in the serum and reduce the abdominal fat rate of chickens.

[0027] An embodiment of the present invention also provides the application of the liver-specific secretion factor FETUB as a biomarker in the identification of the abdominal fat rate of chickens.

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

[0029] An embodiment of the present invention also provides the application of the liver-specific secretion factor FETUB as a biomarker in the preparation of products for identifying the abdominal fat rate of chickens.

[0030] An embodiment of the present invention also provides the application of the liver-specific secretion factor FETUB in the cultivation of chicken strains with a low abdominal fat rate.

[0031] An embodiment of the present invention also provides a method for cultivating chicken strains with a low abdominal fat rate, including the following steps: detecting the content of FETUB protein in the serum of individual chickens, and selecting and retaining 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 roosters) 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-EGFP (a liver-specific recombinant adeno-associated virus empty vector with an EGFP tag), AAV-FETUB (a liver-specific recombinant adeno-associated virus vector for the FETUB gene) were purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.; TG enzyme-linked immunosorbent assay kit; FETUB enzyme-linked immunosorbent assay kit was 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 roosters, and randomly divide them into an experimental group and a control group (6 each), 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 house of Henan Agricultural University and were allowed to freely feed and drink under the same conditions. The nutritional levels during the feeding 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 standard 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. When there was a significant difference in body weight between the experimental group and the control group, slaughter was carried out. After slaughter, the abdominal fat weight was measured and the abdominal fat rate was calculated.

[0044] 2.4 Determination of serum biochemical indexes

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

[0046] 2.5 Result statistics

[0047] Table 1 Comparison of biochemical indexes in serum

[0048]

[0049] Table 1 shows the comparison of biochemical indexes 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 content of FETUB in the serum of the experimental group increased significantly. Therefore, FETUB was successfully overexpressed in the chicken liver and the secretion of its encoded protein increased significantly. And the significant increase in the level of TG in the circulation can provide sufficient substrates for adipocytes. Then, TG is decomposed into fatty acids by LPL, and after being ingested by adipocytes, more and larger lipid droplets will be formed.

[0050] Table 2 Statistics of body weight changes after injection of 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 rate and body weight gain, indicating that FETUB promotes the body weight gain of chickens.

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

[0054]

[0055] Table 3 is a comparison of the body fat traits of 12 Gushi chickens in the experimental group and the control group after slaughter. As shown in Table 3, the experimental group showed higher abdominal fat weight and abdominal fat rate (P < 0.01).

[0056] The inventors also constructed a FETUB gene silencing vector, randomly selected 12 8-week-old Gushi roosters, injected the FETUB gene silencing vector in situ into the liver, and raised them according to the above method. The test individuals were slaughtered according to the standard and serum biochemical indicators were measured. The results showed that after the FETUB gene silencing vector was injected in situ into the chicken liver, the FETUB content in the serum of the test individuals was significantly reduced, and the body weight and abdominal fat rate were also significantly reduced. This shows that low levels of FETUB in the blood circulation of Gushi chickens are significantly correlated with low abdominal fat rates.

[0057] Example 2

[0058] 16 sera from Guangxi Yao chickens with high and low abdominal fat rates were selected, and the FETUB content of the two groups of sera was determined using the FETUB enzyme-linked immunosorbent assay kit. The results are shown in Table 4.

[0059] Table 4 FETUB content in serum of Guangxi Yao chicken

[0060]

[0061] Table 5 Specific Gramm of Guangxi Yao Chicken Liver

[0062]

[0063] As shown in Table 4, the FETUB content was detected in the serum of Guangxi Yao chicken groups. The results showed that there were significant differences in FETUB between chicken groups with high and low abdominal fat rates, and there was no difference in liver proportion between the two groups, indicating that there was a positive correlation between FETUB content and chicken abdominal fat rate.

[0064] In summary, FETUB can be used as a serum marker to predict the abdominal fat rate of chickens. According to the FETUB serum content, the chickens to be tested can be selected and bred to select excellent breeds. Specifically, individuals with low FETUB content in chicken serum can be selected to reduce the abdominal fat rate of chickens and improve economic performance; individuals with high FETUB content in serum can be eliminated.

[0065] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating 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 replacements, 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 chicken abdominal fat rate.

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

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

4. Application of liver-specific secretory factor FETUB as a biomarker in identifying chicken abdominal fat rate.

5. The application according to claim 4, wherein Detecting the content of FETUB protein in the serum of individual chickens, the abdominal fat rate 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 chicken abdominal fat rate.

7. Application of liver-specific secretory factor FETUB in breeding chicken strains with low abdominal fat rate.

8. A method for cultivating a chicken strain with a low abdominal fat percentage, 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 the chicken serum.

Images