Method for efficiently obtaining homozygous red crucian carp

Abstract

The invention belongs to the field of fish genetic breeding, and discloses a method for efficiently obtaining homozygous red crucian carp, which comprises the following steps: (1) carrying out artificial false fertilization on eggs of the red crucian carp to obtain fertilized eggs; (2) performing gene editing on the fertilized eggs; (3) incubating the fertilized eggs subjected to gene editing for 18-32 minutes, and then carrying out cold treatment; and (4) incubating the fertilized eggs after cold treatment to obtain the homozygous red crucian carp. According to the method disclosed by the invention, gene editing and gynogenesis methods are comprehensively utilized, the hatching rate, the survival rate and the homozygous rate are high while the high gene editing efficiency is maintained through the early cold shock treatment time and the proper cold shock treatment time, and the gynogenesis red crucian carp mutant with a high proportion can be obtained.

Description

Technical Field

[0001] The invention belongs to the field of fish genetic breeding, and in particular relates to a method for efficiently obtaining homozygous red crucian carp. Background Art

[0002] The red crucian carp (Carassius auratus red var.) is a variant of the carp, belonging to the Cyprinidae, subfamily Cyprininae, genus Carassius. It boasts vibrant coloration, strong adaptability, robust fecundity, and a prolific egg-laying capacity. It also spawns multiple times annually. As a key resource for fish genetic improvement, it has been utilized in the development of diverse high-quality diploid, triploid, and tetraploid fish strains. Some diploid hybrids have yielded significant economic benefits due to their rapid growth, tender meat, and robust resistance to stress. Therefore, the red crucian carp is a crucial fish species for both genetic resources and genetic improvement.

[0003] Improving fish traits can provide high-quality parents for accelerating the creation of superior fish. Gene editing, as a mature, efficient, and precise genetic improvement technology, has been widely used in fish genetic breeding. In molecular precision breeding of fish based on gene editing, gene editing is performed on fertilized eggs (genetic material comes from both female and male parents), and the offspring must be continuously hybridized and selected to finally obtain gene-edited homozygous offspring. This method usually requires three or even more generations of breeding, which is time-consuming and costly. Therefore, how to efficiently shorten the creation time of gene-edited homozygous offspring and simultaneously obtain a large number of homozygous mutants is of great production significance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a method for efficiently obtaining homozygous red crucian carp.

[0005] In order to solve the above technical problems, the technical solution proposed by the present invention is: A method for efficiently obtaining homozygous red crucian carp, characterized by comprising the following steps: (1) Artificially fertilize the eggs of red crucian carp to obtain fertilized eggs; (2) Gene editing of fertilized eggs; (3) Incubate the gene-edited fertilized eggs for 18-32 minutes and then cool them; (4) The cold-treated fertilized eggs were incubated to obtain homozygous red crucian carp.

[0006] Gynogenesis is an important reproductive mode dominated solely by the female nucleus. Unlike gynogenesis in polyploid fish, the gynogenesis process in diploid fish requires the complete doubling of haploid chromosomes to develop into normal diploid individuals. Compared to chromosome doubling achieved by inhibiting the expulsion of secondary bodies (meiotic gynogenesis, resulting in a heterozygous genotype), individuals formed by inhibiting the first cleavage of fertilized eggs (gynogenesis) are homozygous. Therefore, artificial gynogenesis has been widely used in fish genetic breeding. Furthermore, mature eggs of red crucian carp are highly sticky, and the first cleavage of fertilized eggs occurs a long time after fertilization (typically over an hour). Compared to injection followed by fertilization, microinjection of fertilized eggs is easier, has a controllable injection time, and has a very high success rate. Therefore, the present invention utilizes gene editing and gynogenesis (inhibition of the first cleavage) technologies for red crucian carp, an important economic fish that produces sticky eggs, to accelerate the preparation of homozygous red crucian carp mutants and provide an important source of homozygous mutant gametes for the rapid preparation of other diploid and triploid fish with improved traits.

[0007] This method artificially fertilizes red crucian carp eggs to produce fertilized eggs containing only the genetic material of the egg. These fertilized eggs are then gene-edited and then subjected to cold shock treatment (cold treatment) to double the chromosomes during the first cleavage of the fertilized eggs. Compared to other chromosome doubling treatments (such as heat shock), this method achieves a higher survival rate for the fertilized eggs and makes it easier to obtain homozygous red crucian carp. Furthermore, the earlier timing of cold treatment ensures a higher survival rate of highly edited mutants while also allowing for the efficient production of homozygous mutants.

[0008] Preferably, in step (3), the cold treatment time is 35-45 minutes, preferably 40±3 minutes. The cold treatment time needs to ensure the effect of chromosome doubling while minimizing the impact on the activity of the fertilized egg.

[0009] Preferably, in step (1), the eggs of the red crucian carp are pseudo-fertilized with inactivated amblycephala bream sperm. Preferably, the amblycephala bream semen is diluted with Hank's solution and then inactivated by irradiation with ultraviolet light. Preferably, the semen diluted with Hank's solution is placed in a frozen culture dish, and then the culture dish is placed on a low-temperature shaker for shaking while being inactivated by irradiation with ultraviolet light.

[0010] Preferably, in step (2), gene editing is performed on the fertilized eggs by microinjection. Preferably, in step (2), gene editing is completed within 4 minutes after the fertilized eggs are fertilized. As the egg membrane of the fertilized red crucian carp eggs develops, it gradually hardens. If the diameter of the microinjection needle is small, it is difficult to pierce the egg membrane. If the diameter of the needle is large, the egg membrane wound is large, and the egg mortality rate is extremely high. The injection time should not be too long. Therefore, the injection of the fertilized eggs is started immediately after 2 minutes of fertilization, and it is ensured that 100-150 fertilized eggs are microinjected within 2 minutes.

[0011] Preferably, in step (3), the cold treatment is carried out in cold water at 4-6°C.

[0012] Preferably, in step (3), the gene-edited fertilized eggs are incubated for 20-30 minutes and then cold-treated. Early cold-treatment intervention ensures a high survival rate of high-editing efficiency mutants while also efficiently obtaining homozygous mutants. However, premature intervention may result in the second polar body not yet being expelled.

[0013] Preferably, in step (3), the incubation is carried out in water at 22-24°C.

[0014] Preferably, in step (4), the fish are incubated in water at 22-24° C. until they are 6 months old, and homozygous red crucian carp are screened out.

[0015] In certain embodiments of the present invention, gene editing is to knock out the tyr gene of red crucian carp, that is, to obtain a gynogenetic tyr red crucian carp mutant with complete lack of melanin.

[0016] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes a combination of gene editing and gynogenetic methods. By utilizing an early cold shock treatment timing and an appropriate cold shock treatment duration, the method maintains a high gene editing efficiency while also achieving high hatching rates, survival rates, and homozygosity rates. Furthermore, a high proportion of gynogenetic red crucian carp mutants can be obtained. The diploid homozygous mutants obtained by this method can provide homozygous gametes for breeding trait-improved diploid and triploid fish. Triploid fish are sterile, thus maximizing social, economic, and ecological benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 The control group self-pollinated bull Mutants and gynogenesis in red crucian carp bull Appearance diagram (complete lack of melanin) and chromosome map of the red crucian carp mutant (inhibition of the expulsion of the second-stage body); Figure 2 The control group self-pollinated bull Mutants and gynogenesis in red crucian carp bull Appearance diagram (eyes retain some melanin) and chromosome map of the red crucian carp mutant (inhibited excretion of secondary bodies); Figure 3 Gynogenetic bull Appearance and chromosome map of the red crucian carp mutant (inhibition of the first cleavage); Figure 4 Gynogenetic bull Genotype of the targeted gene in the red crucian carp mutant (inhibition of the first cleavage). DETAILED DESCRIPTION

[0019] To facilitate understanding of the present invention, the present invention will be described in more comprehensive and detailed form below in conjunction with the accompanying drawings and preferred embodiments. However, the protection scope of the present invention is not limited to the following specific embodiments.

[0020] Unless otherwise defined, all technical terms used hereinafter have the same meanings as those generally understood by those skilled in the art. The technical terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0021] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0022] Example 1: The experimental design of this embodiment includes: Control group 1: Cultivation of self-crossed tyr red crucian carp mutants; Control group 2: Fostering gynogenesis by inhibiting the extrusion of the second polar body bull Red crucian carp mutant; Experimental group: Gynogenetic development was achieved by inhibiting the first cleavage bull Red crucian carp mutants, among which the experimental group not only studied the differences in the effects of inhibiting the first cleavage by cold shock or heat shock treatment, but also studied the timing and time of cold shock.

[0023] Specific experimental methods: A method for efficiently obtaining homozygous red crucian carp comprises the following steps: (1) Selection and cultivation of parents: During the breeding season (April-June), separate healthy female red crucian carp with large egg loads and healthy male red crucian carp / bighead bream with abundant semen for one week, paying close attention to the vitality of the selected parents to ensure smooth spawning and sperm production. (2) Self-pollination bull Mutants and gynogenesis in red crucian carp bull Preparation and cultivation of red crucian carp mutants: The female red crucian carp selected in step (1) was used as the female parent and the red crucian carp / grass bream was used as the male parent. Artificial induction of labor was performed by injecting a mixed oxytocin of luteinizing hormone-releasing hormone analogue and human chorionic gonadotropin to obtain mature eggs and semen respectively. The round and clear mature eggs and the semen with strong vitality were selected under an optical microscope for subsequent use.

[0024] The mixture of Hank's diluted amblycephala semen treated with ultraviolet light was treated. The ratio of the mixture was based on semen concentration and sperm motility, with the ratio of Hank's to semen being 1:10. The mixture was spread flat on a frozen culture dish and placed on a flat ice pack wrapped in a towel. After irradiation, the number of amblycephala sperm activated with water was observed under an optical microscope to be 10% of normal sperm, and the swimming time was 70-80% of normal sperm.

[0025] The artificial dry insemination method was used to fertilize the red crucian carp eggs and the red crucian carp semen, and the red crucian carp eggs and the inactivated amblycephala sperm, respectively, and then spread them in 23℃ water to obtain self-fertilized red crucian carp fertilized eggs and gynogenetic red crucian carp fertilized eggs. bull To target genes, bull The gene target and Cas9 protein are mixed to prepare a mixture.

[0026] Control group 1: In self-pollination bull In the construction of red crucian carp mutants, the fertilized eggs were injected 2 minutes after self-fertilization. The mixture was injected into the fertilized eggs by microinjection. The injected eggs were then placed in 23℃ water for incubation to obtain self-fertilized eggs. bull Red crucian carp mutant.

[0027] Control group 2: In female nucleus development bull In the construction of red crucian carp mutant (inhibition of second-stage body discharge), after 2 minutes of fertilization, the female nuclear development fertilized eggs were immediately placed in cold water at 4-6℃ for 30 minutes, and then taken out and continued to be incubated in 23℃ water to obtain female nuclear development. bull Red crucian carp mutant (inhibited second-stage body excretion).

[0028] Experimental group: In gynogenesis bullIn the construction of red crucian carp mutants (inhibition of first cleavage), injection of the fertilized eggs was started immediately 2 minutes after fertilization of a portion of gynogenetic fertilized eggs, and microinjection of 100-150 fertilized eggs was completed within 2 minutes. The injected eggs were then placed in 23°C water for a specific incubation time to complete the expulsion of the second polar body. The specific time was set to 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, and 40 minutes. The injected eggs were immediately placed in 4-6°C cold water after incubation for a specific time to inhibit the first cleavage of the fertilized eggs and induce chromosome doubling to obtain gynogenetic red crucian carp mutants. The specific time was set to 10 minutes, 20 minutes, 30 minutes, 40 minutes, and 50 minutes. Finally, all injected eggs were placed in 23°C water for incubation to obtain cold shock-mediated gynogenetic mutants (inhibition of first cleavage). At the same time, 2 minutes after the fertilization of another part of the female nuclear development fertilized eggs, the fertilized eggs were immediately injected, and it was ensured that 100-150 fertilized eggs were microinjected within 2 minutes. The injected eggs were then placed in 23°C water for a specific incubation time to complete the discharge of the second polar body. The specific time was set to 10 minutes, 20 minutes, 30 minutes, 40 minutes, and 50 minutes respectively. The injected eggs after incubation were immediately placed in a specific temperature for heat shock treatment. The specific temperature was set to 38°C, 39°C, and 40°C in water, and the treatment time was 1.5 minutes and 2 minutes respectively to induce chromosome doubling. Finally, all the injected eggs were placed in 23°C water for incubation to obtain heat shock-mediated female nuclear development. bull Red crucian carp mutant (inhibition of the first cleavage).

[0029] All the above fertilized eggs were placed in the same 23℃ flowing water incubation environment for incubation. During the incubation period, moldy and turbid fertilized eggs were promptly picked out and cleaned. bull Injected individuals and female nucleus development of red crucian carp bull Red crucian carp injection individuals (inhibition of secondary body excretion) and female nuclear development bull The fertilization rate, hatching rate and survival rate of injected individuals of red crucian carp (first cleavage inhibition) are shown in Tables 1 and 2.

[0030] The results in Table 1 and Table 2 show that self-pollination bull The fertilization rate, hatching rate and survival rate of injected red crucian carp were 98%, 90% and 85% respectively. bull All injected red crucian carp (inhibiting the expulsion of the second stage body) failed to develop to the gastrula stage and died. bullThe fertilization rate, hatching rate and survival rate of injected individuals of red crucian carp (inhibiting the discharge of the second stage body) were 70%, 33.2% and 36.5% respectively, while the female nuclear development bull The fertilization rate, hatching rate and survival rate of injected red crucian carp (inhibiting the first cleavage) were significantly reduced. It was also confirmed that the use of incubation time of 20 minutes, 25 minutes, 30 minutes and cold shock treatment time of 40 minutes can obtain better survival female nuclear development. bull The proportion of red crucian carp individuals.

[0031] Table 1. Fertilization rate, hatching rate and survival rate of the control group

[0032] Table 2. Fertilization rate, hatching rate and survival rate of experimental groups

[0033] (3) Genotype, phenotype, editing efficiency, and homozygous efficiency of self-fertilized red crucian carp mutants and gynogenetic red crucian carp mutants: Selfing bull Mutants and gynogenesis in red crucian carp bull The red crucian carp mutants (inhibited excretion of the second-stage body) (incubation time of 5 min and 10 min) both contained two body color variation traits: red body surface (complete lack of melanin) and red body (eyes retain some melanin), e.g. Figure 1 and Figure 2 As shown; while gynogenesis bull The red crucian carp mutant (inhibition of the first cleavage) (experimental group, treatment method 15–50) all showed red body surface (complete lack of melanin), such as Figure 3 shown.

[0034] Wait for the survival self-cross in step (2) bull Mutants and gynogenesis in red crucian carp bull Red crucian carp mutant (inhibition of secondary body expulsion), gynogenesis bull The red crucian carp mutant (inhibited first cleavage) was cultured to 6 months old, and chromosomes of the three mutants were prepared using the kidney chromosome preparation method. If the number of metaphase chromosomes was 100, it was verified as a qualified diploid self-fertilization. bull Mutants and gynogenesis in red crucian carp bull Crucian carp mutant (inhibited expulsion of secondary bodies) ( Figure 1 and Figure 2 ) and diploid gynogenesis bull Crucian carp mutant (inhibition of first cleavage) ( Figure 3 ).

[0035] Subsequently, the tail vein blood method was used, and the red mutant blood DNA was used as a template. The target region specific amplification primers of the target gene were used to amplify the target gene by PCR. The amplified product was recovered and connected to the pMD19-T vector for monoclonal sequencing. 36 monoclones were selected for each targeted gene in each mutant. The editing efficiency standard was that more than 30 monoclones of the 36 monoclones had mutant sequences, and the homozygous efficiency standard was that 36 monoclones of the 36 monoclones had mutant sequences. The gynogenetic development was also tested. bull The red crucian carp mutant (inhibition of first cleavage) (50 tails) was genotyped and the mutant genotypes were counted ( Figure 4 , as shown in SEQ ID NO: 1 to 4). Finally, the self-pollination bull Mutants and gynogenesis in red crucian carp bull Red crucian carp mutant (inhibition of the expulsion of the second stage body, gynogenesis bull The mutation efficiency of the red crucian carp mutant (inhibition of the first cleavage) was statistically analyzed to obtain the editing efficiency and homozygosity rate (Table 3). bull Mutants and gynogenesis in red crucian carp bull The editing efficiency and homozygosity of the red crucian carp mutant (inhibiting the expulsion of the second-order body) were 84.0%, 0 and 82.0%, 0, respectively, while the gynogenetic bull The gradient-specific temperature editing efficiency and homozygosity of the red crucian carp mutant (inhibition of the first cleavage) ranged from 82.0% to 100.0% and from 0 to 20.0%, respectively. When the incubation time was between 15 and 40 min, the homozygosity ranged from 2.0% to 20.0%, and the homozygosity increased significantly, confirming that the editing efficiency and homozygosity of the red crucian carp F0 mutant can be significantly improved by combining gene editing and gynogenesis (inhibition of the first cleavage).

[0036] Table 3. Gene editing efficiency and homozygosity rate in the control and experimental groups

[0037] Note: The two numbers in the first column of data in Table 2 and Table 3 represent the incubation time and cold treatment time, respectively. For example, "40-50" means the incubation time is 40 minutes and the cold treatment time is 50 minutes.

[0038] Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and spirit of the present invention, all of which fall within the scope of protection defined by the present invention.

Claims

1. A method for efficiently obtaining homozygous red crucian carp, characterized in that: The steps include: (1) Artificially fertilize the eggs of red crucian carp to obtain fertilized eggs; (2) Gene editing of fertilized eggs; (3) Incubate the gene-edited fertilized eggs for 18-32 minutes and then cool them; (4) The cold-treated fertilized eggs were incubated to obtain homozygous red crucian carp.

2. The method according to claim 1, characterized in that In step (3), the cold treatment time is 35-45 minutes.

3. The method according to claim 1, characterized in that In step (1), the eggs of the red crucian carp are pseudo-fertilized by inactivated sperm of the amblycephala bream.

4. The method according to claim 3, characterized in that The semen of Megalobrama amblycephala was diluted with Hank's solution and then inactivated by irradiation with ultraviolet light.

5. The method according to claim 1, characterized in that In step (2), the fertilized egg is gene-edited by microinjection.

6. The method according to claim 1, characterized in that In step (3), the mixture is cold treated in cold water at 4-6°C.

7. The method according to claim 1, characterized in that In step (3), the gene-edited fertilized eggs are incubated for 20-30 minutes and then cold treated.

8. The method according to claim 1, characterized in that In step (3), incubate in water at 22-24°C.

9. The method according to claim 1, characterized in that In step (2), gene editing is completed within 4 minutes after the fertilized egg is fertilized.

10. The method according to claim 1, characterized in that In step (4), the fish are incubated in water at 22-24°C until they are 6 months old, and homozygous red crucian carp are screened out.

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