ShRNA targeting and inhibiting expression of chicken cdkn2a gene and application thereof

Abstract

This invention discloses a shRNA that targets and inhibits the expression of the chicken CDKN2A gene and its application, belonging to the fields of molecular biology and poultry breeding technology. The invention provides shRNA1076 with a target nucleotide sequence as shown in SEQ ID NO.3, and its sense and antisense strand sequences as shown in SEQ ID NO.8-9, respectively. This shRNA is constructed into a lentiviral vector to obtain a lentiviral interference vector. This lentiviral interference vector is applied to chicken melanocytes to inhibit the expression of chicken CDKN2A mRNA, while simultaneously downregulating the expression of CDKN1A and SFN mRNA, and upregulating the expression of MITF and DCT mRNA, thus affecting the cell cycle progression of chicken melanocytes and influencing their melanin synthesis status. This shRNA can effectively inhibit the expression level of the chicken CDKN2A gene, with an inhibition efficiency of 70%.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and poultry genetics and breeding technology, and in particular to a shRNA that targets and inhibits the expression of the chicken CDKN2A gene and its application. Background Technology

[0002] Melanin is a naturally occurring pigment widely found in animals, distributed and deposited to varying degrees in the skin, muscles, bones, and feathers of poultry, especially chickens. In livestock farming and poultry breeding, melanin deposition (i.e., blackness) is a crucial economic indicator of the quality of Silkie chickens. Melanocytes are the main cells involved in melanin synthesis, and their functional state directly affects pigmentation in poultry skin, feathers, and other tissues. The CDKN2A gene (cyclin-dependent kinase inhibitor 2A) is a key gene in cell cycle regulation, participating in the p53 signaling pathway and influencing cell proliferation and apoptosis. Recent studies have shown that CDKN2A may be involved in regulating the expression of cell cycle and melanin synthesis-related genes in melanocytes. However, functional studies on the chicken CDKN2A gene are still limited, and the lack of efficient and specific RNA interference tools restricts its application in regulating chicken melanocyte function and improving poultry pigmentation traits.

[0003] RNA interference (RNAi) is a highly efficient and specific gene silencing technique that has emerged in recent years. Short hairpin RNA (shRNA), as an important tool for RNA interference, can be stably integrated into the host cell genome via viral vectors (such as lentiviral vectors). After intracellular processing, it produces small interfering RNA (siRNA), thereby achieving sustained silencing of target genes. Compared with transient siRNA, shRNA has advantages such as long-lasting silencing effects, stable inheritance, and suitability for long-term in vivo studies. However, no shRNA has been reported that effectively inhibits the expression of genes related to chicken melanin synthesis. Therefore, there is an urgent need to provide an shRNA that inhibits the expression of genes related to chicken melanin synthesis to achieve efficient regulation of chicken melanin synthesis. Summary of the Invention

[0004] The purpose of this invention is to provide a shRNA that targets and inhibits the expression of the chicken CDKN2A gene and its application, thereby solving the problems existing in the prior art. The shRNA interference sequence of this invention can effectively inhibit the expression level of the chicken CDKN2A gene, and has broad application prospects for the regulation of chicken melanocyte function and the improvement of poultry pigment traits.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides an shRNA that targets and inhibits the expression of the chicken CDKN2A gene. The shRNA is shRNA1076, and the target nucleotide sequence of shRNA1076 is shown in SEQ ID NO.3.

[0006] Further, the sense strand sequence of the shRNA1076 is prepared as shown in SEQ ID NO.8, and the antisense strand sequence is shown in SEQ ID NO.9.

[0007] The present invention also provides a vector comprising the shRNA as described in claim 1 or 2.

[0008] Furthermore, the backbone vector of the vector is the lentiviral vector PGMLV-SC5.

[0009] The present invention also provides the application of the above-mentioned shRNA or the above-mentioned vector in the preparation of a product that inhibits the expression of the chicken CDKN2A gene.

[0010] The present invention also provides the application of the above-mentioned shRNA or the above-mentioned vector in the preparation of products that regulate chicken melanin synthesis.

[0011] The present invention also provides a product for regulating chicken melanin synthesis, with the above-mentioned shRNA or the above-mentioned vector as the active ingredient.

[0012] The present invention also provides the application of the above-mentioned shRNA, vector, or product in the improvement of chicken pigmentation traits.

[0013] The present invention discloses the following technical effects: This invention proposes an shRNA to inhibit the expression of the chicken CDKN2A gene and constructs a corresponding lentiviral interference vector for the shRNA. This vector can efficiently and specifically inhibit the expression level of chicken CDKN2A gene mRNA, with an inhibition efficiency of up to 70%. The interference plasmid obtained by constructing the shRNA that inhibits chicken CDKN2A gene expression on a lentiviral vector can be used to study the function of CDKN2A in chicken melanocytes and can also be used to regulate chicken melanin deposition, possessing potential genetic breeding application value. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1A schematic diagram of shRNA target design for inhibiting chicken CDKN2A gene expression; where a is a schematic diagram of shRNA interference sites; b is a diagram of different shRNA target sequences and sequencing peaks; Figure 2 A schematic diagram of the shRNA lentiviral vector structure for inhibiting chicken CDKN2A gene expression; Figure 3 The image shows the electrophoresis pattern of the vector digested with enzymes; lane 1 is the DL15000 DNA ladder (bands from top to bottom are: 15000bp, 10000bp, 7500bp, 5000bp, 2500bp, 1000bp, 250bp), and lanes 2-4 are linear vectors digested with BamHI and EcoRI. Figure 4 This is a diagram showing the sequencing results of a positive clone plasmid. Figure 5 Figure 1 shows the results of transfection and activity verification of the CDKN2A-shRNA plasmid vector in DF-1 cells; where a is the fluorescence image of DF-1 cells after transfection (200×); b is the expression of the CDKN2A gene after plasmid transfection into DF-1 cells. Figure 6 The results of the morphological observation of melanocytes in chicken embryo skin (100×) (a) and its indirect immunofluorescence identification (b); Figure 7 The image shows the fluorescence of the Chicken_CDKN2A-shRNA1076 plasmid vector successfully transfected into chicken melanocytes. Figure 8 Figure showing the results of the inhibition of CDKN2A mRNA expression by Chicken_CDKN2A-shRNA1076 and the statistical results of the expression of genes related to cell cycle and melanin synthesis. Detailed Implementation

[0016] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] Example 1: Design and synthesis of chicken CDKN2A gene shRNA sequence and construction of its interference vector 1. shRNA sequence design The mRNA sequence of the chicken CDKN2A gene was retrieved from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Based on shRNA design principles, three shRNA sequences targeting the chicken CDKN2A gene were designed. These three shRNAs target three sites on the chicken CDKN2A gene (NM_204434.1) at 159bp, 883bp, and 1076bp, respectively, with a length of 21 bases. The three shRNAs were named shRNA-159, shRNA-883, and shRNA-1076, respectively. Figure 1 ).

[0022] The target sequence for shRNA-159 interference is: 5'-ATAAGGTGCACCGTCTGCCTT-3', SEQ ID NO.1; The target sequence for shRNA-883 interference is: 5'-CGATGCTCGCCGTATTCTTAA-3', SEQ ID NO.2; The target sequence for shRNA-1076 interference is: 5'-GTTCGACACTGACAGAAATTA-3', SEQ ID NO.3.

[0023] Based on the interference target sequences of the above three shRNAs, the corresponding sense and antisense strand DNA sequences of the shRNAs were designed and synthesized: shRNA-159 positive strand: 5'-GATCCATAAGGTGCACCGTCTGCCTTCTCGAGAAGGCAGACGGTGCACCTTATTTTTTT-3', SEQ ID NO.4; shRNA-159 antisense strand: 5'-AATTAAAAAATAAGGTGCACCGTCTGCCTTCTCGAGAAGGCAGACGGTGCACCTTATG-3', SEQ ID NO.5; shRNA-883 positive strand: 5'-GATCCGCGATGCTCGCCGTATTCTTAACTCGAGTTAAGAATACGGCGAGCATCGTTTTTT-3', SEQ ID NO.6; shRNA-883 antisense strand: 5'-AATTAAAAAACGATGCTCCGCCGTATTCTTAACTCGAGTTAAGAATACGGCGAGCATCGCG-3', SEQ ID NO.7; shRNA-1076 positive strand: 5'-GATCCGTTCGACACTGACAGAAATTACTCGAGTAATTTCTGTCAGTGTCGAACTTTTTT-3', SEQ ID NO.8; shRNA-1076 antisense strand: 5'-AATTAAAAAAGTTCGACACTGACAGAAATTACTCGAGTAATTCTGTCAGTGTCGAACG-3', SEQ ID NO.9; It contains a loop sequence (CTCGAG).

[0024] The 5' end of the above-mentioned sense strand template was supplemented with GATC, which is complementary to the sticky end formed after BamHI digestion; the 5' end of the antisense strand template was supplemented with AATT, which is complementary to the sticky end formed after EcoRI digestion. The designed shRNA primer sequences were sent to Beijing Qingke Biotechnology Co., Ltd. for synthesis.

[0025] 2. Construction of interference vector The synthesized oligo-single-stranded DNA containing the interfering sequence was annealed and paired to generate a double-stranded shRNA oligo. This oligo was then directly ligated into the digested PGMLV-SC5 lentiviral interference vector (Jiman Biotechnology Co., Ltd.) via the restriction enzyme sites at both ends. A schematic diagram of the vector structure is shown below. Figure 2The ligation product was transferred into prepared competent bacterial cells (E. coli strain stbl3). The resulting single-clone colonies were sent to a sequencing company for sequencing identification. The clones that matched correctly were considered to be successfully constructed RNA interference vectors for the target gene.

[0026] 2.1 Annealing of shRNA Dilute the primers with sterile TE buffer to a final concentration of 100 µM. Mix 10 µL of each primer pair, pipette to mix thoroughly, and transfer to a PCR tube for annealing, following the procedure below: 95℃ 30 s, 72℃ 2 min, 37℃ 2 min, 25℃ 2 min.

[0027] After completion, place on ice for a few minutes before connecting directly or freeze at -20°C for storage.

[0028] 2.2 Enzyme digestion and recovery of PGMLV-SC5 vector The enzyme digestion system is as follows: shRNA vector 5 µg, 10×Buffer 5 µL, BamH I 2 µL, EcoR I 2 µL, add ddH2O to 50 µL.

[0029] Digest the enzyme at 37℃ for approximately 30 min. During this time, prepare a 0.8% agarose gel and perform nucleic acid electrophoresis after digestion. After electrophoresis, cut off the strip containing the target fragment. Weigh the gel using a balance and subtract the weight of the empty tube to calculate the gel weight. Calculate the gel volume based on 100 mg being approximately 100 µL, and add one volume of Bingding Solution. Place the gel in a 65℃ water bath to completely dissolve the gel. Gently shake the EP tube during this process to accelerate gel dissolution. Transfer all the liquid to a filter column and centrifuge at 13000 rpm for 30 s (this can be repeated once). Discard the liquid in the tube, add 500 µL of Wash Solution to the column, and centrifuge at 13000 rpm for 30 s. Discard the liquid in the tube again, add 500 µL of Wash Solution to the column, and centrifuge at 13000 rpm for 30 s (this can be repeated once). Then centrifuge for 3 min. Place the filter column in a new 1.5 mL EP tube and allow it to air dry at room temperature. Finally, add 35 µL of ddH2O to the column, let stand for 5 min, and centrifuge at 13000 rpm for 1.5 min. To improve the recovery rate, the dissolved DNA can be added back to the column and centrifuged for one minute. Discard the column; this yields the recovered linear vector fragment. Determine the concentration. The agarose gel electrophoresis image of the enzyme digestion product is shown below. Figure 3 .

[0030] 2.3 Ligation of linearized PGMLV-SC5 vector with primers The connection system is as follows: 3 µL (50 ng) of recovered vector, 1 µL (0.5 µM) of shRNA primer, 1.5 µL of T4 DNA ligase buffer, 1 µL of T4 DNA ligase, and ddH2O to make up to 15 µL.

[0031] Incubate in a 25°C water bath for 30 minutes.

[0032] 2.4 Transformation After thawing the competent cells naturally on ice, add all the ligation product to the competent cells and incubate on ice for 20 min, followed by heat shock in a 42°C water bath for 90 s. Then, quickly place them on ice for 2-3 min. Add 1000 µL of antibiotic-free LB culture medium and incubate at 37°C with shaking at 150 rpm for 45 min. Centrifuge at 3000 rpm for 2 min, discard approximately 850 µL of supernatant, disperse the bacterial culture at the bottom of the tube by pipetting, add it to a culture dish containing the appropriate antibiotic, spread evenly using a sterile spreader, and incubate overnight at 37°C.

[0033] 2.5 Preparation of recombinant plasmids Select several single colonies and perform small-scale shaking culture.

[0034] 2.6 Sequencing to identify positive clones Sequencing results of the shRNA interference vector are as follows: Figure 4 As shown.

[0035] After comparison, the inserted fragment sequence in the recombinant clone was found to be completely consistent with the designed oligo sequence, so the vector was successfully constructed.

[0036] Example 2: Transfection of DF1 cells with chicken CDKN2A lentiviral interference vector and verification of vector activity DF-1 cells (chicken embryo fibroblasts) were provided by Novizan Biotechnology Co., Ltd. The cell suspension was prepared at a concentration of 1×10⁻⁶ cells / mL. 6Cells were evenly distributed to each well of 6-well and 24-well culture plates at a concentration of [cell density] / mL, ensuring a consistent cell count per well. The plates were then placed in an incubator at 37°C and 5% CO2. Cells were allowed to adhere and reach approximately 60%-70% confluence. After reaching the appropriate stage, the complete culture medium was gently aspirated from each well. Each well was then washed with sterile PBS to remove residual culture medium and metabolic products. After washing, approximately 2 mL of preheated complete culture medium was added to each well to meet the requirements for subsequent transfection experiments. Transfection was performed according to the Lipofectamine™ 3000 reagent instructions. Chicken CDKN2A interference vector transfection was performed in the 24-well plates, with cells transfected with the empty vector serving as a control group. The transfected cell plates were returned to the incubator at 37°C and 5% CO2 for another 24 hours. After culture, fluorescence microscopy was used to observe the expression of fluorescent proteins. The results showed that the transfected DF-1 cells exhibited obvious green fluorescence signals under a fluorescence microscope, indicating that the vector was successfully transfected into DF-1 cells. Figure 5 (a)

[0037] Cell samples were collected, and RNA was extracted from DF-1 cells in both the experimental and control groups. cDNA was synthesized via reverse transcription. The expression level of the CDKN2A gene was detected using real-time quantitative PCR to verify the vector interference efficiency. The primers for CDKN2A quantitative PCR detection are as follows: cCDKN2A-F1: CCCGTAGAGCTTTGCAGTTC, SEQ ID NO.10; cCDKN2A-R1: ACATGCAGGAACGGATCTTC, SEQ ID NO.11; cCDKN2A-F2:GACATTGTCGGGGGACTCTA, SEQ ID NO.12; cCDKN2A-R2:TGGTGTGAACACTGGGAAGA, SEQ ID NO. 13.

[0038] The product lengths of cCDKN2A-F1 and cCDKN2A-R1 are 228 bp, while the product lengths of cCDKN2A-F2 and cCDKN2A-R2 are 155 bp.

[0039] Real-time quantitative PCR showed that all three chicken CDKN2A interference vectors were effective and significantly inhibited CDKN2A gene mRNA expression; in particular, the shRNA1076 interference site showed the best interference effect on CDKN2A, with an inhibition efficiency of approximately 70%. Figure 5 (b)

[0040] Example 3 Chicken_CDKN2A-shRNA1076 transfected into chicken melanocytes 1. Isolation and culture of chicken melanocytes (1) Crack the air cell of eggs aged 12.5-18.5 days and remove the chicken embryo with sterile forceps. First, open the abdominal cavity of the chicken embryo with sterile surgical scissors, remove the peritoneum, and place it in preheated PBS + 2% penicillin-antibody solution. Then remove the feathers on the back of the chicken embryo, cut off the skin on the back, and place it in preheated PBS + 2% penicillin-antibody solution as well.

[0041] (2) The peritoneum and dorsal skin were rinsed three times in a PBS + 2% double antibody solution preheated at 37°C to remove hair, mesentery and grease and other impurities.

[0042] (3) Place the cleaned peritoneum and skin in a 10mL penicillin bottle and cut it into a paste-like consistency using sterile surgical scissors.

[0043] (4) Place the minced tissue into a 50mL centrifuge tube and add 0.2% neutral collagenase II and 0.25% trypsin-EDTA at a tissue:enzyme ratio of 1:3 (if the tissue is 5mL, add 7.5mL neutral protease II and 7.5mL trypsin-EDTA). Incubate at 37℃ for one hour, inverting the tube every 15 minutes to mix, until the tissue becomes viscous.

[0044] (5) After the tissue becomes viscous, gently pipette over and over the tissue repeatedly with a 5 mL pipette to separate the melanocytes from the tissue as much as possible. Finally, add an equal volume of DMEM complete medium (7.5 mL fetal bovine serum + 500 µL penicillin-antibody + 42 mL DMEM medium) to stop the digestion, and gently pipette over and over the tissue repeatedly to mix.

[0045] (6) After digestion is terminated, the viscous tissue is first filtered out with a 200-mesh cell sieve, and then cell clumps are removed with 70µm and 40µm filters respectively. The filtered liquid is black.

[0046] (7) Then centrifuge at 1500 rpm for 5 min and discard the supernatant. Add PBS preheated to 37℃ with 2% penicillin antibody, gently pipette, centrifuge at 1500 rpm for 5 min and discard the supernatant. Repeat this step three times until the supernatant becomes clear and transparent.

[0047] (8) Resuspend the cell pellet in complete human epidermal melanocyte culture medium, seed the cells into 6-well plates according to the cell quantity, and incubate in a 5% CO2, 37°C incubator. After 12 hours, aspirate the culture medium, remove the non-adherent cells and supernatant, and change the medium every 2 days thereafter.

[0048] Observe the cell growth status, and when the cells reach 70% confluence, digest and passage them with trypsin containing EDTA to obtain purified chicken melanocytes. Figure 6 (a)

[0049] 2. Identification of chicken melanocytes The expression of tyrosinase (TYR), tyrosinase-associated protein 1 (TYRP1), and dopachrome tautomerase (DCT) directly affects melanin synthesis. Therefore, by detecting their protein expression in in vitro cell culture, melanocytes can be identified to some extent, and their activity status can be indicated. The specific identification steps for labeling melanocytes using TRP2 / DCT antibodies are as follows: (1) Plating: Place the cell spreaders, which have been disinfected with alcohol, into a 24-well plate, and then place the melanocytes into the 24-well plate with the spreaders.

[0050] (2) Fixation: Fix with 4% paraformaldehyde for 5 min. Wash with PBST 3 times / 5 min.

[0051] (3) Permeabilization: Permeabilize the membrane with 0.5% Triton X-100 at room temperature for 15 min. Wash with PBST 3 times for 5 min each time.

[0052] (4) Blocking: Add 200 μL of blocking solution to the 24-well plate (to ensure that all samples are soaked) and let stand at room temperature for 1 hour.

[0053] (5) Primary antibody incubation: Use a pipette to remove residual water droplets from the 24-well plate, add 200 μL of diluted TRP2 / DCT primary antibody, and incubate overnight at 4°C.

[0054] (6) Rinse primary antibody: Use a pipette to draw an appropriate amount of PBST to wash 3 times / 5min.

[0055] (7) Secondary antibody incubation: Use a pipette to remove residual water droplets from the wells, add 200 μL of diluted secondary antibody into the 24-well plate, and incubate at room temperature for 2 hours or in a 37°C incubator for 1 hour.

[0056] (8) Rinse the secondary antibody: Use a pipette to draw an appropriate amount of PBST to rinse 3 times, 5 min each time.

[0057] (9) Add DAPI: Use a pipette to remove the residual water droplets from the well, then add an appropriate amount of DAPI and incubate at 4°C in the dark for 5 minutes.

[0058] (10) Photographing and mounting: Add anti-quenching agent to the slide, then invert the slide onto the slide to prevent air bubbles from forming. Finally, take a photograph under a fluorescence microscope.

[0059] The results showed that all isolated melanocytes expressed DCT protein, confirming that the cells isolated in this invention were melanocytes. Figure 6 (b)

[0060] 3. Transfection of chicken melanocytes The CDKN2A interference vector transfection experiment included a corresponding empty vector group as a control. Cell suspensions were prepared at 1×10⁻⁶. 5 Cells / mL were evenly distributed into each well of a 24-well culture plate, ensuring a consistent cell count per well. The plate was then placed in a 37°C, 5% CO2 incubator. Once the cells adhered and reached approximately 60%-70% confluence, the chicken melanocyte culture medium was gently aspirated from each well. Each well was then washed with sterile PBS to remove residual culture medium and metabolic products. After washing, approximately 0.5 mL of preheated chicken melanocyte culture medium was added to each well. Transfection experiments were performed according to the Lipofectamine™ 3000 reagent instructions, with three replicates per transfection. The stabilized DNA-liposome complex was evenly added to the 24-well plate, and the plate was gently shaken to ensure thorough mixing. The plate was returned to the 37°C, 5% CO2 incubator and cultured for another 24-48 hours. After culture, images were taken using a fluorescence microscope. The transfected melanocytes exhibited a strong green fluorescence signal. Figure 7 This indicates that the Chicken_CDKN2A-shRNA1076 plasmid vector was successfully transfected into chicken melanocytes.

[0061] Example 4: Inhibitory effect of Chicken_CDKN2A-shRNA1076 on chicken CDKN2A expression and its influence on the expression of genes related to cell cycle and melanin synthesis. Chicken melanocyte samples transfected with the Chicken_CDKN2A-shRNA1076 vector and empty vector control samples were collected. Total RNA was extracted from the cells using the TRIzol kit, and its concentration was measured using a nucleic acid analyzer. Qualified RNA samples were sent to Wuhan Bena Technology Co., Ltd. for RNA-seq library construction and sequencing. Changes in the expression of CDKN2A and downstream cell cycle-related genes, as well as the expression of melanin synthesis-related specific genes in melanocytes, were analyzed. Two sequencing experimental groups were set up: the Chicken_CDKN2A-shRNA1076 interference group and the blank control group, with three biological replicates in each group.

[0062] The results show that ( Figure 8Chicken_CDKN2A-shRNA1076 can effectively inhibit the expression of the target gene CDKN2A at the mRNA level, with an inhibition efficiency of 70%. CDKN1A and SFN are downstream target genes of CDKN2A in the cell cycle pathway and can cause cell cycle arrest. In this example, the expression of CDKN2A in chicken melanocytes was inhibited, and the expression levels of CDKN1A and SFN were significantly downregulated, indicating that cell cycle arrest in melanocytes was affected. MITF and DCT are melanin synthesis-related genes. The expression levels of MITF and DCT were significantly upregulated when the expression of CDKN2A in chicken melanocytes was inhibited, indicating that melanin synthesis was promoted.

[0063] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A shRNA that targets and inhibits the expression of the chicken CDKN2A gene, characterized in that, The shRNA is shRNA1076, and the target nucleotide sequence of shRNA1076 is shown in SEQ ID NO.

3.

2. The shRNA as described in claim 1, characterized in that, The sense strand sequence of shRNA1076 is shown in SEQ ID NO.8, and the antisense strand sequence is shown in SEQ ID NO.

9.

3. A carrier, characterized in that, The vector contains the shRNA as described in claim 1 or 2.

4. The carrier as described in claim 3, characterized in that, The backbone vector of the vector is the lentiviral vector PGMLV-SC5.

5. The use of the shRNA as described in claim 1 or 2 or the vector as described in claim 3 or 4 in the preparation of a product that inhibits the expression of the chicken CDKN2A gene.

6. The use of the shRNA as described in claim 1 or 2 or the vector as described in claim 3 or 4 in the preparation of a product that regulates chicken melanin synthesis.

7. A product for regulating melanin synthesis in chickens, characterized in that, The active ingredient is the shRNA as described in claim 1 or 2 or the vector as described in claim 3 or 4.

8. The use of the shRNA as described in claim 1 or 2, the vector as described in claim 3 or 4, or the product as described in claim 7 in the improvement of chicken pigment traits.

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