A transcription factor molecular marker based on regulation of fat metabolism of avian myoblast cells and application thereof

Abstract

The application discloses a kind of transcription factor molecular marker based on regulating poultry myoblast fat metabolism and its application, the transcription factor is RXRA gene, the molecular marker is located on the chromosome where RXRA gene is, including the nucleotide sequence of the C5952908T mutation site and the upstream and downstream base group of the chromosome, the mutation site is linked with the C5952944T, A5952945G and T5952953C mutation site of the coding region of the subsequent chromosome.This application can judge poultry high feed efficiency individual by early molecular marker screening by identifying the genotype of transcription factor molecular marker based on regulating poultry myoblast fat metabolism existing in poultry genome, provides direct technical means for poultry feed efficiency selection, reduces feeding cost by early selection, and genetically improves feed conversion rate, accelerates the genetic progress of selection.

Description

Technical Field

[0001] This invention relates to the field of molecular marker technology, specifically to a transcription factor molecular marker based on regulating lipid metabolism in avian myoblasts and its application. Background Technology

[0002] Feed costs constitute the largest proportion of expenses in poultry farming, accounting for approximately 50-70% of total costs. Due to competition for grains between humans and poultry, and with rising feed costs, feed consumption has become the most significant factor impacting poultry farming. Therefore, improving feed efficiency and avoiding feed energy waste has become a core task in poultry breeding in recent years. Lipid metabolism is a major factor causing changes in feed efficiency. Therefore, effectively preventing excessive intracellular fat deposition and avoiding energy consumption and increased feed consumption due to lipid metabolism is an effective way to improve poultry feed efficiency.

[0003] Nuclear receptors (NRs) primarily function as transcriptional regulators, controlling tissue development, homeostasis, and metabolism. Retinoic acid X receptor Alpha (RXRA), a member of the NR superfamily, is mainly involved in lipid metabolism, cell differentiation, glucose and energy metabolism. Previous studies have found that β-hydroxybutyrate (β-hydroxybutyrate) inhibits fatty acid oxidation and ketone body production in sheep by suppressing RXRA signaling. The constructed lncRNA DANCR, binding to RXRA, enhances PI3K / AKT signaling and breast cancer development by regulating GSK3β to increase serine 49 / 78 phosphorylation. miR-27a enhances the proliferation and inhibits the differentiation of sheep preadipocytes by targeting and inhibiting RXRA expression. During chicken preadipocyte differentiation, RXRA promotes PLIN1 gene transcription in a PPARG-independent manner, thereby promoting chicken preadipocyte differentiation. However, whether and how RXRA affects poultry feed utilization remains unreported. Summary of the Invention

[0004] The purpose of this invention is to provide a molecular marker based on the transcription factor (RXRA) that regulates lipid metabolism in avian myoblasts and its application. The expression level of RXRA in myocytes is directly related to the intracellular lipid metabolism process, and the level of intracellular RXRA activity directly affects cellular lipid metabolism. This invention analyzes the impact of RXRA on poultry feed utilization efficiency. By studying the linkage combination of multiple polymorphic sites in the RXRA coding region leading to changes in amino acids and conformational changes in protein active sites, the factors influencing the regulation of downstream lipid synthesis and metabolism gene expression are obtained, and a molecular marker is developed based on this. This molecular marker is closely linked to poultry feed utilization efficiency. By identifying the type of this molecular marker, high feed efficiency individuals in poultry can be identified at an early stage through molecular marker screening.

[0005] The present invention achieves the above objectives through the following technical solutions:

[0006] A molecular marker based on a transcription factor that regulates lipid metabolism in avian myoblasts, wherein the transcription factor is the RXRA gene, and the molecular marker is located on the chromosome containing the RXRA gene, including the C5952908T mutation site on the chromosome and the nucleotide sequence consisting of its upstream and downstream bases, wherein the mutation site is linked to the C5952944T, A5952945G and T5952953C mutation sites in the coding region of the subsequent chromosome.

[0007] Furthermore, the molecular marker is a nucleotide sequence containing the sequence shown in SEQ ID NO.1 or a mutated sequence thereof, wherein the mutated sequence refers to one or more mutations in C103T, C139T, A140G, and T148C of SEQ ID NO.1, and C103T, C139T, A140G, and T148C correspond to the C5952908T, C5952944T, A5952945G, and T5952953C mutation sites in the coding region of the chromosome.

[0008] This invention also provides the application of this molecular marker in identifying traits related to feed conversion efficiency in poultry.

[0009] Furthermore, if the molecular marker is type C, it indicates that the poultry feed conversion efficiency is high; if the molecular marker is type T, it indicates that the poultry feed conversion efficiency is average.

[0010] This invention also provides a method for identifying poultry feed conversion efficiency traits using this molecular marker, comprising the following steps:

[0011] (1) Collect wing vein blood from poultry and extract total DNA;

[0012] (2) Design specific amplification primers based on the molecular marker as the target fragment, use total DNA as a template, and perform PCR amplification using the specific amplification primers to obtain the amplification product;

[0013] (3) Determine the molecular marker type of the amplified product. If it is T-type, it indicates that the conversion efficiency of poultry feed is high. If it is C-type, it indicates that the conversion efficiency of poultry feed is average.

[0014] Furthermore, the method for determining the molecular marker type of the amplification product in step (3) is as follows:

[0015] If the genotype corresponding to the molecular marker type is TT homozygous, it indicates that the poultry feed conversion efficiency is high.

[0016] If the genotype corresponding to the molecular marker type is CT heterozygous, it indicates that the poultry feed conversion efficiency is high.

[0017] If the genotype corresponding to the molecular marker type is CC homozygous, it indicates that the feed conversion efficiency of poultry is generally low.

[0018] Furthermore, the polymorphisms and distribution of the RXRA gene coding region containing the amplified products were analyzed by PCR amplification and enzyme digestion sequencing. Haplotypes were identified based on the linkage relationships of the polymorphic sites, and the feed conversion efficiency of individual poultry was determined based on the haplotypes.

[0019] If the haplotype is TTTTGGCC, it indicates that the poultry feed conversion efficiency is high;

[0020] If the haplotype is TCTCGATC or TCTCGGCC, it indicates that the poultry feed conversion efficiency is relatively high.

[0021] If the haplotype is CCCCAATT, it indicates that the poultry feed conversion efficiency is average.

[0022] Furthermore, the sequences of the specific amplification primers are as follows:

[0023] F:GTCAGACCTGAGGGCACAA

[0024] R:CAATTCTGCTCACCGCAA.

[0025] The beneficial effects of this invention are as follows: By identifying the genotypes of transcription factor molecular markers that regulate lipid metabolism in avian myoblasts in the avian genome, this invention can screen for high feed efficiency individuals in avian birds at an early stage through molecular marker screening, providing a direct technical means for the breeding of avian birds with high feed efficiency. Through early breeding, feeding costs are reduced while feed conversion rate is genetically improved, accelerating the genetic progress of breeding. Attached Figure Description

[0026] Figure 1 Agarose gel electrophoresis results for identifying feed conversion efficiency traits in poultry using molecular markers. Detailed Implementation

[0027] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0028] 1. Materials

[0029] Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0030] 2. Methods

[0031] 1000 meat ducks were raised in the same batch, individually caged, and their weight and feed intake were recorded from 21 to 42 days of age. The average daily feed intake and final body weight were measured, and the average daily weight gain, metabolic weight and feed conversion ratio were calculated. The residual feed intake (RFI) of each duck was also estimated.

[0032] Collect venous blood from each duck's wing and extract total DNA from the blood. Use a blood DNA extraction kit manufactured by Dalian Takara Bio Co., Ltd. to extract total DNA from the venous blood samples from the duck's wing. The extraction steps should be performed according to the kit's instructions. Alternatively, other tissues such as duck feathers can be used as samples for total DNA extraction.

[0033] Using the partial nucleotide sequence of the retinoic acid X receptor Alpha (RXRA) chromosome shown in SEQ ID NO.1 as a template, specific amplification primers were designed. The primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3, namely:

[0034] F:GTCAGACCTGAGGGCACAA

[0035] R:CAATTCTGCTCACCGCAA

[0036] PCR was performed using 2×TaqPCRMasterMix (KT121221, TIANGEN). The reaction system consisted of 1 μl of P2-F, 1 μl of P2-R, 1 μl of whole genomic DNA, 7 μl of ddH2O, and 10 μl of 2×TaqPCRMasterMix, for a total of 20 μl.

[0037] The PCR amplification conditions were as follows: denaturation at 95℃ for 30 seconds, annealing at 59-64℃ for 30 seconds, extension at 72℃ for 35 seconds, for a total of 30-40 cycles; extension at 72℃ for 10 minutes; storage at 4℃; and the PCR amplification product was obtained.

[0038] The PCR amplification products were detected by agarose gel electrophoresis. The PCR amplification products were digested with BfaI enzyme to obtain the following results: Figure 1 The bands shown are given. Based on the agarose gel electrophoresis results of the target bands, the genotypes corresponding to the molecular markers can be preliminarily determined: the homozygous TT type shows one 700bp band, the homozygous CC type produces two bands of 592bp and 108bp after enzyme digestion, and the heterozygous CT type shows three bands of 700bp, 592bp, and 108bp.

[0039] Meanwhile, the DNA recovery kit from Dalian Takara Bio Co., Ltd. can be used to recover PCR amplification products, and the PCR products can be sent to Shanghai Sangon Biotech for sequencing to obtain specific sequence information. Haploview software can then be used to analyze haplotype linkage relationships at polymorphic sites.

[0040] 3. Results

[0041] The association analysis results between different haplotypes and various traits are shown in Table 1:

[0042] Table 1: Correlation analysis between linked SNPs and feed traits of Qiangying ducks

[0043]

[0044] As shown in Table 1, the molecular marker C5952908T of this invention is linked to the subsequent coding regions C5952944T, A5952945G, and T5952953C mutation sites, exhibiting four different haplotypes. Among them, the residual feed intake of ducks with the CC type (CCCCAATT), CT type (TCTCGATC, TCTCGGCC), and TT type (TTTTGGCC) decreased sequentially. The feed conversion ratio of the CC type (CCCCAATT) was significantly higher than the other groups, indicating that the feed utilization rate of ducks containing the C type molecular marker was significantly lower than that of the T type, and the feed utilization rate of the CT heterozygous type was slightly higher than that of the CC homozygous type. In conclusion, these tightly linked genes determine the feed conversion efficiency of poultry individuals, and the molecular marker based on the C5952908T mutation site developed accordingly can achieve the purpose of early screening and identification of poultry individuals with high feed efficiency.

[0045] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A transcription factor molecular marker based on the regulation of lipid metabolism in avian myoblasts, wherein the transcription factor is the RXRA gene, characterized in that, The molecular marker is a nucleotide sequence containing the sequence shown in SEQ ID NO.

1. The molecular marker is located on the chromosome where the RXRA gene is located, including the C5952908T mutation site of the chromosome. The poultry is a meat duck.

2. The transcription factor molecular marker based on regulating lipid metabolism in avian myoblasts according to claim 1, characterized in that, The mutation site C5952908T of the molecular marker is linked to the mutation sites C5952944T, A5952945G, and T5952953C in the subsequent chromosome coding region.

3. The application of the molecular marker as described in any one of claims 1-2 in identifying traits related to feed conversion efficiency in poultry.

4. The application of the molecular marker according to claim 3 in identifying traits related to feed conversion efficiency in poultry, characterized in that, If the molecular marker is T-type, it indicates that the poultry feed conversion efficiency is high; if the molecular marker is C-type, it indicates that the poultry feed conversion efficiency is average.

5. A method for identifying poultry feed conversion efficiency traits using molecular markers as described in any one of claims 1-2, characterized in that, Includes the following steps: (1) Collect venipuncture blood from birds' wings and extract total DNA; (2) Design specific amplification primers based on the molecular marker as the target fragment, use total DNA as a template, and perform PCR amplification using the specific amplification primers to obtain the amplification product; (3) Determine the molecular marker type of the amplified product. If it is T-type, it indicates that the conversion efficiency of poultry feed is high. If it is C-type, it indicates that the conversion efficiency of poultry feed is average.

6. The method for identifying poultry feed conversion efficiency traits according to claim 5, characterized in that, The method for determining the molecular marker type of the amplification product in step (3) is as follows: If the genotype corresponding to the molecular marker type is TT homozygous, it indicates that the poultry feed conversion efficiency is high. If the genotype corresponding to the molecular marker type is CT heterozygous, it indicates that the poultry feed conversion efficiency is high. If the genotype corresponding to the molecular marker type is CC homozygous, it indicates that the feed conversion efficiency of poultry is generally low.

7. The method for identifying poultry feed conversion efficiency traits according to claim 6, characterized in that, The method for determining the molecular marker type of the amplified product is as follows: PCR amplification and restriction enzyme digestion sequencing are used to analyze the site polymorphism and distribution of the RXRA gene coding region where the amplified product is located. Haplotypes are then determined based on the linkage relationships of the polymorphic sites, and the feed conversion efficiency of individual poultry is assessed based on the haplotype. If the haplotype is TTTTGGCC, it indicates that the poultry feed conversion efficiency is high; If the haplotype is TCTCGATC or TCTCGGCC, it indicates that the poultry feed conversion efficiency is relatively high. If the haplotype is CCCCAATT, it indicates that the poultry feed conversion efficiency is average.

8. The method for identifying poultry feed conversion efficiency traits according to claim 5, characterized in that, The sequence of the specific amplification primers is as follows: F: GTCAGACCTGAGGGCACAA R: CAATTCTGCTCACCGCAA.

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