Application of IGFBP1 gene in regulation or evaluation of reproductive performance of hens

Abstract

The invention provides application of an IGFBP1 gene in regulation or evaluation of reproductive performance of hens, and belongs to the field of biotechnology and genetic breeding. According to the invention, the tissue-specific IGFBP1 recombinant adeno-associated virus vector is constructed, and the tissue-specific IGFBP1 recombinant adeno-associated virus vector is utilized to promote the IGFBP1 gene expression of hens, so that the content of IGFBP1 protein in serum of hens is increased, and the reproductive performance of hens is further improved. Experiments prove that the IGFBP1 gene has influence on the egg laying performance and HPG axis hormone expression level of hens, can increase the total egg yield, ovary index, small white follicles and large white follicles of the hens and increase the contents of LH, E2, AMH and FSH in serum, can be used as a candidate gene or a genetic marker of egg laying traits of the hens, enriches serum judgment indexes of the reproductive performance of the hens, and has a good application prospect. A valuable reference basis is provided for differential diagnosis of the reproductive performance of the hens.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and genetic breeding, and in particular relates to the application of IGFBP1 gene in regulating or evaluating the reproductive performance of hens. Background Art

[0002] my country is the world's largest producer and consumer of eggs. Egg-laying traits are one of the important economic traits of poultry (chickens) and have always been a focus of attention in poultry breeding programs. Egg-laying traits are regulated by multiple genes and are complex traits with low heritability. Revealing the biological functions of related genes is conducive to the effective selection of poultry reproductive traits.

[0003] Egg-laying trait is the most important one in the reproductive performance of poultry (chicken). Its essence is the ovarian ovulation ability, which is regulated by the hypothalamus-pituitary-gonad axis (HPG axis) secreted into the peripheral circulation of multiple sex hormones. Therefore, at present, the serum indicators for evaluating the reproductive performance of hens are mostly concentrated on hormones regulated by the HPG axis, such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrogen (E2), prolactin (PRL), progesterone (PROG) and anti-Mullerian hormone (AMH). However, with the continuous development of sequencing technology and omics research, the molecules that affect the formation and regulation of egg-laying traits are constantly being explored, which not only enriches the candidate genes related to egg-laying traits, but also provides many new biomarkers for evaluating egg-laying traits.

[0004] The present invention also hopes to provide a new biomarker for evaluating poultry reproductive performance and assisting poultry breeding programs. Summary of the invention

[0005] The purpose of the present invention is to provide a biomarker capable of evaluating the reproductive performance of hens, which can be used to improve the reproductive performance of hens and expand the use of the IGFBP1 gene.

[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 improving reproductive performance of hens.

[0008] Preferably, the method for improving the reproductive performance of hens 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 hens, increase the IGFBP1 protein content in the hen serum, and thereby improve the reproductive performance of the hens.

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

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

[0011] Preferably, the improving the reproductive performance of hens includes improving the total egg production of hens, improving the ovarian index, increasing the number of small white follicles and large white follicles, increasing the levels of LH, E2, AMH and FSH in serum, and reducing the level of PRL in serum.

[0012] The invention provides application of IGFBP1 protein in screening hens with high reproductive performance.

[0013] The invention also provides application of IGFBP1 protein in evaluating reproductive performance of hens.

[0014] Beneficial Effects

[0015] After long-term experimental research, the present invention found that the IGFBP1 gene can significantly affect the egg-laying performance of hens and the expression level of HPG axis hormones. Overexpression of the IGFBP1 gene can significantly increase the egg production of hens, improve the ovarian index, and increase the number of small white follicles and large white follicles, which is helpful to improve the reproductive performance of hens. It can be used as a candidate gene or a genetic marker for egg-laying traits of chickens, providing a valuable reference basis for the differential diagnosis of hen reproductive performance, and enriching the basis for serum judgment indicators of hen reproductive performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 To compare the total egg production of hens in different groups;

[0017] Figure 2 This is a comparison of the average egg production of hens in different groups;

[0018] Figure 3 To compare the ovarian weight and ovarian index of hens in different groups;

[0019] Figure 4 Comparison of the number of pre-grade follicles in different groups of hens;

[0020] Figure 5 The comparison of serum IGFBP1 and reproductive hormone levels in different groups of hens;

[0021] Figure 6 To analyze the correlation between IGFBP1 content in serum of hens in different groups and the number of pre-ovarian follicles and reproductive hormone content. DETAILED DESCRIPTION

[0022] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0023] Example 1 Construction of tissue-specific IGFBP1 recombinant adeno-associated virus vector

[0024] 1. pHBAAV-TBG-3flag-P2A-ZsGreen vector digestion

[0025] Add each reagent in the order shown in Table 1, gently pipette to mix, and place in a 37°C water bath to react for 1 to 2 hours; after the enzyme digestion is completed, perform agarose gel electrophoresis to recover the target fragment.

[0026] Table 1 Vector restriction enzyme digestion system

[0027] Reagents Volume (μL) Vector DNA (1ug / uL) 1 10*buffer 4 <![CDATA[DdH2O]]> 32 EcoRI 1.5 B H 1.5 total 40

[0028] 2. Acquisition of IGFBP1 gene CDS sequence

[0029] According to the NCBI online website (https: / / www.ncbi.nlm.nih.gov / ), the CDS sequence information of the IGFBP1 gene (NM_001001294.1) was obtained, and the CDS sequence was amplified by PCR. The following system was prepared, gently mixed, and placed in a PCR instrument for reaction (primers and reaction system are shown in Tables 2 and 3, and the reaction program is shown in Table 4).

[0030] Table 2 Primers

[0031]

[0032] Table 3 PCR amplification system

[0033]

[0034]

[0035] Table 4 PCR program

[0036]

[0037] 3. Recombinant plasmid ligation, transformation and extraction

[0038] One-step cloning ligation system: prepare the following reaction system in an ice-water bath. After the ligation reaction solution reacts at 50°C for 30 minutes, it is placed on ice for 5 minutes and immediately transformed. The reaction system is shown in Table 5.

[0039] Table 5 HB infusion TM One-step cloning and ligation system

[0040] Component name Volume (μL) Target gene fragment 1(100ng / μL) Linearized vector 1(50ng / μL) 2×HBinfusionTMMastermix 10 <![CDATA[ddH2O]]> 8 Total volume 20

[0041] The culture was shaken at 37°C and 230 rpm for 50 min. After overnight culture on LB solid medium, positive clones were selected for colony PCR. The positive clones corresponding to the correct band were selected and inoculated into LB medium containing the corresponding antibiotics for 5 hours of shaking. The bacterial solution was sent to Shangya Co., Ltd. for further sequencing and identification. 500 μL of the bacterial solution with matching identification sequence was extracted and inoculated into 100 mL LB liquid medium (containing 10 μg ampicillin) for 14 hours of shaking. Plasmid DNA was extracted from the bacterial solution according to the instructions of Tiangen Plasmid Extraction Kit (DP103).

[0042] 4. Tissue-specific IGFBP1 adeno-associated virus packaging

[0043] Subculture AAV-293 cells into 100 mm dishes for transfection. After the operation, place in an incubator at 37°C, 5% CO2 and 95% relative humidity. Confirm that the cell density reaches about 80-90% confluence before transfection (5μg AAV-IGFBP1 plasmid). Opti MEM needs to be preheated in a 37°C water bath, and LipofiterTM transfection reagent needs to be restored to room temperature before use. Shake well before use.

[0044] 6h after transfection, replace with fresh complete medium containing 10% fetal bovine serum (FBS). 72h after transfection, gently scrape the cells containing AAV particles with a cell scraper, collect them in a 15mL centrifuge tube, centrifuge at 150×g for 3min to collect the cells, remove the culture supernatant, wash once with PBS, and finally resuspend the cells with 300μL PBS. Prepare a 37℃ constant temperature water bath and liquid nitrogen, and freeze and thaw the centrifuge tube containing cells three times in liquid nitrogen and a 37℃ water bath. 4℃, 2000×g, 5min, remove cell debris, and collect the lysis supernatant containing AAV particles.

[0045] 5. Tissue-specific IGFBP1 AAV purification

[0046] Add 0.1 μL of Benonase enzyme to each 1 mL of crude virus extract, and place in a 37°C water bath for 1 hour to remove the cell genome and residual plasmid DNA in the virus solution. Centrifuge at 600×g, 4°C for 10 minutes and take the supernatant. Perform column purification according to the Biomiga adeno-associated virus purification kit (V1469-01), add the 4 mL AAV virus sample liquid obtained by column purification to the ultrafiltration tube, and centrifuge at 1400×g for 30 minutes to obtain about 1 mL AAV. Collect the purified virus and store it at -80°C.

[0047] Example 2. Animal experiment design

[0048] 1. Gushi chickens were raised to 17 weeks of age after hatching. In this experiment, 180 healthy Gushi hens with similar body weights in the experimental group and the control group at 17 weeks of age were selected, 145 chickens in the no treatment group (Blank), 15 chickens in the control group (AAV-NC), and 20 chickens in the experimental group (AAV-IGFBP1). 12 vg / mL titer of adeno-associated virus was injected in situ into the liver and then the animals were raised normally until 31 weeks old. During this period, the egg production of each group was counted. The total egg production was as follows: Figure 1 The average egg production is shown in Figure 2 As shown. After being raised to 31 weeks of age, slaughter experiments were conducted. Ten individuals were randomly slaughtered in the Blank group, and all individuals in the control and experimental groups were slaughtered. Body weight was measured before slaughter, and ovarian weight and ovarian index were measured after slaughter (the results are shown in Figure 3 As shown), the number of pre-rank follicles was counted (the results are shown Figure 4 As shown, SWF: small white follicle; LWF: large white follicle; SYF: small yellow follicle; LYF: large yellow follicle).

[0049] 2. Serum reproductive hormone level detection

[0050] Serum was collected from the subwing vein at 17, 21, 27, and 31 weeks of age to detect the level of reproductive hormones in the serum. The detection kits were purchased from Shanghai ELISA Biotechnology Co., Ltd. According to the instructions of chicken IGFBP1 ELISA kit (YJ33484), FSH ELISA kit (YJ33314), LH ELISA kit (YJ33288), E2 ELISA kit (YJ33317), PROG ELISA kit (YJ33209), PRL ELISA kit (YJ33312), and AMH kit (YJ33412), the absorbance (OD value) of each sample was measured at a wavelength of 450nm using an enzyme label analyzer Infinite F50. In the Excel worksheet, the standard concentration was used as the horizontal axis and the corresponding OD value was used as the vertical axis to draw the linear regression curve of the standard. The concentrations of IGFBP1, FSH, LH, E2, PROG, PRL, and AMH in the serum of each sample were calculated according to the curve equation. The statistical results are shown in the figure. Figure 5 The correlation analysis results are shown in Figure 6 shown.

[0051] Results: Compared with the Blank and AAV-NC groups, liver-specific IGFBP1 adeno-associated virus (AAV-IGFBP1) injected orally into the liver significantly increased the total egg production of hens (19-31 weeks of age) (see Figure 1 ), egg production of hens at 25, 27, 28, 30 and 30 weeks of age also increased significantly (see Figure 2); In situ injection of AAV-IGFBP1 into the liver had no significant effect on the ovarian weight of 31-week-old hens, but significantly increased the ovarian index and the number of small and large white follicles (see Figure 3 and 4 ).

[0052] In situ liver injection of AAV-IGFBP1 not only increased the serum IGFBP1 content in 21, 27 and 31-week-old Gushi hens, but also induced a significant increase in the serum LH, E2, AMH and FSH contents, and significantly reduced the serum PRL content (see Figure 5 ).

[0053] Further correlation analysis showed that serum IGFBP1 level was significantly positively correlated with the number of pre-grade follicles, serum LH, E2, AMH and PROG levels (R = 0.304, P = 0.043; R = 0.874, P = 9.22 × 10 -58 ; R = 0.456, P = 1.14 × 10 -10 ; R = 0.479, P = 9.52 × 10 -12 ; R = 0.221, P = 0.003) (see Figure 6 ).

[0054] In summary, the IGFBP1 gene and its related materials can significantly affect the egg-laying performance of hens and the expression level of HPG axis hormones. They can be used to increase the egg production and ovarian index of hens, increase the number of small and large white follicles, and induce an increase in the levels of LH, E2, AMH and FSH in serum, thereby improving the reproductive performance of hens.

[0055] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of IGFBP1 gene in improving the reproductive performance of hens.

2. The use according to claim 1, characterized in that: The method for improving the reproductive performance of hens 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 hens, increase the IGFBP1 protein content in the hen serum, and thus improve the reproductive performance of the hens.

3. The use according to claim 2, characterized in that: The hen is 17 to 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, and the injection volume is 280-320 μL / chicken, (0.8-1.2)×10 12 vg / mL titer.

5. The use according to claim 4, characterized in that: The method for improving the reproductive performance of hens includes improving the total egg production of hens, improving the ovarian index, increasing the number of small white follicles and large white follicles, increasing the levels of LH, E2, AMH and FSH in serum, and reducing the level of PRL in serum.

6. Application of IGFBP1 protein in screening hens with high reproductive performance.

7. Application of IGFBP1 protein in evaluating the reproductive performance of hens.

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