Application of leptinA and leptinB genes of cynoglossus semilaevis in inhibiting growth of ovary cells of cynoglossus semilaevis

Abstract

The invention relates to application of leptinA and leptinB genes of cynoglossus semilaevis in inhibition of growth of ovary cells of cynoglossus semilaevis, and belongs to the field of molecular biology, the application method is to inhibit development of the ovary cells of cynoglossus semilaevis by interfering expression of the leptinA or leptinB genes in the body of cynoglossus semilaevis, the gene sequence of the leptinA is shown in GenBank No: KY432435, the gene sequence of the leptinB is shown in GenBank No: KY432435, and the gene sequence of the leptinB is shown in GenBank No: KY432435. And the gene sequence of the leptin B is shown as GenBank No: MF784434. The invention also provides siRNA (small interfering ribonucleic acid) for interfering expression of the leptinA or leptinB gene, and the siRNA can inhibit development of ovary cells of the cynoglossus semilaevis, so that the growth speed of the cynoglossus semilaevis is increased.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology, and particularly relates to application of half-smooth tongue sole leptinA and leptinB genes in inhibiting the growth of half-smooth tongue sole ovarian cells. Background Art

[0002] Cynoglossus semilaevis is a large offshore benthic flatfish and an important aquaculture variety of marine fish in my country. It has male and female dimorphism, that is, the size and growth rate of female fish are significantly faster than those of male fish. Aquaculture operators often choose female seedlings for aquaculture to improve production efficiency. However, it is found in the production process that female aquacultured Cynoglossus semilaevis generally has the phenomenon of precocity, that is, the ovaries have developed before reaching the market specifications (weight less than 500g), and a visible bulge is formed on the body surface. In this process, the quota of body energy used for ovarian development increases, while the quota used for growth decreases, causing growth to slow down or stagnate, extending the aquaculture cycle, causing the cost of aquaculture inputs such as feed to increase, and the market price of commercial fish with premature ovaries is reduced due to abnormal appearance (bulge in the ovary), which seriously affects the aquaculture efficiency and becomes a bottleneck restricting the healthy development of the aquaculture industry. The technical problem to be solved by the present invention is to provide a method for promoting the rapid growth of Cynoglossus semilaevis by regulating the expression level of the leptin gene. Summary of the invention

[0003] In view of the above technical problems, the present invention provides an application of half-smooth tongue sole leptinA and leptinB genes in inhibiting the growth of half-smooth tongue sole ovarian cells. The application can inhibit the development of half-smooth tongue sole ovarian cells by interfering with the expression of half-smooth tongue sole leptinA and leptinB genes, thereby increasing the growth rate of half-smooth tongue sole.

[0004] The present invention is achieved through the following technical solutions:

[0005] The invention discloses an application of leptinA and leptinB genes of half-smooth tongue sole in inhibiting the development of ovarian cells of half-smooth tongue sole. The application method is to interfere with the expression of leptinA or leptinB gene in half-smooth tongue sole, thereby inhibiting the development of ovarian cells of half-smooth tongue sole. The gene sequence of leptinA is shown in GenBank No: KY432435; the gene sequence of leptin B is shown in GenBank No: MF784434.

[0006] The present invention also provides a growth-promoting biological preparation for half-smooth tongue sole, which contains siRNA derived from leptinA or leptinB gene, wherein the sequence of the siRNA is F: 5'-ccuguuuaacaaggcaccutt-3', R: 5'-aggugccuuguuaaacaggtt-3' designed based on the leptinA gene; and F: 5'-gcugcucagucagaucacatt-3', R: 5'-ugugaucugacugagcagctt-3' designed based on the leptinB gene.

[0007] The beneficial effects of the present invention compared with the prior art are as follows:

[0008] The present invention first discovered that the half-smooth tongue sole leptinA and leptinB genes are related to the growth and development of ovarian cells, and the transcription and translation levels of the leptinA and leptinB genes positively regulate the expression of leptin receptors, JAK2 and STAT3. When the expression of the leptinA and leptinB genes is interfered with by siRNA, the growth and migration of ovarian cells can be inhibited, and the apoptosis of ovarian granulosa cells can be promoted, so that more nutrients can be used for the growth of the half-smooth tongue sole. The present invention experimentally confirms that after regulation, the growth rate of the half-smooth tongue sole is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a diagram showing the knockdown efficiency of leptinA and leptinB in ovarian cells of Semilaevis semilaevis;

[0010] Figure 2 The figure shows the effect of knocking down leptinA and leptinB on the expression of genes related to signaling pathways and steroid hormone synthesis in the ovarian cells of Cynoglossus semilaevis. A is the expression of related genes when leptinA is knocked down; B is the expression of related genes when leptinB is knocked down;

[0011] Figure 3 This is a diagram showing the effects of knocking down leptinA and leptinB genes on cell proliferation and viability in ovarian cells of Semilaevis semilaevis;

[0012] Figure 4 Figure 2 shows that knockdown of leptinA and leptinB significantly affected the migration ability and wound healing process of cells in the ovarian cells of Cynoglossus semilaevis. A shows the migration of ovarian cells 24 hours and 48 hours after knockdown of leptinA and leptinB, and B reveals that at 48 hours, the wound healing rate of knockdown cells was significantly reduced compared with the control group.

[0013] Figure 5This is the sequence and key domain map of the leptin receptor gene;

[0014] Figure 6 It is the relevant information and restriction site map of pcDNA3.1(+) vector;

[0015] Figure 7 The results of the semi-smooth tongue sole recombinant proteins leptinA and leptinB activating transient transfected STAT3-luciferase activity by binding to leptin receptor / GPER / AR. A shows different concentrations of recombinant leptinA binding to leptin receptor and activating STAT3 signal; B shows different concentrations of recombinant leptinB binding to leptin receptor and activating STAT3 signal; C shows different concentrations of recombinant leptinA binding to G protein-coupled estrogen receptor (GPER) and activating STAT3 signal; D shows different concentrations of recombinant leptinB binding to androgen receptor (AR) and activating STAT3 signal; E shows SDS-PAGE and Western-blot of purified recombinant protein leptinA; F shows SDS-PAGE and Western-blot of purified recombinant protein leptinB;

[0016] Figure 8 This is a graph showing apoptosis analysis of ovarian cells of Semilaevis semilaevis after knocking down the leptinA and leptinB genes. A is the ovarian cell si-NC of the control group; B is the apoptosis of ovarian cells in the leptinA knockdown group (si_lepA); C is the apoptosis of ovarian cells in the leptinB knockdown group (si_lepB);

[0017] Fig. 9 This is the detection diagram of estradiol secretion in ovarian cells of Semilaevis semilaevis after knocking down the leptinA and leptinB genes. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.

[0019] Example 1 Full-length and siRNA design and primer design and synthesis

[0020] The mRNA sequences of leptinA and leptinB of Cynoglossus semilaevis were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / gene / ) (leptin A (lepA) GenBank No: KY432435; leptin B (lepB) GenBank No: MF784434). Three pairs of siRNAs of lepA and lepB of Cynoglossus semilaevis and control siRNA were designed using GenScript siRNA Target Finder (https: / / www.genscript.com / siRNA_target_finder.html) and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The specific sequences are shown in Table 1.

[0021] Table 1 siRNA sequences

[0022]

[0023] Example 2 Isolation, culture, inoculation and transfection of Cynoglossus semilaevis ovarian cells

[0024] Primary culture: (1) Ten female half-laevis semilaevis soles weighing about 110 g were selected and anesthetized with MS-222. The ovaries were removed under sterile conditions and washed three times with 1% double antibody PBS solution; (2) the ovarian surface membrane and blood vessels were removed, the ovaries were cut into 1 mm pieces, and then washed again with 1% double antibody PBS solution for three times; (3) EDTA digestion enzyme equivalent to 3 times the volume of the ovaries was added, digested for 15 minutes, and centrifuged at 100 rpm for 15 minutes under low temperature conditions; (4) the supernatant was discarded, and the cells were resuspended in incomplete culture medium containing 1% triple antibody, and this step was repeated twice; (5) the cells were resuspended in complete culture medium (containing 20% ​​fetal bovine serum, 1% triple antibody, 1% human growth factor and DMEM culture medium), the cell concentration was adjusted to 1×10^5 / mL, and then inoculated into T25 culture flasks and cultured in a 25°C incubator; (6) when the cells adhered to the wall and reached 80-90% confluence, they were digested with digestive pancreatic enzymes and passaged.

[0025] Transfection: (1) When the cells were in good condition and the confluence reached 70-80%, the siRNA described in Example 1 was transfected according to the instructions of Invitrogen's Lipofectamine 3000 transfection reagent, with 3 replicates per group; (2) After culturing in a 25°C incubator for 24 hours, the medium was replaced with complete medium, and the cells were collected after 48 hours. The collected cells were quickly frozen in liquid nitrogen and then stored at -80°C for subsequent experiments.

[0026] Example 3 Total RNA extraction

[0027] Refer to the operating instructions of Takara's total RNA extraction kit to extract tissue or cell RNA. The specific operating steps are as follows: (1) Sample processing: Take the cells collected in Example 2, first remove the culture supernatant, wash twice with pre-cooled PBS, then add 1 mL of RNAiso Plus, shake gently to ensure that the lysate is evenly distributed on the cell surface, transfer the lysate containing the cells to a centrifuge tube, use a pipette to repeatedly blow and aspirate until there is no obvious precipitation in the lysate, transfer it to an RNase-free centrifuge tube, let it stand at room temperature for 5 minutes, and then separate RNA from the nuclear protein; (2) Add 200 μL of chloroform, vortex and incubate at room temperature for 5 minutes; (3) Centrifuge at 4°C, 12,000g for 15 minutes; (4) Pipette 500 μL of the upper aqueous phase after stratification and transfer it to a new EP tube, add an equal volume of isopropanol, turn the centrifuge tube upside down to mix thoroughly, and let it stand at room temperature for 10 minutes; (5) Centrifuge at 12,000g at 4°C for 10 minutes and discard the filtrate; (6) Add 1 mL Wash the bottom precipitate with 75% ethanol and centrifuge at 7500g for 5 minutes at 4°C; (7) Repeat step (6) once; (8) Discard the supernatant and keep the precipitate. After the bottom of the centrifuge tube is dry, add 30uL DEPC water, dissolve it completely, and store it in a -80°C refrigerator.

[0028] After the total RNA extraction is completed, the following steps are performed to evaluate the quality of the RNA sample: (1) Take 1 μL of RNA solution sample and measure the absorbance ratio OD of the sample using an ultra-micro nucleic acid protein analyzer. 260 / OD 280 ; (2) Evaluation results: a. If OD 260 / OD 280 If the value is between 1.8 and 2.0, it means that the RNA sample is of suitable purity and can be used for subsequent experiments; b. If the OD 260 / OD 280 If the OD value is lower than 1.8, it may indicate protein contamination in the sample; c. 260 / OD 280 Values ​​above 2.0 may indicate contamination by other nucleic acids in the sample.

[0029] Example 4 mRNA reverse transcription and RT-qPCR

[0030] The reverse transcription of mRNA was performed according to the Takara reverse transcription kit (PrimeScript TMRT reagent Kit with gDNA Eraser) instruction manual, reaction system: (1) 5×gDNA Eraser Buffer 2μL, gDNA Eraser 1μL, Total RNA 1ug, calculate the sample addition amount and add the sample, add water (RNase-free water) to the total volume of 10μL, incubate at 42℃ for 2 minutes; (2) (1) reaction solution, PrimeScript RT Enzyme Mix I 1μL, RT Primer Mix 1μL, 5×PrimeScript Buffer 24μL, RNase Free dH 2 O 4μL, total volume 20μL. Reaction procedure: incubate at 37℃ for 15min, 85℃ for 5s, and store at 4℃. After reverse transcription, the reaction product was stored at -20℃.

[0031] Using RT-qPCR technology, TB Premix Ex Taq TM II kit to detect the expression level of mRNA. Reaction system: TB Green Premix Ex Taq II 10μL, PCR Forward Primer (10μM) 0.8μL, PCR Reverse Primer (10μM) 0.8μL, cDNA template 2μL, sterile water 6.4μL. Reaction procedure: 95℃ pre-denaturation for 30 seconds; 95℃ 5s, 60℃ 20s, repeated 40 cycles. Each sample was tested 3 times. The experimental results were obtained using 2 -ΔΔct Methods The relative expression levels of sample mRNA were calculated.

[0032] The present invention uses synthetic siRNA to conduct in vitro cell level experiments (inhibition / control experiments) to study the function of leptin in the ovarian cells of Cynoglossus semilaevis. The qPCR method is used to detect its effect, such as Figure 1 As shown, the knockdown efficiency of si_lepA2 and si_lepB3 was detected.

[0033] The results showed that the inhibition efficiency of transfected si_lepA2 and si_lepB3 was about 60% and 75% compared with si_NC. The following examples used these two pairs of siRNA.

[0034] Example 5 qPCR detection of the effects of si-lepA and si-lepB on JAK2 and STAT3 transcription levels

[0035] Genes related to signaling pathways and estradiol synthesis, such as JAK2, STAT3, lepA, lepB, star, p450ssc, p450c17, p450arom, 3β-hsd, 17β-hsd, and housekeeping genes 18s and β-actin, were synthesized based on previous research sequences. Primers are shown in Table 2:

[0036] Table 2. Primer sequences

[0037]

[0038] After knocking out the leptinA and leptinB genes, genes related to estrogen synthesis were detected at the RNA level by RT-qPCR. Knocking out leptinA or leptinB down-regulated the mRNA expression of Jak2 and Stat3, and also down-regulated the mRNA expression of Star, 3β-HSD, and P450arom ( Figure 2 ). P450c17 and P450ssc were significantly upregulated after leptinA or leptinB were knocked out. Only when leptinB was knocked out, the expression of 17β-HSD was significantly upregulated. That is, from the RNA level, interfering with lepB may affect estrogen synthesis.

[0039] Example 6 CCK8 detection of the effect of interfering with lepA and lepB expression on the growth of Cynoglossus semilaevis ovarian cells

[0040] In order to evaluate the growth and viability of ovarian cells and normal ovarian cells with knockdown of lepA and lepB. The cell counting kit (CCK-8) was used for detection according to the instructions. The specific method is: the primary ovarian cells were inoculated in 96-well plates according to the above method, and si-lepA and si-lepB were used to transfect three types of cells (si_NC, si_lepA, si_lepB) according to the method of Example 1, with 6 replicates per group. After transfection for 24 hours, 10 μL CCK-8 was added to each well, incubated at 24 ° C for 1-4 hours, and the culture medium was used as a blank control. Finally, the absorbance of each well was detected at a wavelength of 450 nm.

[0041] In order to study the function of lepA and lepB genes, these two genes were knocked out in tongue sole ovarian cells by siRNA transfection. The results showed that after 48 hours, the proliferation rate of ovarian cells was significantly reduced after knocking down lepA and lepB ( Figure 3 ).

[0042] Example 7 Effects of interfering with lepA and lepB on migration of ovarian cells of Cynoglossus semilaevis

[0043] Knockdown of lepA and lepB in primary ovarian cells by the above transfection method. 24 hours after transfection, vertical lines were drawn with a sterile 200 μL pipette tip, and then washed three times with PBS to remove detached cells. Add medium without FBS, and take pictures at 0h, 24h and 48h. Finally, ImageJ software was used to analyze the scratch area and average scratch width.

[0044] The results showed that the migration speed of ovarian cells was significantly reduced after knocking down lepA and lepB ( Figure 4 ).

[0045] Example 8 Dual luciferase gene reporter assay

[0046] Through the dual luciferase gene reporter experiment, we further explored whether lepA and lepB activated the activity of the JAK-STAT signaling pathway by binding to the leptin receptor. The specific experimental steps are as follows:

[0047] 1. Obtain target gene sequence fragments:

[0048] The mRNA information of the Cynoglossus semilaevis LepR (Gene ID: XM_025065894.1) gene was queried from NCBI. The gene sequence and key structural domains are shown in the figure below. Figure 5 , such as transmembrane domain, JAK2 box1, JAKbox2 and STATbox binding region in the membrane, etc.; designed primers and sent them to Sangon Biotech Co., Ltd. for synthesis. The details are shown in Table 3.

[0049] Table 3. Designed primer sequences

[0050]

[0051] Use the annealing method to obtain the target gene sequence fragment: Dissolve the synthesized LepR oligo to a concentration of 100 μM. Prepare the annealing reaction system according to Table 4;

[0052] Table 4. Annealing reaction system

[0053]

[0054] Annealing was performed on a PCR instrument according to the following procedure: 98°C for 10 sec; 61°C for 15 sec; 72°C for 30 sec (33 cycles); and stored at 4°C. After annealing, a dsDNA template with a concentration of 10 μM was obtained. The obtained template solution was diluted 50 times to a final concentration of 200 nM for ligation reaction.

[0055] 2. Clone the target gene sequence fragment into the vector

[0056] (1) The dual luciferase reporter gene plasmid uses pcDNA3.1 plasmid, and the map information is as follows Figure 6 As shown;

[0057] (2) The vector pcDNA3.1 was digested with EcoRⅠ and XbaⅠ, and the digestion reaction system was prepared according to Table 5. The digestion was carried out at 37°C for 1.5 h;

[0058] Table 5. Enzyme digestion reaction system

[0059]

[0060] (3) After electrophoresis, the double-digested pcDNA3.1 linear vector was recovered using a DNA gel recovery kit;

[0061] (4) The pcDNA3.1 vector band obtained by Fusion ligation and the target gene dsDNA template were connected at 50°C for 15 min. The connection system is shown in Table 6;

[0062] Table 6 shows the connection system

[0063]

[0064] 3. Conversion

[0065] (1) Place the competent cells on ice, add 10 μL of the ligation product, mix well, and place on ice for 30 min;

[0066] (2) Place in a 42°C water bath for 45 seconds, then immediately transfer to an ice bath and cool for 2 minutes; (3) Add 450 μL LB culture medium (without antibiotics) to the centrifuge tube, mix well, and incubate at 37°C shaker at 250 rpm for 1 hour; (4) Take 40 μL and 80 μL of the incubated competent cells, respectively, and spread them on LB solid culture medium plates containing 50 μg / mL ampicillin; (5) Place the cells upside down in a 37°C incubator overnight.

[0067] 4. Bacteria culture and sequencing verification

[0068] (1) Select a colony from the cultured plate and place it in a test tube containing 500 μL of LB medium (containing 50 μg / mL ampicillin) at 37°C and 250 rpm for 4 h;

[0069] (2) 400 μL of bacterial solution was taken for sequencing. After the sequencing result was aligned with the target sequence, the bacterial solution containing the lepR-pcDNA3.1 plasmid was obtained and the bacterial solution was cultured in large quantities.

[0070] 5. Plasmid extraction

[0071] Plasmid extraction was performed from the bacterial solution containing the lepR-pcDNA3.1 plasmid according to the instructions of the plasmid mini-extraction (Tiangen, China). The specific plasmid extraction is as follows:

[0072] (1) Take 5 mL of overnight cultured bacterial solution and add it to a centrifuge tube. Centrifuge it at 12,000 rpm for 1 min using a conventional desktop centrifuge and remove the supernatant.

[0073] (2) Add 150 μL of solution P1 (25 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50 mM glucose) to the centrifuge tube containing the bacterial precipitate (add RNase A and TIANRed before use. Mix according to TIANRed: solution P1 = 1:200, and mix thoroughly by inversion. The mixed solution is clear red. Add the mixed solution to the collected bacteria and mix thoroughly. Due to the presence of the bacteria, the mixed solution is turbid red). Use a pipette to thoroughly suspend the bacterial precipitate;

[0074] (3) Add 150 μL of solution P2 (250 mM NaOH, 1% SDS) to the centrifuge tube, and gently turn it upside down 6-8 times to fully lyse the bacteria. (Mix gently, do not shake violently to avoid contaminating genomic DNA. At this time, the bacterial solution should become clear and viscous. If it does not become clear, it may be due to excessive bacteria and incomplete lysis. The amount of bacteria should be reduced. Since TIANRed is used, after adding solution P2 and mixing thoroughly, the color of the solution is clear purple. If there is turbid red mixed in the purple, it means that the lysis is not sufficient. Continue mixing until the color of the solution completely turns clear purple.);

[0075] (4) Add 350 μL of solution P5 (potassium acetate and acetic acid) to the centrifuge tube, and immediately mix by inverting up and down 12-20 times. Mix thoroughly and a flocculent precipitate will appear. Centrifuge at 12000 rpm for 2 min. (Mix immediately after adding solution P5, and mix quickly by inverting up and down to avoid local precipitation. A small amount of tiny white precipitate in the supernatant will have no effect on subsequent experiments. If there are a large number of tiny white precipitates in the supernatant, centrifuge again and take the supernatant. Since TIANRed is used, after adding solution P5 and mixing thoroughly, the solution is clear yellow. If there is purple mixed in the yellow, it means that the renaturation is insufficient. Continue mixing until the color of the solution completely changes to clear yellow);

[0076] (5) Use a pipette to transfer the supernatant collected in the previous step to the adsorption column CP3 (the adsorption column is placed in the collection tube). Centrifuge at 12000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube;

[0077] (6) Add 300 μL of rinsing solution PWT (Tris-HCl and ethanol) to the adsorption column CP3 (add anhydrous ethanol before use), centrifuge at 12000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CP3 in the collection tube;

[0078] (7) Place the adsorption column CP3 in a collection tube and centrifuge at 12,000 rpm for 1 min to remove the residual rinse solution in the adsorption column;

[0079] (8) Place the adsorption column CP3 in a clean centrifuge tube, add 15 μL of enzyme-free water to the middle of the adsorption membrane, centrifuge at 12,000 rpm for 30 seconds to collect the plasmid solution into the centrifuge tube, and finally measure the concentration using Nanodrop.

[0080] 6. Cell transfection and dual luciferase activity detection

[0081] Cell transfection: (1) COS-7 cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin-gentamicin at 37°C and 5% CO 2 . One day before the transfection experiment, cells were seeded in 96-well plates. The cell fusion rate at the time of transfection was 70%-80%; (2) 100ng lepR-pcDNA3.1, 6ng pRL-TK reporter gene plasmid (Biyuntian, China) and 100ng STAT3-LUC plasmid (Biyuntian, China) were co-transfected into COS-7 cells, with 6 replicates in each group. 48h after transfection, rlepA and rlepB were diluted from 10-12M and added to 96-well plates respectively, and incubated for 3 hours (the negative control was only COS-7 cells, and the positive control was transfected with lepR-pcDNA3.1, pRL-TK and STAT3-LUC respectively).

[0082] Dual-luciferase activity detection: Fluorescence detection was performed using the Dual-Luciferase reporter gene detection kit (Quanshijin, China). The details are as follows:

[0083] (1) Remove the cell culture medium, carefully rinse twice with 1xPBS, add 20 μL 1xCell Lysis Buffer, and fully lyse at room temperature for 10 minutes;

[0084] (2) Add 100 μL of Luciferase Reaction Reagent equilibrated to room temperature into a 96-well plate, shake horizontally to mix, and detect the activity of the firefly luciferase reporter gene in a luminometer;

[0085] (3) Pipette 100uL of Luciferase Reaction Reagent II equilibrated to room temperature and add it to the above 96-well plate. Mix by horizontal shaking and detect the activity of Renilla luciferase reporter gene in a chemiluminescence instrument.

[0086] 7. Dual luciferase gene reporter assay for other receptors

[0087] G protein estrogen-coupled receptor (GPER, GenBank No: XM_008329491.3) and androgen receptor (AR, GenBank No: XM_008332997.2) were ligated to pcDNA3.1 vector at EcoRⅠ and XbaⅠ restriction sites using the homology arm method, and transfection and dual luciferase assay were performed according to the above method.

[0088] The molecular weights of the purified rlepA and rlepB recombinant proteins were approximately 14 kDa and 15 kDa, respectively. Figure 7 E and F). Meanwhile, in lane 2 ( Figure 7 E and F) were further confirmed by Western-blot, and their concentrations were adjusted to different concentration gradients (10 -7 -10 -12 M). LepRpcDNA3.1 / AR-pcDNA3.1 / GPER-pcDNA3.1, STAT3-LUC plasmid and pRL-TK reporter gene plasmid were co-transfected into COS7 cells, and different concentration gradients of rlepA and rlepB were added and cultured for 48 hours. Reporter gene assay showed that rlepA binds to lepR and activates the STAT3 signaling pathway, and the activation of rlepA is dose-dependent ( Figure 7 Although rlepB can also activate lepR, its biological activity in activating luciferase is not as strong as that of rlepA ( Figure 7 In addition, rlepA can activate GPER but not AR ( Figure 7 In contrast, rlepB can activate AR but not GPER ( Figure 7 D).

[0089] Example 9 Interference with lepA and lepB promotes apoptosis of ovarian cells of Semilaevis semilaevis

[0090] The original ovarian cells were planted in 6-well culture plates, and after the cells grew to a density of 70% to 80%, siRNA, including si_NC, si_lepA and si_lepB, were transfected. After 48 hours of transfection, the cells were treated with trypsin to detach from the culture plate and rinsed three times with phosphate buffered saline (PBS). Subsequently, the cells were centrifuged at 1200rpm for 5 minutes and rinsed three times with PBS again. Next, the cells were resuspended with 100μL of AnnexinV binding buffer, and 5μL of AnnexinV-FITC and 5μL of propidium iodide (PI) were added respectively. According to the manufacturer's guidelines, the cells were placed in a dark environment and incubated for 15 minutes. For the control group, cells resuspended only with binding buffer were used as negative controls, while cells mixed with PI or AnnexinV-FITC alone were used as positive controls to eliminate spectral overlap. Finally, 400 μL of AnnexinV binding buffer was added to the cells and detected using a flow cytometer (Beckman Coulter CytoFLEX, USA) using FITC and PI channels.

[0091] The present invention transfects si_lepA, si_lepB and si_NC into cultured semilaevis semilaevis ovarian cells to determine whether leptin plays a role in regulating ovarian cell apoptosis.

[0092] AnnexinV FITC / PI staining was used to detect the apoptosis rate of ovarian cells using flow cytometry. The apoptosis rate was detected by flow cytometry after PI and AnnexinV-FITC staining. The results are shown in Figure 8 As shown in Figure 2, interference with lepA and lepB promoted apoptosis of ovarian cells in Semilaevis semilaevis. Figure 8 It can be seen that the apoptosis rate of ovarian cells transfected with si_lepA was significantly higher than that of the si_NC group (P<0.05); the apoptosis rate of ovarian cells transfected with si_lepB was also significantly higher than that of the si_NC group (P<0.05). The above results indicate that lepA and lepB may have an anti-apoptotic effect on ovarian cells of semilaevis semilaevis and are a pro-apoptotic factor in ovarian cells.

[0093] Example 10 Effect of interfering with lepA and lepB on the secretion of sex steroid hormones

[0094] In order to better understand the effect of lepA and lepB on estradiol synthesis in primary ovarian cells, ovarian cells were collected after si_NC, si_lepA and si_lepB transfection for 48h, and estradiol (E2) ELISA kit (Cayman, USA) was used for detection according to the manufacturer's instructions. The absorbance value at 450 nanometer wavelength was then detected with an enzyme-linked immunosorbent assay reader, and the standard curve was fitted by a four-parameter Logistic curve using Origin software (OriginLab, USA). Finally, the concentration of E2 in ovarian cells was calculated by the standard curve formula. The specific method of ELISA is as follows:

[0095] (1) Sample preparation: After cell lysis, centrifuge and collect the supernatant (e.g., 100-500 g, 5 minutes);

[0096] (2) Preparation before testing: a. After taking the test kit out of the refrigerator, place it at room temperature (25-28°C) for 20 minutes to equilibrate; after each test, store the remaining reagent at 4°C. b. Prepare an appropriate amount of washing solution: dilute the washing solution (20X) to 1X with double distilled water or deionized water. For example, 10ml of washing solution (20X) is mixed with 190ml of water to obtain 1X washing solution. c. Take 5 clean 1.5ml centrifuge tubes, pre-add 250μl of sample diluent to each tube, and refer to Figure 2 The standard was diluted in multiples to obtain 6 standard concentrations of 1000, 500, 250, 125, 62.5, and 31.25 pg / ml. The diluted standard was added to the pre-coated plate wells in sequence. The standard dilution was directly used as 0 pg / ml concentration, for a total of seven standard concentrations. d. Prepare horseradish peroxidase labeled estradiol solution: according to the volume marked on the label, take the sample dilution solution into 1 bottle of horseradish peroxidase labeled estradiol and place it at 25-28°C.

[0097] (3) Specific operation steps: a. Plate setting: 2 blank plates (BLK), 2 non-specific binding wells (NSB), 2 maximum binding wells (BO) and an eight-point standard curve. b. Addition of reagents: Add samples and standards of different concentrations to the corresponding wells at 90 μl / well, add sample dilution as 0 concentration standard, and then add prepared horseradish peroxidase estradiol at 10 μl / well, mix thoroughly for 10 seconds, seal the reaction wells with a plate sealer (white), and incubate at room temperature in the dark for 120 minutes. c. Wash the plate 3 times, and pat dry on thick absorbent paper for the last time. d. Add 100 μl / well of the color developer TMB solution, seal the reaction wells with a plate sealer (white), and incubate at room temperature in the dark for 15-20 minutes. When the room temperature is low, the incubation time needs to be appropriately extended. At this time, the standard can be incubated until a very significant color change occurs. If the sample concentration is high enough, a significant color change will also occur. e. Add 50 μl / well of stop solution, mix well and measure the A450 value immediately.

[0098] To better understand the effect of knocking down lepA and lepB on estradiol at the protein level, we measured estradiol concentrations in vitro (in culture medium of primary ovarian cells after knocking down lepA and lepB). Although the E2 concentrations detected in vitro were low, the concentration of E2 secreted by ovarian cells of Semilaevis semilaevis was still significantly increased after interfering with lepB compared with NC ( Fig. 9 ). However, after interfering with lepA, the concentration of E2 did not change significantly compared with NC.

[0099] The embodiments described above are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. The use of leptinA and leptinB genes of Cynoglossus semilaevis in inhibiting the development of ovarian cells of Cynoglossus semilaevis, characterized in that: The application method is to interfere with the expression of leptinA or leptinB gene in the half-smooth tongue sole, thereby inhibiting the development of half-smooth tongue sole ovarian cells. The gene sequence of leptinA is shown in GenBank No: KY432435; the gene sequence of leptin B is shown in GenBank No: MF784434.

2. A biological agent for promoting growth of Cynoglossus semilaevis, characterized in that: The biological preparation contains siRNA derived from leptinA or leptinB gene, wherein the sequences of the siRNA are F: 5'-ccuguuuaacaaggcaccutt-3' and R: 5'-aggugccuuguuaaacaggtt-3' designed based on the leptinA gene; and F: 5'-gcugcucagucagaucacatt-3' and R: 5'-ugugaucugacugagcagctt-3' designed based on the leptinB gene.

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