Shrna effectively inhibiting expression of yak fgf5 gene and application thereof

Abstract

The present application relates to the technical field of genetic engineering, and particularly relates to effective inhibition of the growth of yak FGF5 The shRNA for gene expression and application thereof. The shRNA is any one of (1) or (2): (1) FGF5-sh1, the nucleotide sequence is shown as SEQ ID NO:1; (2) FGF5-sh2, the nucleotide sequence is shown as SEQ ID NO:2. The present application successfully clones the shRNA sequence to a lentivirus interference vector through reverse PCR combined with homologous recombination technology, and constructs a recombinant lentivirus interference plasmid. The vector system not only carries a green fluorescent protein marker, which is convenient for real-time tracking of transfection and infection efficiency, but also has a puromycin resistance gene, which is beneficial to the screening and long-term culture of a stable cell strain, and provides a stable, efficient and traceable molecular tool for subsequent yak FGF5 Gene function research.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more particularly to an effective method for inhibiting yak... FGF5 shRNAs for gene expression and their applications. Background Technology

[0002] yak ( Bos grunniens The yak is an important livestock species on the Qinghai-Tibet Plateau, providing local herders with crucial resources such as meat, milk, and wool. To adapt to the hypoxic, low-temperature, and intense ultraviolet radiation environment of high-altitude regions, the yak has evolved a unique skin and hair structure: its skin contains primary hair follicles (producing coarse hair) and secondary hair follicles (producing downy hair). The secondary hair follicles are crucial for thermoregulation and are the key morphological basis for the yak's cold resistance. Yak hair follicles exhibit a distinct seasonal growth cycle (anagen, catagen, and telogen phases), which is synergistically influenced by photoperiod, environmental stress, and genetic regulation. Elucidating the molecular mechanisms of hair follicle cycle transformation is of great significance for understanding the yak's adaptation to the plateau and its genetic selection.

[0003] Fibroblast growth factor 5 (FGF5) FGF5 FGFR1-4 is a member of the FGF family and regulates cell growth by binding to the receptor FGFR1-4 to transmit signals. This gene was initially discovered in human tumor cells, encoding 268 amino acids, and has since been identified in multiple species, including mice, cattle, humans, rats, goats, cats, and dogs. FGF5 It has the function of inhibiting hair growth and promoting the transformation of hair follicles into the resting phase. Studies have shown that... FGF5 It can accelerate the transition of cashmere goat hair follicles to the regression phase, thereby reducing cashmere yield; while inhibiting its expression can slow down the hair follicle cycle process, improving cashmere yield and quality. In species such as goats and dogs, FGF5 Natural mutations in genes can lead to loss or reduction of protein function, thereby prolonging the hair growth phase and producing a long-haired phenotype. In Tianzhu white yaks, FGF5 The gene g.166858 T>A site was significantly associated with head hair length, back hair length, and hair production.

[0004] shRNA is a small RNA molecule with a hairpin structure that can silence target gene expression via the RNAi pathway. Unlike short-acting siRNA, shRNA can be introduced into cells via vectors and continuously expressed under the drive of the U6 or H1 promoter, making it a highly efficient tool for gene function research. (Specifically for yak...) FGF5 Designing specific shRNA sequences and constructing corresponding lentiviral expression systems will provide a basis for in-depth analysis of yak genetics. FGF5 This lays the foundation for gene function. This tool can be widely applied to yaks. FGF5 Related cell research has significant application prospects and economic value. Summary of the Invention

[0005] The purpose of this invention is to provide an effective method for controlling yak populations. FGF5 shRNAs for gene expression and their applications.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an shRNA that effectively inhibits the expression of the FGF5 gene in yaks, wherein the shRNA is any one of the following (1) or (2): (1) FGF5-sh1, whose nucleotide sequence is shown in SEQ ID NO:1; (2) FGF5-sh2, whose nucleotide sequence is shown in SEQ ID NO:2.

[0007] Preferably, the shRNA comprises a 21nt siRNA sense strand, a 7nt loop, and a 21nt siRNA antisense strand.

[0008] This invention provides the application of the shRNA that effectively inhibits the expression of the FGF5 gene in yaks: Applications in the preparation of products that inhibit FGF5 gene expression in yaks, or Applications in the preparation of kits for detecting the function of the FGF5 gene in yaks, or Applications in molecular breeding of yaks or in improving the quality of yak wool.

[0009] Preferably, the product is an shRNA lentiviral interference vector.

[0010] Preferably, the shRNA lentiviral interference vector is obtained by inserting the shRNA into the lentiviral interference vector through reverse PCR combined with homologous recombination.

[0011] Preferably, the lentiviral interference vector is the pLKO.1-U6-EF1a-copGFP-T2A-puro lentiviral interference vector.

[0012] This invention provides an shRNA lentiviral interference vector that effectively inhibits the expression of the yak FGF5 gene, wherein the shRNA lentiviral interference vector contains the shRNA.

[0013] The present invention provides a host cell containing the shRNA lentiviral interference vector described above.

[0014] This invention provides a method for effectively inhibiting the expression of the FGF5 gene in yaks, comprising the following steps: ligating the shRNA to a lentiviral interference vector to construct an shRNA lentiviral recombinant vector; and transfecting the shRNA lentiviral recombinant vector into 293T cells.

[0015] Preferably, the shRNA is ligated to a lentiviral interference vector via reverse PCR and homologous recombination.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides two specific targeting yak FGF5 The shRNA sequence of the gene, verified by real-time quantitative PCR, can significantly downregulate yak... FGF5 The mRNA transcription level of genes, among which FGF5 The suppression efficiency of -sh1 reached 40.71%. FGF5 The inhibition efficiency of -sh2 was 32.27%, indicating that both shRNAs had good target gene silencing effects. Furthermore, this invention successfully cloned the above-mentioned shRNA sequences into [the target gene] using reverse PCR combined with homologous recombination technology. pLKO.1 - U6 - EF1a - copGFP - T2A - puro A lentiviral interference vector was used to construct a recombinant lentiviral interference plasmid. This vector system not only carries a green fluorescent protein (copGFP) marker for real-time tracking of transfection and infection efficiency, but also possesses a puromycin resistance gene, which is beneficial for stabilizing cell lines and enabling long-term culture. This paves the way for subsequent cytokine-based cell line studies at the cellular and even individual levels. FGF5 Gene function research provides a stable, efficient, and traceable molecular tool.

[0017] More importantly, compared to artificially synthesized siRNA molecules, the one constructed in this invention... FGF5 -shRNA lentiviral interference vectors possess significant technical advantages. On one hand, lentiviral vectors can mediate the continuous and stable transcriptional expression of shRNA in host cells, avoiding the rapid decay of the effect after transient siRNA transfection, thus achieving long-term and persistent gene silencing in both in vivo and in vitro experiments. On the other hand, this vector can infect various difficult-to-transfect cell types through lentiviral packaging, greatly expanding its application range. Based on these advantages, this invention not only provides a deeper understanding of yak gene silencing but also... FGF5 This study provides a key experimental tool for understanding the molecular mechanisms of genes in hair follicle cycle regulation, down growth, and high-altitude adaptation. It also provides potential technical reserves for the genetic improvement of superior hair traits in yak molecular breeding, and has important basic research value and industrial application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 To suppress yaks FGF5 A schematic diagram of the structure of the FGF5-sh1 lentiviral vector for gene expression.

[0020] Figure 2 To suppress yaks FGF5 A schematic diagram of the structure of the FGF5-sh2 lentiviral vector for gene expression.

[0021] Figure 3 The image shows the fluorescence observation results of 293T cells 48 hours after co-transfection with the interference plasmid. In the image: A: FGF5 -sh1 interference vector transfected into 293T cells, B: FGF5 -sh2 interference vector transfected into 293T cells, C: empty vector transfected into 293T cells.

[0022] Figure 4 FGF5 -shRNA pairs FGF5 Quantitative results of gene interference. Detailed Implementation

[0023] The following embodiments are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.

[0024] Main reagents and instruments, equipment

[0025] Clean bench (Sujing Group, SW-CJ-2FD)

[0026] Gene Amplification Instrument (KUBO·TECH, q225)

[0027] Electrophoresis apparatus (Junyi, JY600E)

[0028] Low-temperature refrigerated centrifuge (Xiangyi, TGL-16M)

[0029] Micro-volume ultraviolet spectrophotometer (QSPEC, NanoUV-3000)

[0030] 293T cells

[0031] DMEM (Gibco, 12800-017)

[0032] Tiypsin-EDTA (Gibco, 25200-072)

[0033] Opti-MEM (Gibco, 31985-070)

[0034] Fetal-Bovine-Serum (Excell, FSP500)

[0035] FItran transfection reagent (Huijun Biotechnology, FA1802)

[0036] Micro-agarose gel DNA recovery kit (Sangon Biotech B518131-0100)

[0037] Plasmid miniprep kit (Sangon Biotech B518191-0050)

[0038] PrimeSTAR Max Premix(2X)(Takara R045A)

[0039] TaKaRa Taq (Takara R001B)

[0040] ClonExpress II One Step Cloning Kit ligase (Novizan C112-2)

[0041] DH5α competent cells (Huijun Biotechnology)

[0042] pLKO.1-U6-EF1a-copGFP-T2A-puro vector

[0043] Hieff qPCR SYBR Green Master Mix (no ROX) (YEASEN, 11201ES08)

[0044] HifairIII 1stStrand cDNA Synthesis SuperMix for qPCR (YEASEN, 11141ES10)

[0045] Example 1

[0046] The method steps of the present invention are as follows: I. Yak FGF5 Construction of gene shRNA interference vector According to data on yaks published in the NCBI database FGF5The ORF sequence of the gene mRNA [XM_005909150.2] was artificially designed, synthesized, and screened to select two different target sites as target sequences (597bp-617bp and 574bp-594bp). These sequences were then designed into shRNA structures containing a siRNA sense strand, a loop (CTCGAG), and an siRNA antisense strand. The specific sequence is as follows: (1) FGF5 -sh1 sequence: 5'-AGATCTACCCGGATGGCAAAGCTCGAGCTTTGCCATCCGGGTAGATCT-3'; (SEQ ID NO: 1) (2) FGF5 -sh2 sequence: 5'-AGTGGGCATCGGTTTCCATCTCTCGAGAGATGGAAACCGATGCCCACT-3'; (SEQ ID NO: 2) Note: Thymine (T) in shRNA transcripts is replaced with uracil (U). 2. Take 50 ng of lentiviral interference vector, add 0.2 µL of upstream primer, 0.2 µL of downstream primer, and 5 µL of high-fidelity enzyme PrimeSTAR Max (2×), and make up to 10 µL with sterile ultrapure water. Perform PCR reaction with the following program: 95℃ for 5 min; 95℃ for 20 s; 60℃ for 30 s; 72℃ for 1 min; 72℃ for 10 min.

[0047] FGF5 -sh1 upstream primer: 5'-AAGCTCGAGCTTTGCCATCCGGGTAGATCTTTTTTGAATTCTCGACCTCGAGACAAATG-3' (SEQ ID NO: 3) FGF5 -sh1 downstream primer: 5'-GGATGGCAAAGCTCGAGCTTTGCCATCCGGGTAGATCTCCGGTGTTTCGTCCTTTCCAC-3' (SEQ ID NO: 4) FGF5 -sh2 upstream primer: 5'-TCTCTCGAGAGATGGAAACCGATGCCCACTTTTTTGAATTCTCGACCTCGAGACAAATG-3' (SEQ ID NO:5) FGF5 -sh2 downstream primer: 5'-GGTTTCCATCTCTCGAGAGATGGAAACCGATGCCCACTCCGGTGTTTCGTCCCTTTCCAC-3' (SEQ ID NO:6) 3. PCR product recovery After electrophoresis, the PCR product is cleaved from the agarose gel, and the target gene fragment is recovered and purified. The specific steps are as follows: 1) After the enzyme digestion products are subjected to 1% gel electrophoresis, the gel strips containing the target fragment and the vector are cut off under UV light using a scalpel and transferred to clean 1.5 mL EP tubes. Buffer B2 is added at 3-6 times the weight of the gel block. 2) Incubate in a 50℃ water bath for 10 minutes until the gel is completely dissolved, shaking and mixing 3 times during the water bath. 3) Transfer the solution to the adsorption column, centrifuge at 8,000×g for 30 s, and discard the liquid in the collection tube; 4) Add 500µL Wash Solution to the column, centrifuge at 9,000×g for 30 s, and discard the liquid in the collection tube; 5) Repeat the previous operation once; 6) Centrifuge the empty column at 9,000 rpm for 1 min to completely remove any residual liquid from the purification column; 7) Place the column on a new 1.5 mL EP tube, add 35 µL of Elution Buffer to the center of the column, let it stand at room temperature for 1 min, then centrifuge for 1 min. Centrifuge at 13,400 × g for 1 min to elute the DNA.

[0048] 4. Conversion of the linker products

[0049] 1) Add 10 µL of the ligation product to 100 µL of stbl3 competent cells while in an ice bath. Gently swirl to mix and incubate on ice for 30 min.

[0050] 2) 42℃ water bath heat shock for 60s.

[0051] 3) Quickly transfer the tube to an ice bath and incubate for 2-3 minutes.

[0052] 4) Add 300µL of LB medium, mix well, and incubate at 37℃ and 200 rpm for 1 h with shaking.

[0053] 5) In a clean bench, spread the bacterial solution evenly on an LB agar plate containing Amp antibiotic (100µg / mL) and leave it at room temperature until the liquid is absorbed.

[0054] 6) Invert the plate and transfer it to a 37°C biochemical incubator for overnight incubation.

[0055] 5. Colony PCR verification

[0056] Positive clones obtained from colony PCR identification were sequenced for verification. After sequencing, the sequencing results were compared with those obtained by software. Sequencing primer 5'-TGTGGGCGATGTGCGC-3' (SEQ ID NO: 7).

[0057] (1) Preparation of bacterial detection PCR system

[0058] Table 1 PCR system

[0059] 2) Prepare the mixture according to the above system and add it to the sterile eight-tube bundles respectively; 3) In the clean bench, select eight individual colonies and mark them. Use a clean pipette tip to gently touch one side of the colony on the plate. Transfer the pipette tip with bacteria to the tube containing the mixed liquid. Gently shake the pipette tip to disperse the bacteria into the liquid. Discard the pipette tip.

[0060] 4) PCR amplification program

[0061] Table 2 PCR amplification program

[0062] 5) After amplification, add 2µL of 10×loading buffer to the PCR tube, take 10µL of sample and load it onto a 1% gel for electrophoresis, and add 5µL of marker as a reference.

[0063] 6) Based on the marker size, select the correct colony, shake it, and sequence it.

[0064] 6. Plasmid extraction: Use a plasmid extraction kit to extract plasmids according to the instructions. The specific steps are as follows: 1) Collect 3 mL of bacterial culture that was correctly sequenced in step 5 using a 2 mL EP tube, centrifuge at 8,000×g for 2 min to collect the bacterial cells, and discard the culture medium; 2) Add 250µL Buffer P1 to resuspend the bacterial cells; 3) Add 250µL Buffer P2, gently invert and mix 6 times, and let stand at room temperature for 2 min; 4) Add 350µL Buffer P3 and gently mix by inverting the container 6 times. 5) Centrifuge at 12,000×g for 10 min, carefully aspirate the supernatant into the DNA purification column, centrifuge at 8,000 g for 30 s, and discard the liquid in the collection tube; 6) Add 500µL Wash Solution, centrifuge at 8,000×g for 30 s, and discard the liquid in the collection tube.

[0065] 7) Add 500µL of solution W1 to the column, centrifuge at 13,000 rpm for 1 min, centrifuge at 8,000×g for 30 s, and discard the liquid in the collection tube; 8) Repeat step 7) once; 9) Centrifuge the empty column at 8,000 × g for 1 min; 10) Remove the column and place it in a new 1.5 mL EP tube. Add 50-100 µL of Elution Buffer to the center of the adsorption membrane. Let it stand at room temperature for 1 min, then centrifuge for 1 min to elute the plasmid.

[0066] II. Transfection of 293T cells with shRNA interference vector

[0067] 1.293T cells passaged and seeded into plates

[0068] 1) Remove the cryovials and thaw them quickly in a 37°C water bath. Transfer the cell cryopreservation solution to 7-8 mL of preheated complete culture medium, centrifuge at 800 rpm for 5 min, remove the supernatant, resuspend the cells in 1 mL of complete culture medium, and place them in a culture flask. Incubate in a 37°C CO2 incubator.

[0069] 2) Remove the cell culture flask, aspirate the old culture medium, and wash the cells 1-2 times with PBS.

[0070] 3) Add 1 mL of Trypsin-EDTA, gently shake the bottle to ensure that the Trypsin-EDTA is evenly distributed to the cells at the bottom of the bottle. When the cells are about to separate and appear granular (observe under an inverted microscope), add an amount of complete culture medium corresponding to the amount of Trypsin-EDTA to stop the reaction.

[0071] 4) Transfer the liquid into an EP tube and centrifuge at 800 rpm for 5 min; remove the supernatant, resuspend the cells in 1 mL of complete culture medium, seed the cells at an appropriate ratio, and shake the bottle to distribute the cells evenly. 5) Place it in a CO2 incubator (culture conditions: 5% CO2, temperature: 37℃) and continue to expand the culture as needed for the experiment.

[0072] 2. Plasmid transfection of 293T cells

[0073] 1) Once the cells have completely adhered to the wall and grown to a density of about 60%, they can be transfected. Before transfection, replace each well with 2 mL of fresh DMEM complete medium containing 10% fetal bovine serum and incubate in an incubator for 1 h. 2) Put 2 strips FGF5293T cells were transfected with the shRNA vector. 3 μg of plasmid was dissolved in 150 µL of Opti-MEM medium to prepare solution A. 12 µL of transfection reagent was dissolved in 150 µL of Opti-MEM medium to prepare solution B. Solutions A and B were mixed and allowed to stand for 5 min. Solution B was then mixed into solution A and allowed to stand for 10 min before being added to cell culture plates. The cells were cultured in an incubator at 37°C, 5% CO2, and 95% relative humidity. Once the cells reached confluence, they were collected for RT-qPCR detection.

[0074] III. shRNA ​ Evaluation of the effect of gene transcription level repression

[0075] RNA extraction was performed according to the Triozol instructions provided by Life Technologies. The procedure is as follows: 1) Add 1 mL of TRIzol to the cell culture plate, pipette it several times, then aspirate it and place it in a 1.5 mL centrifuge tube; 2) Add 0.2 mL of chloroform, tighten the centrifuge tube cap, invert and mix for 60 s (do not vortex or shake violently), let stand at room temperature for 3 min, centrifuge at 12,000×g, 4℃ for 15 min, and place on ice; 3) The solution separates into three layers. The RNA is dissolved in the aqueous phase. Carefully pipette 500µL of the aqueous phase into another new RNase-free EP tube. 4) Add 500µL isopropanol, incubate at -20℃ for 1 h, centrifuge at 12,000×g at 4℃ for 10 min. After centrifugation, RNA precipitate will appear at the bottom of the tube. Discard the supernatant. 5) Add 1 mL of 75% ethanol, gently invert by hand, centrifuge at 12,000×g for 5 min, and discard the supernatant; 6) Dry the sample on a clean bench for 10 min, then add an appropriate amount of DEPC water to dissolve the RNA. Add 40 µL of RNase-free water to dissolve the precipitate.

[0076] 2. RNA quality testing

[0077] RNA concentration was determined using a UV spectrophotometer.

[0078] 3. Genomic DNA removal

[0079] (1) Prepare the reaction system on ice according to the table below, with a total volume of 15µL. To ensure the accuracy of the reaction solution preparation, first prepare a premixed system according to the reaction number + 2, then dispense it into each reaction tube, and finally add the RNA sample.

[0080] Table 3 Reaction System

[0081] (2) Briefly centrifuge to collect the solution on the tube wall to the bottom of the tube.

[0082] (3) Incubate at 42℃ for 2 min.

[0083] (4) After the reaction is complete, briefly centrifuge and place on ice to cool.

[0084] 4. cDNA Synthesis and Reverse Transcription

[0085] (1) Prepare the reaction system on ice. Please prepare the reaction solution on ice.

[0086] Table 4 Reaction System

[0087] (2) Mix well and centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.

[0088] (3) cDNA synthesis reaction conditions: 25℃ for 5 min, 55℃ for 15 min, 85℃ for 5 min.

[0089] (4) After the reaction is complete, centrifuge briefly and place on ice before performing quantitative PCR. If long-term storage is required, place at -20℃.

[0090] 5. Real-time quantitative PCR

[0091] Each sample was used in parallel experiments with 3 target genes and 3 internal control genes. The PCR reaction volume was 10 μL, and the reaction system was as follows: Table 5 Reaction System

[0092] The reaction conditions are: Table 6 Reaction conditions

[0093] Table 7 Primer sequences

[0094] After the cycle is completed, the temperature is increased from 60℃ to 98℃ to obtain the melting curve.

[0095] Before performing quantitative PCR, a preliminary test should be conducted to assess the quality of the cDNA sample. Real-time PCR should be performed on the cDNA sample using the internal reference gene GAPDH, and the cDNA sample quality should be evaluated based on the Ct value. Samples with Ct values ​​between 18 and 25 can proceed directly to the later stages of the experiment; if Ct ≥ 30, the cDNA sample concentration is too low, and reverse transcription needs to be repeated; if there are no amplification results, the cDNA quality is problematic, and reverse transcription needs to be repeated.

[0096] Change the cDNA template from 10x to 10x 5 The probes were serially diluted and used as templates for quantitative real-time PCR reactions of each probe pair. A standard curve was constructed using five gradient reactions of the internal control gene GAPDH. The slopes and R values ​​of the curves for the other primer pairs were compared. 2 Compare with the internal reference gene to ensure consistent amplification efficiency.

[0097] 5. Statistical analysis of real-time PCR data

[0098] The standardized statistical analysis of the quantitative real-time PCR data involved correcting for differences in initial template amounts and reverse transcription efficiencies among different samples using the GAPDH gene as an internal reference. For each reaction, the target gene and internal reference gene for each sample were repeated three times to obtain the average Ct value. The ΔΔCt value for each sample was calculated using Excel software, and the relative expression level of the target gene and the inhibition efficiency of each shRNA fragment were calculated using the 2-ΔΔCt method. Interference efficiency (%) = [(expression level of expression control group - expression level of shRNA group) / expression level of expression control group] × 100%.

[0099] As can be seen from the above, both shRNAs of the present invention are effective against... ​ It has a good inhibitory effect, among which ​ The suppression efficiency of -sh2 is 32.27%. ​ -sh1 showed the highest suppression efficiency, reaching 40.71%.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A shRNA capable of inhibiting the expression of the gene of FGF5 of Bos Taurus, characterized in that, The shRNA is either (1) or (2) below: (1) FGF5-sh1, whose nucleotide sequence is shown in SEQ ID NO:1; (2) FGF5-sh2, whose nucleotide sequence is shown in SEQ ID NO:

2.

2. The shRNA capable of inhibiting the expression of the FGF5 gene of the yak according to claim 1, wherein, The shRNA comprises a 21nt siRNA sense strand, a 6nt loop, and a 21nt siRNA antisense strand.

3. The application of the shRNA described in claim 1 or 2 that effectively inhibits the expression of the yak FGF5 gene: Applications in the preparation of products that inhibit FGF5 gene expression in yaks, or Applications in the preparation of kits for detecting the function of the FGF5 gene in yaks, or Applications in molecular breeding of yaks or in improving the quality of yak wool.

4. Use according to claim 3, characterized in that, The product is an shRNA lentiviral interference vector.

5. Use according to claim 4, characterized in that, The shRNA lentiviral interference vector was obtained by inserting the shRNA into the lentiviral interference vector through reverse PCR combined with homologous recombination.

6. Use according to claim 5, characterized in that, The lentiviral interference vector is pLKO.1-U6-EF1a-copGFP-T2A-puro lentiviral interference vector.

7. A shRNA lentivirus interference vector effectively inhibiting the expression of the FGF5 gene of the yak, characterized in that, The shRNA lentiviral interference vector contains the shRNA as described in claim 1 or 2.

8. A host cell, characterized in that, The host cell contains the shRNA lentiviral interference vector as described in claim 7.

9. A method for effectively inhibiting the expression of a Bos taurus FGF5 gene, the method comprising administering to a Bos taurus an effective amount of the double-stranded RNA of claim 1. The procedure includes the following steps: linking the shRNA described in claim 1 or 2 to a lentiviral interference vector to construct an shRNA lentiviral recombinant vector; and transfecting the shRNA lentiviral recombinant vector into 293T cells.

10. The method of claim 9, wherein, The shRNA was ligated to a lentiviral interference vector via reverse PCR and homologous recombination.

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