Fish gonadotropin II in-vivo expression vector as well as construction method and application thereof

Abstract

The invention discloses a fish gonadotropin II in-vivo expression vector as well as a construction method and application thereof, and belongs to the technical field of bioengineering. The fish gonadotropin II in-vivo expression vector is constructed by adopting a primer bridging method and specifically comprises the following steps: connecting a beta subunit and an alpha subunit of epinephelus coioides gonadotropin II to the carboxyl terminal of human chorionic gonadotropin to form a GTHII beta-CTP-alpha target base sequence fragment in a single-chain form; and the target base sequence fragment of the GTHII beta-CTP-alpha is inserted into a multiple cloning site of a pcDNA3.1 / Zeo (+) no-load plasmid. The method has the beneficial effects that in-vivo expression of the gonadotropin II gene of the fish is realized, early ovarian development of the fish can be induced, injection is performed once every 10 days and only needs to be performed three times, the injection times are reduced, the workload is reduced, the cost is reduced, and more importantly, the ovarian development induction efficiency is improved to about 95%.

Description

Technical Field

[0001] The present invention relates to an expression vector and a construction method and application thereof, in particular to an in vivo expression vector of fish gonadotropin II and a construction method thereof and application thereof in inducing fish ovarian development, belonging to the technical field of bioengineering. Background Art

[0002] In fish production, the development of male and female reproductive organs is asynchronous or the gonads develop slowly, necessitating artificial induction of gonadal development. Currently, drugs commonly used to induce gonadal development in fish include fish pituitary extract, human chorionic gonadotropin (hCG), and luteinizing hormone-releasing hormone analogue (LHRHa). Fish pituitary extract is derived from the fish pituitary gland, while hCG is derived from the urine of pregnant women. Both require artificial extraction, a complex process that can also be contaminated by other hormones, potentially interfering with research results. LHRHa, on the other hand, requires artificial synthesis and is relatively expensive.

[0003] In vivo expression vectors can bring exogenous genes into the somatic cells of organisms to achieve the expression of exogenous genes in the organisms, without the need for large-scale integration into the genome or changes in the immune system. In fish, somatic gene transfer technology is mainly used for immune prevention of fish (for example: carp, Atlantic salmon, zebrafish, black sea bass, orange-spotted grouper, etc.). Chinese invention patent CN114807231A (Fish follicle-stimulating hormone somatic gene transfer vector and its construction method and application) applied somatic gene transfer technology to fish reproduction for the first time. By constructing a fish follicle-stimulating hormone somatic gene transfer vector and injecting it into the skeletal muscle of fish, the induction of early ovarian development in fish was achieved with the assistance of a gene introduction instrument. However, it still has the following shortcomings:

[0004] 1. Injection is required once every 7 days, a total of 4 injections are required, which is a large number of injections, a large workload and a high cost;

[0005] 2. The efficiency of ovarian development induction has reached about 85%, which needs to be further improved. Summary of the Invention

[0006] To address the deficiencies of the prior art, the present invention aims to provide an in vivo expression vector for fish gonadotropin II that requires fewer injections and has a higher efficiency in inducing ovarian development, as well as a method for constructing the in vivo expression vector and a method for inducing fish ovarian development using the in vivo expression vector.

[0007] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0008] The fish gonadotropin II in vivo expression vector is composed of a carboxy terminal of human chorionic gonadotropin, a beta subunit and an alpha subunit of Epinephelus coioides gonadotropin II and a pcDNA3.1 / Zeo(+) empty plasmid, wherein the beta subunit and the alpha subunit of the Epinephelus coioides gonadotropin II are connected to the carboxy terminal of the human chorionic gonadotropin to form a GTHIIβ-CTP-α target base sequence fragment in a single chain form, and the GTHIIβ-CTP-α target base sequence fragment is integrally inserted into a multiple cloning site of the pcDNA3.1 / Zeo(+) empty plasmid.

[0009] The construction method of the aforementioned fish gonadotropin II in vivo expression vector is characterized by adopting a primer bridging method and comprising the following steps.

[0010] Step 1: The beta subunit and the alpha subunit of the Epinephelus coioides gonadotropin II are connected by using the carboxy terminal of the human chorionic gonadotropin to construct a GTHIIβ-CTP-α target base sequence fragment in a single chain form, and specifically:

[0011] (1) The beta subunit of the gonadotropin II is amplified by using the primer β-F and the primer β-R, the alpha subunit of the gonadotropin II is amplified by using the primer α-F and the primer α-R, and the carboxy terminal of the human chorionic gonadotropin is self-amplified by using the primer CTP-F and the primer CTP-R, wherein the sequences of the primer β-F and the primer β-R are shown in SEQ ID NO: 3 and SEQ ID NO: 4, the sequences of the primer α-F and the primer α-R are shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the sequences of the primer CTP-F and the primer CTP-R are shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0012] (2) The alpha-CTP sequence is amplified by using the primer α-R and the primer CTP-F with the alpha subunit of the gonadotropin II and the carboxy terminal of the human chorionic gonadotropin as templates;

[0013] (3) The GTHIIβ-CTP-α target base sequence fragment is amplified by using the primer α-R and the primer β-F with the alpha-CTP and the beta subunit of the gonadotropin II as templates;

[0014] Step 2: The GTHIIβ-CTP-α target base sequence fragment is integrally inserted into a multiple cloning site of a pcDNA3.1 / Zeo(+) empty plasmid to construct a fish gonadotropin II in vivo expression vector.

[0015] Preferably, in Step 2, the process of integrally inserting the GTHIIβ-CTP-α target base sequence fragment into the multiple cloning site of the pcDNA3.1 / Zeo(+) empty plasmid is specifically as follows:

[0016] (1) Using EcoR I, Kpn I and 10×M buffer, pcDNA3.1 / Zeo(+) empty plasmid and GTHIIβ-CTP-α target base sequence fragment are respectively double enzyme cutting treatment;

[0017] (2) The enzyme cutting product is electrophoresed by agarose, and the GTHIIβ-CTP-α target base sequence fragment is recovered, and the GTHIIβ-CTP-α target base sequence fragment is connected to the multiple cloning site of the pcDNA3.1 / Zeo(+) empty plasmid by T4 DNA ligase.

[0018] The application of the aforementioned fish gonadotropin II in vivo expression vector is used to induce early ovary development of Epinephelus coioides, and the induction method is specifically as follows:

[0019] The aforementioned fish gonadotropin II in vivo expression vector is injected into the skeletal muscle of the gonad-undeveloped Epinephelus coioides at a dose of 400 ng / g.bw, and the injection is performed every 10 days, and immediately after each injection, the gene introduction instrument is used for electric shock, and the parameters of the gene introduction instrument are set as follows: the pulse number is 4, the pulse voltage is 100 V, the pulse duration is 20 ns, and the pulse width is 100 ns; immediately after the electric shock is completed, the Epinephelus coioides is placed into new water equipped with an oxygen pump, and the early ovary development of the Epinephelus coioides is induced after three times of injection.

[0020] The application has the advantages that:

[0021] (1) The fish gonadotropin II in vivo expression vector provided by the application is easy to obtain, low in cost, and free from other hormone pollution;

[0022] (2) The fish gonadotropin II in vivo expression vector is introduced into the fish body by using the somatic cell gene transfer technology, so that the fish gonadotropin II gene of the fish itself is expressed in vivo, and the early ovary development of the fish is induced by injection once every 10 days and only three times, so that the injection frequency is reduced, the workload is reduced, the cost is reduced, and more importantly, the ovary development induction efficiency is obviously improved, and the ovary development induction efficiency reaches about 95%. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is a schematic diagram of the construction process of the gonadotropin II in vivo expression vector of Epinephelus coioides;

[0024] Figure 2Figure is a comparison chart of E2 secreted by Epinephelus coioides in different groups, wherein, IC group is initial control group (fish sampling before experiment), Con group is blank control group (injected with PBS), pcDNA3.1 group is negative control group (injected with pcDNA3.1 / Zeo(+) empty plasmid), gpGTHII group is experimental group (injected with gpGTHII vector), and data is shown as mean ± standard error, n = 17-20, and different letters represent statistically significant difference (p < 0.05);

[0025] Figure 3 Figure is a comparison chart of early ovary development of Epinephelus coioides in different groups, wherein, IC group is initial control group (fish sampling before experiment), Con group is blank control group (injected with PBS), pcDNA3.1 group is negative control group (injected with pcDNA3.1 / Zeo(+) empty plasmid), gpGTHII group is experimental group (injected with gpGTHII vector), OI represents I phase oocyte, and OII represents II phase oocyte. DETAILED DESCRIPTION

[0026] The following will be specifically introduced by taking Epinephelus coioides as an example in combination with the drawings and specific embodiments.

[0027] I. Structure of fish GTHII in vivo expression vector

[0028] The fish GTHII in vivo expression vector provided by the application is composed of a carboxy terminal (CTP) of human chorionic gonadotropin (hCG), a β subunit and an α subunit of fish GTHII and a pcDNA3.1 / Zeo(+) empty plasmid, wherein, the β subunit and the α subunit of fish GTHII are connected to the carboxy terminal (CTP) of human hCG to form a single-chain GTHII β-CTP-α structure, and the single-chain GTHII β-CTP-α structure is inserted into a multiple cloning site of the pcDNA3.1 / Zeo(+) empty plasmid to form the fish GTHII in vivo expression vector, which is denoted as gpGTHII-pcDNA3.1 transgenic vector, and is abbreviated as gpGTHII vector.

[0029] II. Construction method of fish GTHII in vivo expression vector

[0030] The primer bridging method is used to construct the gpGTHII vector, as shown in Figure 1, and the construction process is specifically as follows: Figure 1

[0031] ​Step 1: The β subunit and α subunit of GTHII of Epinephelus malabaricus were connected by the carboxyl terminal (CTP) of human hCG to construct the GTHII β-CTP-α target base sequence fragment in single chain form.

[0032] (1) Design of primers

[0033] The specific primer sequences for constructing the recombinant expression vector are shown in Table 1.

[0034] Table 1 Specific primers for constructing gpGTHII vector

[0035]

[0036] (2) The β subunit (GTHIIβ) of GTHII was amplified by using the primer β-F and β-R and the α subunit (GTHIIα) of GTHII was amplified by using the primer α-F and α-R, and CTP was synthesized by self-amplification using the primer CTP-F and CTP-R, with the pituitary cDNA of Epinephelus malabaricus as the template.

[0037] Table 2 GTHIIβ, GTHIIα, and CTP gene amplification reaction system

[0038]

[0039]

[0040] PCR reaction conditions: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 20 s, 56℃ annealing for 20 s, 72℃ extension for 30 s, 35 cycles; 72℃ for 1 min; 4℃ storage.

[0041] (3) The α-CTP sequence was amplified by using the primer α-R and CTP-F, with GTHIIα and CTP as the template.

[0042] Table 3 GTHIIα subunit and CTP gene ligation amplification reaction system

[0043] Ingredients Amount added ddH2O 3.6 μL ExTaq buffer 5.0 μL 10 μM CTP-F 0.2 μL 10 μM α-R 0.2 μL GTHII α cDNA 0.5 μL CTP cDNA 0.5 μL Total volume 10.0 μL

[0044] PCR reaction conditions: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 20 s, 56℃ annealing for 20 s, 72℃ extension for 30 s, 35 cycles; 72℃ for 1 min; 4℃ storage.

[0045] (4) The GTHIIβ-CTP-α target base sequence fragment was amplified by using the primer α-R and β-F, with α-CTP and GTHIIβ as the template.

[0046] Table 4 GTHIIβ and α-CTP gene ligation amplification reaction system

[0047]

[0048]

[0049] PCR reaction conditions: 94°C pre-denaturation 3 min; 94°C denaturation 20 s, 56°C annealing 20 s, 72°C extension 1 min, 35 cycles; 72°C keep 3 min; 4°C keep.

[0050] Step 2: The GTHII β-CTP-α target base sequence fragment was inserted into the multiple cloning site of the pcDNA3.1 / Zeo(+) empty plasmid to construct the gpGTHII vector.

[0051] (1) Double enzyme digestion

[0052] The pcDNA3.1 / Zeo(+) empty plasmid containing EcoR I and Kpn I two enzyme digestion sites and the GTHII β-CTP-α target base sequence fragment containing EcoR I and Kpn I two enzyme digestion sites were subjected to double enzyme digestion using EcoR I (1040A), Kpn I (1068A) and 10×M buffer (TaKaRa Company).

[0053] The double enzyme digestion conditions are as follows:

[0054] Table 5 Double enzyme digestion reaction system of pcDNA3.1 / Zeo(+) empty plasmid

[0055]

[0056]

[0057] Table 6 Double enzyme digestion reaction system of GTHII β-CTP-α target base sequence fragment

[0058] Ingredients Amount added ddH2O 10.0 μL 10 x M buffer 5.0 μL EcoRI 2.5 μL KpnI 2.5 μL GTHII β-CTP-α DNA 40.0 μL Total volume 60.0 μL

[0059] The two double enzyme digestion reaction systems given in Tables 5 and 6 were mixed and subjected to enzyme digestion at 37°C for 1.5 h.

[0060] (2) Connection and transformation of GTHII β-CTP-α target base sequence fragment and large-scale amplification and extraction of gpGTHII-pcDNA3.1

[0061] The enzyme digestion product was subjected to electrophoresis with agarose, and the GTHII β-CTP-α target base sequence fragment was recovered and connected to the multiple cloning site of the pcDNA3.1 / Zeo(+) empty plasmid using T4 DNA ligase (TransGen Biotech Company).

[0062] Table 7 DNA ligation reaction system

[0063]

[0064]

[0065] After ligation at 16°C for 15±1h, the product was transformed into DH5α competent E. coli, and positive clones were screened and evenly spread on LB Amp+ plates for expansion culture.

[0066] EndoFree Mini Plasmid Kit II (TIANGEN Biotech) was used to extract endotoxin-free plasmids, laying the foundation for in vivo injection experiments.

[0067] 3. Application of Fish Gonadotropin II In Vivo Expression Vector

[0068] 1. Experimental fish

[0069] Eighty immature groupers with an average body weight of 100±6g (about 3 months old) were randomly divided into four groups (20 in each group), namely:

[0070] Initial control group (IC group): the fish were sacrificed before the experiment and no injection was given;

[0071] Blank control group (Con group): injected with PBS;

[0072] Negative control group (pcDNA3.1 group): injected with pcDNA3.1 / Zeo(+) empty plasmid; experimental group (gpGTHII group): injected with gpGTHII vector.

[0073] 2. Anesthesia

[0074] MS-222 (ethyl m-aminobenzoate methanesulfonate) was used to anesthetize each group of Epinephelus coioides. Specifically, an appropriate amount of MS-222 was directly dissolved in clean seawater containing experimental fish to anesthetize the Epinephelus coioides. The anesthesia time should not be too long.

[0075] 3. Injection

[0076] Injections were performed once on the 1st, 11th and 21st days of the experiment using a sterile hypodermic syringe with a caliber of 4.5 and an injection depth of 4mm-6mm in the thicker muscle between the lateral line and the dorsal fin.

[0077] The injection dose of PBS each time was 1 μL / g.bw.

[0078] The injection dose of pcDNA3.1 / Zeo(+) empty plasmid and gpGTHII vector was 400 ng / g.bw each time.

[0079] 4. Electric shock

[0080] Immediately after each injection, the gene gun was used to electric shock the each group of Epinephelus coioides with pre-set parameters.

[0081] The parameters of the gene gun were set as follows: the number of pulses was 4, the pulse voltage was 100 V, the pulse duration was 20 ms, and the pulse width was 100 ms.

[0082] Studies have shown that the use of a gene gun around the injection site using a four-electrode pulse method can improve the absorption of plasmid.

[0083] 5. Revival

[0084] Immediately after each electric shock, each group of Epinephelus coioides was placed in fresh water equipped with an oxygen pump, and the fish were revived.

[0085] 6. Estradiol (E2) analysis

[0086] 48 hours after the last injection, blood was taken from the tail vein of the experimental fish, and then the blood was incubated in a 4°C refrigerator for 6 hours. After centrifugation at 3000 rpm and 4°C for 10 minutes, the serum was separated and stored at -80°C (for estrogen E2 detection experiment).

[0087] The E2 content was determined using a fish E2 ELISA analysis kit (MLbio, Shanghai, China), and the experimental method is described in the instruction manual.

[0088] After detection and statistics, the secretion of estrogen E2 in different groups of Epinephelus coioides was as shown in Table 1. Figure 2 Table 1

[0089] 7. Preparation of ovarian tissue paraffin sections

[0090] 48 hours after the last injection, the ovarian tissue of the experimental fish was taken and paraffin sections were prepared. The preparation of paraffin sections mainly refers to the "Practical Pathological Tissue Staining Technology", and the reaction time of each step is adjusted according to the actual situation. First, the purchased paraffin was placed in a 60°C constant temperature box for at least 24 hours, and the ovarian tissue fixed in Bouin's fixative for 24 hours was taken out and stored in 70% alcohol at room temperature.

[0091] (1) Dehydration and wax immersion

[0092] The detailed steps of dehydration and wax immersion are shown in Table 8.

[0093] Table 8 Dehydration and wax immersion steps

[0094] Step Reagent Treatment time 1 70% (v / v) ethanol 1h 2 80% (v / v) ethanol 1h 3 95% (v / v) ethanol 1.5h 4 95% (v / v) ethanol 1h 5 Anhydrous ethanol 30 min 6 Anhydrous ethanol 30 min 7 Ethanol and xylene mixture (1:1 by volume) 30 min 8 Xylene 30 min 9 Xylene and paraffin mixture (1:1 by volume) 1 h (in a 60°C oven) 10 Paraffin 2 h (in a 60°C oven) 11 Paraffin 2 h (in a 60°C oven)

[0095] (2) Embedding

[0096] Embedding was performed on a heating plate. The ovary tissue block was placed in the middle of a paper box, and the wax was poured. After the surface formed a coagulation, the paper box was placed in water to cool. After 24 h at room temperature, the next step was performed or it was temporarily stored for later use. The excess paraffin without ovary tissue was cut off, and the wax block was trimmed into a trapezoidal or square shape. Then the wax block was cut into a 7 mm thick slice on a microtome; the slice was placed in sterile deionized water cooled to 42°C for spreading. The spread slice was then moved to a glass slide, baked at 42°C, and placed in a room temperature drying place after the slice was dried, ready for use. Then the glass slide was deparaffinated (treated with xylene for 10 min, twice).

[0097] (3) Rehydration

[0098] The detailed steps of rehydration are shown in Table 9.

[0099] Table 9 Rehydration steps

[0100] Step Reagent Treatment time 1 Xylene and ethanol mixture (1:1 by volume) 3 min 2 Anhydrous ethanol 5 min 3 Anhydrous ethanol 5 min 4 95% (v / v) ethanol 5 min 5 80% (v / v) ethanol 3 min 6 70% (v / v) ethanol 3 min 7 50% (v / v) ethanol 1 min 8 Distilled water 1 min 9 Distilled water 1 min

[0101] (4) HE staining

[0102] The detailed steps of HE staining are shown in Table 10.

[0103] Table 10 HE staining steps

[0104] Step Reagent Treatment time 1 Haematoxylin stain 1 min 2 Tap water rinse 8 min 3 1% hydrochloric acid alcohol differentiation 5s 4 Distilled water 1 min 5 Distilled water 1 min 6 70% (v / v) ethanol 3 min 7 80% (v / v) ethanol 3 min 8 95% alcohol-soluble eosin stain 3 min 9 95% (v / v) ethanol 5 min 10 95% (v / v) ethanol 5 min 11 Anhydrous ethanol 5 min 12 Anhydrous ethanol 5 min 13 Xylene 10 min 14 Xylene 10 min 15 Dry in fume cupboard 30 min

[0105] (5) Mounting

[0106] After the slice was dried, a drop of neutral gum was dropped on the ovary tissue, and a cover glass was placed on it. After 3 h or so, the gum was dried, and the specimen was placed in a specimen box for storage.

[0107] The cell morphology of the ovary tissue of Epinephelus coioides in different groups was observed by optical microscopy, as shown in Figure 3 .

[0108] The development of the ovary of Epinephelus coioides in different groups (stage I, stage II) was counted, as shown in Table 11.

[0109] Table 11 Counting table of the development of the ovary of Epinephelus coioides in different groups

[0110] IC group Con group pcDNA3.1 group gpGTHII group Total number of Epinephelus coioides 20 fish 20 fish 20 fish 20 fish Number of ovaries in stage I 20 fish 18 fish 18 fish 1 fish Number of ovaries in stage II 0 fish 2 fish 2 fish 19 fish

[0111] From Figure 2 and Figure 3And it can be seen from Table 11 that injection of the gpGTHII vector provided by the application can stimulate E2 secretion of Epinephelus coioides and induce early ovarian development of Epinephelus coioides, and the induction efficiency reaches about 95%.

[0112] It should be noted that the above examples are merely examples for clearly illustrating the present application, and are not intended to limit the implementation modes of the present application. Based on the above description, other different forms of changes or variations can also be made by those skilled in the art. Here, all the implementation modes cannot be exhausted. Any obvious changes or variations derived from the technical scheme of the present application are still within the protection scope of the present application.

Claims

1. A fish gonadotropin II in vivo expression vector, characterized in that: The invention is composed of the carboxyl terminus of human chorionic gonadotropin, the β subunit and α subunit of gonadotropin II of grouper coioides, and a pcDNA3.1 / Zeo(+) empty plasmid. The β subunit and α subunit of gonadotropin II of grouper coioides are connected to the carboxyl terminus of human chorionic gonadotropin to form a single-chain GTHIIβ-CTP-α target base sequence fragment. The GTHIIβ-CTP-α target base sequence fragment is entirely inserted into the multiple cloning site of the pcDNA3.1 / Zeo(+) empty plasmid.

2. The method for constructing the fish gonadotropin II in vivo expression vector according to claim 1, characterized in that: The primer bridging method includes the following steps: Step 1: Use the carboxyl terminus of human chorionic gonadotropin to connect the β subunit and α subunit of grouper gonadotropin II to construct a single-chain GTHIIβ-CTP-α target base sequence fragment. Specifically: (1) Using pituitary cDNA from grouper as a template, primers β-F and β-R were used to amplify the β subunit of gonadotropin II, primers α-F and α-R were used to amplify the α subunit of gonadotropin II, and primers CTP-F and CTP-R were used to self-amplify and synthesize the carboxyl terminus of human chorionic gonadotropin, wherein the sequences of primers β-F and β-R are shown in SEQ ID NO: 3 and SEQ ID NO: 4, the sequences of primers α-F and α-R are shown in SEQ ID NO: 1 and SEQ ID NO: 2, and the sequences of primers CTP-F and CTP-R are shown in SEQ ID NO: 5 and SEQ ID NO: 6; (2) Using the α-subunit of gonadotropin II and the carboxyl terminus of human chorionic gonadotropin as templates, primers α-R and CTP-F were used to amplify the α-CTP sequence; (3) Using α-CTP and gonadotropin II β subunit as templates, primers α-R and β-F were used to amplify the target base sequence fragment of GTHIIβ-CTP-α; Step 2: Insert the entire GTHIIβ-CTP-α target base sequence fragment into the multiple cloning site of the pcDNA3.1 / Zeo(+) empty plasmid to construct an in vivo expression vector for fish gonadotropin II.

3. The construction method according to claim 2, characterized in that In Step 2, the process of inserting the entire GTHIIβ-CTP-α target base sequence fragment into the multiple cloning site of the pcDNA3.1 / Zeo(+) empty vector plasmid is as follows: (1) The pcDNA3.1 / Zeo(+) empty vector plasmid and the target base sequence fragment of GTHIIβ-CTP-α were double-digested using EcoR I, Kpn I, and 10×M buffer; (2) The enzyme-digested product was subjected to agarose electrophoresis to recover the target base sequence fragment of GTHIIβ-CTP-α, and the target base sequence fragment of GTHIIβ-CTP-α was ligated to the multiple cloning site of pcDNA3.1 / Zeo(+) empty plasmid using T4 DNA ligase.

4. The use of the fish gonadotropin II in vivo expression vector according to claim 1, characterized in that: Used to induce early ovarian development in Epinephelus coioides. The induction method is as follows: The fish gonadotropin II in vivo expression vector is injected into the skeletal muscle of the gonad-undeveloped grouper at a dose of 400 ng / g.bw, once every 10 days. After each injection, electric shock is immediately performed using a gene transfer instrument. The parameters of the gene transfer instrument are set as follows: the number of pulses is 4, the pulse voltage is 100 V, the pulse duration is 20 ns, and the pulse width is 100 ns. After the electric shock is completed, the grouper is immediately placed in fresh clean water equipped with an oxygen pump. After completing three injections, early ovarian development in the grouper can be induced.

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