Use of igfbp1 gene in regulating or evaluating reproductive performance of hens

Abstract

The application provides application of an IGFBP1 gene in regulation or evaluation of hen reproductive performance, and belongs to the field of biotechnology and genetic breeding. The application promotes expression of the IGFBP1 gene of a hen by constructing a tissue-specific IGFBP1 recombinant adeno-associated virus vector and using the tissue-specific IGFBP1 recombinant adeno-associated virus vector to promote expression of the IGFBP1 gene of the hen, and improves the IGFBP1 protein content in serum of the hen, and further improves the reproductive performance of the hen. The application proves by experiments that the IGFBP1 gene has an influence on the egg production performance of a hen and the expression level of an HPG axis hormone, can improve the total egg production of a hen, an ovary index, the number of small white follicles, the number of large white follicles, and the content of LH, E2, AMH and FSH in serum, can be used as a candidate gene or a genetic marker of egg production performance of a hen, enriches the serum judgment index of hen reproductive performance, and provides a valuable reference basis for differential diagnosis of hen reproductive performance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and genetic breeding, and particularly relates to application of IGFBP1 gene in regulation or evaluation of hen reproductive performance. BACKGROUND

[0002] China is the world's largest egg producer and consumer. Egg laying traits are one of the important economic traits of poultry (chickens) and have been the focus of 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 beneficial to the effective selection of poultry reproductive traits.

[0003] Egg laying traits are the most important of poultry (chicken) reproductive performance, and the essence is the ovulation ability of the ovary, which is regulated by multiple sex hormones secreted by the hypothalamic-pituitary-gonadal axis (HPG axis) into the peripheral circulation. Therefore, the serum indicators for evaluating the reproductive performance of hens at this stage are mainly focused on hormones such as follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrogen (E2), prolactin (PRL), progesterone (PROG), and anti-Mullerian hormone (AMH) regulated by the HPG axis. However, with the continuous development of sequencing technology and omics research, molecules affecting the formation and regulation of egg laying traits are being continuously 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 application also aims to provide a new biomarker for evaluating poultry reproductive performance and assisting poultry breeding programs. SUMMARY

[0005] The present application aims to provide a biomarker for evaluating the reproductive performance of hens, which can be used to improve the reproductive performance of hens and expand the use of IGFBP1 gene.

[0006] To achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides the application of IGFBP1 gene in improving the reproductive performance of hens.

[0008] Preferably, the method for improving the reproductive performance of hens is to construct a tissue-specific IGFBP1 recombinant adeno-associated virus vector and use the tissue-specific IGFBP1 recombinant adeno-associated virus vector to promote the expression of IGFBP1 gene in hens, increase the content of IGFBP1 protein in the serum of hens, and thus improve the reproductive performance of hens.

[0009] Preferably, the hens are 17-31 weeks old.

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

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

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

[0013] The application also provides application of IGFBP1 protein in evaluation of the reproductive performance of hens.

[0014] Beneficial effects

[0015] The application is the result of long-term experimental research, and it is found that the IGFBP1 gene can significantly affect the egg production performance of hens and the expression level of hormones in the HPG axis. Overexpression of the IGFBP1 gene can significantly improve the egg production of hens, improve the ovary index, increase the number of small white follicles and large white follicles, and help to improve the reproductive performance of hens, and can be used as a candidate gene or a genetic marker of egg production traits of hens, thereby providing a valuable reference basis for the differential diagnosis of the reproductive performance of hens, and enriching the basis for the serum judgment index of the reproductive performance of hens. BRIEF DESCRIPTION OF DRAWINGS

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

[0017] Figure 2 Comparison of the average egg production of hens in different groups;

[0018] Figure 3 Comparison of the ovary weight and ovary index of hens in different groups;

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

[0020] Figure 5 Comparison of the content of IGFBP1 and reproductive hormones in serum of hens in different groups;

[0021] Figure 6 Correlation analysis of the content of IGFBP1 in serum, the number of pre-grade follicles and the content of reproductive hormones of hens in different groups. DETAILED DESCRIPTION

[0022] The technical solutions provided by the application will be described in detail below in combination with the embodiments, but they should not be understood as limiting the scope of protection of the application.

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

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

[0025] According to the order in Table 1 below, each reagent was added in turn, mixed gently, and placed in a 37°C water bath for 1-2h; after the digestion was completed, agarose gel electrophoresis was performed, and the target fragment was recovered.

[0026] Table 1 Vector digestion system

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

[0028] 2. Acquisition of IGFBP1 gene CDS sequence

[0029] The CDS sequence of the IGFBP1 gene (NM_001001294.1) was obtained from the NCBI online website (https: / / www.ncbi.nlm.nih.gov / ), and the CDS sequence was amplified by PCR. The following system was prepared, mixed gently, and placed in a PCR instrument for reaction (primers and reaction system as shown in Tables 2 and 3, and reaction program as 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, the following reaction system was prepared in ice water bath. After the ligation reaction solution was reacted at 50°C for 30 min, it was placed on ice for 5 min, and then immediately transformed. The reaction system is shown in Table 5.

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

[0040] 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

[0041] Cultivate with shaking at 37°C and 230 rpm for 50 minutes. After overnight incubation 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 Extraction Kit (DP103).

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

[0043] 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.

[0044] 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 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 particles.

[0045] 5. Purification of tissue-specific IGFBP1 adeno-associated virus

[0046] 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 solution. Centrifuge at 600 × g at 4°C for 10 minutes and collect the supernatant. Perform column purification using the Biomiga Adeno-Associated Virus Purification Kit (V1469-01). Add 4 mL of AAV viral sample obtained by column purification to an ultrafiltration tube and centrifuge at 1400 × g for 30 minutes to obtain approximately 1 mL of AAV. Collect the final purified virus and store at -80°C.

[0047] Example 2. Animal experiment design

[0048] 1. Gushi chickens were raised to 17 weeks of age after hatching. In this study, 180 healthy Gushi hens with similar body weights in the experimental and control groups at 17 weeks of age were selected. There were 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 into the liver in situ and the animals were raised normally until 31 weeks. During this period, the egg production of each group was counted. The total egg production was as follows: Figure 1 As shown, the average egg production is 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 follicles before the statistical level (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 test

[0050] Serum was collected from the subwing vein at 17, 21, 27, and 31 weeks of age to measure reproductive hormone levels. Detection kits were purchased from Shanghai ELISA Biotechnology Co., Ltd. The absorbance (OD) of each sample was measured at a wavelength of 450 nm using an Infinite F50 enzyme-linked immunosorbent assay (ELISA) according to the instructions for the 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). A linear regression curve was drawn in an Excel worksheet with the standard concentration as the abscissa and the corresponding OD value as the ordinate. 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 table below. 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) in situ liver injection 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) ; AAV-IGFBP1 injection in situ in the liver had no significant effect on the ovary weight of 31-week-old hens, but significantly increased the ovary index, and significantly increased the number of small white follicles and large white follicles (see Figure 3 and 4 ).

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

[0053] Further correlation analysis found that the content of IGFBP1 in the serum was significantly positively correlated with the number of pre-grade follicles, the content of LH, E2, AMH and PROG in the serum (R=0.304, P=0.043; R=0.874, P=9.22x10 -58 ; R=0.456, P=1.14x10 -10 ; R=0.479, P=9.52x10 -12 ; R=0.221, P=0.003) (see Figure 6 ).

[0054] As can be seen from the above, IGFBP1 gene and related materials can significantly affect the laying performance of hens and the expression level of HPG axis hormones, and can be used to improve the laying performance of hens and the ovary index, increase the number of small white follicles and large white follicles, induce an increase in the content of LH, E2, AMH and FSH in the serum, and further improve the reproductive performance of hens.

[0055] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. IGFBP1 The application of genes in improving the reproductive performance of hens 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 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 reproductive performance of hens. IGFBP1 Gene expression, increasing the IGFBP1 protein content in hen serum, thereby improving the reproductive performance of hens.

3. The use according to claim 2, characterized in that The hens 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. The use according to claim 4, characterized in that The improving of 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. The use of IGFBP1 protein in screening hens with high reproductive performance is characterized in that: The sequence of the gene encoding the IGFBP1 protein is shown in SEQ ID NO.

3.

7. Application of IGFBP1 protein in evaluating reproductive performance of hens, characterized in that: The sequence of the gene encoding the IGFBP1 protein is shown in SEQ ID NO.3.

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