A method for cultivating high androgenic Nile tilapia

Abstract

The invention discloses a method for cultivating high-androgenic Nile tilapia, belonging to the technical field of aquaculture sex control. The invention finds that Kdm6bb_tv1 is the male sex determining gene of Nile tilapia; by overexpressing the Kdm6bb_tv1 gene, the sex reversal rate of female Nile tilapia can be increased. Further combined with high temperature-assisted means, the sex reversal rate of all-female genotype (XX) fry can be further increased.

Description

Technical Field

[0001] The invention relates to the technical field of aquaculture sex control, and in particular to a method for cultivating highly androgenic Nile tilapia. Background Art

[0002] Tilapia has excellent characteristics such as omnivorous diet, fast growth, strong reproduction, firm meat and few intramuscular spines. It is an important farmed fish recommended by the Food and Agriculture Organization of the United Nations (FAO) to countries around the world. In recent years, my country's tilapia farming output has increased year by year, with an annual farming output of about 1.7 million tons, making it the world's largest tilapia producer and exporter. Nile tilapia is an important farmed species of tilapia. GIFT Tilapia (Genetic Improvement of Farmed Tilapia) is an excellent variety of artificially cultivated Nile tilapia. It has the characteristics of fast growth, good body shape, and tender meat, and is deeply loved by farmers. Male Nile tilapia grows faster than female fish. All-male or high-male rate farming can greatly increase production and overcome the problem of over-breeding in mixed male and female farming. Therefore, all-male or high-male rate seedling cultivation is an important demand of the tilapia industry.

[0003] At present, there are three main methods for breeding high-androgenicity tilapia: hormone method, super-androgenic method and hybridization method. However, all three methods have certain defects. For example, the hormone method involves food safety and environmental safety issues; the super-androgenic method is difficult to mass-produce due to the cumbersome production of super-male broodstock; and the hybridization method is currently limited to the application in Nile hybrid tilapia. Therefore, it is still necessary to find an efficient and environmentally friendly method for breeding all / high-androgenicity Nile tilapia.

[0004] Nile tilapia is a dioecious fish with an XX / XY sex determination system. The sex differentiation of Nile tilapia is regulated by both genetics and temperature, and belongs to the Genetic Sex Determination plus Temperature Effect (GSD+TE) type. At present, gene editing technology has been established in a variety of farmed fish. Gene editing technology can not only study the function of genes, but also be used for variety improvement. Studies have shown that multiple gene knockouts in Nile tilapia can lead to sex reversal, such as knockout of Dmrt1 and Gsdf, which leads to sex reversal from male to female; knockout of Foxl2 and Cypl9ala, which leads to sex reversal from female to male. However, these genes are downstream genes for sex determination. Identifying the upstream genes that control their expression and using the identified new genes to target and cultivate transgenic tilapia with a high male rate will have very important application value. Summary of the invention

[0005] In view of the above prior art, the purpose of the present invention is to provide a method for breeding high androgenic Nile tilapia. The present invention has found that the endogenous Kdm6bb_tv1 gene of Nile tilapia is involved in the sex determination of Nile tilapia; by overexpressing the Kdm6bb_tv1 gene, the sex reversal rate of female Nile tilapia can be increased. Further combined with high temperature-assisted means, the sex reversal rate of female Nile tilapia can be further increased.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides the use of the Kdm6bb_tv1 gene in any one of the following (1)-(3):

[0008] (1) Induce sex reversal in female Nile tilapia;

[0009] (2) Improve the male rate of Nile tilapia;

[0010] (3) Cultivating highly androgenic Nile tilapia;

[0011] The nucleotide sequence of the Kdm6bb_tv1 gene is shown in SEQ ID NO.1.

[0012] Preferably, the increasing the male rate of Nile tilapia is achieved by inducing sex reversal in female fish.

[0013] The second aspect of the present invention provides the use of a recombinant expression vector containing the Kdm6bb_tv1 gene in increasing the male rate of Nile tilapia.

[0014] A third aspect of the present invention provides a method for cultivating high androgenic Nile tilapia, comprising the following steps:

[0015] (1) introducing a recombinant expression vector containing the Kdm6bb_tv1 gene into fertilized eggs of Nile tilapia, incubating them, selecting positive individuals and raising them to sexual maturity as the F0 generation;

[0016] (2) mating the F0 generation individuals with wild-type Nile tilapia to obtain the F1 generation;

[0017] (3) After the F1 generation individuals reach sexual maturity, they are mated with each other to obtain the F2 generation, and the positive individuals are screened out from the F2 generation to obtain Nile tilapia with overexpression of the Kdm6bb_tv1 gene.

[0018] Preferably, in step (1), the recombinant expression vector containing the Kdm6bb_tv1 gene is constructed by inserting the Kdm6bb_tv1 gene into a pTol2 (pT2AL200R150G) vector.

[0019] Preferably, in step (1), the incubation temperature is 28° C., and after 30 days of incubation, positive individuals are screened using RT-PCR and Western Blot techniques.

[0020] Furthermore, the method also includes the step of subjecting the Nile tilapia with overexpressed Kdm6bb_tv1 gene to high temperature treatment during the heat-sensitive period.

[0021] Preferably, the temperature of the high temperature treatment is 36° C. and the treatment time is 12 days.

[0022] Beneficial effects of the present invention:

[0023] (1) The present invention finds that Kdm6bb_tv1 is the male sex-determining gene of Nile tilapia; by overexpressing the Kdm6bb_tv1 gene, the male differentiation pathway is opened, thereby greatly improving the male rate of Nile tilapia.

[0024] (2) The combination of Kdm6bb_tv1 gene overexpression and high temperature assisted cultivation has a synergistic promoting effect, which can significantly increase the sex reversal rate of female Nile tilapia and further increase the male rate of Nile tilapia. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 :EF1α:Kdm6bb_tv1 myc Schematic diagram of the overexpression vector construction method.

[0026] Figure 2 :The molecular identification results of F2 generation individuals with Kdm6bb_tv1 overexpression, wherein Figure a shows the RT-PCR identification results; Figure b shows the Western-blot identification results.

[0027] Figure 3 :The histological structure of gonads of 6-month-old Nile tilapia (HE staining). Figure a shows the ovarian structure of XX genotype female fish; Figure b shows the testis structure of XY male fish; Figure c shows the testis structure of pseudo-male fish induced by 36℃ high temperature; Figure d shows the testis structure of F2 pseudo-male fish with overexpression of Kdm6bb_tv1; the scale of the picture is 5μm.

[0028] This indicates that high temperature treatment at 36°C or overexpression of Kdm6bb_tv1 can induce the gonads of XX female fish to develop into testes, and the development is not significantly different from that of the testes of XY male fish. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0030] As mentioned above, the ratio of male to female in the natural population of Nile tilapia is close to 1:1; and male Nile tilapia grows faster than females, so all-male or high-male ratio breeding can greatly increase production and overcome the problem of over-breeding in mixed breeding of males and females. Therefore, all-male or high-male ratio seed breeding is an important demand of the tilapia industry.

[0031] However, the traditional methods have many problems in breeding high / all-male tilapia fry, such as low efficiency, environmental pollution, and species restrictions.

[0032] Based on this, the present invention starts from the perspective of gene editing, finds out the gene that determines the male sex of Nile tilapia, and improves the sex reversal rate of female Nile tilapia through genetic engineering, thereby breeding seedlings with a high male sex rate.

[0033] The present invention finds that the endogenous Kdm6bb_tv1 of Nile tilapia is the male sex determining gene of Nile tilapia. The nucleotide sequence of the Kdm6bb_tv1 gene is shown in SEQ ID NO.1, which is as follows:

[0034]

[0035] An expression vector was constructed and transferred into the genome of Nile tilapia. By increasing the expression of the Kdm6bb_tv1 gene, the male differentiation pathway was opened, greatly improving the male rate of Nile tilapia.

[0036] In one embodiment of the present invention, a method for increasing the male rate of Nile tilapia by overexpressing the Kdm6bb_tv1 gene is provided, comprising the following steps:

[0037] (1) Obtaining the Kdm6bb_tv1 gene sequence: Total RNA was extracted from Nile tilapia and reverse transcribed to synthesize cDNA; specific PCR primers were designed, and the CDS sequence of the Kdm6bb_tv1 gene was amplified by PCR technology.

[0038] (2) Construction of Kdm6bb_tv1 overexpression vector: The Kdm6bb_tv1 gene fragment was inserted into a transgenic vector pT2AL200R150G (referred to as pTol2) to construct EF1α:Kdm6bb_tv1 myc Overexpression vector.

[0039] (3) Microinjection and acquisition of F2 individuals overexpressing Kdm6bb_tv1: EF1α:Kdm6bb_tv1 myc The overexpression vector and in vitro transcribed transposase mRNA (pCS-TP plasmid as template, in vitro transcribed synthesis) were co-injected into 1-cell stage fertilized eggs, and the injected fertilized eggs were incubated at 28°C. After 30 days of incubation, part of the tail fin was cut and genomic DNA was extracted. The positive individuals (F0) were detected and selected using RT-PCR technology. F0 individuals were raised to sexual maturity, and positive female individuals were selected to mate with XX pseudo-male fish to obtain the F1 generation. The positive individuals in the F1 generation had both phenotypic females and phenotypic males, and they were mated to obtain the F2 generation.

[0040] (5) Feeding and male rate statistics of Kdm6bb_tv1 overexpressing F2 individuals: Kdm6bb_tv1 overexpressing F2 individuals were raised in 28°C water until sexual maturity (180 days). HE staining technology was used to distinguish sperm and ovaries, and the male rate was counted.

[0041] Furthermore, Kdm6bb_tv1 overexpressing fish were combined with high temperature to assist in the production of male fish: Kdm6bb_tv1 overexpressing F2 generation individuals were transferred to 36°C water temperature for 12 days 9 days after fertilization (the first day when sperm and egg combined), and then the water temperature was restored to 28°C and continued to be raised until sexual maturity.

[0042] The results of the gonadal histology (HE staining) of 6-month-old Nile tilapia are as follows: Figure 3As shown, Figure a shows the ovarian structure of XX genetic type female fish; Figure b shows the testis structure of XY male fish; Figure c shows the testis structure of pseudo-male fish induced by 36°C high temperature; Figure d shows the testis structure of F2 generation pseudo-male fish with Kdm6bb_tv1 overexpression; the picture scale is 5μm.

[0043] This indicates that high temperature treatment at 36°C or overexpression of Kdm6bb_tv1 can induce the gonads of XX female fish to develop into testes, and the development is not significantly different from that of the testes of XY male fish.

[0044] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0045] The test materials used in the examples of the present invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without detailed conditions are carried out according to conventional test methods or the operating instructions recommended by the supplier.

[0046] The Nile tilapia used in the embodiments of the present invention is Tilapia GIFA.

[0047] Example 1: Obtaining F2 individuals with overexpression of Kdm6bb_tv1

[0048] 1. Total RNA extraction and cDNA synthesis of Nile tilapia:

[0049] Sterile Nile tilapia gonads, about 0.02 g, were placed in a mortar, and an appropriate amount of liquid nitrogen was quickly added for grinding. When it was ground into powder, it was transferred to a 1.5 mL centrifuge tube; 1 mL Trizol reagent was added to the centrifuge tube and placed at room temperature for 15 min; centrifuged at 12,000 rpm for 10 min, and the precipitate was discarded; the supernatant was transferred to another centrifuge tube, 200 μL chloroform was added, oscillated for 30 s, and placed at room temperature for 15 min; centrifuged at 4°C and 12,000 rpm for 15 min, the upper aqueous phase was aspirated and transferred to another centrifuge tube; isopropanol was added at a ratio of 0.5 mL isopropanol / mL Trizol reagent, and mixed, and placed at -20°C for 30 min; centrifuged at 4°C and 12,000 rpm for 15 min, the supernatant was discarded, and the precipitate was dried at room temperature; RNA was dissolved in 50 μL NRA enzyme-free water; 1 μL was taken and used for Nan ND-1000 nucleic acid analyzer was used to measure the OD value of the extracted RNA. The remaining RNA was used for reverse transcription to synthesize the first-strand cDNA. The synthesis steps are as follows: 1 μg RNA, 2 μL gDNA Eraser buffer, gDNA Eraser, and RNA-free water were added to a 0.2 mL centrifuge tube in sequence; the mixture was gently pipetted and mixed, and placed in a PCR instrument at 42°C for 2 min, and immediately placed on ice; the following components were added in sequence: 4 μL 5× PrimeScript buffer, 1 μL RT Primer Mix, 1 μL PrimeScript RT Enzyme Mix I, and RNA-free water were added to 10 μL; the mixture was gently mixed at 37°C for 15 min; the enzyme activity was inactivated at 85°C for 5 s, and the cDNA obtained by reverse transcription was stored in a -20°C refrigerator.

[0050] 2. Primer design and synthesis:

[0051] Gene amplification primers with homology arms were designed using biological software. The primer sequences are as follows:

[0052] Kdm6bb_tv1-F:5'-CAAAGAATTCCTCGACGGATCCACCGGTCGCCACCATGTATCACTCAGTAGAGCTCTAC-3'; (SEQ ID NO. 2)

[0053] Kdm6bb_tv1-R:5'-GTCTGGATCATCATCGATTTACAGATCCTCTTCAGAGATGAGTTTCTGCTCCTTTGAGAAGGGAGTTGGAGTCAG-3'. (SEQ ID NO.3)

[0054] 3. Construction of Kdm6bb_tv1 overexpression vector:

[0055] Nile tilapia cDNA was used as a template for PCR amplification, and the above primers were used for PCR amplification and recovered and purified through agarose gel; the pTol2 plasmid was double-digested with BamH1 and Cla1, and the enzyme digestion system was as follows: plasmid 1μg, 10×Kbuffer 2μL, BamH1 (30U / μL) 1.0μL, Cla1 (100U / μL) 1.0μL, and RNA-free water was added to 20μL. The above mixture was placed in a 0.2mL centrifuge tube and incubated at 30℃ for 2h; the pTol2 plasmid digestion product and PCR product were gel-recovered; the PCR product was connected to the pTol2 vector using Takara's In-fusion kit. The connection system is as follows: 2μL of linearized pTol2 plasmid, 2μL of gel-purified PCR product, 2μL of 5X In-Fusion HD Enzyme Premix, and RNA-free water to 10μL. The above mixture was placed in a 0.2mL centrifuge tube and gently mixed, and incubated at 50℃ for 30min; transformation of the recombinant plasmid: add 10μL of the connection product to 50μL of Escherichia coli competent cells, mix well, and ice bath for 30min; heat shock at 42℃ for 30s, and immediately place on ice for 2min; add 500μL of LB medium, 180rpm Incubate at 37℃ for 1h; centrifuge at 4,000rpm for 1min, discard part of the supernatant, and resuspend the remaining 150μL of bacteria, apply it to LB plate (containing 100mg / L Amp); culture at 37℃ overnight. Pick a single clone and culture it in 1mL LB medium (containing 100mg / L Amp) at 37℃, 180rpm for 8h. After sequencing and alignment, extract the recombinant plasmid (EF1α:Kdm6bb_tv1 myc ) and stored in a -20℃ refrigerator.

[0056] 4. Microinjection and acquisition of Kdm6bb_tv1 overexpressing F2 individuals:

[0057] EF1α:Kdm6bb_tv1 myc The expression vector and transposase mRNA were mixed to a final concentration of 50 ng / μL, and injected into 1-cell fertilized eggs by microinjection. The injected fertilized eggs were incubated at 28°C. After 30 days of incubation, part of the tail fin was cut and genomic DNA was extracted. The positive individuals (F0) were detected and selected using RT-PCR technology. The F0 individuals were raised to sexual maturity, mated with the wild type to obtain the F1 generation, and the F1 generation was mated with the same type to obtain the F2 generation. The positive individuals were detected and selected using RT-PCR and Western-bolt technology to obtain Kdm6bb_tv1 overexpressing fish.

[0058] Example 2: Feeding of Kdm6bb_tv1 overexpressing fish and male rate statistics

[0059] 1. Rearing of Kdm6bb_tv1 overexpressing fish:

[0060] 800 XX Kdm6bb_tv1 overexpressing fry 9 days after fertilization (Kdm6bb_tv1 overexpressing fish were obtained according to the method of Example 1, and XX Kdm6bb_tv1 overexpressing fry were obtained by mating XX Kdm6bb_tv1 overexpressing phenotype female fish and XX Kdm6bb_tv1 overexpressing phenotype male fish) were placed in a glass tank with 360L water, and the water temperature was controlled at 28±1°C. During this period, powdered eel feed was fed, artificial aeration was performed, and the dissolved oxygen in the water was maintained at 4.8mg / L, and the pH was 8.24. The fry were continuously raised to 2 months of age, and the fry were transferred to a cement pond for breeding. The pond specifications were 7.5m*1.5m*2m, and floating granular feed was fed.

[0061] 2. Male rate statistics:

[0062] The above fry were cultured to 6 months of age, and the male and female fish were counted (dissected and observed for sex identification), where there were 520 males and 280 females, with a male rate of 65.0%.

[0063] Example 3: Kdm6bb_tv1 overexpression fish combined with high temperature to assist in producing high male fish

[0064] 1. Kdm6bb_tv1 overexpression fish combined with high temperature assistance:

[0065] 800 XX Kdm6bb_tv1 overexpressing fry 9 days after fertilization (Kdm6bb_tv1 overexpressing fish were obtained according to the method of Example 1, and XX Kdm6bb_tv1 overexpressing fry were obtained by mating XX Kdm6bb_tv1 overexpressing phenotype female fish and XX Kdm6bb_tv1 overexpressing phenotype male fish) were placed in a glass tank with 360L water, and the water temperature was controlled at 36±1°C. During this period, powdered eel feed was fed, artificial aeration was performed, and the dissolved oxygen in the water was maintained at 4.8mg / L, and the pH was 8.24. The fish were raised continuously for 12 days; then the water temperature was controlled to 28°C, the dissolved oxygen and pH of the water remained unchanged, and the fish were raised until 2 months old. The fry were transferred to a cement pond for breeding, the pond size was 7.5m*1.5m*2m, and floating granular feed was fed.

[0066] 2. Male rate statistics:

[0067] The above fry were cultured to 6 months of age, and the male and female fish were counted (dissected and observed for sex identification), where there were 724 males and 76 females, with a male rate of 90.5%.

[0068] Comparative example: High temperature induced masculinization of Nile tilapia

[0069] 1. High temperature treatment and feeding management:

[0070] 800 XX-type Nile tilapia fry, which were 9 days after fertilization, were taken and placed in a glass tank with 360 L of water. The water temperature was controlled at 36±1° C. During this period, powdered eel feed was fed and artificial aeration was performed to maintain the dissolved oxygen of the water at 4.8 mg / L and the pH of the water at 8.24. The fry were raised continuously for 12 days. Then the water temperature was controlled to 28° C., the dissolved oxygen and pH of the water remained unchanged, and the fry were continued to be raised until they were 2 months old. The fry were transferred to a cement pond with a size of 7.5 m*1.5 m*2 m for raising, and floating granular feed was fed.

[0071] 2. Male rate statistics:

[0072] The above fry were cultured to 6 months of age, and the male and female fish were counted (dissected and observed for sex identification), where there were 504 males and 296 females, with a male rate of 63.0%.

[0073] Comparison example:

[0074] 1. Feeding and management:

[0075] 800 XX-type Nile tilapia fry, 9 days after fertilization, were taken and placed in a 360L glass tank with water temperature controlled at 28±1°C. During this period, powdered eel feed was fed and artificial aeration was used to maintain the dissolved oxygen of the water at 4.8mg / L and the pH value at 8.24. The fry were raised continuously until they were 2 months old. The fry were then transferred to a cement pond with a size of 7.5m*1.5m*2m and fed with floating granular feed.

[0076] 2. Male rate statistics:

[0077] The above fry were cultured to 6 months of age, and the male and female fish were counted (female / male fish can be distinguished based on the genital pores). Among them, there were 163 male fish and 637 female fish, with a male rate of 20.4%.

[0078] Based on the above experimental results, the following conclusions can be drawn:

[0079] Overexpression of the Kdm6bb_tv1 gene can significantly increase the sex reversal rate of XX genotype female fish; Kdm6bb_tv1 overexpression fish combined with high temperature auxiliary treatment has a synergistic effect, and the male rate of the cultivated all-XX genotype fry is as high as over 90%.

[0080] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of the Kdm6bb_tv1 gene in any of the following (1)-(3): (1) Induce sex reversal in female Nile tilapia; (2) increasing the male rate of Nile tilapia in the offspring of Kdm6bb_tv1 overexpressing fish; (3) Cultivating highly androgenic Nile tilapia; The nucleotide sequence of the Kdm6bb_tv1 gene is shown in SEQ ID NO.

1.

2. Use of a recombinant expression vector containing the Kdm6bb_tv1 gene in increasing the male rate of Nile tilapia, wherein increasing the male rate of Nile tilapia refers to increasing the male rate of Nile tilapia in the mating offspring of Kdm6bb_tv1 overexpressing fish.

3. A method for breeding highly androgenic Nile tilapia, characterized in that: The following steps are involved: (1) introducing a recombinant expression vector containing the Kdm6bb_tv1 gene into fertilized eggs of Nile tilapia, incubating them, selecting positive individuals and raising them to sexual maturity as the F0 generation; (2) mating the F0 generation individuals with wild-type Nile tilapia to obtain the F1 generation; (3) After the F1 generation individuals reach sexual maturity, they are mated with each other to obtain the F2 generation. Positive individuals are screened from the F2 generation to obtain Nile tilapia with stable overexpression of the Kdm6bb_tv1 gene.

4. The method according to claim 3, characterized in that In step (1), the recombinant expression vector containing the Kdm6bb_tv1 gene is constructed by inserting the Kdm6bb_tv1 gene into the pT2AL200R150G vector.

5. The method according to claim 3, characterized in that: In step (1), the incubation temperature is 28° C. After 30 days of incubation, positive individuals are screened using RT-PCR and Western bolt technology, and the individuals are raised to 6 months of age, and the male rate is statistically analyzed.

Images