CN114891832B

CN114891832B - Construction method and application of recombinant lentivirus for over-expressing bovine TUSC5a and TUSC5b genes

Info

Publication number: CN114891832B
Application number: CN202210587879.9A
Authority: CN
Inventors: 周扬; 夏晗; 李凡; 彭玲伟; 杜玉琴; 滑国华; 杨利国; 郭爱珍
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2023-10-27
Anticipated expiration: 2042-05-27
Also published as: CN114891832A

Abstract

The application discloses a construction method and application of an over-expression bovine TUSC5a and TUSC5b gene recombinant slow virus, wherein the over-expression recombinant slow virus can regulate and control fat deposition as an invasive tool or as an artificial agent, the expression of TUSC5a can obviously promote fat synthesis and accumulation, and the expression of TUSC5b can obviously inhibit fat synthesis and accumulation. The construction method comprises (1) cloning TUSC5a and TUSC5b sequences; (2) Double-enzyme cutting TUSC5a and TUSC5b sequences carrying HIS labels with slow virus vectors respectively by using endonuclease, and connecting the products after glue recovery by using ligase to obtain the over-expression recombinant slow virus; (3) a step of verifying the over-expression recombinant lentivirus; (4) a step of packaging the over-expressed recombinant lentivirus. The application can correctly recognize the regulation network of TUSC5 on fat deposition to reach the level of splicing isomers, and the two packaged TUSC5 splicing isomer slow viruses can be used as a tool for manually regulating the fat deposition capacity of beef cattle, thereby realizing the dosage regulation of the fat deposition capacity.

Description

Construction method and application of recombinant lentivirus for over-expressing bovine TUSC5a and TUSC5b genes

Technical Field

The application relates to the technical field of genetic engineering, in particular to a construction method and application of a recombinant lentivirus for overexpressing bovine TUSC5a and TUSC5b genes.

Background

Fat deposition ability is an important factor in determining the economic value of beef cattle. The selling prices of the beef fat tissues at different positions are greatly different. Subcutaneous and intraperitoneal fat deposition (tallow) costs around 6 yuan/kg; if fat is deposited in the muscle, snowflake beef can be formed, the beef quality is obviously improved, and the price is about 1500 yuan/kg. Therefore, the ability to control fat deposition at different locations is now a key to scientific research and to increase the economic value production of beef cattle. TUSC5 has been shown to be an important gene regulating fat deposition in humans, mice and cattle. The gene is worth noting that the gene is specifically and highly expressed in adipose tissues, the characteristic can ensure that the gene can not influence the growth and development of other tissues under the premise of manual regulation, and the tissue-specific start expression is not needed to be considered when preparing a relevant bioengineering regulation tool, so that the operation is simpler and more convenient. The above demonstrates that TUSC5 has great potential in achieving artificial regulation of fat deposition.

It was found that there are two different alternative splice isomers of TUSC5, TUSC5a and TUSC5b, but it is not clear at present whether both have a regulatory effect on fat deposition, and whether the extent of the regulatory effect of both on fat deposition is consistent. Previous studies demonstrated that alternative splicing would result in different transcripts produced from the same gene would have different and even opposite functions. Therefore, resolving the regulatory effect of two different splice isomers of TUSC5 on fat deposition is essential for its use in artificial regulation of bovine fat deposition.

The slow virus can integrate the genome sequence of the slow virus into a host cell by infecting cells or tissues, thereby realizing the effect of continuous expression of a target gene in target tissues, further regulating the development of the cells or tissues, realizing the purpose of artificial regulation, and being an effective genetic engineering means, and having wider application at present. By constructing and packaging lentiviruses that overexpress TUSC5a and TUSC5b, on the one hand, the regulatory capacity of both on fat deposition can be studied in adipocyte cell lines; on the other hand, on the basis of defining the regulation of fat deposition ability by two different splice isomers, a potential effective tool with the capacity of manually regulating fat deposition can be developed.

Therefore, the research builds and packages the TUSC5a and TUSC5b lentivirus by searching proper conditions, analyzes the difference of the capacity of the TUSC5a and TUSC5b on fat deposition on the basis, lays a foundation for further developing an effective biological tool for manually regulating and controlling the fat deposition capacity of beef cattle in the future, and provides necessary materials for rapidly improving the economic value of beef cattle.

Disclosure of Invention

The first object of the present application is to provide the use of the bovine TUSC5a and TUSC5b genes in the regulation of fat deposition in beef cattle, the TUSC5a and TUSC5b genes being two splice isomers of the bovine TUSC5 gene.

The second object of the present application is to provide the use of recombinant lentiviruses overexpressing the bovine TUSC5a and TUSC5b genes, which are two splice isomers of the bovine TUSC5 gene, in the regulation of fat deposition in beef cattle.

A third object of the present application is to provide an artificial formulation for regulating fat deposition in beef cattle.

The fourth object of the present application is to provide a method for constructing recombinant lentiviruses over-expressing bovine TUSC5a/TUSC5b genes, the TUSC5a and TUSC5b genes being two splice isomers of the bovine TUSC5 gene.

In order to achieve the above purpose, the application adopts the following technical scheme:

the application of the bovine TUSC5a and TUSC5b genes in the regulation and control of fat deposition of beef cattle, the expression of TUSC5a obviously promotes the synthesis and accumulation of fat, the expression of TUSC5b obviously inhibits the synthesis and accumulation of fat, the TUSC5a sequence is shown as SEQ ID No.1, and the TUSC5b sequence is shown as SEQ ID No. 2. Specifically, a molecular tool for editing TUSC5a and TUSC5b genes can be constructed into a virus for targeting infected bovine cells, and the target can enter a bovine body through intravenous injection or oral administration and the like to be integrated into a bovine genome for expression, so that the purposes of regulating and controlling fat deposition capacity and regulating and controlling beef quality are achieved.

The application of the recombinant slow virus of the over-expressed bovine TUSC5a and TUSC5b genes in beef cattle fat deposition regulation, the slow virus integrates the TUSC5a and TUSC5b genes into a genome to express and regulate fat deposition, the expression of the TUSC5a obviously promotes fat synthesis and accumulation, the expression of the TUSC5b obviously inhibits fat synthesis and accumulation, the TUSC5a sequence is shown as SEQ ID No.1, and the TUSC5b sequence is shown as SEQ ID No. 2. Specifically, the recombinant lentiviruses of the overexpression bovine TUSC5a and TUSC5b genes can be taken as a molecular tool to enter the bovine body for integration into the bovine genome for expression by intravenous injection, oral administration or the like.

An artificial preparation for regulating fat deposition in beef cattle, comprising an over-expression slow virus vector for over-expressing bovine TUSC5a/TUSC5b genes. The artificial preparation has the potential for manually regulating fat deposition capacity or parts, further regulating beef quality, and can be used for manually regulating beef fat deposition and production of high-grade beef.

The construction method of the over-expression bovine TUSC5a/TUSC5b gene recombinant lentivirus comprises the steps of respectively carrying out double enzyme digestion on TUSC5a/TUSC5b sequences carrying HIS labels and lentivirus vectors by using endonuclease, and connecting the products after glue recovery by using ligase to obtain the over-expression bovine TUSC5a/TUSC5b gene recombinant lentivirus;

the lentiviral vector is pCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro, and the sequence added to the lentiviral vector connected by the ligase is as follows:

5'-ccg (protecting base) +GAATTC (ECOR 1) +ATG+TUSC5a/TUSC5b sequence + CATCATCACCATCACCAT (HIS tag sequence) +CCTAGG (BAMH 1) +cgc (protecting base) -3',

the TUSC5a sequence is shown as SEQ ID No.1, the TUSC5b sequence is shown as SEQ ID No.2, the sequence added on the lentiviral vector after the TUSC5a sequence is connected is shown as SEQ ID No.3, and the sequence added on the lentiviral vector after the TUSC5b sequence is connected is shown as SEQ ID No. 4.

Still further, the method further comprises the step of cloning TUSC5a/TUSC5b sequence, specifically:

cDNA obtained from bovine adipose tissue, a first fragment of TUSC5a was amplified using TUSC5a-F and TUSC5a-overlap-R as primers, and a second fragment of TUSC5a was amplified using TUSC5a-overlap-F and TUSC5a-RED1-R2 as primers; after the PCR product gel is recovered, TUSC5a-F, TUSC5a-RED1-R2 is used as a template, and is used as a primer for amplification to obtain a TUSC5a sequence; TUSC5a-F, TUSC a-RED1-R1 is used as a primer to amplify to obtain a TUSC5b sequence, after HindI and KpnI are subjected to double enzyme digestion, gel recovery products are subjected to T4 ligase to construct TUSC5a-RED1 and TUSC5b-RED1;

wherein, the sequences of the primers are shown in the following table:

still further, the method further comprises the step of adding an HIS tag to the TUSC5a/TUSC5b sequence, specifically:

using TUSC5a-RED1 as template, using primer TUSC5a-F, TUSC5a-R to amplify TUSC5a sequence carrying HIS tag;

using TUSC5b-RED1 as template, using primer TUSC5b-F, TUSC5b-R to amplify TUSC5b sequence carrying HIS tag;

wherein, the sequences of the primers are shown in the following table:

the PCR amplification system (20. Mu.L) of TUSC5a-RED1 was as follows:

the PCR amplification system (20. Mu.L) of TUSC5b-RED1 was as follows:

further, the method comprises the step of integrating the recombinant lentivirus of the over-expressed bovine TUSC5a/TUSC5b gene into a host chromosome to construct a TUSC5a/TUSC5b stable transgenic cell line.

Still further, the method also comprises the step of packaging the recombinant lentivirus of the over-expressed bovine TUSC5a/TUSC5b gene, wherein the packaging system of the recombinant lentivirus of the over-expressed bovine TUSC5a/TUSC5b gene and the three plasmids Pspax2 and PMD2.G is used for packaging the lentivirus by using 293T cells as packaging cells.

Still further, pspax2: pmd2.G: the volume ratio of the recombinant lentivirus of the over-expressed bovine TUSC5a/TUSC5b gene is 2:3: 4.

The application has the beneficial effects that: according to the application, the pCDH-CMV-MCS-EF 1-copGGFP-T2A-Puro (LV 34) lentivirus is taken as a vector to construct two over-expression recombinant lentiviruses of TUSC5a and TUSC5b, the two over-expression recombinant lentiviruses are taken as a tool with infectivity or as an artificial preparation to regulate and control fat deposition, the expression of TUSC5a obviously promotes fat synthesis and accumulation, and the expression of TUSC5b obviously inhibits fat synthesis and accumulation. The application has the advantages that the regulation network of TUSC5 on fat deposition is correctly known to go deep into the level of splicing isomers, and the reverse regulation effect of TUSC5a and TUSC5b on fat deposition is found, and the two packaged TUSC5 splicing isomer slow viruses can be used as tools for manually regulating the fat deposition capacity of beef cattle, so that the dosage regulation of the fat deposition capacity is realized, and the application has important significance for quickly and manually promoting the economic value improvement of beef cattle in China.

Drawings

FIG. 1 is a graph showing the results of TUSC5a and TUSC5b gel electrophoresis;

FIG. 2 is a schematic diagram of construction of a recombinant lentivirus over-expressing the bovine TUSC5a, TUSC5b gene;

FIG. 3 is a graph showing the results of TUSC5a and TUSC5b vector enzyme-resolved gel electrophoresis;

FIG. 4 is a TUSC5a, TUSC5b vector sequencing verification result;

FIG. 5 is a fluorescent image of TUSC5a, TUSC5b lentiviral infected 293T cells;

FIG. 6 is a graph showing the results of the expression verification of TUSC5a, TUSC5b lentiviral infection 293T;

FIG. 7 is a graph showing the results of C3H10T1/2 induced differentiation of the TUSC5a and TUSC5b groups;

FIG. 8 is a graph of results of C3H10T1/2 induced differentiation oil red O staining over-expressing TUSC5a, TUSC5b groups;

FIG. 9 is a graph showing changes in gene expression during induced differentiation of C3H10T1/2 cells overexpressing TUSC5a, TUSC5b groups.

Detailed Description

The following examples are provided to further illustrate the application but are not to be construed as limiting the application and any modifications and alterations made thereto are within the scope of the application without departing from the spirit and scope thereof.

Unless otherwise stated, the technical means involved in the following examples are conventional techniques commonly used by those skilled in the art of graminess.

1. Construction of vectors

1. Cloning of TUSC5a and TUSC5b sequences

cDNA obtained from bovine adipose tissue, a first fragment of TUSC5a was amplified using TUSC5a-F and TUSC5a-overlap-R as primers, and a second fragment of TUSC5a was amplified using TUSC5a-overlap-F and TUSC5a-RED1-R2 as primers; after the PCR product gel is recovered, TUSC5a-F, TUSC5a-RED1-R2 is used as a template, and is used as a primer for amplification to obtain a TUSC5a sequence; TUSC5b sequence was obtained by amplification using TUSC5a-F, TUSC5a-RED1-R1 as a primer, and the size was verified by gel electrophoresis. The results of TUSC5a and TUSC5b gel electrophoresis are shown in FIG. 1. The amplified product gel electrophoresis results showed consistent band sizes for the TUSC5a and TUSC5b sequences. The TUSC5a sequence is shown as SEQ ID No.1, and the TUSC5b sequence is shown as SEQ ID No. 1. The sequences of the primers are shown in Table 1 below.

TABLE 1 TUSC5a and TUSC5b sequence cloning procedure each primer sequence

Overlapping amplification first step strategy (35 cycles):

overlapping amplification second step (35 cycles):

after double cleavage by Hind I and KpnI, the gel recovery products were stored by T4 ligase to construct TUSC5a-RED1 and TUSC5b-RED 1.

2. Construction of recombinant lentiviruses overexpressing TUSC5a, TUSC5b

The primer TUSC5a-F, TUSC5a-R was used to amplify the TUSC5a sequence carrying the HIS tag using TUSC5a-RED1 as template. TUSC5b sequence carrying the HIS tag was amplified using the primer TUSC5b-F, TUSC5b-R using TUSC5b-RED1 as a template.

Primer sequences to which HIS tags are added to the TUSC5a and TUSC5b sequences are shown in table 2. The PCR amplification system (20. Mu.L) of TUSC5a-RED1 is shown in Table 3. The PCR amplification system (20. Mu.L) of TUSC5b-RED1 is shown in Table 4.

TABLE 2 primer sequences to which HIS tags are added to TUSC5a and TUSC5b sequences

TABLE 3 PCR amplification System of TUSC5a-RED1 (20. Mu.L)

Amplification strategy:

TABLE 4 PCR amplification System of TUSC5b-RED1 (20. Mu.L)

Amplification strategy:

the TUSC5a and TUSC5b sequences carrying the HIS tag are respectively digested with pCDH-CMV-MCS-EF 1-copGGFP-T2A-pur (LV 34) lentiviral vector by ECOR1 and BAMH1 endonucleases, and the products after gel recovery are respectively connected by T4 DNA ligase to obtain the recombinant lentivirus of the overexpressed bovine TUSC5a and TUSC5b genes. FIG. 2 is a schematic diagram showing construction of a recombinant lentivirus over-expressing the bovine TUSC5a, TUSC5b gene.

Wherein T4 DNA ligase was used, incubated at 37℃for 2h and the ligation system (20. Mu.L) was as shown in Table 5.

TABLE 5 ligation System Using T4 DNA ligase (20. Mu.L)

Sequence addition to lentiviral vector cases: 5'-ccg (protecting base) +GAATTC (ECOR 1) +ATG+TUSC5a/TUSC5b sequence + CATCATCACCATCACCAT (his sequence) +CCTAGG (BAMH 1) +cgc (protecting base) -3'. The sequence added by the recombinant TUSC5a on the slow virus vector is shown as SEQ ID No.3, and the sequence added by the recombinant TUSC5b on the slow virus vector is shown as SEQ ID No. 4.

3. Verification of recombinant lentivirus overexpressing TUSC5a, TUSC5b

By utilizing the infection integration characteristic of the slow virus, the recombinant slow viruses of the over-expressed bovine TUSC5a and TUSC5b genes are respectively integrated on a host chromosome to achieve persistent expression, and TUSC5a and TUSC5b stable transgenic cell strains are constructed and used for cell function research of genes.

The method comprises the following specific steps:

(1) Taking 50 mu L of competent cells, adding 1 mu L of a connecting product (overexpressing bovine TUSC5a and TUSC5b gene recombination slow virus), gently mixing, and carrying out ice bath for 30min;

(2) Transferring to 42 ℃ for 90s, rapidly taking out and putting on ice for 2min, wherein the centrifuge tube cannot be shaken in the process;

(3) Adding 500 mu L of LB liquid medium without antibiotics, uniformly mixing, placing at 37 ℃ for 200r/min, and vibrating and culturing for 1h to revive bacteria;

(4) Taking 20 mu L of the mixture after gentle mixing, uniformly coating the mixture on LB solid medium containing ampicillin by using a coater, placing the flat plate in a constant temperature incubator at 37 ℃, inverting the flat plate after 30min, and continuously culturing for 12-14 h;

(5) On an ultra-clean workbench, single colonies are selected and inoculated into 3mL of LB liquid medium containing 100 mug/mL ampicillin, and the shaking culture is carried out for 10 hours at 37 ℃ at 220 r/min;

(6) Inoculating the culture on an ultra-clean workbench according to the proportion of 1/1000, and culturing in 200mL LB liquid medium containing 100 mug/mL ampicillin at 37 ℃ for 220r/min under shaking overnight;

(7) Centrifuging and collecting thalli at 8000r/min at 4 ℃ to extract the over-expression recombinant lentivirus;

(8) After the over-expression recombinant lentivirus is extracted, double enzyme digestion verification sequences are carried out, and each tube of reaction system is as follows:

and (3) carrying out enzyme digestion at 37 ℃, carrying out gel electrophoresis to obtain products, finding out areas corresponding to the sizes of the bands obtained by enzyme digestion and the target bands, and determining positive clones, wherein the gel electrophoresis result diagram of enzyme digestion of TUSC5a and TUSC5b vectors is shown in figure 3, and the gel electrophoresis result shows that TUSC5a and TUSC5b sequences are detected in enzyme digestion products, and the over-expression recombinant lentivirus is successfully constructed.

To further verify the construction of the recombinant lentiviruses of the overexpressed bovine TUSC5a and TUSC5b genes, the extracted recombinant lentiviruses were sequenced, and the sequence results were compared with the sequences of TUSC5a and TUSC5b, as shown in FIG. 4, which shows the results of the sequencing verification of the recombinant lentiviruses of the overexpressed bovine TUSC5a and TUSC5b genes, and the results show that the sequences of the recombinant lentiviruses are consistent with the sequences of TUSC5a and TUSC5b, and the construction of the recombinant lentiviruses of the overexpression is completed.

(9) And (3) culturing the over-expression recombinant slow virus again in a large quantity by using the corresponding positive bacterial liquid, and then extracting to obtain the over-expression recombinant slow virus for later use.

2. Packaging of recombinant lentiviruses over-expressing bovine TUSC5a, TUSC5b genes

1. Packaging of TUSC5a, TUSC5b overexpressed recombinant lentiviruses

The lentivirus is packaged by using 293T cells as packaging cells through a lentivirus core plasmid (namely over-expression recombinant lentivirus obtained by extracting a large amount of positive bacterial liquid in the steps), pspax2 and PMD2.G three-plasmid lentivirus packaging system. Because of the tag of the promoter COPGFP with independent CMV in the core plasmid, 293T cells expressed green fluorescent protein upon lentiviral packaging, which could be observed under a microscope.

293T cell inoculation: cells that had been passaged after resuscitation were used for culture and their growth was observed. Selecting cells in the logarithmic phase with normal morphology and no pollution. They were inoculated in six well plates (cell number of about 5X 105 cells per well) and placed in a 5% CO2 incubator at 37℃for culture.

Preparation of plasmids and transfection reagents: three plasmid DNA solutions, according to pmd2.G: psPAX2: the core plasmid is 2:3:4, and adding PBS to a constant volume of 200 mu L, and standing at room temperature for 30min.

Plasmid transfection: usingin vitro DNA transfection kit (Polyplus-transfer Co.) lentiviral expression systems were introduced into 293T cells, and specific steps were performed with reference to the instructions:

(1) Transfection is performed when 293T cells reach 70% -90% confluency;

(2) Fresh culture solution is replaced 1h to 2h before transfection, and 293T cell morphological changes are stimulated;

(3) Preparation of transfection complexes per well: 200 mu L jet PRIME Buffer is added to 3 sterile EP pipes of 1.5mL respectively, 9 mu g of pre-incubated lentiviral expression system mixed plasmid is added to each pipe respectively, the mixture is gently blown and mixed, 9 mu L of jet PRIME transfection reagent is added, and the mixture is gently blown and mixed to form a transfection complex;

(4) Incubating for 15min at room temperature;

(5) Adding the DNA-liposome complex into a cell culture solution, and shaking to make the cell culture solution uniform;

(6) Cells were cultured at 37℃for 8 hours, and the culture medium was changed.

(7) Collecting culture solution supernatants of 24h, 48h and 72h respectively, mixing, centrifuging the collected supernatant (4 ℃,4000, 10 min), and collecting the centrifuged supernatant; the supernatant was filtered on a super clean bench with a 0.45 μm filter screen.

As shown in FIG. 5, which shows fluorescence diagrams of TUSC5a, TUSC5b lentiviral infected 293T cells, the TUSC5a, TUSC5b, control groups were observed under a fluorescence microscope as apparent green fluorescence expression after 24h transfection of 293T cells with a lentiviral core plasmid, pspax2, PMD2.G three plasmid lentiviral packaging system.

2. Detection of recombinant lentiviruses over-expressing bovine TUSC5a and TUSC5b genes

After 293T cells were transfected with TUSC5a, TUSC5b, control lentiviral core plasmid, pspax2, PMD2.G three-plasmid lentiviral packaging system for 24h, cellular RNA and protein were extracted for detection, as shown in FIG. 6, which shows graphs of results of verification of expression of TUSC5a, TUSC5b lentiviral infection 293T cells, and the results showed that TUSC5a, TUSC5b, control group 293T cells were significantly improved on average at mRNA level and protein level after lentiviral infection.

RNA extraction:

(1) Removing the culture medium in the cell plate to be treated, gently washing the cells twice with PBS, sucking the waste liquid, adding 1mL of Trizol reagent into each hole, gently shaking the cell culture plate to ensure that the Trizol uniformly covers the cell surface, and incubating for 5min at room temperature;

(2) The cells were blown with RNase-free tips to complete lysis and the fluid was transferred to a 1.5mL EP tube;

(3) Adding 200 mu L of precooled chloroform into each tube, gently reversing for mixing for several times, and standing at room temperature for 10min;

(4) Labeling, sealing, centrifuging at 12000r/min at 4deg.C for 15min, standing at room temperature for 5min, and layering sample (upper water phase, middle layer is protein layer, lower phenol phase, and RNA is in upper water phase);

(5) Sucking the upper water phase (about 400 mu L) into a new 1.5mL EP tube, adding equal amount of pre-cooled isopropanol at-20deg.C, mixing thoroughly, and standing at room temperature for 10min;

(6) Labeling and sealing, and centrifuging at 12000r/min for 10min at 4 ℃ by using a centrifuge, wherein white RNA precipitation at the bottom of the tube can be seen (no obvious precipitation can be observed when the RNA amount is small);

(7) Removing supernatant, adding 1mL of 75% absolute cold ethanol into the precipitate, rinsing for 2 times (note that 75% absolute ethanol is prepared by DEPC water and is used at present), centrifuging for 5min at 8000r/min at 4deg.C, and drying excessive water in the tube by using clean cotton towel for the last time;

(8) Drying at room temperature for 5-10 min, making the precipitate semitransparent, adding appropriate amount of DEPC water, and dissolving in ice for 30min;

(9) 2 mu L of the concentration is measured by separate filling, 3 mu L of the gel is run to detect whether degradation exists, and the rest is stored at the temperature of minus 80 ℃ for standby.

Protein extraction:

(1) Preparing lysate (1 mL system): preparing 1000. Mu.L of high-strength RIPA lysate, adding 20. Mu.L of 50 XCocktail protease inhibitor, adding 10. Mu.L of phosphorylation protease inhibitor A, B solution and 10. Mu.L of PMSF reagent;

(2) Collecting a cell protein sample: the cells were washed 3 times with pre-chilled PBS and the residual solution was finally blotted thoroughly. 120-150. Mu.L of protein lysate was added to each well of cells, and the cell culture plate was placed on ice for 5min during which time the plate was repeatedly shaken to bring the lysate into full contact with the cells. Cells and lysates were collected in 1.5mL EP tubes with a clean cell scraper; the ice bath was repeated several times with 200. Mu.L of a pipette every 10min during 30min to ensure complete cell lysis. Centrifuging at 12000r/min for 5min at 4 ℃, and collecting the supernatant into a new EP tube to obtain the cellular protein.

3. Effect of overexpression of TUSC5a, TUSC5b on C3H10T1/2 induced differentiation

1. TUSC5a and TUSC5b overexpressing C3H10T1/2 cells induce differentiation

Maintenance was performed using reagent A (1.0. Mu. Mol/L rosiglitazone, 1.0. Mu. Mol/L dexamethasone, 10mg/L insulin, 0.5mmol/L IBMX) and 48h later with replacement of reagent B (1.0. Mu. Mol/L rosiglitazone, 10mg/L insulin). Lipogenesis was induced in TUSC5a, TUSC5B, control group C3H10T1/2 cells in an alternating fashion with reagent A and reagent B.

At 4d, the cells were stained with oil red O, as shown in FIG. 7, which shows the results of the induced differentiation of TUSC5a and TUSC5bC3H 10T1/2, as shown in FIG. 8, which shows the results of the induced differentiation of C3H10T1/2 of the groups TUSC5a and TUSC5b, which showed that the number and size of lipid droplets deposited in the TUSC5a group were higher than those in the TUSC5b and Control group was higher than those in the TUSC5b group, and it was found that the overexpression of TUSC5a increased the capacity of the induced differentiation of C3H10T1/2 compared with Control group, while the overexpression of TUSC5b group decreased the capacity of the induced differentiation of fat compared with Control group. In addition, apoptosis was found in C3H10T1/2 overexpressing TUSC5b when differentiation was induced for 4 d.

2. Gene expression changes during TUSC5a and TUSC5b overexpressing C3H10T1/2 cells induced differentiation

PPARgamma, FASN, FABP4 and SCD1 are important marker genes in fat deposition process, and we quantitatively detect and analyze the expression of the PPARgamma, FASN, FABP4 and SCD1 in the process of cell differentiation. As shown in FIG. 9, the results of the over-expression TUSC5A and TUSC5b C H10T1/2 induced differentiation oil red O staining are shown, the expression levels of PPARgamma, FASN, FABP4 and SCD1 are gradually increased within 0-6 days, the TUSC5A group is significantly higher than Control, and the TUSC5b group shows that the expression is significantly inhibited relative to Control. The expression level of PPARgamma, FASN, FABP4 and SCD1 was slowly decreased in 6-10 days. This reflects that expression of TUSC5a significantly promoted fat synthesis and accumulation, and expression of TUSC5B significantly inhibited fat synthesis and accumulation.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the application.

Sequence listing

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aagccgtcca agtccctctc agggcctctg gacctggagc agaatggcca cagcctgccc 180

ttcaaggtga tatccgagga gcaccgggaa gccttgcacc ccctctcatc ctcccgggcc 240

agctccagga gggcgtcctc cacggccacc acctcctatg cccaggacgg agaagttccc 300

aaagattatc tcatccttgc catcgcctcc tgcttctgcc ccgtctggcc cctcaacctc 360

atgcccctca tcttttccat catgtctcga agtagcgtgc aacaggggga cctggacggg 420

gcccggaggc tgggccgcct ggccgggacg ctcagcatca ccttcatcat catggggatc 480

atcatcatca tcgtggctgt gactgtcaac tttgcagttc cgaagaaa 528

<210> 3

<211> 654

<212> DNA

<213> cow (Bovine)

<400> 3

ccggaattca tggccaaccc cggacagcct cagttttccg cggcgcggga gccaggcact 60

gcctcacccc tgaacctgcc ggagatggag aagctccttg tgcaggtcgg gggccaggat 120

gacaagtccc tgaagccgtc caagtccctc tcagggcctc tggacctgga gcagaatggc 180

cacagcctgc ccttcaaggt gatatccgag gagcaccggg aagccttgca ccccctctca 240

tcctcccggg ccagctccag gagggcgtcc tccacggcca ccacctccta tgcccaggac 300

ggagaagttc ccaaagatta tctcatcctt gccatcgcct cctgcttctg ccccgtctgg 360

cccctcaacc tcatgcccct catcttttcc atcatgtctc gaagtagcgt gcaacagggg 420

gacctggacg gggcccggag gctgggccgc ctggccggga cgctcagcat caccttcatc 480

atcatgggga tcatcatcat catcgtggct gtgactgtca actttgcagg aatcaaaacc 540

ccaaatcctc caaaaaggga ccgggagacc ctgtgttctg agctccgtcg tgctatcgct 600

gttcttttct ttacagttcc gaagaaacat catcaccatc accatcctag gcgc 654

<210> 4

<211> 567

<212> DNA

<213> cow (Bovine)

<400> 4

ccggaattca tggccaaccc cggacagcct cagttttccg cggcgcggga gccaggcact 60

gcctcacccc tgaacctgcc ggagatggag aagctccttg tgcaggtcgg gggccaggat 120

gacaagtccc tgaagccgtc caagtccctc tcagggcctc tggacctgga gcagaatggc 180

cacagcctgc ccttcaaggt gatatccgag gagcaccggg aagccttgca ccccctctca 240

tcctcccggg ccagctccag gagggcgtcc tccacggcca ccacctccta tgcccaggac 300

ggagaagttc ccaaagatta tctcatcctt gccatcgcct cctgcttctg ccccgtctgg 360

cccctcaacc tcatgcccct catcttttcc atcatgtctc gaagtagcgt gcaacagggg 420

gacctggacg gggcccggag gctgggccgc ctggccggga cgctcagcat caccttcatc 480

atcatgggga tcatcatcat catcgtggct gtgactgtca actttgcagt tccgaagaaa 540

catcatcacc atcaccatcc taggcgc 567

Claims

1. The application of the bovine TUSC5a and TUSC5b genes in the regulation and control of fat deposition in beef cattle is characterized in that: the regulation is as follows: expression of TUSC5a significantly promotes fat synthesis and accumulation, expression of TUSC5b significantly inhibits fat synthesis and accumulation, TUSC5a sequence is shown in SEQ ID No.1, and TUSC5b sequence is shown in SEQ ID No. 2.

2. The use according to claim 1, characterized in that: the regulation of beef fat deposition by the bovine TUSC5a and TUSC5b genes is performed by overexpressing recombinant lentiviruses of the bovine TUSC5a and TUSC5b genes, which integrate TUSC5a and TUSC5b into the genomic gene expression and thereby regulate fat deposition.

3. The use according to claim 2, characterized in that: the recombinant slow virus of the over-expressed bovine TUSC5a/TUSC5b genes is used for preparing an artificial preparation for regulating and controlling fat deposition of beef cattle.

4. The use according to claim 2, characterized in that: the construction method of the recombinant lentivirus for over-expressing the bovine TUSC5a/TUSC5b genes comprises the following steps: the method comprises the steps of carrying out double enzyme digestion on TUSC5a/TUSC5b sequences carrying HIS labels and lentiviral vectors respectively by using endonucleases, and connecting products after glue recovery by using ligases to obtain the recombinant lentivirus of the overexpressed bovine TUSC5a/TUSC5b genes;

5' -ccg +GAATTC+ATG+TUSC5a/TUSC5b sequence +

CATCATCACCATCACCAT+CCTAGG+cgc-3'，

5. The use according to claim 4, characterized in that: the method also comprises the step of cloning TUSC5a/TUSC5b sequence, specifically comprising the following steps:

wherein, the sequences of the primers are shown in the following table:

6. the use according to claim 5, characterized in that: the method also comprises the step of adding an HIS tag to the TUSC5a/TUSC5b sequence, and specifically comprises the following steps:

wherein, the sequences of the primers are shown in the following table:

7. the use according to claim 6, characterized in that:

the PCR amplification system (20. Mu.L) of TUSC5a-RED1 was as follows:

the PCR amplification system (20. Mu.L) of TUSC5b-RED1 was as follows:

8. the use according to claim 4, characterized in that: the method also comprises the step of integrating the recombinant lentivirus of the over-expressed bovine TUSC5a/TUSC5b gene into a host chromosome to construct a TUSC5a/TUSC5b stable transgenic cell line.

9. The use according to claim 4, characterized in that: the method also comprises the step of packaging the bovine TUSC5a/TUSC5b gene recombinant lentivirus by over-expressing the bovine TUSC5a/TUSC5b gene recombinant lentivirus and a Pspax2 and PMD2.G three-plasmid lentivirus packaging system, and the 293T cells are used as packaging cells for packaging the lentivirus.

10. The use according to claim 9, characterized in that: pspax2: pmd2.G: the volume ratio of the recombinant lentivirus of the over-expressed bovine TUSC5a/TUSC5b gene is 2:3: 4.