A chicken adipogenesis gene 3apos; -UTR variants and uses thereof

Abstract

The invention provides a chicken adipogenesis gene 3 '-UTR variant and application thereof, and belongs to the technical field of molecular biology, and chicken adipogenesis genes comprise BMP2 and PDK1. The invention finds that adipogenesis genes BMP2 and PDK1 have different 3 '-UTR long and short transcript expressions in the chicken preadipocyte differentiation process and the association between the dynamic change of the genes BMP2 and PDK13'-UTR and adipogenesis for the first time, and further provides the application of the 3 '-UTR variants of the genes BMP2 and PDK1 in preadipocyte differentiation identification and analysis. The invention provides a material and a method for genetic breeding of broiler chickens, and has a good application prospect in improvement of chicken quality breeding.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and in particular to chicken lipogenesis gene 3'-UTR variants and their application in preadipocyte differentiation identification and analysis, wherein the chicken lipogenesis gene includes BMP2 (bone morphogenetic protein-2) and PDK1 (pyruvate dehydrogenase kinase 1), providing new information for chicken quality genetic breeding research. Background Art

[0002] Adipocytes, derived from mesenchymal stem cells, are primarily divided into three categories: white adipocytes, brown adipocytes, and beige adipocytes. The entire adipocyte differentiation process is divided into six stages: mesenchymal precursors, preadipocytes, growth-arrested preadipocytes, mitotic clonal expansion, terminal differentiation, and mature adipocytes. This is valuable for in vitro investigation of the mechanisms of cell proliferation, differentiation, adipokine secretion, and gene expression.

[0003] Chickens, as an economically important agricultural poultry species, have been considered an ideal experimental model animal in biological research. Studies have found that while fat synthesis in mammals primarily occurs in adipose tissue, 90% of fat synthesis in poultry occurs in the liver. The mechanisms of adipocyte differentiation and synthesis differ significantly between the two species. For example, the transcription factor SREBP1 has two isoforms (SREBP1α and SREBP1c) in humans and mice, while chickens have only one isoform, SREBP1α. Studies have also found that the level of lipogenesis in chicken liver and fat deposition in muscle are important factors determining chicken quality and health. Therefore, studying the changes in fat metabolism genes during chicken adipocyte differentiation is beneficial for research on chicken quality genetics and breeding.

[0004] The 3'-UTR plays a crucial role in gene expression regulation, playing a crucial role in mRNA post-transcriptional modification, intracellular localization and transport, maintaining mRNA stability, and ensuring translation efficiency. With the development of 3'-UTR sequencing technology, research on the regulation of gene expression by 3'-UTRs has become a hot topic. Studies have found that mutations in the 3'-UTR region of the HMGCR gene in Beijing oily chickens significantly affect total cholesterol and LDL cholesterol levels in chicken serum. Furthermore, polymorphisms in the Sirt2 and WNT10B genes have been shown to be associated with backfat thickness in pigs, with WNT10B being significantly associated with hip and shoulder thickness. However, few studies have examined the role of 3'-UTRs in chicken preadipocyte differentiation. Summary of the Invention

[0005] In view of the technical problems existing in the background technology, the present invention uses chicken preadipocytes as experimental objects to reveal the adipogenic genes in the differentiation process of chicken preadipocytes. BMP2 and PDK1 The dynamic change rules of 3'-UTR of chicken were obtained, and the acquisition and application of 3'-UTR variants were provided, providing a theoretical basis for further research on the molecular regulatory mechanism of chicken fat production.

[0006] The technical solutions of the present invention are as follows: Application of 3'-UTR variants of chicken adipogenic genes in the identification and analysis of chicken preadipocyte differentiation, including chicken adipogenic genes BMP2 and PDK1 .

[0007] In the previous study on the dynamic changes of 3'-UTR of the whole transcriptome during the differentiation of animal fat cells, the inventors found that the 3'-UTR of lipid metabolism genes related to the differentiation of chicken, duck and mouse preadipocytes had multiple different transcripts; further using chicken preadipocytes as experimental subjects, the inventors discovered for the first time that the adipogenic genes in the differentiation of chicken preadipocytes BMP2 、 PDK1 as well as LPL (lipoprotein lipase) has different 3'-UTR length transcripts, especially the gene BMP2 and PDK1 Different 3'-UTRs are expressed significantly differently during differentiation, so the gene BMP2 and PDK1 The 3'-UTR can be used to identify chicken preadipocyte differentiation-related genes.

[0008] In some embodiments of the present invention, the chicken preadipocytes are immortalized chicken preadipocytes (ICP1), and the chicken preadipocyte differentiation is induced by oleic acid.

[0009] Preferably, in the above scheme, the chicken adipogenic gene 3'-UTR variant is obtained by 3'-RACE and nested PCR, and the primers used include: B-GSP1 / 2 with sequences as shown in SEQ ID NO.29-30, and P-GSP1 / 2 with sequences as shown in SEQ ID NO.31-32.

[0010] More preferably, in the above nested PCR, the first PCR reaction program is: 94°C 3 min; 94°C 30 s, 56°C 30 s, 72°C 30 s, 35 cycles; 72°C 10 min; save at 4°C; the second PCR reaction program is: 94°C 3 min; 94°C 30 s, 60-62°C 30 s, 72°C 30 s, 35 cycles; 72°C, 10 min; save at 4°C.

[0011] Preferably, in the above scheme, during the differentiation of preadipocytes, genes BMP2 and PDK1 The 3'-UTR of the gene changes dynamically, and BMP2 Contains 5 transcripts, gene PDK1 Contains 4 transcripts.

[0012] More preferably, the genes are analyzed by semi-quantitative analysis and qPCR detection. BMP2 and PDK1 The expression of each 3'-UTR variant during cell differentiation was investigated, and the primers used included: P7-F / R as shown in SEQ ID NOs. 11-12, BMP2-FA and BMP2-R-1 as shown in SEQ ID NOs. 13-14, BMP2-F / R-2 as shown in SEQ ID NOs. 15-16, BMP2-F / R-3 as shown in SEQ ID NOs. 17-18, P8-F / R as shown in SEQ ID NOs. 19-20, PDK1-FA and PDK1-R-1 as shown in SEQ ID NOs. 21-22, PDK1-F / R-2 as shown in SEQ ID NOs. 23-24, PDK1-F / R-3 as shown in SEQ ID NOs. 25-26, and PDK1-F / R-4 as shown in SEQ ID NOs. 27-28.

[0013] Experimental data show that in the early stage of chicken preadipocyte differentiation, BMP2 and PDK1 The expression of the long variant is better than that of the short variant. BMP2 In the first 72 hours, it mainly exists in the form of long transcripts. In the later expression, long and short transcripts coexist. PDK1 The long transcript was mainly expressed in the first 24 h of differentiation, and both the long and short transcripts were expressed at 48 h, 72 h, 96 h, and 120 h.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This study first discovered the adipogenic gene in the differentiation process of chicken preadipocytes BMP2 and PDK1There are transcripts with different 3'-UTR lengths, and the expression of 3'-UTRs varies significantly during the differentiation process. Specifically, during the differentiation of chicken preadipocytes, BMP2 and PDK1 The 3'-UTR of the gene was dynamically changed, and 5 and 4 transcripts of different lengths were detected, respectively. BMP2 and PDK1 The expression levels of the long variants in the early stages of chicken preadipocyte differentiation were better than those of the short variants. The research results of the present invention provide a theoretical basis for further research on fat differentiation in poultry and an important reference for improving chicken quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] To more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0016] Figure 1 Graph showing the results of Oil Red O staining of chicken preadipocytes at 0 h and 120 h of induction differentiation in Example 1 of the present invention; Figure 2 The gene in the process of inducing differentiation of ICP1 cells in Example 1 of the present invention PPARγ 、 FABP4 、 BMP2 、 PDK1 、 LPL and LDAH The expression change results diagram; Figure 3 For the gene in Example 1 of the present invention BMP2 、 LPL and PDK1 3'-RACE gel electrophoresis; lanes 1 and 2 are 3'-RACE amplification products, and lane M is D2000 Marker; Figure 4 For the gene in Example 1 of the present invention BMP2 、 LPL and PDK1 Schematic diagram of 3'-RACE and standard PCR experiments; Figure 5 is a gel electrophoresis diagram of transcript fragments of different lengths of the gene in Example 1 of the present invention; wherein A is BMP2 Gene, B PDK1 Gene; Figure 6 In the process of cell induction differentiation in Example 2 of the present invention BMP2 (A) and PDK1Figure 2. Expression detection results of different transcripts of genes (B). Figure 7 In the process of cell induction differentiation in Example 2 of the present invention BMP2 (A) and PDK1 (B) Relative expression levels of different transcripts of the gene. DETAILED DESCRIPTION

[0017] The following embodiments of the technical solution of the present invention will be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only examples and are not intended to limit the scope of protection of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains; the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the present invention; the term "include" and any variations thereof herein are intended to cover non-exclusive inclusions.

[0019] In the present invention, 3'-UTR refers to the 3' non-coding region of a transcript starting after the stop codon and ending at the polyadenylation site (poly-A site); transcript refers to the mRNA product transcribed from the same gene, including its coding region (CDS) and non-coding regions (such as 5'-UTR, 3'-UTR); 3'-UTR variants refer to different transcripts derived from the same gene but with length or sequence differences in the 3'-UTR region; total isoforms refer to the sum of all detectable 3'-UTR transcript types of the target gene.

[0020] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product specifications were used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased commercially.

[0021] In the following examples, chicken immortalized preadipocyte cell line was used as experimental material.

[0022] Example 1 This example shows BMP2 and PDK1 The changes in 3'-UTR length of genes during chicken preadipocyte differentiation include the following: (1) Culture and differentiation of chicken preadipocytes.

[0023] When the confluence of the ICP1 cells in the cell culture flask reaches 90%, discard the cell culture medium from the flask and rinse the cells twice with 5 mL of PBS. Then, add 480 μL of trypsin and digest the cells at 37°C for 50-60 seconds. Once the cells begin to flow like a quicksand, add 20 mL of complete culture medium to the flask. After agitating the cell suspension, transfer the cell suspension to a six-well plate and plate evenly (add 2 mL of cell suspension to each well). Incubate in a 5% CO2, 37°C incubator. After 24 hours, when the cells reach 50-60% confluence, replace the culture medium with induction medium containing oleic acid (160 μM) to induce cell differentiation. Thereafter, change the oleic acid induction medium every 24 hours.

[0024] (2) Study on the expression patterns of related genes during ICP1 cell differentiation.

[0025] After a large number of experiments, it was found that compared with other genes, BMP2 and PDK1 Gene expression levels showed obvious regular changes as ICP1 cells differentiated. The specific experimental steps and results are as follows: ① Oil red O staining.

[0026] After differentiation is complete, the medium was discarded, the cells were washed three times with PBS, and fixed with 1 mL of 4% paraformaldehyde solution at 4°C for 30 min. Oil Red O working solution was prepared by mixing the original Oil Red O solution with deionized water in a ratio of 3:2, then filtered. The fixative was discarded, the cells were washed three times with PBS, and dried in a 65°C oven. 1 mL of Oil Red O working solution was added and stained in the dark for 30 min. The Oil Red O working solution was discarded, the cells were washed three times with PBS, 0.5 mL of 60% isopropanol was added and discarded within 20 seconds, and the cells were washed three times with PBS. 1 mL of PBS was added and the cells were photographed under a microscope. The PBS was discarded, 1 mL of 100% isopropanol was added, and the cells were shaken on a horizontal shaker for 15 min. The OD value was measured at 510 nm using a microplate reader.

[0027] Oil Red O results showed that compared with 0h, the lipid droplets in cells at 120h increased significantly ( Figure 1 ), the cells were induced to differentiate successfully.

[0028] ②Detection of target gene expression level.

[0029] Extraction of total cell RNA: remove the culture medium, wash three times with PBS, add an appropriate amount of 1 mL RNAiso Plus, lyse on ice for 15 minutes, gently pipette to mix, and transfer to an RNase-free 1.5 mL EP tube; add 200 μL chloroform to the EP tube, shake vigorously for 15 seconds, let it stand at room temperature for 5 minutes, and then centrifuge at 4°C, 12000 rpm for 15 minutes; carefully aspirate the supernatant and place it in a new RNase-free 1.5 mL EP tube, add an equal volume of pre-cooled isopropanol to the supernatant, shake vigorously for 15 seconds, let it stand at room temperature for 10 minutes; then centrifuge at 4°C, 12000 rpm for 10 minutes; discard the supernatant, add 1 mL of pre-cooled 75% ethanol, shake for about 15 seconds, and centrifuge at 4°C, 7500 rpm for 5 minutes (2 times); completely discard the supernatant, place the EP tube on filter paper to dry at room temperature, and add 30 μL The precipitate was dissolved in ddH2O; the integrity and concentration of RNA were determined by agarose gel electrophoresis and NanoDrop, and then stored in a -80℃ refrigerator.

[0030] Reverse transcription: Perform the RevertAid First Strand cDNA Synthesis Kit. The reaction system is shown in Tables 1 and 2. The reaction conditions are: 65°C for 5 min, 25°C for 5 min, 42°C for 60 min, and 70°C for 5 min. The synthesized cDNA was stored at −20°C.

[0031] Table 1 Reverse transcription reaction system 1

[0032] Table 2 Reverse transcription reaction system 2

[0033] Fluorescence quantitative PCR reaction: The primers shown in Table 10 were used and the reaction system shown in Table 3 was used for the reaction. Figure 2 As shown, marker genes PPARγ The expression level of Figure 2 A), and marker genes FABP4 The expression is first rising and then falling ( Figure 2 B); while observing genes BMP2 、 PDK1 、 LPL and LDAH Expression was found that during the differentiation of preadipocytes, BMP2 and PDK1 The gene expression level in the early stage (24h-48h) was significantly higher than that in the later stage; LPL and LDA The expression of the H gene gradually increased, reached the maximum level at 48 h, and then gradually decreased ( Figure 2CF).

[0034] Table 3 qPCR reaction system

[0035] (3) Genes in the process of chicken preadipocyte differentiation BMP2 、 PDK1 and LPL Changes in the 3'-UTR.

[0036] ① 3'-RACE sequencing analysis of target genes.

[0037] 3'-RACE cDNA first strand synthesis: According to the NCBI chicken BMP2 、 PDK1 and LPL Design 3'-RACE primers based on the gene reference sequence, as shown in Table 10. Perform 3'-RACE first-strand cDNA synthesis using the SMARTer RACE 5' / 3' Kit User Manual. First, prepare the buffer mix, 3'-RACE cDNA synthesis reaction solution, and 3'-RACE-Ready cDNA synthesis master mix (see Tables 4-6). Add the 3'-RACE-Ready cDNA synthesis master mix to the 3'-RACE cDNA synthesis reaction tube for a total volume of 20 μL. Incubate the reaction at 42°C for 90 minutes and then at 70°C for 10 minutes. Dilute the first-strand cDNA synthesis reaction product with Tricine-EDTA buffer and store at -20°C.

[0038] Table 4 Buffer Mix composition

[0039] Table 5 Composition of 3'-RACE cDNA synthesis reaction solution

[0040] Table 6 Composition of 3'-RACE-Ready cDNA Synthesis Master Mix

[0041] 3'-RACE amplification of the target gene: Nested PCR was performed using a 50 μL reaction system (see Tables 7 and 8). The first reaction was performed at 94°C for 3 minutes, followed by 35 cycles (94°C for 30 seconds, 56°C for 30 seconds, and 72°C for 30 seconds), followed by 72°C for 10 minutes, and then stored at 4°C. The second reaction was performed at 94°C for 3 minutes, followed by 35 cycles (94°C for 30 seconds, 60-62°C for 30 seconds, and 72°C for 30 seconds), followed by 72°C for 10 minutes, and then stored at 4°C. The second PCR primers were located downstream of the first PCR primers, and the product from the first PCR was diluted 50-fold and used as the template for amplification. Gene-specific primer GSP1 was used to identify and amplify known regions of the target gene and, together with the universal primers, to obtain preliminary amplification products of unknown sequences. Gene-specific primer GSP2 was used to further improve the specificity and accuracy of amplification, thereby obtaining complete or nearly complete cDNA sequences of the target gene.

[0042] Table 7 First PCR system

[0043] Table 8 Second PCR system

[0044] PCR product recovery and sequencing verification: PCR products were subjected to 2% agarose gel electrophoresis, and the target fragment was recovered according to the instructions for the AxyPrep DNA Gel Recovery Kit (Shanghai Kemin Biotechnology Co., Ltd.). A ligation-reverse transformation experiment was then performed according to the instructions for the pEASY-Blunt Simple Cloning Kit. Approximately 12 individual colonies were selected and placed in EP tubes containing 900 μL of LB liquid medium containing ampicillin and cultured in a shaking incubator at 37°C overnight. Colony PCR verification was then performed using the reaction system shown in Table 9. The PCR products were then subjected to 2% agarose gel electrophoresis, and bacterial cultures with a consistent size and high brightness were selected and sent to Jinweizhi Biotechnology Co., Ltd. for sequencing. Finally, the returned sequencing results were compared with the target gene fragment to verify the identity of the PCR product. The bacterial culture PCR reaction program was as follows: 94°C, 5 min; 32 cycles {94°C, 30 s; 58°C, 30 s; 72°C, 1 min}; 72°C, 5 min; and save at 4°C.

[0045] Table 9 Bacterial liquid PCR system

[0046] The results of agarose gel electrophoresis of PCR products are shown in Figure 3 , BMP2The gene obtained three relatively clear bands and a relatively fuzzy band (250bp-500bp) ( Figure 3 A); PDK1 The gene obtained two relatively clear bands and two relatively fuzzy bands (about 750bp) ( Figure 3 B); LPL The first 3'-RACE experiment of the gene obtained a relatively clear band and two relatively fuzzy bands (500bp-750bp and about 1500bp). The second 3'-RACE experiment obtained two relatively clear bands ( Figure 3 C). APA (alternative polyadenylation) variants were found in these three genes.

[0047] In addition, sequencing results revealed BMP2 The gene has five cDNA sequences of 286 bp, 347 bp, 592 bp, 617 bp, and 1096 bp (named B-APA1, B-APA2, B-APA3, B-APA4, and B-APA5, respectively); PDK1 There are four cDNA sequences with lengths of 281 bp, 523 bp, 646 bp, and 840 bp (named P-APA1, P-APA2, P-APA3, and P-APA4, respectively); LPL The gene has three cDNA sequences of 352bp, 734bp and 809bp (named L-APA1, L-APA2 and L-APA3, respectively).

[0048] ②Analysis of 3'-UTR length differences.

[0049] According to the sequence information obtained by sequencing, BMP2 The forward primer BMP2-FA and three different reverse primers that bind to different regions of the 3'-UTR were designed for the gene. PDK1 Gene design: forward primer PDK1-FA and four different reverse primers binding to different regions of the 3'-UTR, representing different 3'-UTR lengths ( Figure 4 ).

[0050] To verify BMP2 and PDK1 The changes in 3'-UTR length of genes during cell differentiation were investigated by different primer combinations (BMP2-FA + BMP2-R1, BMP2-FA + BMP2-R2, BMP2-FA + BMP2-R3 and PDK1-FA + PDK1-R1, PDK1-FA + PDK1-R2, PDK1-FA + PDK1-R3, PDK1-FA + PDK1-R4). BMP2 and PDK1 It should be noted that based on the results of agarose gel electrophoresis and sequencing, BMP2 The gene had only three variants with clear electrophoresis bands and stable expression, so this step only performed quantitative analysis on the 3'-UTRs corresponding to these three bands.

[0051] The results are as follows Figure 5 As shown, it can be seen from the brightness of the stripes that BMP2 At 0h and 24h of preadipocyte differentiation, only the shorter products generated by BMP2-R1 and BMP2-R2 were expressed, and the long 3'-UTR was almost non-existent. On the contrary, the product generated by BMP2-R3 was significantly expressed at 96h and 120h. BMP2 Genes tend to express transcripts with shorter 3'-UTR at 0h, 24h, 48h, and 72h ( Figure 5 A). PDK1 The product generated by PDK1-R4 was almost not expressed at 0h and 24h of preadipocyte differentiation, but was significantly expressed at 48h, 72h and 96h. It is speculated that the transcripts with shorter 3'-UTR tend to be expressed at 0h, 24h and 120h than at 48h, 72h and 96h ( Figure 5 B).

[0052] The primers used in this example are shown in Table 10.

[0053] Table 10 PCR primers and gene-specific primer sequences

[0054] Example 2 Using the primers in Example 1, this example further determined the expression of the 3'-UTR of the target genes BMP2 and PDK1 at different differentiation stages during preadipocyte differentiation by qPCR experiments.

[0055] The forward and reverse primers used for the short fragments and the reverse primer used for the long fragments were primers that have been used to analyze 3'-UTR length variation. The amplified products of BMP2-R1 and PDK1-R1 were used to detect the expression of total isoforms (indicated by S). BMP2The primers used for detection include P7-F / R, BMP2-FA and BMP2-R-1, BMP2-F / R-2, and BMP2-F / R-3 (see Table 10 for specific sequences). Among them, P7-F and P7-R are forward and reverse primers of the TBP internal reference gene, used for data normalization (2-ΔΔCT), BMP2-FA and BMP2-R-1 are used to detect the expression of the total isomer S of BMP2, BMP2-F-2 and BMP2-R-2 are used to detect the expression of the longer transcript L-er of BMP2, and BMP2-F-3 and BMP2-R-3 are used to detect the expression of the longest transcript L-est of BMP2. PDK1 The primers used for detection include P8-F / R, PDK1-FA and PDK1-R-1, PDK1-F / R-2, PDK1-F / R-3, and PDK1-F / R-3 (see Table 10 for specific sequences), among which P8-F / R is the forward and reverse primer of the TBP internal control and is used for data normalization (2-ΔΔCT), PDK1-FA and PDK1-R-1 are used to detect the expression of the total PDK1 isoform S, PDK1-F / R-2 is used to detect the expression of the longer PDK1 transcript L-er-1, PDK1-F / R-3 is used to detect the expression of the longer PDK1 transcript L-er-2 (length L-er-2>L-er-1), and PDK1-F / R-4 is used to detect the expression of the longest PDK1 transcript L-est.

[0056] Test results such as Figure 6 As shown in the figure, L-er represents the longer transcript, L-est represents the longest transcript, S represents the total isoform, and the vertical axis represents the relative expression of the gene (TBP gene is used as the internal reference, and the original CT value is converted to the relative gene expression using the 2-ΔΔCT method). Figure 6 As shown in A, there is no difference in the expression of L-er and S in BMP2 gene at 0h, 24h, 48h and 72h of differentiation of chicken preadipocytes, indicating that it mainly exists in the form of long transcripts; while there is no difference in the expression of L-er and L-est at 96h and 120h of differentiation, but both are significantly lower than the expression of S, indicating that long and short transcripts exist at the same time and have the same expression level. Figure 6 As shown in Figure 2, the PDK1 gene mainly expresses the long transcript at 0h and 24h of adipocyte differentiation, and the expression levels of the long and short transcripts are the same at 48h, 72h, 96h, and 120h of differentiation.

[0057] Furthermore, by comparing the expression levels of the total isoform relative to the longer 3'-UTR isoform ( Figure 7 ), the results showed that compared with before adipocyte differentiation induction (0h), BMP2 and PDK1 genes tended to express transcripts with longer 3'-UTR at 48h.

[0058] In summary, the dynamic changes of the 3'-UTR of the adipogenic genes BMP2 and PDK1 during chicken preadipocyte differentiation are related to adipogenesis, which can be used for the identification and analysis of chicken preadipocyte differentiation, providing materials and methods for conventional genetic breeding of chicken quality, and has application prospects.

[0059] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the present invention, other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present invention.

Claims

1. Application of a chicken lipogenic gene 3'-UTR variant in chicken preadipocyte differentiation identification and analysis, characterized in that: The chicken adipogenic gene includes BMP2 and PDK1 .

2. The use according to claim 1, characterized in that The chicken preadipocytes are chicken immortalized preadipocytes.

3. The use according to claim 1, characterized in that The chicken preadipocyte differentiation is induced by oleic acid.

4. The use according to claim 2, characterized in that The chicken adipogenic gene 3'-UTR variant is obtained by 3'-RACE and nested PCR.

5. The use according to claim 4, characterized in that The gene was obtained by the following primers BMP2 and PDK1 3'-UTR variants: B-GSP1 / 2 whose sequences are shown in SEQ ID NOs. 29-30, and P-GSP1 / 2 whose sequences are shown in SEQ ID NOs. 31-32.

6. The use according to claim 5, characterized in that In the nested PCR, the first PCR reaction program is: 94°C 3 min; 94°C 30 s, 56°C 30 s, 72°C 30 s, 35 cycles; 72°C 10 min; 4°C save; the second PCR reaction program is: 94°C 3 min; 94°C 30 s, 60-62°C 30 s, 72°C 30 s, 35 cycles; 72°C, 10 min; 4°C save.

7. The use according to claim 2, characterized in that Genes in the preadipocyte differentiation process BMP2 and PDK1 The 3'-UTR of the gene changes dynamically, and the BMP2 Contains 5 transcripts, the gene PDK1 Contains 4 transcripts.

8. The use according to claim 7, characterized in that qPCR detection and gene analysis BMP2 and PDK1 Expression of various 3'-UTR variants during cell differentiation.

9. The use according to claim 8, characterized in that The primers used in the qPCR include The following: P7-F / R as shown in SEQ ID NOs. 11-12, BMP2-FA and BMP2-R-1 as shown in SEQ ID NOs. 13-14, BMP2-F / R-2 as shown in SEQ ID NOs. 15-16, BMP2-F / R-3 as shown in SEQ ID NOs. 17-18, P8-F / R as shown in SEQ ID NOs. 19-20, PDK1-FA and PDK1-R-1 as shown in SEQ ID NOs. 21-22, PDK1-F / R-2 as shown in SEQ ID NOs. 23-24, PDK1-F / R-3 as shown in SEQ ID NOs. 25-26, and PDK1-F / R-4 as shown in SEQ ID NOs. 27-28.

10. The use according to claim 8, characterized in that In the early stage of preadipocyte differentiation, the expression levels of the long variants of BMP2 and PDK1 were higher than those of the short variants.

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