An induction method for efficiently producing triploid Nibea albiflora males

Abstract

The present invention discloses an induction method for efficiently producing triploid male Nibea albiflora, belonging to the technical field of triploid induction of male Nibea albiflora, and specifically relates to nutritional regulation and photothermal regulation of sexually mature pseudo-male Nibea albiflora and female Nibea albiflora, sperm and eggs collection; artificial insemination to obtain fertilized eggs, which are immersed in seawater for treatment and then incubated, and finally triploid male Nibea albiflora is induced and cultivated; when the fertilized eggs are immersed in seawater during artificial insemination and incubation, purine derivatives can be added to the seawater to treat the fertilized eggs, thereby improving the success rate of triploid induction of Nibea albiflora. The method of the present invention has a high success rate of triploid induction of Nibea albiflora, and triploid male Nibea albiflora is successfully obtained under subsequent induction.

Description

Technical Field

[0001] The present invention belongs to the technical field of inducing triploid of Nibea albiflora, and particularly relates to an induction method for efficiently producing triploid Nibea albiflora with all male sex. Background Art

[0002] Artificially inducing fish chromosome triploidization and sex control can change corresponding biological traits, such as growth rate, male and female sex, gonadal development, etc., and are important technical means for fish genetic improvement. Especially in some fish species with sexual dimorphism in growth, the combined use of chromosome triploidization and sex control technologies can further accelerate the improvement of performance such as growth and increase the aquaculture benefit. At present, these two technologies have been applied in fish species such as Nile tilapia in Mozambique and rainbow trout. Through the analysis of growth performance, research shows that triploidization and sex control of fish with sexual dimorphism can significantly improve their aquaculture benefit. Therefore, in fish farming, in order to obtain higher aquaculture benefit, research and application of triploidization and sex control can be carried out on various fish species with characteristics such as sexual dimorphism in growth. In addition, triploid fish have sterile gonads and are ideal recipients in germ cell transplantation operations. Therefore, artificially inducing fish triploid also has important application value in germ cell transplantation. At the same time, currently, the transplantation recipients are mainly larvae and juveniles, but at this stage, the sex cannot be distinguished from the external morphology, and after transplantation, the sex of the donor offspring produced is determined by the sex of the recipient. Therefore, in order to accurately obtain offspring of the expected sex, it is also necessary to further control the sex of the triploid recipient.

[0003] Nibea albiflora has typical sexual dimorphism in growth characteristics, and the growth rate of female fish is significantly higher than that of male fish. Therefore, triploidization and sex control of Nibea albiflora not only have potential economic benefits in the aquaculture production of Nibea albiflora, but also have important application value in research such as germ cell transplantation and gene editing breeding of Sciaenidae fish. This method establishes an induction method for triploid Nibea albiflora with all male sex, opening up a new way for sex control breeding and polyploid breeding of Sciaenidae fish such as Nibea albiflora. At the same time, this method can also be extended to other fish species to promote the research and application of triploid breeding and sex control of marine fish. Summary of the Invention

[0004] The purpose of the present invention is to provide an induction method for efficiently producing triploid Nibea albiflora with all male sex, which has a high success rate of inducing triploid and good effects.

[0005] The technical solution adopted by the present invention to achieve the above purpose is as follows:

[0006] An induction method for efficiently producing triploid Nibea albiflora males, comprising: nutritionally regulating and regulating light and temperature of mature pseudo-male Nibea albiflora and female Nibea albiflora, extracting sperm and eggs; obtaining fertilized eggs by artificial insemination, immersing them in seawater for treatment and then incubating and culturing, and finally inducing and culturing triploid Nibea albiflora males; immersing the fertilized eggs in seawater at 3-12 °C for 5-20 min, then transitioning in seawater at 13-16 °C for 3-10 min, and finally putting them into seawater at 20-25 °C for hatching and culturing; using a compound feed containing the aromatase inhibitor letrozole during the induction and culturing.

[0007] Preferably, the seawater for treating the fertilized eggs contains purine derivatives.

[0008] More preferably, the purine derivative is methoxypurine ester or methoxypurine isohydroxamic acid; the methoxypurine ester is prepared by reacting 6-methoxypurine with ethyl 2-bromoacetate; the methoxypurine isohydroxamic acid is prepared by reacting the methoxypurine ester with hydroxylamine hydrochloride.

[0009] Further preferably, in the preparation of the methoxypurine ester, 6-methoxypurine is added to DMF, potassium carbonate is added, ethyl 2-bromoacetate is added, and the reaction is carried out at 20-40 °C for 8-24 h. After the reaction is completed, the pH is adjusted to neutral, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried with magnesium sulfate, evaporated to dryness, and purified by silica gel column chromatography to obtain the methoxypurine ester.

[0010] When the triploid Nibea albiflora is induced and prepared by the present invention, at a temperature of 3-12 °C, after adding a trace amount of purine derivatives, the induction success rate of triploid Nibea albiflora can be improved, and the induction success rate of purine derivatives with isohydroxamic acid groups is higher. When there is a hydroxyl group on the purine of the purine derivative, the induction success rate of triploid Nibea albiflora can also be improved. When there is a hydroxyl group on the purine of the purine derivative and an isohydroxamic acid group at the same time, the success rate of the obtained triploid Nibea albiflora is higher.

[0011] Even more preferably, in the preparation of the methoxypurine ester, the usage amount of 6-methoxypurine is 8-12 wt% of DMF.

[0012] Even more preferably, in the preparation of the methoxypurine ester, the usage amount of potassium carbonate is 10-15 wt% of DMF.

[0013] Even more preferably, in the preparation of the methoxypurine ester, the usage amount of ethyl 2-bromoacetate is 100-150 wt% of 6-methoxypurine.

[0014] Even more preferably, in the preparation of the methoxypurine ester, the column chromatography eluent is a mixture of ethyl acetate and petroleum ether in a mass ratio of 1:5-20.

[0015] Further preferably, in the preparation of methoxypurine isohydroxamic acid, hydroxylamine hydrochloride is added to anhydrous methanol, then sodium methoxide is added, and the reaction is carried out at 20 - 40 °C for 20 - 60 min. After the reaction is completed, it is cooled, and the solid is filtered off. The methoxypurine ester is added to the filtrate, and the reaction is carried out at 50 - 60 °C for 4 - 12 h. After the reaction is completed, the pH is adjusted to 5 - 6, concentrated and stirred, and purified by silica gel column chromatography to obtain methoxypurine isohydroxamic acid.

[0016] Even more preferably, in the preparation of methoxypurine isohydroxamic acid, the amount of hydroxylamine hydrochloride used is 0.5 - 2 wt% of anhydrous methanol.

[0017] Even more preferably, in the preparation of methoxypurine isohydroxamic acid, the amount of sodium methoxide used is 80 - 120 wt% of hydroxylamine hydrochloride.

[0018] Even more preferably, in the preparation of methoxypurine isohydroxamic acid, the amount of methoxypurine ester used is 150 - 300 wt% of hydroxylamine hydrochloride.

[0019] Even more preferably, in the preparation of methoxypurine isohydroxamic acid, the column chromatography eluent is a mixture of methanol and ethyl acetate in a mass ratio of 1:1 - 3.

[0020] More preferably, the purine derivative is a hydroxy purine ester or a hydroxy purine isohydroxamic acid; the hydroxy purine ester is prepared by reacting 6 - hydroxy purine with ethyl 2 - bromoacetate; the hydroxy purine isohydroxamic acid is prepared by reacting the hydroxy purine ester with hydroxylamine hydrochloride.

[0021] Further preferably, the present invention can also prepare a hydroxy purine ester. Compared with the preparation of methoxypurine ester, the difference lies in that 6 - methoxypurine is replaced by 6 - hydroxy purine.

[0022] The present invention can also prepare a hydroxy purine isohydroxamic acid. Compared with the preparation of methoxypurine isohydroxamic acid, the difference lies in that the methoxypurine ester is replaced by the hydroxy purine ester.

[0023] More preferably, the content of the purine derivative in the seawater for treating fertilized eggs is 0.02 - 0.2 wt%.

[0024] Preferably, in nutritional regulation, nereis and squid are used as bait for cultivation, and 3 - 5 wt% of vitamin C and 1 - 2 wt% of vitamin E are added to the bait.

[0025] Preferably, in light and temperature regulation, the light intensity is 60 - 650 lux, the light time is 12 - 16 hours per day, and the water temperature is 18 - 22 °C.

[0026] Preferably, the amount of aromatase inhibitor letrozole in the compound feed is 5 - 20 mg / kg.

[0027] Preferably, in the cultivation of pseudo - male Nibea albiflora and female Nibea albiflora, select sexually mature 2 - year - old pseudo - male fish and normal female fish and place them in a cement pond. Conduct nutritional regulation and light - temperature regulation on the normal female fish.

[0028] More preferably, in the cultivation of pseudo - male Nibea albiflora and female Nibea albiflora, nutritional regulation means cultivating with nereis and squid as bait, adding 3 - 5 wt% vitamin C and 1 - 2 wt% vitamin E to the bait, and feeding 2 - 3 times a day.

[0029] More preferably, in the cultivation of pseudo - male Nibea albiflora and female Nibea albiflora, light - temperature regulation is to control the surface illumination of the broodstock pond with a 60 - 70 W incandescent lamp, the illumination intensity is 60 - 650 lux, the illumination time is 12 - 16 hours / day, and the water temperature is 18 - 22 °C.

[0030] More preferably, in the cultivation of pseudo - male Nibea albiflora and female Nibea albiflora, when they reach sexual maturity, inject luteinizing hormone - releasing hormone - A2 into the coelom of the female fish for artificial induced spawning. Use the abdominal extrusion method to collect the eggs of the female fish and the sperm of the pseudo - male fish respectively. The collected sperm and eggs are placed in a dry beaker for standby.

[0031] Preferably, in artificial insemination and hatching cultivation, dry insemination is adopted. Drop the semen of the collected pseudo - male fish into the eggs, gently shake and mix evenly, and then add a small amount of seawater for fertilization; 2 - 4 minutes after insemination, immerse the fertilized eggs in seawater at 3 - 12 °C for 5 - 20 minutes, take out the treated fertilized eggs and transfer them in seawater at 13 - 16 °C for 3 - 10 minutes, and then put them into seawater at 20 - 25 °C to prepare for hatching. 20 - 30 days after hatching, transfer the fry to a breeding bucket and adapt to cultivate for 3 - 7 days.

[0032] Preferably, in artificial insemination and hatching cultivation, purine derivatives can be added to the seawater, and the content of methoxypurine ester is 0.02 - 0.2 wt%. The purine derivatives include methoxypurine ester or methoxypurine isohydroxamic acid or hydroxypurine ester or hydroxypurine isohydroxamic acid.

[0033] Preferably, in the induced cultivation of all - male triploid Nibea albiflora, the all - female Nibea albiflora fry after hatching cultivation are fed with compound feed for 30 - 90 days, feeding 2 - 3 times a day. After the compound feed feeding, use ordinary feed to cultivate until they become adult fish.

[0034] More preferably, in the induced cultivation of all - male triploid Nibea albiflora, compared with the ordinary feed, the difference in the compound feed is that aromatase inhibitor letrozole is added to the compound feed, and the amount of aromatase inhibitor letrozole is 5 - 20 mg / kg.

[0035] More preferably, in the induction and cultivation of all-male triploid Nibea albiflora, letrozole, an aromatase inhibitor, is dissolved in a small amount of 95% ethanol and then mixed with ordinary feed to obtain compound feed.

[0036] More preferably, in the induction and cultivation of all-male triploid Nibea albiflora, water is changed regularly every day, and the amount of water changed each time is not less than 200%.

[0037] Preferably, in artificial insemination and hatching cultivation, piperazine sulfate can be added to seawater, and the content of piperazine sulfate is 0.02 - 0.1 wt%. In the exploration of the induction of triploid Nibea albiflora, it is found that when a small amount of piperazine sulfate is used in combination with purine derivatives, better effects can be achieved, and the induction success rate of triploid Nibea albiflora can be improved.

[0038] The present invention discloses a purine derivative, which includes methoxypurine ester or methoxypurine isohydroxamic acid or hydroxypurine ester or hydroxypurine isohydroxamic acid; methoxypurine ester is prepared by reacting 6-methoxypurine with ethyl 2-bromoacetate; methoxypurine isohydroxamic acid is prepared by reacting methoxypurine ester with hydroxylamine hydrochloride; hydroxypurine ester is prepared by reacting 6-hydroxypurine with ethyl 2-bromoacetate; hydroxypurine isohydroxamic acid is prepared by reacting hydroxypurine ester with hydroxylamine hydrochloride.

[0039] The present invention discloses the use of the above purine derivative in the preparation of a reagent for improving the induction success rate of triploid Nibea albiflora.

[0040] In the present invention, through nutritional regulation and photothermal regulation of sexually mature pseudo-male Nibea albiflora and female Nibea albiflora, sperm and eggs are taken; fertilized eggs are obtained through artificial insemination, immersed in seawater for treatment and then hatched and cultivated, and finally the induction and cultivation of all-male triploid Nibea albiflora are carried out; when the fertilized eggs are immersed in seawater during artificial insemination and hatching cultivation, purine derivatives can be added to the seawater to treat the fertilized eggs, improving the induction success rate of triploid Nibea albiflora. Therefore, the present invention has the following beneficial effects: the induction success rate of triploid Nibea albiflora is high, and all-male triploid Nibea albiflora is successfully obtained under subsequent induction. Therefore, the present invention aims to provide an induction method for efficiently producing all-male triploid Nibea albiflora with a high induction success rate and good effects. Description of the Drawings

[0041] Figure 1 is an infrared spectrogram;

[0042] Figure 2 is a triploid induction success rate graph;

[0043] Figure 3 is a comparison graph of gonad tissue sections;

[0044] Figure 4 is a DNA relative content and chromosome karyotype analysis graph. Detailed implementation manners

[0045] The technical solution of the present invention will be further described in detail below in conjunction with the specific implementation manners and the accompanying drawings:

[0046] The common feed of the present invention is composed of 25wt% soybean cake, 25wt% cottonseed hull, 20wt% sorghum, 15% distillers' grains, 12% wheat bran, 2.5% bone meal and 0.5% folium isatidis. It is dry weight and does not contain water.

[0047] Example 1:

[0048] An induction method for efficiently producing triploid Nibea albiflora with all males

[0049] Cultivation of pseudo-male Nibea albiflora and female Nibea albiflora: Select 2-year-old pseudo-male fish and normal female fish that have reached sexual maturity and place them in a cement pond. Conduct nutritional regulation and light and temperature regulation on the normal female fish. Nutritional regulation means cultivating with nereis and squid as bait, adding 4wt% vitamin C and 1.5wt% vitamin E to the bait, and feeding twice a day; Light and temperature regulation is to control the surface light of the broodstock pond with a 65W incandescent lamp, the light intensity is 300 lux, the light time is 12 hours / day, and the water temperature is 20°C. When they reach sexual maturity, inject luteinizing hormone-releasing hormone-A2 into the body cavity of the female fish for artificial induced spawning. The eggs of the female fish and the sperm of the pseudo-male fish are collected respectively by the abdominal extrusion method, and the collected sperm and eggs are placed in a dry beaker for standby. Luteinizing hormone-releasing hormone-A2 is purchased from Ningbo No. 2 Hormone Factory.

[0050] Artificial insemination and hatching and cultivation: Adopt dry insemination, drop the collected semen of the pseudo-male fish into the eggs, gently shake and mix evenly, and then add a small amount of seawater for fertilization; 2.5 minutes after insemination, immerse the fertilized eggs in seawater at 8°C for 10 minutes, take out the treated fertilized eggs and transfer them in seawater at 15°C for 5 minutes, and then put them into seawater at 22°C to prepare for hatching. 25 days after hatching, transfer the fry to a breeding bucket and adapt to cultivate for 5 days.

[0051] Induced cultivation of all-male triploid Nibea albiflora: The all-female Nibea albiflora fry after hatching and cultivation are fed with compound feed for 40 days, twice a day. After the compound feed is fed, they are cultivated with common feed until they become adult fish. Compared with the common feed, the difference in the compound feed is that an aromatase inhibitor letrozole is added to the compound feed, and the amount of the aromatase inhibitor letrozole is 10 mg / kg. The aromatase inhibitor letrozole is dissolved in a small amount of 95% ethanol and then mixed with the common feed to obtain the compound feed. Change the water on time every day, and the amount of water changed each time is not less than 200%.

[0052] Example 2:

[0053] An induction method for efficiently producing triploid male Nibea albiflora. Compared with Example 1, the difference in this example is that methoxypurine ester is used in the seawater during artificial insemination and hatching cultivation.

[0054] Preparation of methoxypurine ester: Add 6-methoxypurine to DMF, add potassium carbonate, add ethyl 2-bromoacetate, react at 30 °C for 12 h, adjust the pH to neutral after the reaction is completed, extract with ethyl acetate, wash with saturated sodium chloride solution, dry with magnesium sulfate, spin dry, and purify by silica gel column chromatography to obtain methoxypurine ester. The usage amount of 6-methoxypurine is 10 wt% of DMF, the usage amount of potassium carbonate is 13 wt% of DMF, and the usage amount of ethyl 2-bromoacetate is 120 wt% of 6-methoxypurine. The column chromatography eluent is a mixture of ethyl acetate and petroleum ether in a mass ratio of 1:10.

[0055] Artificial insemination and hatching cultivation: Adopt dry insemination. Drop the semen of pseudo-male fish collected into the eggs, gently shake and mix evenly, and then add a small amount of seawater for fertilization; 2.5 minutes after insemination, immerse the fertilized eggs in seawater containing methoxypurine ester at 8 °C for 10 minutes, take out the treated fertilized eggs and transfer them in seawater at 15 °C for 5 minutes, and then put them into seawater at 22 °C to prepare for hatching. 25 days after hatching, transfer the fry to a breeding bucket and acclimatize and cultivate for 5 days. In the seawater containing methoxypurine ester, the content of methoxypurine ester is 0.06 wt%.

[0056] Example 3:

[0057] An induction method for efficiently producing triploid male Nibea albiflora. Compared with Example 1, the difference in this example is that methoxypurine ester is used in the compound feed during the induction cultivation of triploid male Nibea albiflora.

[0058] Preparation of methoxypurine ester: Add 6-methoxypurine to DMF, add potassium carbonate, add ethyl 2-bromoacetate, react at 30 °C for 12 h, adjust the pH to neutral after the reaction is completed, extract with ethyl acetate, wash with saturated sodium chloride solution, dry with magnesium sulfate, spin dry, and purify by silica gel column chromatography to obtain methoxypurine ester. The usage amount of 6-methoxypurine is 10 wt% of DMF, the usage amount of potassium carbonate is 13 wt% of DMF, and the usage amount of ethyl 2-bromoacetate is 120 wt% of 6-methoxypurine. The column chromatography eluent is a mixture of ethyl acetate and petroleum ether in a mass ratio of 1:10.

[0059] Preparation of methoxypurine hydroxamic acid: Hydroxylamine hydrochloride was added to anhydrous methanol, and then sodium methoxide was added. The reaction was carried out at 30 °C for 30 min. After the reaction was completed, it was cooled, and the solid was removed by filtration. Methoxypurine ester was added to the filtrate, and the reaction was carried out at 55 °C for 8 h. After the reaction was completed, the pH was adjusted to 6, concentrated and stirred, and purified by silica gel column chromatography to obtain methoxypurine hydroxamic acid. The usage amount of hydroxylamine hydrochloride was 1 wt% of anhydrous methanol, the usage amount of sodium methoxide was 100 wt% of hydroxylamine hydrochloride, and the usage amount of methoxypurine ester was 200 wt% of hydroxylamine hydrochloride. The eluent for column chromatography was a mixture of methanol and ethyl acetate in a mass ratio of 1:2.

[0060] Artificial insemination and hatching cultivation: Dry insemination was adopted. The semen of pseudo-male fish collected was dropped into the eggs, gently shaken and mixed evenly, and then a small amount of seawater was added for fertilization; 2.5 min after insemination, the fertilized eggs were immersed in seawater containing methoxypurine hydroxamic acid at 8 °C for 10 min, and the treated fertilized eggs were taken out and transitioned in seawater at 15 °C for 5 min, and then put into seawater at 22 °C to prepare for hatching. 25 days after hatching, the fry were transferred to a breeding bucket and adapted for cultivation for 5 days. In the seawater containing methoxypurine hydroxamic acid, the content of methoxypurine hydroxamic acid was 0.06 wt%.

[0061] Example 4:

[0062] An induction method for efficiently producing triploid male Nibea albiflora. Compared with Example 2, the difference in this example is that in the compound feed in the induction cultivation of triploid male Nibea albiflora, methoxypurine ester was replaced by hydroxypurine ester.

[0063] The preparation of hydroxypurine ester is different from that of methoxypurine ester in that 6-methoxypurine was replaced by 6-hydroxypurine.

[0064] Example 5:

[0065] An induction method for efficiently producing triploid male Nibea albiflora. Compared with Example 3, the difference in this example is that in the compound feed in the induction cultivation of triploid male Nibea albiflora, methoxypurine hydroxamic acid was replaced by hydroxypurine hydroxamic acid.

[0066] The preparation of hydroxypurine ester is different from that of methoxypurine ester in that 6-methoxypurine was replaced by 6-hydroxypurine.

[0067] The preparation of hydroxypurine hydroxamic acid is different from that of methoxypurine hydroxamic acid in that methoxypurine ester was replaced by hydroxypurine ester.

[0068] Example 6:

[0069] An induction method for efficiently producing all-male Nibea albiflora triploids. Compared with Example 3, the difference in this example is that piperazine sulfate is used in the compound feed during the induction cultivation of all-male triploid Nibea albiflora.

[0070] Artificial insemination and hatching cultivation: Dry insemination is adopted. The semen of pseudo-male fish collected is dropped into the eggs, gently shaken and mixed evenly, and then a small amount of seawater is added for fertilization. 2.5 minutes after insemination, the fertilized eggs are immersed in seawater at 8 °C containing methoxy purine isohydroxamic acid and piperazine sulfate for 10 minutes. The treated fertilized eggs are taken out and transferred in seawater at 15 °C for 5 minutes, and then put into seawater at 22 °C for hatching preparation. 25 days after hatching, the fry are transferred to a breeding bucket and acclimated for 5 days. In the seawater containing methoxy purine isohydroxamic acid and piperazine sulfate, the content of methoxy purine isohydroxamic acid is 0.06 wt%, and the content of piperazine sulfate is 0.04 wt%.

[0071] Example 7:

[0072] An induction method for efficiently producing all-male Nibea albiflora triploids. Compared with Example 5, the difference in this example is that piperazine sulfate is used in the compound feed during the induction cultivation of all-male triploid Nibea albiflora.

[0073] Artificial insemination and hatching cultivation: Dry insemination is adopted. The semen of pseudo-male fish collected is dropped into the eggs, gently shaken and mixed evenly, and then a small amount of seawater is added for fertilization. 2.5 minutes after insemination, the fertilized eggs are immersed in seawater at 8 °C containing hydroxy purine isohydroxamic acid and piperazine sulfate for 10 minutes. The treated fertilized eggs are taken out and transferred in seawater at 15 °C for 5 minutes, and then put into seawater at 22 °C for hatching preparation. 25 days after hatching, the fry are transferred to a breeding bucket and acclimated for 5 days. In the seawater containing hydroxy purine isohydroxamic acid and piperazine sulfate, the content of hydroxy purine isohydroxamic acid is 0.06 wt%, and the content of piperazine sulfate is 0.04 wt%.

[0074] Test example:

[0075] 1. Infrared characterization

[0076] Test sample: Hydroxy purine isohydroxamic acid prepared in Example 5.

[0077] The infrared spectrum of the hydroxy purine isohydroxamic acid prepared by the present invention is as Figure 1 shown. Among them, at 3348 cm -1 is the infrared absorption peak of the hydroxyl group, at 3082 cm -1 is the infrared absorption peak of the nitrogen-hydrogen on the amide, at 2867 cm -1 is the infrared absorption peak of the methylene group, and at 1711 cm -1The place is the infrared absorption peak of the carbonyl group, 1629 cm -1 The place is the infrared absorption peak of the conjugation of carbon-carbon double bonds, indicating that hydroxypurine isohydroxamic acid is obtained.

[0078] 2. Triploid induction success rate

[0079] Use CyFlow Ploidy Analyser and chromosome karyotype analysis to detect the ploidy of juvenile fish, and conduct test analysis on Nibea albiflora obtained by the methods of each example. If the DNA content and chromosome number of Nibea albiflora obtained in the example are 1.5 times that of normal diploids, it proves that triploid induction is successful.

[0080] The triploid induction success rates of Nibea albiflora induced in each example are as Figure 2 shown, where S1 is Example 1, S2 is Example 2, S3 is Example 3, S4 is Example 4, S5 is Example 5, S6 is Example 6, S7 is Example 7. The method of the present invention first conducts nutritional regulation and photothermal regulation on pseudo-male Nibea albiflora and female Nibea albiflora, then takes sperm and eggs, then performs artificial insemination and hatching cultivation, and finally conducts the induction cultivation of all-male triploid Nibea albiflora. In the artificial insemination and hatching cultivation of the present invention, when the fertilized eggs are immersed in seawater, purine derivatives can be added to the seawater to treat the fertilized eggs, improving the triploid induction success rate of Nibea albiflora. Through comparison, it can be found that purine derivatives containing isohydroxamic acid groups are superior to purine derivatives without isohydroxamic acid groups, and when the purine molecular formula in the purine derivatives contains hydroxyl groups, the effect is relatively better than that without hydroxyl groups. When the purine molecular formula in the purine derivatives contains hydroxyl groups and isohydroxamic acid groups, its triploid induction success rate for Nibea albiflora is the highest.

[0081] 3. Detection of all-male triploid Nibea albiflora

[0082] After dissecting out the gonads of Nibea albiflora, fix them with Bouin's fluid and observe by making tissue sections.

[0083] Taking the all-male triploid Nibea albiflora cultivated by the method of Example 5 as a test example, setting a control group. The control group was not subjected to cold shock induction and was always fed with ordinary feed. Samples of Nibea albiflora in the two groups were taken at 75 days old, 90 days old, 120 days old, 150 days old, 180 days old, 210 days old, 270 days old, 300 days old, and 360 days old, and the sex was identified by tissue sections. After microscopic observation and analysis, males were selected. The Nibea albiflora in the test groups of the aromatase inhibitor letrozole and hydroxypurine isohydroxamic acid all showed the same testis structure as that of the normal group males, but the testis development lagged behind that of diploids and mature sperm could not be generated, indicating that the inclusion of letrozole and hydroxypurine isohydroxamic acid can effectively induce the production of all-male triploid Nibea albiflora. The gonad tissue sections of Nibea albiflora at 300 days old are as Figure 3As shown, where A is the test group and B is the control group. Meanwhile, during the above time period, the ploidy of all-male triploids and diploids was detected using a ploidy analyzer and chromosome karyotype analysis. The detection results showed that both the DNA content and chromosome number of all-male triploids were 1.5 times that of diploids. The relative DNA content and chromosome karyotype analysis of Nibea albiflora at 300 days old are as Figure 4 shown, where A is the DNA content and chromosome number, 2n is the DNA of diploids, and 3n is the DNA of triploids.

[0084] The above embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Those of ordinary skill in the art can also make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the patent protection scope of the present invention shall be defined by the claims.

Claims

1. An induction method for efficiently producing triploid all-male Nibea albiflora, comprising: Perform nutritional regulation and photothermal regulation on sexually mature pseudo-Atrobucca nibe and female Nibe croaker, and collect sperm and eggs; Obtain fertilized eggs through artificial insemination, immerse them in seawater for treatment and then incubate and cultivate them, and finally conduct the induction cultivation of all-male triploid Nibe croaker; the fertilized eggs are immersed in seawater at 3-12°C for 5-20 minutes, then transitioned in seawater at 13-16°C for 3-10 minutes, and finally placed in seawater at 20-25°C for incubation and cultivation; a compound feed containing the aromatase inhibitor letrozole is used in the induction cultivation; The seawater for treating the fertilized eggs contains purine derivatives; The purine derivatives are methoxypurine ester or methoxypurine isohydroxamic acid or hydroxypurine ester or hydroxypurine isohydroxamic acid; the methoxypurine ester is prepared by reacting 6-methoxypurine with ethyl 2-bromoacetate; the methoxypurine isohydroxamic acid is prepared by reacting methoxypurine ester with hydroxylamine hydrochloride; the hydroxypurine ester is prepared by reacting 6-hydroxypurine with ethyl 2-bromoacetate; the hydroxypurine isohydroxamic acid is prepared by reacting hydroxypurine ester with hydroxylamine hydrochloride; The content of the purine derivatives in the seawater for treating the fertilized eggs is 0.02-0.2 wt%; 2. An induction method for efficiently producing triploid all-male Nibea albiflora according to claim 1, characterized in that: In the nutritional regulation, nereis and squid are used as baits for cultivation, and 3-5 wt% of vitamin C and 1-2 wt% of vitamin E are added to the baits; 3. An induction method for efficiently producing triploid all-male Nibea albiflora according to claim 1, characterized in that: In the photothermal regulation, the light intensity is 60-650 lux, the light time is 12-16 hours per day, and the water temperature is 18-22°C; 4. An induction method for efficiently producing triploid Nibea albiflora with all male individuals according to claim 1, characterized in that: The amount of the aromatase inhibitor letrozole in the compound feed is 5-20 mg / kg; 5. Use of a purine derivative in the preparation of a reagent for improving the induction success rate of triploid Nibe croaker, the purine derivative including methoxypurine isohydroxamic acid or hydroxypurine ester or hydroxypurine isohydroxamic acid; the methoxypurine ester is prepared by reacting 6-methoxypurine with ethyl 2-bromoacetate; the methoxypurine isohydroxamic acid is prepared by reacting methoxypurine ester with hydroxylamine hydrochloride; the hydroxypurine ester is prepared by reacting 6-hydroxypurine with ethyl 2-bromoacetate; the hydroxypurine isohydroxamic acid is prepared by reacting hydroxypurine ester with hydroxylamine hydrochloride.

Images