Dairy goat FTO gene and its application in regulating lipid accumulation in mammary cells of dairy goats

Abstract

The present invention belongs to the field of dairy goat breeding genetic engineering, and specifically relates to a dairy goat FTO gene and its application in regulating lipid accumulation in dairy goat mammary cells. By cloning and overexpressing the FTO gene in dairy goat mammary epithelial cells, it is found that overexpression of the gene can downregulate the expression of genes related to fatty acid synthesis, desaturation, and triglyceride synthesis metabolic pathways, and upregulate the expression of key genes for triglyceride degradation. At the same time, the content of intracellular triglycerides is significantly reduced. Based on this technical effect, it provides an important theoretical basis for further developing high-quality milk production technology, regulating the content of triglycerides in goat milk from the genetic level, and implementing molecular assisted breeding.

Description

Technical Field

[0001] The invention relates to the technical field of dairy goat breeding genetic engineering, and in particular to a dairy goat FTO gene and an application thereof in a dairy goat mammary epithelial cell lipid accumulation regulation process. Background Art

[0002] Goat milk has a high total fat content and contains a rich variety of fatty acids, such as short-chain and medium-chain fatty acids, unsaturated fatty acids, and conjugated acids. Goat milk has a good preventive effect on human diseases such as arteriosclerosis, coronary heart disease, and liver cirrhosis. It can also be used as an auxiliary treatment measure for metabolic diseases such as nutrient absorption disorder syndrome, small intestinal dysfunction, neonatal malnutrition, and gallstones. It is a natural drink for nutrition and health care. Studies have shown that the quality of goat milk is closely related to the composition and content of fatty acids and the content of triglycerides. Therefore, clarifying the function of key genes in fatty acid metabolism provides new ideas for the production of high-quality goat milk and provides molecular materials and theoretical basis for the implementation of molecular breeding measures to improve the fatty acid content composition of goat milk.

[0003] The fat and obesity-associated gene (FTO) is a transcriptional coactivator that encodes a 2-hydroxyglutarate-dependent nuclease that is involved in DNA repair and fatty acid metabolism. As the earliest discovered N6-methyladenosine (m6A) demethylase, FTO is associated with fatty acid synthase (FASN) and lipid accumulation. FTO protein has been shown to regulate the expression of downstream molecules through post-transcriptional modifications or participate in adipogenesis and tumorigenesis by targeting the mammalian target protein rapamycin (mTOR). Studies have shown that knockdown of FTO can significantly increase the level of m6A in total RNA, thereby reducing lipid accumulation in porcine adipocytes and 3T3-L1 preadipocytes. In addition, in the hepatocytes of mice fed a high-fat diet, overexpression of FTO reduced the level of m6A in total RNA, accompanied by an increase in the expression of genes related to lipogenesis. In goats, the FTO gene is located on chromosome 18 and is highly expressed in the spleen, lung, and adipose tissue of goats. The widespread expression of FTO in animal tissues suggests that it may play an important role in regulating energy metabolism.

[0004] Therefore, in-depth research on the function of the FTO gene in dairy goats will help change the milk fat content at the genetic level, provide a basis for improving the quality of goat milk, and have important theoretical and practical significance for the healthy development of the dairy goat industry. Summary of the invention

[0005] The object of the present invention is to solve at least one of the technical drawbacks.

[0006] To this end, an object of the present invention is to propose a dairy goat FTO gene and its application in regulating lipid accumulation in dairy goat mammary cells, so as to solve the problems mentioned in the background technology and overcome the shortcomings of the prior art.

[0007] In order to achieve the above-mentioned object, the present invention provides a dairy goat FTO gene, the nucleotide sequence of which is 1518 bp in length and is shown in Sequence 1.

[0008] Preferably, the amino acid sequence of the FTO gene has a homology greater than 99% with that of sheep, cattle and buffalo.

[0009] The method for overexpressing the FTO gene in dairy goats comprises the following steps:

[0010] The first step is to connect FTO to the pcDNA3.1 overexpression vector using a seamless cloning kit based on the CDS region of the dairy goat FTO gene to obtain the overexpression vector pcDNA3.1-FTO;

[0011] In the second step, the overexpression vector pcDNA3.1-FTO and the empty vector pcDNA3.1 were transfected into goat mammary epithelial cells using X-tremeGENE HP DNA Transfection Reagent.

[0012] Preferably, the primers for seamless cloning in the first step are:

[0013] F: 5'-CTGGCTAGCGTTTAATGAAGCGGACCCCGACG-3';

[0014] R: 5'-TTAAACGGGTCTAGACCTAGGGCCTGGTCTCCAGAA-3'.

[0015] Preferably, the reaction system in the first step is: 2 μL of 5×SE Cloning Buffer, 1.68 μL of pcDNA3.1, 0.63 μL of target fragment, 1 μL of SE Recobinase, and water is added to make up to 10 μL.

[0016] Preferably, the method down-regulates the expression of genes related to de novo fatty acid synthesis, fatty acid desaturation, and triglyceride synthesis.

[0017] Preferably, the method upregulates the expression of genes related to triglyceride hydrolysis.

[0018] The application of the dairy goat FTO gene is applied to the process of regulating lipid accumulation in dairy goat mammary epithelial cells.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] The present invention involves cloning and overexpressing the FTO gene in mammary epithelial cells of dairy goats. It is found that overexpression of the gene can downregulate the expression of genes related to fatty acid synthesis, desaturation, and triglyceride synthesis metabolism pathways, and upregulate the expression of key genes for triglyceride degradation. At the same time, the content of intracellular triglycerides is significantly reduced. Based on this technical effect, it provides an important theoretical basis for further developing high-quality milk production technology, regulating the content of triglycerides in goat milk at the genetic level, and implementing molecular assisted breeding.

[0021] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0023] Figure 1 Cloning of the sheep FTO gene;

[0024] Figure 2 Comparison of amino acid sequences of FTO genes in different species;

[0025] Figure 3-1 Construction of pCDNA3.1-FTO overexpression vector;

[0026] Figure 3-2 PCR verification of pCDNA3.1-FTO overexpression vector bacterial solution;

[0027] Figure 4-1 Detection of the overexpression effect of FTO in mammary epithelial cells;

[0028] Figure 4-2 Effects of FTO overexpression on genes related to de novo fatty acid synthesis in goat mammary epithelial cells;

[0029] Figure 4-3 Effects of FTO overexpression on genes related to triglyceride synthesis in goat mammary epithelial cells;

[0030] Figure 4-4 Effects of FTO overexpression on genes related to triglyceride degradation in goat mammary epithelial cells;

[0031] Figure 5 Effect of FTO overexpression on triglyceride content in mammary gland cells. DETAILED DESCRIPTION

[0032] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0033] The present invention is further explained below with reference to examples, but the examples do not limit the present invention in any form.

[0034] Unless otherwise stated, the experimental methods or analytical methods involved in the following examples are conventional methods.

[0035] The following reagents, unless otherwise specified, can be obtained from commercial sources. Before introducing the specific examples, we first introduce the basic information of the biological materials, experimental reagents, experimental instruments, etc. involved in the examples:

[0036] Cell and plasmid sources:

[0037] Dairy goat mammary epithelial cells and pcDNA3.1 plasmid were donated by He Qiuya from the College of Animal Science and Technology of Northwest Agriculture and Forestry University; dry period and peak lactation mammary gland tissues were donated by Shi Hengbo from the Dairy Science Institute of Zhejiang University; and pMD-19T vector was purchased from Takara Biotechnology Co., Ltd.

[0038] Experimental reagents:

[0039] Epidermal growth factor, X-tremeGENE HP DNA Transfection Reagent, restriction endonuclease, RNA extraction kit, LB medium, seamless cloning kit, reverse transcription kit, gel recovery kit, TBE electrophoresis fluid, plasmid extraction kit, ampicillin, fetal bovine serum, tissue cell glycerol assay kit, DMEM / F-12 medium, etc. are all commonly used materials in molecular biology experiments.

[0040] Experimental instruments:

[0041] The present invention mainly uses the following instruments: PCR instrument, gel imaging system, Bio-Rad fluorescence quantitative instrument (CFX-96), electrophoresis instrument, Eppendorf pipette, and carbon dioxide incubator purchased from Beijing Liuyi Instrument Factory.

[0042] The present invention is further described below with reference to specific embodiments.

[0043] Example 1

[0044] 1. Cloning and bioinformatics analysis of the CDS region of sheep FTO gene

[0045] According to the sheep FTO gene sequence (KR013052.1) included in GenBank, the cloning primers of the FTO gene were designed using Premier 5.0, the upstream primer was 5'-AGGCGGCTATATCGGCAG-3' (sequence 2), and the downstream primer was 5'-TCTAGGGCCTGGTCTCCAG-3' (sequence 3).

[0046] The upstream of the real-time quantitative primer is 5'-TGTCGCCTGTCAACCTTCC 3' (sequence 4),

[0047] The downstream primer was 5'-CAATCTCATCGTGCCCATCA-3' (SEQ ID NO: 5).

[0048] The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0049] 2. PCR amplification using cDNA from breast tissue during peak lactation as template

[0050] PCR sample loading system: PrimeSTAR Max Premix 25 μL, F-Primer 1 μL, R-Primer 1 μL, cDNA 1 μL, sterile water 221 μL, total volume 50 μL.

[0051] PCR reaction conditions: 98°C, 10 sec; 57°C, 5 sec; 72°C, 5 sec; 72°C, 10 min; 4°C, storage. The obtained sheep FTO gene CDS region 1518 bp was cloned into the pMD-19T vector ( Figure 1 ) and sequenced.

[0052] 3. Predict the molecular weight, theoretical isoelectric point, protein hydrophobicity and transmembrane domain of dairy goat FTO protein

[0053] Clustal Omega (https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) was used to perform multiple alignments of FTO amino acid sequences among species, and ProParam (http: / / www.expasy.org / tools / protparam.html), ExPASy Proteomics Serve (http: / / us.expasy.org / cgi-bin / protscale.p) and TMHMM (http: / / www.cbs.dtu.dk / services / TMHMM / ) were used to predict the molecular weight, theoretical isoelectric point, protein hydrophobicity and transmembrane domain of dairy goat FTO protein.

[0054] The amino acid sequence of the FTO gene CDS region of dairy goats is more than 99% homologous to that of sheep (NM_001104931.1), cattle (NM_001098142.1) and buffalo (XM_006043050.4). Figure 2 ), which indicates that the FTO gene is highly conserved in ruminants. ExPASy ProtParam tool online analysis showed that the molecular weight of FTO protein is 58.55ku and the theoretical isoelectric point is 5.28. Protein hydrophobicity prediction by ExPASy Proteomics Server found that the maximum hydrophobicity of FTO protein is 2.000 and the minimum is -3.222, which shows that the protein has strong hydrophobicity. TMHMM analysis found that the protein does not have a transmembrane region.

[0055] Example 2 Construction of sheep FTO gene overexpression vector and its effect on fatty acid metabolism related genes in cells

[0056] According to the FTO gene CDS region obtained above, FTO was connected to the pcDNA3.1 overexpression vector using a seamless cloning kit.

[0057] Primers for seamless cloning were designed using Primer 5.0:

[0058] F: 5'-CTGGCTAGCGTTTAATGAAGCGGACCCCGACG-3' (sequence 6)

[0059] R: 5'-TTAAACGGGTCTAGACCTAGGGCCTGGTCTCCAGAA-3' (sequence 7)

[0060] Perform PCR amplification of the target fragment and construct the overexpression vector pcDNA3.1-FTO using the seamless cloning kit. The reaction system is as follows: 2 μL 5×SE Cloning Buffer, 1.68 μL pcDNA3.1, 0.63 μL target fragment, 1 μL SERecobinase, and add water to make up to 10 μL. After mixing, place in a 37°C water bath for 5-30 minutes, then transfer to ice or store at -20°C for competent cell transformation experiments. Culture in a 37°C incubator overnight, pick positive clones, expand the culture, and verify the bacterial solution by PCR. Figure 3-1 and Figure 3-2 As shown, the FTO vector was successfully constructed, and the positive clones were sent for sequencing.

[0061] On this basis, X-tremeGENE HP DNA Transfection Reagent was used to transfect pcDNA3.1-FTO and pcDNA3.1 empty vectors into goat mammary epithelial cells. After incubation for 48 hours, the expression level of the FTO gene in the cells was detected using real-time fluorescence quantitative technology. Figure 4-1 As shown, the real-time quantitative results showed that compared with the control group, the expression level of the FTO gene was significantly upregulated by about 4 times after transfection of the overexpression vector, so subsequent experiments could be carried out.

[0062] The specific operations are as follows:

[0063] 1. Cell culture

[0064] The basic culture medium of goat mammary epithelial cells is: 10% fetal bovine serum and 90% DMEM / F-12, as well as 5μg / mL insulin, 10ng / mL epidermal growth factor, 100U / mL penicillin / streptomycin and 1μg / mL hydrocortisone. The cells are cultured in a constant temperature cell culture incubator at 37°C and 5% CO2. The cells are passaged when they are full. Since the cells grow fast, they are passaged at a ratio of 1:3 and fresh culture medium is replaced every day.

[0065] 2. Cell transfection

[0066] Refer to the X-tremeGENE HP DNA Transfection Reagent manual for transfection experiments. Select a 24-well plate and set up three replicates. First, prepare the transfection complex with a ratio of transfection reagent to vector of 3:1. Use DMEM / F-12 to make up the remaining volume. Mix gently and incubate at room temperature for 20 minutes. Then add the incubated transfection complex dropwise to the 24-well plate. Mix gently horizontally. Collect cells 48 hours after transfection.

[0067] 3. Extraction of total cell RNA

[0068] The RNA extraction method refers to the total RNA extraction kit for cultured cells / bacteria of Tiangen Biochemical Technology Co., Ltd. First, remove the cell culture medium and add an appropriate amount of lysis solution RL. Transfer all the solutions to the filter column CS, centrifuge at 12000rpm for 2min, and collect the filtrate. Add 1 volume of 70% ethanol to the filtrate, mix well, and transfer the resulting solution and precipitate to the adsorption column CR3, centrifuge at 12000rpm for 30sec, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. Add 350μL of deproteinization solution RW1 to the adsorption column CR3, centrifuge at 12000rpm for 30sec, and pour out the waste liquid in the collection tube. Add 500μL of rinsing solution RW to the adsorption column CR3, let it stand at room temperature for 2min, centrifuge at 12000rpm for 30sec, pour out the waste liquid in the collection tube, and repeat the wash again. Then centrifuge the empty tube for 3min. Place the adsorption column CR3 in a new centrifuge tube, add 30-100 μL RNase-Free ddH2O, place at room temperature for 2 minutes, and centrifuge at 12000 rpm for 2 minutes to obtain RNA solution. Detect RNA concentration and purity.

[0069] 4. Reverse transcription

[0070] First, remove the genomic DNA and prepare the reaction mixture on ice according to the following ingredients: 5×gDNAEraser Buffer, 2.0μL; gDNA Eraser, 1.0μL; Total RNA, up to 1μg; RNase Free ddH2O added to 10μL. After adding, mix well and react at room temperature for 30 minutes.

[0071] Preparation of reverse transcription reaction solution: reaction solution for removing genomic DNA, 10.0 μL; PrimerScript RTEnzyme Mix I, 1.0 μL; RT Primer Mix or miRNA29d-3p RT primer, 1.0 μL; 5× PrimeScript Buffer 2, 4.0 μL; RNase Free dH2O, 4.0 μL.

[0072] The reaction procedure was: 37°C, 15 min; 85°C, 5 sec; 4°C, storage.

[0073] 5. Real-time fluorescence quantitative PCR

[0074] RT-qPCR was performed using UXT and MRPL39 genes as internal reference genes. The reference design of the 20 μL system was as follows: SYBRGreen Mix, 10.0 μL; PCR Forward Primer, 0.8 μL; PCR Reverse Primer, 0.8 μL; DNA template, 2.0 μL; sterile water, 6.0 μL.

[0075] PCR reaction program: 95℃ pre-denaturation for 30s; 95℃ for 5s; 60℃ for 30s; 40 cycles. The primers used are shown in Table 1 below:

[0076] Table 1 Primer sequence list

[0077]

[0078] Real-time fluorescence quantitative detection found that the mRNA expression of SREBP1, a key transcription factor in milk fat synthesis, was significantly downregulated (P < 0.05), the expression of FASN, a gene involved in de novo fatty acid synthesis, was significantly downregulated (P < 0.05), and the expression of SCD1, a fatty acid desaturase, did not change significantly. The expression of DGAT2, a gene involved in triglyceride synthesis, was significantly downregulated (P < 0.05), and the expression of HSL, a gene involved in triglyceride hydrolysis, was significantly upregulated (P < 0.05).

[0079] Example 3 Effect of overexpression of FTO gene on triglyceride content in mammary epithelial cells of dairy goats

[0080] The cell transfection operation was the same as in Example 2.

[0081] Discard the cell culture medium and rinse three times with PBS. The operation method refers to the tissue cell triglyceride (TAG) enzymatic assay kit of Beijing Pulilai Gene Technology Co., Ltd. Add an appropriate amount of lysate to each well, lyse on a shaker for 30 minutes, scrape the cells with a cell scraper, and transfer the cells and lysate to a 1.5mL centrifuge tube. Then heat the lysate at 70°C for 10 minutes, centrifuge at 2000rpm for 5 minutes at room temperature, and take the supernatant for enzymatic assay. The working solution is prepared in a 4:1 ratio. Add 10μL of the sample to be tested, react at 37°C or 25 degrees Celsius for 10 minutes, and the color is stable within 60 minutes after the reaction is balanced. First, adjust the blank tube with distilled water and the working tube to zero, and then use an enzyme reader to detect the absorbance at a wavelength of 550nm. Finally, construct a triglyceride standard curve, then determine the concentration of triglycerides in the sample, and finally correct the triglyceride content with the total protein concentration per mg cell.

[0082] The results showed that after overexpression of FTO gene in mammary epithelial cells of dairy goats, the triglyceride content in the cells was significantly downregulated (P<0.05).

[0083] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0084] It is not difficult for those skilled in the art to understand that the present invention includes any combination of the invention content and specific implementation methods of the above specification and the various parts shown in the drawings. Due to the limited space and to make the specification concise, the various schemes composed of these combinations are not described one by one. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0085] Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and cannot be understood as limiting the present invention. Those skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An application of the FTO gene of a dairy goat in regulating lipid accumulation in mammary epithelial cells of a dairy goat, wherein after overexpressing the FTO gene in the mammary epithelial cells of a dairy goat, the triglyceride content in the cells is significantly reduced, characterized in that: The steps include: The first step is to connect FTO to the pcDNA3.1 overexpression vector using a seamless cloning kit based on the CDS region of the dairy goat FTO gene to obtain the overexpression vector pcDNA3.1-FTO; In the second step, the overexpression vector pcDNA3.1-FTO and the empty vector pcDNA3.1 were transfected into goat mammary epithelial cells using X-tremeGENE HP DNA Transfection Reagent; The length of the nucleotide sequence of the FTO gene is 1518 bp, and its nucleotide sequence is shown in Sequence 1.

2. The use of the dairy goat FTO gene as described in claim 1 in regulating lipid accumulation in dairy goat mammary epithelial cells, characterized in that: This method downregulated the expression of genes involved in de novo fatty acid synthesis, fatty acid desaturation, and triglyceride synthesis.

3. The application of the dairy goat FTO gene as described in claim 1 in regulating lipid accumulation in dairy goat mammary epithelial cells, characterized in that: This approach upregulated the expression of genes involved in triglyceride hydrolysis.

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