Application of igfbp1 gene in regulating chicken fat deposition traits

Abstract

The application provides application of an IGFBP1 gene in regulating chicken fat deposition traits, and belongs to the technical fields of biotechnology and genetic breeding. The application constructs a tissue-specific IGFBP1 recombinant adeno-associated virus vector, and uses the tissue-specific IGFBP1 recombinant adeno-associated virus vector to promote expression of the IGFBP1 gene of a chicken, promotes expression of the IGFBP1 gene of the chicken by promoting expression of the IGFBP1 gene of the chicken, increases the content of IGFBP1 protein in serum of the chicken, and further increases the content of intramuscular fat of the chicken, reduces the weight of abdominal fat of the chicken and the abdominal fat rate of the chicken. Through long-term experimental research, it is found that the chicken IGFBP1 can significantly affect the differential deposition of chicken IMF and AbF, the expression level of serum IGFBP1 protein is significantly negatively correlated with the weight of abdominal fat and the abdominal fat rate of the chicken, and is significantly positively correlated with the content of intramuscular fat of the chicken, and can be used as a basis for a serum judgment index of chicken fat deposition traits, and can also be used for screening chicken breeds with high intramuscular fat and low abdominal fat rate.

Description

Technical Field

[0001] The invention belongs to the fields of biotechnology and genetic breeding, and particularly relates to application of an IGFBP1 gene in regulating chicken fat deposition traits. Background Art

[0002] Intramuscular fat (IMF) content in livestock and poultry plays a crucial role in determining muscle tenderness, water-holding capacity, and flavor, and is a key indicator of meat quality. Excessive abdominal fat (AbF) deposition not only reduces feed efficiency, carcass yield, and reproductive performance, but also increases the difficulty of meat processing. Therefore, increasing IMF content and reducing AbF deposition are urgent issues for breeders. Applying modern breeding techniques using molecular markers to selectively select for differential fat deposition in chickens can effectively improve breeding efficiency.

[0003] Adipogenesis in animals is a highly organized and complex process involving proliferation, differentiation, and evolution, and is strictly regulated. Adipose tissue development is mediated by multiple factors, including environmental and genetic factors. Numerous functional genes or gene families participate in this regulatory process, among which the insulin-like growth factor (IGF) system plays a crucial biological role. The IGF system, comprised of IGF1 and 2 (IGF1 and IGF2), IGF1 and 2 receptors (IGF1R and IGF2R), IGF-binding proteins 1-6 (IGFBP1-6), IGF2-binding proteins 1-3 (IGF2BP1-3), and IGFBP-related proteins 1-10 (IGFBP-rP1-10), mediates adipogenesis and differentiation, among other biological functions. IGFBPs are a family of proteins with high affinity for IGFs (IGF1 and IGF2), regulating their bioavailability and also influencing related biological functions independently of IGFs.

[0004] Research has shown that IGFBP1 is involved in the molecular regulation of glucose metabolism and lipid deposition, with serum IGFBP1 levels negatively correlated with total fat mass. It has also been identified as a potential target for blood protein analysis in obesity. However, IGFBP1 has not yet been studied or applied as a serum marker for assessing fat deposition in chickens, either domestically or internationally. Summary of the Invention

[0005] The purpose of the present invention is to provide a scheme capable of evaluating the fat deposition traits of chickens, to obtain poultry varieties with high IMF content and low AbF deposition, and to expand the application of IGFBP1.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides application of IGFBP1 gene in regulating chicken fat deposition traits.

[0008] Preferably, the method for improving the fat deposition traits of chickens is: the method for regulating the fat deposition traits of chickens is: constructing a tissue-specific IGFBP1 recombinant adeno-associated virus vector, and using the tissue-specific IGFBP1 recombinant adeno-associated virus vector to promote the expression of the IGFBP1 gene in chickens, thereby increasing the IGFBP1 protein content in chicken serum by promoting the expression of the chicken IGFBP1 gene, thereby increasing the intramuscular fat content of the chicken and reducing the abdominal fat weight and abdominal fat rate of the chicken.

[0009] Preferably, the chicken is 17 to 31 weeks old.

[0010] Preferably, the dosage form of the tissue-specific IGFBP1 recombinant adeno-associated virus vector is an injection, with an injection volume of 280-320 μL / chicken, (0.8-1.2)×10 12 vg / mL titer.

[0011] The present invention also provides the use of IGFBP1 protein in evaluating chicken fat deposition traits.

[0012] The present invention also provides the use of IGFBP1 in screening chicken breeds with high intramuscular fat and low abdominal fat rates.

[0013] Beneficial effects

[0014] After a long period of experimental research, the present invention found that chicken IGFBP1 can significantly affect the differential deposition of chicken IMF and AbF. The expression level of serum IGFBP1 protein was significantly correlated with the abdominal fat weight (21W, R = -0.538, P = 0.026; 31W, R = -0.670, P = 0.003) and abdominal fat rate (21W, R = -0.521, P = 0.001; 31W, R = -0.612, P = 9.40 × 10 -5 ) was significantly negatively correlated with the intramuscular fat content (TG) at 21 weeks of age (R=0.728, P=0.001). It can not only be used as a basis for serum judgment indicators of chicken fat deposition traits, but also can be used to screen chicken breeds with high intramuscular fat and low abdominal fat rates.

[0015] In summary, the present invention enriches the basis for serum judgment indicators of chicken fat deposition traits and provides a new marker for screening chicken breeds with high intramuscular fat and low abdominal fat rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Serum IGFBP1 content and its effect on abdominal fat weight, abdominal fat rate and intramuscular fat content in hens;

[0017] Figure 2 To analyze the correlation between serum IGFBP1 content and abdominal fat weight, abdominal fat rate and intramuscular fat content (TG) of hens;

[0018] Figure 3 The correlation between serum IGFBP1 content and abdominal fat weight, abdominal fat rate and intramuscular fat content (TG) was verified for high and low abdominal fat weight (rate) groups. DETAILED DESCRIPTION

[0019] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] Example 1 Construction of tissue-specific IGFBP1 recombinant adeno-associated virus vector

[0021] 1. Enzyme digestion of pHBAAV-TBG-3flag-P2A-ZsGreen vector

[0022] Add each reagent in the order shown in the table below, gently pipette to mix, and place in a 37°C water bath for 1.5 hours. After the enzyme digestion is complete, perform agarose gel electrophoresis to recover the target fragment.

[0023] Table 1 Vector enzyme digestion system

[0024] Reagent Volume (μL) Vector DNA (1 ug / uL) 1 10* buffer 4 DdH2O 32 EcoRI 1.5 BamHI 1.5 total 40

[0025] 2. Acquisition of IGFBP1 gene CDS sequence

[0026] The CDS sequence information of the IGFBP1 gene (NM_001001294.1) was obtained from the NCBI online website (https: / / www.ncbi.nlm.nih.gov / ). The CDS sequence was amplified by PCR. The following system was prepared, gently mixed, and placed in a PCR instrument for reaction.

[0027] Table 2 Primers

[0028]

[0029] Table 3 PCR amplification system

[0030]

[0031]

[0032] Table 4 PCR program

[0033]

[0034] 3. Recombinant plasmid ligation, transformation and extraction

[0035] For the one-step cloning and ligation system, prepare the following reaction system in an ice-water bath. Incubate the ligation reaction at 50°C for 30 minutes, place on ice for 5 minutes, and then immediately transform.

[0036] Table 5 HB infusion™ one-step cloning and ligation system

[0037] Component Name Volume (μL) Gene fragment of interest 1 (100 ng / μL) Linearized vector 1 (50 ng / μL) 2 x HB infusion™ Master mix 10 ddH2O 8 Total volume 20

[0038] Cultivate with shaking at 37°C and 230 rpm for 50 minutes. After overnight culture on LB solid medium, select positive clones and perform colony PCR. Select positive clones corresponding to the correct band and inoculate them into LB medium containing the corresponding antibiotics and shake for 5 hours. The culture liquid is then sent to Shangya Co., Ltd. for further sequencing and identification. 500 μL of the culture liquid with a matching sequence is inoculated into 100 mL of LB liquid medium (containing 10 μg of ampicillin) and shaken for 14 hours. Plasmid DNA is then extracted from the culture liquid according to the instructions of the Tiangen Plasmid Mini (Medium) Extraction Kit.

[0039] 4. Tissue-specific IGFBP1 adeno-associated virus packaging

[0040] Passage AAV-293 cells into 100 mm dishes for transfection. Place in an incubator at 37°C, 5% CO2, and 95% relative humidity. Once the cells have reached approximately 80-90% confluency, transfection (5 μg AAV-IGFBP1 plasmid) can be performed. Preheat OptiMEM in a 37°C water bath. Bring Lipofectamine™ transfection reagent to room temperature before use. Shake thoroughly before use.

[0041] 6 hours after transfection, replace the culture medium with fresh complete medium containing 10% fetal bovine serum (FBS). 72 hours after transfection, gently scrape the cells containing AAV-IGFBP1 particles with a cell scraper and collect them in a 15 mL centrifuge tube. Centrifuge at 150 × g for 3 minutes to collect the cells. Remove the culture supernatant, wash once with PBS, and resuspend the cells in 300 μL of PBS. Prepare a 37°C water bath and liquid nitrogen. Repeat the freeze-thaw cycle three times in liquid nitrogen and a 37°C water bath. Centrifuge at 4°C at 2000 × g for 5 minutes to remove cell debris and collect the lysate containing AAV-IGFBP1 particles.

[0042] 5. Purification of tissue-specific IGFBP1 adeno-associated virus

[0043] Add 0.1 μL of Benonase per 1 mL of crude viral extract and incubate in a 37°C water bath for 1 hour to remove the cellular genome and residual plasmid DNA from the viral fluid. Centrifuge at 600 × g at 4°C for 10 minutes, and collect the supernatant. Column purification was performed using the Biomiga Adeno-Associated Virus Purification Kit V1469-01. 4 mL of AAV-IGFBP1 viral sample, obtained by column purification, was added to an ultrafiltration tube and centrifuged at 1400 × g for 30 minutes to obtain approximately 1 mL of AAV-IGFBP1. The resulting purified virus was collected and stored at -80°C.

[0044] Example 2 Animal Test

[0045] Gushi chickens were raised to 17 weeks of age after hatching. In this study, 52 healthy Gushi hens with similar body weights in the experimental and control groups at 17 weeks of age were selected. There were 23 chickens in the control group (AAV-NC) and 29 chickens in the experimental group (AAV-IGFBP1). All chickens were treated with 1×10 12 After in situ liver injection with adeno-associated virus (AAV) at a titer of vg / mL, rats were raised normally until 21 weeks of age, at which time 8 and 9 rats in the control and experimental groups, respectively, were slaughtered. At 31 weeks of age, 15 and 20 rats in the control and experimental groups, respectively, were slaughtered. Serum was collected from the subwing vein at both ages. Body weight was measured before slaughter, and abdominal fat weight was measured after slaughter. Breast muscle tissue was collected and stored at -80°C for the following experiments.

[0046] From a large group of Gushi hens (healthy group, 43 weeks), 8 and 10 healthy individuals with high and low abdominal fat weight (rate) were selected (abdominal fat rate exceeding 20% ​​of the average abdominal fat rate of the large group was considered high, and abdominal fat rate below 20% was considered low). Serum was collected from the subwing vein for separation, and breast muscle tissue was collected and stored at -80℃ for later use to further verify the correlation between serum IGFBP1 content and lipid deposition.

[0047] 1. Serum IGFBP1 level detection

[0048] According to the instructions of the Chicken IGFBP1 ELISA Kit (YJ33484), the absorbance (OD) of each sample was measured at a wavelength of 450 nm using an Infinite F50 enzyme-labeled analyzer. In an Excel worksheet, a linear regression curve equation (y = ax - b) was plotted with the standard concentration as the abscissa and the corresponding OD value as the ordinate. The IGFBP1 concentration in each sample serum was calculated according to the curve equation: y = 137.13x - 8.8512, R 2 =0.9995.

[0049] 2. Determination of muscle IMF content by Soxhlet extraction

[0050] Studies have shown that triglyceride (TG) is the main lipid component of intramuscular fat, and TG content can represent intramuscular fat content. The TG content of breast muscle was determined according to the instructions of the Tissue Cell Triglyceride Enzymatic Assay Kit (E1043). Tissue was lysed with 20 μL of lysis buffer per 1 mg of tissue. Protein in the tissue supernatant was quantified using the BCA Protein Assay Kit (P1511). An appropriate amount of supernatant was heated to 70°C for 10 minutes and centrifuged at 2000 rpm for 5 minutes at room temperature. The supernatant was then used for enzymatic assays. The absorbance (OD value) of each sample was measured at a wavelength of 550 nm using an Infinite F50 enzyme-labeled analyzer. In an Excel worksheet, a linear regression curve of the standard was plotted with the standard concentration as the horizontal axis and the corresponding OD value as the vertical axis. According to the curve equation y = 0.0005x - 0.0464, R 2 =0.9994 to calculate the TG content in each tissue sample.

[0051] Results: Compared with the AAV-NC group, serum IGFBP1 levels at 21 and 31 weeks of age were significantly increased after in situ liver injection of liver-specific adeno-associated virus (AAV-IGFBP1). Figure 1 A), and can significantly reduce the abdominal fat rate of hens in the two time periods ( Figure 1 B corresponds to 21 weeks and Figure 1 C corresponds to 31 weeks), and significantly promoted the intramuscular fat content at 21 weeks ( Figure 1 Correlation analysis showed that serum IGFBP1 levels were significantly negatively correlated with abdominal fat weight and abdominal fat rate at 21 and 31 weeks of age (R = -0.583, P = 0.026; R = -0.670, P = 0.003; R = -0.521, P = 0.001; R = -0.612, P = 9.42 × 10 -5 )( Figure 2 AD), and was significantly positively correlated with intramuscular fat content at 21 weeks of age (R=0.728, P=0.001) ( Figure 2 E).

[0052] In order to further verify the correlation between serum IGFBP1 content and lipid deposition, the abdominal fat weight (rate) difference individuals ( Figure 3 A), compared with individuals with low abdominal fat mass (rate), the serum IGFBP1 content and intramuscular fat content of individuals with high abdominal fat mass (rate) were significantly lower (P<0.01) ( Figure 3 B and C); at the same time, correlation analysis found that serum IGFBP1 content was significantly positively correlated with intramuscular fat content (R = 0.727, P = 6.37 × 10 -4), and abdominal fat weight and abdominal fat percentage were significantly negatively correlated (R = -0.490, P = 0.039; R = -0.487, P = 0.041) Figure 3 D-F).

[0053] In summary, IGFBP1 can significantly affect the differential deposition of chicken intramuscular fat and abdominal fat, is significantly correlated with the differential deposition of fat, and can be used as a candidate gene or genetic marker for chicken fat deposition traits.

[0054] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.

Claims

1. The application of IGFBP1 gene in regulating chicken fat deposition traits is characterized by: described IGFBP1 The sequence of the gene is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that The method for regulating the fat deposition traits of chickens is: constructing a tissue-specific IGFBP1 recombinant adeno-associated virus vector, and using the tissue-specific IGFBP1 recombinant adeno-associated virus vector to promote the expression of the IGFBP1 gene in chickens, thereby increasing the IGFBP1 protein content in the serum by promoting the expression of the chicken IGFBP1 gene, thereby increasing the chicken's intramuscular fat content and reducing the chicken's abdominal fat weight and abdominal fat rate.

3. The use according to claim 2, characterized in that The chickens are between 17 and 31 weeks old.

4. The use according to claim 3, characterized in that The dosage form of the tissue-specific IGFBP1 recombinant adeno-associated virus vector is an injection, with an injection volume of 280-320 μL / chicken, (0.8-1.2)×10 12 vg / mL titer.

5. Application of IGFBP1 protein in evaluating chicken fat deposition traits, characterized in that: The sequence of the gene encoding the IGFBP1 protein is shown in SEQ ID NO.

3.

6. The application of IGFBP1 in screening chicken breeds with high intramuscular fat and low abdominal fat rate is characterized in that: The sequence of the gene encoding the IGFBP1 protein is shown in SEQ ID NO.3.

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