Application of overexpressed carp lpla gene in regulation and control of cyprinid fish tissue related gene expression

Abstract

The invention discloses application of an overexpressed carp lpla gene in regulation and control of expression of cyprinid fish tissue-related genes, and relates to the field of gene engineering, in particular to application of the overexpressed carp lpla gene in regulation and control of cyprinid fish tissue-related genes. The overexpressed carp lpla gene is applied to regulation and control of contents of lipid indexes of total cholesterol TC and triglyceride TG of different tissues of cyprinid fishes. The invention further discloses application of the overexpressed carp lpla gene in regulation and control of fat marker genes lpla, ppar gamma, Fabp3, fast and acaca of livers and muscle tissues of cyprinid fishes. The invention further discloses application of the overexpressed carp lpla gene in regulating and controlling the relative expression quantity of carp liver cell fat metabolism related genes lpla, ppar gamma, Fabp3, fast, acaca, hsl and pnpla2. The overexpressed carp lpla gene can regulate and control lipid deposition in different tissues in zebra fish, and a research model is provided for lpla to regulate and control fat deposition and differentiation. According to the overexpressed carp lpla gene disclosed by the invention, lpla high-expression carp hepatocytes are obtained through a liposome transfection technology, so that fat key gene expression in the carp hepatocytes is changed.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering, in particular, the application of overexpression of the lpla gene of Cyprinus carpio in regulating the expression of tissue-related genes of the family Cyprinidae. BACKGROUND

[0002] With the upgrading of consumption, the comprehensive optimization of the flavor, taste and nutritional value of edible meat has become a new demand in the market; the content of intramuscular fat (IMF) can significantly affect the sensory quality of meat , high IMF can greatly improve the tenderness, flavor and marbling score of meat , and thus increase the economic value-added. The famous Atlantic salmon (Salmo salar) and the Chinook salmon (Oncorhynchus masou) enjoy high market value due to their high natural IMF content. As an important economic fish in China, Cyprinus carpio occupies a core position in freshwater fisheries. The results of key differential gene expression analysis on Cyprinus carpio with different fat contents show that the lpla gene related to fat metabolism has different expression patterns in muscle and abdominal fat, and its expression amount is positively correlated with the IMF content and negatively correlated with the abdominal fat content, indicating that the gene may be involved in the differential regulation of fat deposition in the two tissues of Cyprinus carpio .

[0003] The deposition of IMF in animal body is a manifestation of the dynamic balance between fat synthesis and decomposition, and LPL, as a rate-limiting enzyme in the physiological process of decomposing triglyceride (TG) in lipoprotein, releasing free fatty acids (FFAs) and monoglycerides, directly affects the degree of fat accumulation . In beef cattle, it is found that the expression of LPL has a certain positive regulation on the deposition of IMF in the longissimus dorsi muscle of hybrid beef cattle , and the genes including LPL play an important role in the tenderness and marbling score of beef quality , and it is found that the mutation site of the gene in Beijing black pig population is significantly correlated with the IMF content . The use of LPL gene regulation has an important role in analyzing the molecular mechanism of intramuscular fat deposition, and overexpression of the lpla gene of Cyprinus carpio provides a reference for fish fat deposition and metabolic regulation model. SUMMARY

[0004] The present application provides an application of overexpression of the lpla gene of Cyprinus carpio in regulating the expression of tissue-related genes of the family Cyprinidae.

[0005] The application of overexpressing the carp lpla gene in regulating the content of total cholesterol (TC) and triglyceride (TG) in different tissues of the Cyprinidae.

[0006] The application of overexpressing the carp lpla gene in regulating the content of total cholesterol (TC) and triglyceride (TG) in different tissues of the Cyprinidae.

[0007] The application of overexpressing the carp lpla gene in regulating the content of total cholesterol (TC) and triglyceride (TG) in different tissues of the Cyprinidae.

[0008] The application of overexpressing the carp lpla gene in regulating the content of total cholesterol (TC) and triglyceride (TG) in different tissues of the Cyprinidae.

[0009] I. Taking the empty vector as a template, Vector primers are designed and synthesized, and the sequence of the Vector region of the overexpression recombinant plasmid is prepared by PCR amplification; the empty vector is pDestTol2CG2-Mod plasmid;

[0010] II. According to the sequence information of the carp lpla gene in the Ensembl database, CDS primers containing homologous arms of the coding region of the carp lpla gene are designed and synthesized, and the sequence of the CDS region containing homologous arms is obtained by PCR amplification with carp liver cDNA as a template; the sequence number of the carp lpla gene is ENSCCRT00000164182.1;

[0011] III. According to the sequence information of the carp lpla gene in the Ensembl database, Promoter primers containing homologous arms of the promoter region of the carp lpla gene are designed and synthesized, and the sequence of the Promoter region containing homologous arms is obtained by PCR amplification with carp liver DNA as a template; the sequence number of the carp lpla gene is ENSCCRT00000164182.1;

[0012] IV. The three sequences of the Vector region, the CDS region and the Promoter region are homologously recombined, the obtained competent bacteria are transformed, and the obtained circular plasmid is a recombinant overexpression plasmid of the carp lpla gene.

[0013] Further, the Vector primers in step I are:

[0014] Vector-F: 5'-TCTCGAGCCTCTAGAACTATAG-3',

[0015] Vector-R: 5'-AGATCTGCGAAGATACGG-3'; the sequence fragment of about 4580 bp in the empty vector pDestTol2CG2-Mod plasmid in step one was selected as a template to design the Vector primer.

[0016] Further, the CDS primer in step two is:

[0017] CDS-F: 5'-tcacatacagTTCGATTGAACCAGCGAG-3',

[0018] CDS-R: 5'-atagttctagaggctcgagaGTGAAGGTTACTAGTCTGAATATTC-3';

[0019] Further, the homologous arm region of the designed CDS-R primer can base-pair complementarily with the Vector-F primer and its amplification product, so as to "seamlessly" clone the CDS region and the Vector region.

[0020] Further, the Promoter primer in step three is:

[0021] Promoter-F: 5'-ggccgtatcttcgcagatctAGCACCGGTGTCTTCTAC-3', Promoter-R: 5'-ttcaatcgaaCTGTATGTGAGCGCTTTG-3';

[0022] Further, the homologous arm region of the designed Promoter-F primer can base-pair complementarily with the Vector-R primer and its amplification product, so as to "seamlessly" clone the Promoter region and the Vector region; the homologous arm region of the designed CDS-F primer can base-pair complementarily with the homologous arm region of the Promoter-R primer, so as to "seamlessly" clone the CDS region and the Promoter region.

[0023] Further, the sequence of the homologous arm region of the CDS, Promoter primer is respectively: CDS-F: 5'-tcacatacag-3', CDS-R: 5'-atagttctagaggctcgaga-3', Promoter-F: 5'-ggccgtatcttcgcagatct-3', Promoter-R: 5'-ttcaatcgaa-3'.

[0024] Further, the homologous recombination method in step four is that the fragments of the Vector region, the CDS region and the Promoter region are connected in a proportion of 1:4:2.

[0025] The present application has the following advantages:

[0026] The present application integrates the carp lpla gene into the overexpression vector plasmid through homologous recombination, wherein the Vector, CDS and Promoter primers designed in the plasmid construction can be complementary to each other in base pairs, so as to realize the "seamless" cloning of the three of Vecotr region, CDS region and Promoter region, and obtain the circular recombinant plasmid of the overexpression carp lpla gene. The lpla gene recombinant overexpression plasmid obtained by the present application improves the expression efficiency of the lpla gene in zebrafish, and provides tool support for subsequent lpla regulation of fat deposition. The obtained plasmid can cause high expression of the lpla gene in zebrafish and carp liver cells.

[0027] The present application overexpresses the carp lpla gene, and the constructed recombinant overexpression vector improves the expression amount of lpla in cells and fish, can regulate the deposition of lipids in zebrafish, and provides a fish lipid deposition regulation model, which provides a research model for lpla regulation of fat deposition and differentiation. In addition, the present application uses the "seamless" cloning technology to construct the overexpression recombinant plasmid vector; the overexpression carp lpla gene constructed by microinjection is used to realize high expression of carp liver cells by liposome transfection technology, so that the lipid deposition of zebrafish is changed, and the expression of key genes in the fat of carp liver cells is changed. The present application provides a research platform for exploring the role of the gene in regulating the tissue difference deposition of the fish of the family Cyprinidae, and provides theoretical support for further high-IMF low-abdominal fat high-quality meat breeding. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A schematic diagram of the carp lpla gene recombinant overexpression plasmid is shown in Figure 1.

[0029] Figure 2 The amplification results of the CDS and Promoter regions in the positive clone bacteria are shown in Figure 2.

[0030] Figure 3To test the positive individuals of zebra fish by microinjection of the recombinant overexpression plasmid of the lpla gene of common carp;

[0031] Figure 4 To test the TC and TG contents in the liver and muscle tissues of the transgenic zebra fish overexpressing the lpla gene of common carp and the wild type control;

[0032] Figure 5 To test the relative expression amounts of the fat metabolism marker genes in the liver and muscle tissues of the transgenic zebra fish overexpressing the lpla gene of common carp and the wild type control;

[0033] Figure 6 To test the relative expression amounts of the fat metabolism marker genes after the transfection of the liver cells of common carp with the lpla gene of common carp. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0035] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0036] In the present application, the lpla gene of common carp is integrated into the overexpression vector plasmid by homologous recombination to prepare the overexpression plasmid.

[0037] The method for constructing the recombinant overexpression plasmid of the lpla gene of common carp (the schematic diagram of the recombinant overexpression plasmid of the lpla gene of common carp in the present embodiment is shown in Figure 1 The steps are as follows:

[0038] 1. The Vector primer is designed and synthesized by taking the empty vector as a template, and the sequence of the Vector region of the overexpression recombinant plasmid is prepared by PCR amplification

[0039] The empty vector is pDestTol2CG2-Mod, and the Vector primers are: Vector-F: 5'-TCTCGAGCCTCTAGAACTATAG-3', Vector-R: 5'-AGATCTGCGAAGATACGG-3'. The PCR reaction system is 50 μL: 2x Phanta Flash Master Mix (Dye Plus) (Vazyme, P520-03) high-fidelity enzyme 25 μL, ddH2O 16 μL, empty vector template 5 μL, Vector-F / R primer (2*2) μL. The PCR reaction program is: 98℃ 30 s; 98℃ 10 s, 54℃ 5 s, 72℃ 40 s, 35 cycles; 72℃ 1 min; 4℃ ∞. The PCR product is subjected to agarose gel electrophoresis, and a target band of about 4580 bp is observed. The PCR product is recovered to prepare the sequence of the Vector region of the overexpression recombinant plasmid, and the concentration is determined.

[0040] 2. Design and synthesize CDS and Promoter primers containing homologous arms of the CDS and Promoter region of the carp lpla gene

[0041] a) In the known transcription sequence in the Ensembl database (ENSCCRG00000005349), the CDS region sequence is searched, and part of the non-coding region sequence in the genome of appropriate length is added upstream and downstream of the CDS region sequence, as a sequence template for designing CDS region amplification primers of overexpression recombinant plasmids containing homologous arms. In this embodiment, a sequence containing the CDS region of the carp lpla gene with a length of about 1600 bp is finally selected as a sequence template for designing CDS region amplification primers of overexpression recombinant plasmids containing homologous arms.

[0042] b) A sequence containing the lpla gene promoter with an appropriate length is selected about 2~3 kbp upstream of the CDS region in the database as a sequence template for designing Promoter region amplification primers of overexpression recombinant plasmids containing homologous arms. In this embodiment, a sequence containing the promoter of the carp lpla gene with a length of about 2800 bp is finally selected as a sequence template for designing Promoter region amplification primers of overexpression recombinant plasmids containing homologous arms.

[0043] c) In the NEBuilder website ( https: / / nebuilder.neb.com / !#) The sequence of the Vector region of the overexpression recombinant plasmid amplified in step 1 was input, and self-defined primers were selected, and the primer Vector-F / R was input; the sequence template of the amplification primers of the CDS region and the Promoter region described above was input, and designed primers were selected to obtain CDS primers and Promoter primers containing homologous arms that can "seamlessly" recombine with the Vector region. In this embodiment, the CDS primers and the Promoter primers containing homologous arms are respectively: CDS-F: 5'-tcacatacagTTCGATTGAACCAGCGAG-3', CDS-R: 5'-atagttctagaggctcgagaGTGAAGGTTACTAGTCTGAATATTC-3'; Promoter-F: 5'-ggccgtatcttcgcagatctAGCACCGGTGTCTTCTAC-3', Promoter-R: 5'-ttcaatcgaaCTGTATGTGAGCGCTTTG-3'. Among them, the sequence of the lowercase part is the designed homologous arm region.

[0044] 3. The sequence of the CDS region and the Promoter region containing homologous arms was amplified by PCR using cDNA and DNA of carp liver as templates

[0045] a) Total RNA was extracted from the liver tissue of carp, and reverse transcribed into cDNA to obtain an amplification template for preparing the sequence of the CDS region of the overexpression recombinant plasmid.

[0046] b) The sequence of the CDS region of the overexpression recombinant plasmid was prepared by PCR amplification using the above-mentioned carp cDNA as a template and using CDS primers containing homologous arms. The PCR reaction system was 50 μL: 2*Phanta Flash Master Mix (Dye Plus) (Vazyme, P520-03) high-fidelity enzyme 25 μL, ddH2O 16 μL, carp cDNA template 5 μL, CDS-F / R primer (2*2) μL. The PCR reaction program was: 98℃ 30 s; 98℃ 10 s, 56.2℃ 5 s, 72℃ 40 s, 35 cycles; 72℃ 1 min; 4℃ ∞. The PCR product was subjected to agarose gel electrophoresis, and a target band of about 1600 bp was observed. The PCR product was recovered to obtain the sequence of the CDS region of the overexpression recombinant plasmid, and the concentration was determined.

[0047] c) DNA was extracted from the liver tissue of carp to obtain an amplification template for preparing the sequence of the Promoter region of the overexpression recombinant plasmid.

[0048] d) PCR amplification of the CDS region sequence of the overexpression recombinant plasmid using the above carp DNA as a template and a Promoter primer containing a homologous arm. The PCR reaction system was 50 μL: 2x Phanta Flash Master Mix (Dye Plus) (Vazyme, P520-03) high-fidelity enzyme 25 μL, ddH2O 16 μL, carp DNA template 5 μL, Promoter-F / R primer (2*2) μL. The PCR reaction program was: 98℃ 30 s; 98℃ 10 s, 56.2℃ 5 s, 72℃ 40 s, 35 cycles; 72℃ 1 min; 4℃ ∞. The PCR product was subjected to agarose gel electrophoresis, and a target band of about 2800 bp was observed. The PCR product was recovered to prepare the Promoter region sequence of the overexpression recombinant plasmid, and the concentration was determined.

[0049] 4. Mix the Vector region, CDS region, and Promoter region fragments, and connect the fragments using "seamless" cloning technology

[0050] a) Assemble the vector fragment Vector region and the insert sequence fragments CDS region and Promoter region using the NEBuilder HiFi DNA Assembly Master Mix #E262L kit, set the molar ratio Vector region:CDS region:Promoter region = 1:4:2, and input the corresponding concentrations of each fragment on the NEBuilder website (https: / / nebuildercalculator.neb.com / ) to calculate the required addition volume for connecting each fragment. In this embodiment, the connection system is specifically: NEBuilder HiFi DNA Assembly Master Mix 10 μL, Vector region sequence product 0.5 μL, CDS region sequence product 2.2 μL, Promoter region sequence product 1.5 μL, deionized water 5.8 μL.

[0051] b) Treat the connection system at 50℃ for 1 h and then at 4℃ for 23 h to ensure that each fragment is fully connected.

[0052] 5. Transform the connection product into competent bacteria, test and select positive monoclonal bacteria, expand and extract the plasmid, and obtain the carp lpla gene recombinant overexpression plasmid

[0053] a) Thaw competent bacteria DH5α (Bomaide Biotechnology) on ice in advance. When the cells are just thawed, add 2 μL of ligation system, tap the bottom of the tube with your finger to mix gently, and incubate in ice water for 30 min. Heat shock at 42℃ for 60 s, then immediately incubate in ice water for 2 min. Add 250 μL of sterile LB culture medium and incubate at 37℃ on a shaker at 150~200 rpm for 60 min. Spread 150 μL of bacterial culture on an LB agar plate containing ampicillin and incubate upside down at 37℃ overnight.

[0054] b) Select single clones after overnight culture, dilute with sterile physiological saline to obtain bacterial suspension, and perform bacterial PCR using CDS primers and Promoter primers respectively; perform agarose electrophoresis on the PCR products, and the clones that show the corresponding target size are considered positive clones. Figure 2 The image shows the amplification results of the CDS and Promoter regions in the positive clone bacteria (verification agar diagram), indicating that the recombinant overexpression plasmid of the carp lpla gene was successfully transformed into competent bacteria.

[0055] c) The positive clones selected by amplification culture were used to extract plasmids using a kit to obtain the recombinant overexpression plasmid of the carp lpla gene, which was then frozen at -20℃ for later use.

[0056] Example 2: Application of overexpression of the carp lpla gene

[0057] 1. A transgenic zebrafish model overexpressing the *carp lpla* gene was constructed using the recombinant overexpression plasmid prepared in Example 1. The steps are as follows:

[0058] 1.1 Transposase mRNA synthesis:

[0059] 1.1.1 Escherichia coli containing pCS2-transposase plasmid was amplified in liquid LB medium, and pCS2-transposase plasmid was extracted using the Plasmid Miniprep Kit (Beijing Zhuangmeng International Biotechnology Co., Ltd., ZP101).

[0060] 1.1.2 The pCS2-transposase plasmid was digested with the NotI site using the FastDigest NotI kit (Thermo Fisher Scientific, FD0594).

[0061] 1.1.3 After enzyme digestion, add 1 / 20 volume of 0.5 M EDTA and 1 / 10 volume of 3 M sodium acetate, mix well, then add 2 volumes of pre-cooled anhydrous ethanol, mix well, and place at -20°C for at least 15 min; centrifuge at 4°C and 12500 rpm for 15 min, discard the liquid and recover the DNA precipitate; wash the precipitate with 500 μL of pre-cooled 70% ethanol, centrifuge at 4°C and 12500 rpm for 15 min, discard the liquid and recover the DNA precipitate, and let it dry at room temperature for 10 min; then soak and resuspend the precipitate with an appropriate amount of enzyme-free water, and determine the concentration of linearized plasmid DNA.

[0062] 1.1.4 In vitro transcription of linearized plasmid DNA is performed using the mMESSAGE mMACHINE™ SP6 kit (Thermo, AM1340), and then mRNA is recovered using the RNeasy® Mini Kit (QIAGEN GmbH, 74104), i.e., transposase mRNA, which is stored at -20°C for later use.

[0063] 1.2 Zebrafish microinjection and culture:

[0064] 1.2.1 After mixing the purified carp lpla gene recombinant overexpression plasmid prepared in Example 1 with transposase mRNA at a ratio of 1:1 (the final concentration of the plasmid is 250 ng / μL), the mixture is injected into 1-cell stage zebrafish eggs, and 0.02% phenol red solution (10 mM Tris-HCl pH 8.0) without recombinant plasmid vector and transposon is injected into 1-cell stage zebrafish eggs as a control.

[0065] 1.2.2 The embryos after injection are incubated in a biochemical incubator at 28.5°C for about 6-7 days until the larvae can swim, and then transferred to a zebrafish room for further feeding.

[0066] 1.3 After the above fry grows to 2 months old, fin DNA is extracted, and PCR verification is performed using detection primers to screen out microinjection positive individuals:

[0067] 1.3.1 Fin DNA is extracted by alkaline lysis. For alkaline lysis, add 10 μL of 50 mM NaOH to each appropriate amount of sample and digest at 95°C for 40 min, then add 1 μL of 1 M Tris-HCl (pH 8.0) and vortex to mix to terminate the digestion, centrifuge for 10 s, and take the supernatant to obtain the sample genomic DNA.

[0068] 1.3.2 Detection primers are designed in the CDS region and the Promoter region, and the specific sequences are as follows: X-F: 5’-CCTGCCAAATGACAAGTCCC-3’, and X-R: 5’-GGCTGTGACCTCCAGAA-3’.

[0069] 1.3.3 PCR verification using the DNA from the above step as template and detection primers, and polyacrylamide gel electrophoresis of the PCR product to screen out microinjection positive individuals, i.e. the constructed overexpression of carp lpla gene transgenic zebrafish model. The PCR reaction system is 10 μL: Taq enzyme 5 μL, ddH2O 3.2 μL, zebrafish genomic DNA 1 μL, X-F / R primer (0.4*2) μL. The PCR reaction conditions are: 95°C for 3 min; 95°C for 30 s, 57.2°C for 30 s, 72°C for 30 s, 35 cycles; 72°C for 5 min; 4°C for ∞.

[0070] 1.4 Screening out the corresponding zebrafish with target size bands and positive zebrafish. Figure 3 The positive individuals of microinjection of zebrafish with recombinant overexpression plasmid of carp gene are tested (agarose gel electrophoresis diagram, white box is positive individual), which indicates that the overexpression of carp lpla gene transgenic zebrafish model is successfully constructed.

[0071] 2. Application of overexpression of carp lpla gene in regulating lipid index TC and TG content in different tissues of cyprinid fish.

[0072] The effect of overexpression of carp lpla on lipid index in different tissues of zebrafish is as follows:

[0073] When the above microinjection positive zebrafish grow to 3 months old, the liver and muscle tissues are taken together with wild type control zebrafish, and the contents of total cholesterol TC (Nanjing Jiancheng Biological Engineering Institute, A111-1-1) and triglyceride TG (Nanjing Jiancheng Biological Engineering Institute, A110-1-1) are determined by using the kit. Figure 4 The TC and TG contents in liver and muscle tissues of overexpression of carp lpla gene transgenic zebrafish and wild type control are shown (lpla+ is overexpression of carp lpla gene transgenic zebrafish, L is liver, and M is muscle; *P < 0.05, and ns P > 0.05), the TC content in muscle of overexpression of carp lpla gene is about 7 times higher than that of wild type control, and the TC and TG contents in liver are also increased by more than 1 times, which indicates that overexpression of carp lpla gene causes differential deposition of fat in zebrafish.

[0074] 3. Application of overexpression of carp lpla gene in regulating lipid marker genes lpla, pparγ, fabp3, fasn and acaca in liver and muscle tissues of cyprinid fish.

[0075] The effect of overexpression of carp lpla on lipid marker genes in liver and muscle tissues is as follows:

[0076] When the above microinjection positive zebrafish grow to 3 months old, the liver and muscle tissue of the positive zebrafish and wild type zebrafish are taken, total RNA of the tissue is extracted and reverse transcribed to cDNA, and the relative expression amount of the fat metabolism marker gene is detected. In this experiment, the genes detected are: lpla, pparγ (Peroxisome Proliferators-activated Receptors γ), fasn (Fatty Acid Synthase), fabp3 (Fatty Acid-binding Protein 3) and acaca (Acetyl-CoA Carboxylase alpha), and 18S gene is used as a control. Among them, the corresponding fluorescence quantitative primers are:

[0077] lpla-F: 5'-AGACAGCTTCAACAAAGGGCATG-3',

[0078] lpla-R: 5'-GCCGTGGATTCCGTACAATGAG-3';

[0079] fabp3-F: 5'-CAGACGTTTTTGTTGGCACAT-3',

[0080] fabp3-R: 5'-GATGGTCGTGGGTTTGGTC-3';

[0081] fasn-F: 5'-TCAGCCATTACGTACCACT-3',

[0082] fasn-R: 5'-GTAGGAACAGTGGTGCTTTTCT-3';

[0083] acaca-F: 5'-GGGAGGATCATGGGTCGTCA-3',

[0084] acaca-R: 5'-CTCCAGCTCTTTACGCTCAGA-3';

[0085] pparγ-F: 5'-CCCAAGTTTGAGTTCTCCGTCA-3',

[0086] pparγ-R: 5'-AGTCGGGATGGTTCATCTTCA-3';

[0087] 18S-F: 5'-ACGATCAGATACCGTCGTAGTTCC-3',

[0088] 18S-R: 5’-CTGTCAATCCTTTCCGTGTCCG-3’.

[0089] Figure 5 The relative expression of the corresponding fat metabolism marker genes in the liver and muscle tissue of the transgenic zebrafish overexpressing the carp lpla gene and the wild type control (P < 0.05, ns represents P > 0.05), after overexpression of the carp lpla gene, the gene and pparγ in the liver and muscle were highly expressed, the expression of fabp3 and fasn genes in the two tissues was similar, and the expression of acaca gene was reduced in the liver and increased in the muscle, indicating that overexpression of the carp lpla gene caused different responses between different tissues of zebrafish at the transcription level.

[0090] 4. Application of overexpression of carp lpla gene in regulating the relative expression of fat metabolism marker genes lpla, pparγ, fabp3, fasn, acaca, hsl and pnpla2 in carp hepatocytes.

[0091] 4.1 Transfection of carp hepatocytes with recombinant overexpression plasmid of carp lpla gene, the steps are as follows:

[0092] 4.1.1.1 The carp hepatocyte line was preserved by the Freshwater Fish Breeding National and Local Joint Engineering Laboratory of Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences

[0093] 4.1.1.1 The carp hepatocyte line was preserved by the Freshwater Fish Breeding National and Local Joint Engineering Laboratory of Heilongjiang Fisheries Research Institute, Chinese Academy of Fishery Sciences .

[0094] 4.1.1.2 The frozen cells were quickly placed in a 37 ℃ water bath and shaken constantly until they were thawed, then transferred to a centrifuge tube containing 15% fetal bovine serum DMEM / F12 complete culture medium, centrifuged at 1300 r / min for 5 min, the supernatant was removed, and the cells were resuspended in DMEM / F12 complete culture medium and placed in a pre-coated T25 culture bottle, shaken well and placed in a 5% CO2, 28 ℃ incubator for culture, and the medium was changed after 16 h.

[0095] 4.2 When the cells were cultured to 80% confluence, they were trypsinized and evenly inoculated into 12-well plates. In this experiment, 3.0 μL of LipofectamineTM 3000 transfection reagent (ThermoFisher SCIENTIFIC, catalog number L3000150) and 1.0 μg of recombinant overexpression plasmid of carp lpla gene prepared in Example 1 were added to each well in the culture medium, and the specific transfection operation was completed according to the instructions.

[0096] 4.3 Total RNA of transfected and untransfected common carp liver cells was extracted by using blood / cell / tissue genomic DNA extraction kit (TIANGEN, DP304).

[0097] 4.4 The total RNA was reversely transcribed into cDNA by using PrimeScript™ FAST RT reagent Kit with gDNA Eraser (TaKaRa, RR092A).

[0098] 4.5 The relative expression of fat metabolism marker genes of the cDNA was detected. In this experiment, the detected genes were lpla, pparγ, fabp3, fasn, acaca, hsl (Hormone-Sensitive Lipase) and pnpla2 (Patatin-Like Phospholipase Domain Containing 2), with 18S gene as control. The corresponding fluorescence quantitative primers were as follows:

[0099] hsl-F: 5'-TCAAGGTGGTCCATTCCTGC-3',

[0100] hsl-R: 5'-CCTGAAAAACGCTCCAGTGC-3';

[0101] pnpla2:-F: 5'-CAACTCTGAGGCCTTGTCCAT-3',

[0102] pnpla2-R: 5'-GGGTAAAGTCAAAGGTCAGGTCAG-3'.

[0103] Figure 6 The relative expression of fat metabolism marker genes after overexpression of common carp lpla gene transfected into common carp liver cells is shown (*P < 0.05, ns P > 0.05). The expression of the gene after overexpression of common carp lpla gene was significantly higher than that of untransfected (P < 0.0001), the expression of ACACA gene was significantly lower than that of untransfected (P > 0.0001), and the expression of upstream PPARγ gene had no significant effect (P > 0.05), indicating that lpla gene transfected into common carp liver cells had an effect on cell fat deposition related genes, but overexpression of common carp lpla gene had different effects on the change of fat deposition mode at individual and cell levels of zebrafish.

Claims

1. Application of overexpression of carp lpla gene in regulating lipid index total cholesterol (TC) and triglyceride (TG) content in different tissues of Cyprinidae fish.

2. Application of overexpression of carp lpla gene in regulating lpla, pparγ, fabp3, fasn and acaca, which are fatty marker genes in liver and muscle tissues of Cyprinidae fish.

3. Application of overexpression of carp lpla gene in regulating relative expression of lpla, pparγ, fabp3, fasn, acaca, hsl and pnpla2, which are fatty metabolism related genes in liver cells of carp.

4. A method for constructing an overexpression vector containing the overexpressed carp Ipla gene according to claims 1, 2 and 3, characterized by The method is performed according to the following steps: I. Taking empty vector as template, Vector primers are designed and synthesized, and the sequence of Vector region of overexpression recombinant plasmid is prepared by PCR amplification; the empty vector is pDestTol2CG2-Mod plasmid; II. According to the sequence information of carp lpla gene in Ensembl database, CDS primers containing homologous arms of coding region of carp lpla gene are designed and synthesized, and the sequence of CDS region containing homologous arms is obtained by PCR amplification with carp liver cDNA as template; the sequence number of the carp lpla gene is ENSCCRT00000164182.1; III. According to the sequence information of carp lpla gene in Ensembl database, Promoter primers containing homologous arms of promoter region of carp lpla gene are designed and synthesized, and the sequence of Promoter region containing homologous arms is obtained by PCR amplification with carp liver DNA as template; the sequence number of the carp lpla gene is ENSCCRT00000164182.1; IV. The three sequences of Vector region, CDS region and Promoter region are homologously recombined, and the obtained circular plasmid is the recombinant overexpression plasmid of carp lpla gene.

5. The method for constructing an overexpression vector containing overexpressed carp Ipla gene according to claim 4, wherein In step I, the Vector primers are: Vector-F: 5'-TCTCGAGCCTCTAGAACTATAG-3', Vector-R: 5'-AGATCTGCGAAGATACGG-3'.

6. The method for constructing an overexpression vector containing overexpressed carp Ipla gene according to claim 4, wherein In step II, the CDS primers are: CDS-F: 5'-tcacatacagTTCGATTGAACCAGCGAG-3', CDS-R: 5'-atagttctagaggctcgagaGTGAAGGTTACTAGTCTGAATATTC-3'.

7. The method for constructing an overexpression vector containing overexpressed carp Ipla gene according to claim 4, wherein In step III, the Promoter primers are: Promoter-F: 5'-ggccgtatcttcgcagatctAGCACCGGTGTCTTCTAC-3', 8. The method for constructing an overexpression vector containing overexpressed carp Ipla gene according to claim 4, wherein Promoter-R: 5'-ttcaatcgaaCTGTATGTGAGCGCTTTG-3'. In step IV, the homologous recombination method is that the Vector region, CDS region and Promoter region are connected at a molar ratio of 1:4:2.

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