Use of targeting rbm24a to control fish fertility

Abstract

The present application belongs to the technical field of genetic propagation, and particularly relates to application of targeted Rbm24a control of fish fertility. Specifically, the present application finds that Rbm24a is a new fish germ plasm component, which plays the function of a germ plasm organizer. Loss of function of the maternal protein will lead to the failure of primordial germ cells to form and exhibit a sterile phenotype, but does not affect the development of other tissues and organs, so that rbm24a is a new target gene for controlling fish fertility. Further, the present application also develops a strategy of inducing degradation of maternal Rbm24a protein by treatment with plant growth hormone, thereby achieving control of fish fertility, and also can produce sterile offspring by inducing degradation of Rbm24a through zGrad. The method is convenient, safe and thorough in controlling fish fertility, and therefore has good practical application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic breeding technology, and particularly relates to application of targeted Rbm24a control of fish fertility. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in any country in the world.

[0003] Fish is a high-quality food source for humans, and its production accounts for 52.2% of the total aquaculture production in China (2022 National Fishery Economic Statistics Bulletin), far more than other aquatic species. Therefore, improving the yield and quality of economic fish has very important strategic and economic value. Fish gonad development sometimes has a negative impact on economic traits, because gonad development is very energy-consuming, competing with vegetative growth, resulting in slower growth rate and deteriorated meat quality, and therefore, breeding infertile fish has broad economic value. In addition, control of fertility is also a key to allowing genetically modified fish to be used for production. This is because genetically modified fish with excellent traits may escape from the breeding environment into the natural world, reproduce in large numbers, and cause ecological invasion and destruction. From the perspective of ecological safety, releasing infertile fry for production is a perfect solution to avoid species invasion. Therefore, it is very important to establish a convenient, safe and thorough method for controlling fish fertility.

[0004] Rbm24a is an RNA binding protein, and the inventors found in previous studies that the protein coding gene is not only expressed in the heart and skeletal muscle, but also has very specific expression in the sensory organ basement, especially in the lens. Further studies by tissue sectioning found that the gene is very specifically expressed in the cytoplasm of differentiating primary and secondary fiber cells. And finally it was proved that Rbm24a is a key post-transcriptional regulator of regulating lens terminal differentiation and transparency, which controls the length of mRNA poly(A) tail in the cytoplasm through cytoplasmic polyadenylation mechanism to regulate the translation efficiency of lens-specific mRNA (PNAS, 2020, 117(13): 7245). However, its role in fish fertility has not been reported. SUMMARY

[0005] In order to solve the above technical problems, the application provides an application of targeting Rbm24a in controlling fish fertility. Specifically, the application finds that Rbm24a is a new component of fish germ plasm, which plays a role of a germ plasm organizer. Loss of maternal protein function of Rbm24a will lead to the failure of primordial germ cells to form and exhibit a sterile phenotype, but does not affect the development of other tissues and organs, so that Rbm24a is a new target gene for controlling fish fertility. Further, the application also develops a strategy of inducing degradation of maternal Rbm24a protein by plant growth hormone treatment, so as to realize the control of fish fertility; at the same time, the zGrad can also be used to induce degradation of Rbm24a to produce sterile offspring. Based on the above research results, the application is completed.

[0006] In order to achieve the above technical purposes, the technical solutions adopted by the application are as follows:

[0007] In the first aspect of the application, an application of targeting Rbm24a in controlling fish fertility is provided.

[0008] Specifically, the application finds in zebrafish experimental research that Rbm24a protein is a new component of fish germ plasm, and knockout of rbm24a leads to loss of germ cell specificity and sterility, so that targeting Rbm24a can effectively control fish fertility.

[0009] Therefore, the application specifically shows that knockout of gene rbm24a leads to loss of germ cell specificity and sterility of fish.

[0010] In the second aspect of the application, a control method of fish fertility is provided, which comprises inducing degradation of maternal Rbm24a protein of fish by plant growth hormone treatment.

[0011] Alternatively, the control method can also be to induce degradation of Rbm24a by zGrad to produce sterile offspring, without the need for plant growth hormone treatment.

[0012] In the third aspect of the application, the above control method is applied in any one or more of the following:

[0013] (a) large-scale production of sterile fish;

[0014] (b) breeding of excellent lines of fish and controlling fertility.

[0015] In the application (a), the sterile fish includes sterile fish embryos.

[0016] In the application (b), the control of fertility specifically shows control of fish sterility.

[0017] The above one or more technical solutions have the following beneficial technical effects:

[0018] The above technical solution first discovers that Rbm24a is a new germ plasm component, and loss of maternal protein function of Rbm24a will lead to failure of formation of primordial germ cells and exhibit a sterile phenotype, but does not affect the development of other tissues and organs, and therefore Rbm24a is a new target gene for controlling fish fertility.

[0019] Further development of a strategy of inducing degradation of maternal Rbm24a protein by treatment with a plant growth regulator can achieve control of fish fertility, and this solution has incomparable advantages: first, targeting Rbm24a, the removal of germ cells is complete, and complete sterility can be achieved; second, it has no effect on the health of fish, and specifically removes germ cells without affecting the development of other tissues and organs. Third, human and animal cells do not respond to plant growth regulators, and there is no need to worry about the safety of treatment with a plant growth regulator; fourth, the treatment procedure is simple, the cost is low, and the time is short, and thousands of embryos can be treated on a large scale; and the above technical solution also provides a method for producing sterile offspring by inducing degradation of Rbm24a with zGrad.

[0020] Establishment of an rbm24a-gfp-degron KI strain in an economic fish can achieve large-scale production of sterile embryos for direct use in production, and the rbm24a-gfp-degron KI strain can also be used as a chassis for breeding operations. The excellent strain selected through breeding operations also has the characteristic of controlling fertility, and the sterile embryos thereof can be used for production without worrying about the impact on ecological safety, and therefore has good practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0021] The drawings accompanying the specification of the present application serve to provide further understanding of the present application, and the illustrative embodiments of the present application and the description thereof serve to explain the present application, and do not constitute an improper limitation on the present application.

[0022] Figure 1 In the embodiments of the present application, it is found that Rbm24a is a germ plasm protein component; a, exogenous DNA insertion site of a gene knock-in fish. b, Rbm24a-GFP maternal protein is observed to be specifically located in germ plasm and germ cells in rbm24a-gfp KI embryos. c, at the 4-cell stage, Rbm24a protein is co-localized with germ plasm RNA ddx4, dazl, and germ plasm protein Piwil1. d, at 24-hpf (hours post fertilization), Rbm24a is co-localized with germ plasm Ddx4 in PGCs.

[0023] Figure 2Fig. 1. Knockout of rbm24a maternal product leads to germ cell specific loss and sterility in the present application; a, the method of generating rbm24a maternal mutant. b and c, the eggs produced by the mating of rbm24a maternal mutant male fish with wild type female fish cannot be divided, while the eggs produced by the mating of wild type male fish with wild type female fish are normal. d and e, compared with the testis of wild type fish, which is located on both sides of the swim bladder, the rbm24a maternal mutant male fish has no obvious testis structure. f and g, the rbm24a maternal mutant male fish only contains gonadal somatic cell structure, which appears as a vacuole, and has no germ cells. h, the detection of germ cell markers at 24hpf shows that the germ cells are completely lost in the rbm24a maternal mutant. i and j, in the rbm24a maternal mutant, the germ plasm protein Ddx4 is completely lost. k and l, in the rbm24a maternal mutant, the germ plasm protein Piwil1 is completely lost. m and n, injection of YFP-nanos3 'UTR reporter gene into wild type fish shows obvious PGCs, but no bright fluorescent PGCs are observed in the mutant. o, detection of germ cell marker genes at 6hpf shows that no signal is detected in the rbm24a mutant. p, single cell RNA-seq data shows that the PGC cluster is completely lost in the 6-hpf rbm24a maternal mutant, while other cell clusters have no obvious changes.

[0024] Figure 3 Fig. 2. Construction of rbm24a-gfp-degron KI (a) and realization of auxin-induced germ cell loss in this strain (b) in the present application.

[0025] Figure 4 Fig. 3. Generation of sterile offspring by zGrad-induced degradation of Rbm24a in the present application; a, co-expression of GFP fusion protein and zGrad in zebrafish will trigger ubiquitination and degradation of GFP fusion protein, b, two methods for generating sterile offspring, c, in 6hpf and 24hpf embryos injected with zGrad mRNA, the expression of nanos3, a marker gene of PGCs, is completely lost. DETAILED DESCRIPTION

[0026] It should be noted that the following detailed description is illustrative only and is intended to provide further description of the present application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0027] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. It will be understood that the scope of the present application is not limited to the particular specific embodiments described below; it will be further understood that the terms used in the description of the embodiments are for the purpose of describing the particular embodiments and are not intended to be limiting of the present application.

[0028] In one embodiment of the present application, there is provided an application of targeting Rbm24a in controlling fish fertility.

[0029] Specifically, it is found in zebrafish experimental study that Rbm24a protein is a new germ plasm component in fish, and knocking out rbm24a leads to loss of germ cell specificity and sterility, so it is known that targeting Rbm24a can effectively control fish fertility.

[0030] Therefore, the application specifically shows that knocking out gene rbm24a leads to loss of germ cell specificity and sterility in fish.

[0031] In another embodiment of the present application, there is provided a method for controlling fish fertility, which comprises treating and inducing degradation of maternal Rbm24a protein in fish with plant growth hormone.

[0032] In another embodiment of the present application, the method comprises:

[0033] In the third intron of rbm24a gene, a modified fourth exon of rbm24a is knocked in, and green fluorescent protein and degron sequence are fused to the endogenous Rbm24a protein; at the same time, an E3 ubiquitin ligase gene TIR1 responsive to plant growth hormone is connected next to the rbm24a gene, and the E3 ubiquitin ligase gene TIR1 is driven by a promoter, so that a large amount of TIR1 protein is expressed in eggs and early embryos; through subculture, a rbm24a-gfp-degron KI homozygote strain is selected and bred; then, plant growth hormone is applied to treat early embryos thereof; wherein the promoter is not limited, as long as it can drive the gene to be expressed in a large amount in oocytes, and in one embodiment of the present application, the promoter is zp3b (zpc) promoter.

[0034] The homozygous gene knock-in fish rbm24a-gfp-degron KI is viable and fertile, but when early embryos thereof are treated with auxin, endogenous Rbm24a (with degron tag) is recognized by TIR1 expressed from maternal sources, connected to ubiquitin and specifically degraded, thereby causing germ plasm assembly disorder and sterility.

[0035] In another embodiment of the present application, the auxin is specifically potassium 1-naphthaleneacetate, and the application concentration is 1-5 μM (preferably 3 μM), and the treatment time is 1-10 hours, preferably 6 hours; in one embodiment of the present application, 3 μM potassium 1-naphthaleneacetate is applied to the zygote for 6 hours, and then the PGCs in the embryo are completely absent.

[0036] Further, the step of auxin treatment can be omitted, thus providing another method for controlling the fertility of fish, which is specifically: on the basis of rbm24a-gfp KI, zGrad is co-expressed. The zGrad is an artificial E3 ubiquitin ligase targeting GFP fusion protein, which comprises a nanobody targeting GFP (vhhGFP4) and an F-box domain from the zebrafish F-box and WD repeat domain containing 11b (fbxw11b) gene. Co-expression of GFP fusion protein and zGrad in zebrafish will trigger ubiquitination and degradation of GFP fusion protein; therefore, introduction of mRNA of zGrad into zygotes produced by rbm24a-gfp-degron KI homozygous lines can also effectively deplete Rbm24a protein, resulting in sterility.

[0037] Of course, if the purpose is large-scale production, Tg(ef1α:zGrad) transgenic fish can be constructed, and the homozygous male fish of this line is mated with rbm24a-gfp-degron KI homozygous female fish, and the offspring produced thereby will all be sterile.

[0038] In another embodiment of the present application, the above-mentioned control method is applied in any one or more of the following:

[0039] (a) large-scale production of sterile fish;

[0040] (b) breeding of excellent lines of fish and controlling fertility.

[0041] In the application (a), the sterile fish includes sterile fish embryos.

[0042] In the application (b), the controlling of fertility specifically means controlling the sterility of fish.

[0043] In the present application, the fish is preferably an economic fish, and of course the fish can also be a fish used for basic research, such as zebrafish and the like, which is not specifically limited herein.

[0044] The present application is further explained by the following examples, which do not limit the present application. It should be understood that these examples are merely for the purpose of illustration of the present application and are not intended to limit the scope of the present application. In the following examples, the materials, reagents, vectors, strains, and the like used, if not specifically stated, are obtained from commercial channels.

[0045] Example

[0046] 1. Discovery of Rbm24a as a necessary germ plasm component for primordial germ cells

[0047] To understand the function of maternal Rbm24a protein, we first investigated the subcellular distribution of this protein during early development. Since there is no specific antibody against zebrafish Rbm24a protein, we tried to construct a knock-in strain with a C-terminal GFP tag using an intron genomic editing strategy, which is connected to the endogenous rbm24a coding region Figure 1 a). By replacing the last exon with the version with a GFP tag, we successfully obtained rbm24a-GFP knock-in lines (referred to as rbm24a-GFP KI) F0 embryos with a GFP pattern highly matched with rbm24a mRNA expression. The Rbm24a-GFP protein expressed by zygotes in these embryos was clearly expressed in the lens, heart, skeletal muscle and hair cells, indicating successful knock-in during early cleavage.

[0048] Next, we examined the localization of maternal Rbm24a-GFP in early embryos produced by F1 rbm24a-GFP KI fish, and found that the GFP signal was localized in the germ plasm and germ cells Figure 1 b). At the 1-cell stage, Rbm24a-GFP was localized as tiny dots in the entire cortical layer. They actively migrated and accumulated along the cleavage furrow during the first few cell divisions Figure 1 b). At the 1-somite stage, maternal Rbm24a-GFP protein specifically appeared in germ cells, which were divided into left and right groups by the notochord Figure 1 b). At 24 hours post-fertilization (hpf), maternal Rbm24a-GFP was still visible in the primordial germ cells (PGCs) that had migrated to the genital ridge Figure 1 b). Maternal Rbm24a-GFP was present as large granules around the germ cell nucleus, while zygotic Rbm24a-GFP was almost evenly distributed in the cells in the adjacent myotome Figure 1b) Rbm24a-GFP co-localizes with ddx4, dazl and Piwil1 in early cleavage stage embryos Figure 1 c), and at 24hpf it perfectly co-localizes with the Ddx4 protein in PGCs Figure 1 d). Since homozygous rbm24a-GFP KI lines are viable and fertile, the GFP tag does not affect the function of the endogenous Rbm24a protein. Therefore, the localization pattern of Rbm24a-GFP should represent the status of the endogenous protein. These results indicate that Rbm24a protein is a novel germ plasm component in fish.

[0049] 2. Knockout of rbm24a leads to a completely sterile phenotype

[0050] Since heterozygous mutants of rbm24a exhibit lethal phenotypes such as heart failure, cataracts and hair cell defects, homozygous mutant females and their maternal mutant embryos cannot be obtained by traditional genetic approaches. Heart defects can be rescued by using the cmlc2 promoter to drive the expression of rbm24-gfp, but the resulting fish are still not viable because the larvae exhibit an uncoordinated phenotype. Therefore, to facilitate the screening of maternal mutants, we first constructed a knock-in line that fused RFP to the C-terminus of the Rbm24a protein and simultaneously inserted a zpc:cas9 vector downstream of the rbm24a gene. We named this knock-in line rbm24a-RFP KI zpc:cas9 Similar to the rbm24a-GFP KI line, the rbm24a-RFP KI zpc:cas9 line in the homozygous state is healthy and fertile. The Rbm24a-RFP signal is present in the correct tissues where the endogenous rbm24a gene is expressed. Maternal Rbm24a-RFP also shows germ plasm localization. Meanwhile, the zpc:cas9 insert supports sufficient maternal expression of the Cas9 protein, as injection of bmp2b sgRNA into embryos produced by rbm24a-RFP KI zpc:cas9 females results in a nearly 100% dorsalization phenotype. The high genome editing efficiency of the rbm24a-RFP KI zpc:cas9 line is maintained in three consecutive generations.

[0051] On the other hand, we constructed a transgenic vector that expresses a maternal BFP marker and four highly efficient sgRNAs targeting the rbm24a coding sequence. This sgRNA expression vector was introduced into homozygous rbm24a-RFP KI zpc:cas9 embryos, and finally obtained rbm24a maternal mutants (Mrbm24a) Figure 2a). Mrbm24a mutants were easily selected because of the absence of their Rbm24a-RFP signal Figure 2 a lower right corner inset). Mrbm24a embryos appeared normal and were able to survive to adulthood. Maternal and zygotic mutants of rbm24a (MZrbm24a) exhibited the same lethal phenotype as their zygotic mutants, indicating that maternal Rbm24a protein could not compensate for the zygotic rbm24a function. However, Mrbm24a adult fish exhibited a male-only phenotype. They had mating behavior but could only produce unfertilized eggs Figure 2 b and c), and were thus sterile. In wild-type males, testes were found on both sides of the swim bladder. In contrast, rbm24a maternal mutant fish deposited adipose tissue where testes should be present Figure 2 d and e). Sectioning and HE staining analysis revealed that no sperm and immature male germ cells could be observed in Mrbm24a fish. In contrast, only empty testis tubular structures remained in Mrbm24a mutant fish, very similar to the phenotype of dnd1 deficient fish Figure 2 f and g). Since the lack of germ cells in Mrbm24a adult fish, we hypothesized that the formation of primordial germ cells could have been disrupted. To investigate this possibility, we checked the expression of several germ plasm mRNAs, including nanos3, ddx4, ca15b, tdrd7 and kop, in 24h post-fertilization maternal mutants of rbm24a. Remarkably, the PGC-specific expression of these germ plasm mRNAs was abolished in 24h post-fertilization Mrbm24a embryos Figure 2 h). Consistently, protein markers of PGCs, such as Piwil1 and Ddx4, could not be detected by immunofluorescence in the mutants Figure 2 i-l). We also injected YFP-nanos3-3'UTR mRNA into wild-type and Mrbm24a embryos, but could not detect any YFP-positive PGCs in the latter Figure 2 m and n). The absence of PGCs in the mutants could even be observed at the initial stages of development at 6h post-fertilization Figure 2 o). The results of single-cell sequencing also indicated that the PGC cell type was specifically lost in Mrbm24a embryos Figure 3 p). These results collectively indicate the complete absence of PGCs in rbm24a mutants, which can serve as a target gene to manipulate fish fertility.

[0052] 3. Construction of a zebrafish line in which auxin induces degradation of Rbm24a

[0053] In order to enable Rbm24a to respond to auxin induced protein degradation (AID), we performed a gene knock-in operation at the same position of the third intron of rbm24a, so that the endogenous protein of Rbm24a carries a GFP marker and a degron sequence Figure 3 a) The rbm24a-gfp-degron KI homozygous line was selected by subculture. The fertilized eggs produced by the rbm24a-gfp-degron KI homozygous line were placed in 3 μM K-NAA (1-naphthaleneacetic acid, potassium salt, auxin analog, dissolved in E3 buffer) for 6 hours, and the untreated and treated embryos at 6hpf and 27hpf were fixed, respectively, and nanos3 was used as a probe to detect whether PGCs were present. The results show that PGCs in embryos treated with auxin are completely absent Figure 4 b).

[0054] Among them, the sgRNA sequence for performing gene knock-in at the third intron of the rbm24a gene is: taatacgactcactataGGGCTGCTGTCATGTTGGGTgttttagagctagaa (SEQ ID NO. 1)

[0055] zpc:tir1 KI rbm24a 3rd intron Gene knock-in plasmid sequence information:

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] Among them, the black bold part is the sgRNA target point region, the blue underlined part is the last exon region of rbm24a; the green part is the EGFP sequence; the red underlined part is the degron sequence; the orange part is the 3' untranslated region of rbm24a; the gold underlined part is the zpc promoter sequence; is the Kozak sequence, is the FLAG sequence, the purple underlined part is the tir1 sequence, and the blue italic part is the SV40 poly(A) signal sequence.

[0062] 4. zGrad induces Rbm24a degradation to produce sterile offspring.

[0063] Another approach to controlling Rbm24a degradation is to co-express zGrad on top of rbm24a-gfp KI. zGrad is a synthetic E3 ubiquitin ligase targeting the GFP fusion protein, containing a GFP-targeting nanobody (vhhGFP4) and an F-box domain derived from the zebrafish F-box and WD repeat domain containing 11b (fbxw11b) gene. Co-expression of the GFP fusion protein and zGrad in zebrafish will induce ubiquitination and degradation of the GFP fusion protein. Figure 4 a, eLife, 2019, 10.7554). Therefore, injecting zGrad mRNA (100 pg / embryo) into fertilized eggs produced by the rbm24a-gfp-degron KI homozygous strain can effectively deplete Rbm24a protein, producing a sterile effect. For large-scale production, Tg(ef1α:zGrad) transgenic fish can be constructed, and homozygous males of this strain can be mated with homozygous females of rbm24a-gfp-degron KI; the offspring produced will all be sterile. Figure 4 b). This method eliminates the need for auxin treatment, but requires maintaining two strains. For example... ​ As shown in Figure c, the expression of the PGC marker gene nanos3 was completely absent in 6hpf and 24hpf embryos injected with zGrad mRNA, demonstrating the effectiveness of this strategy.

[0064] The pCS2-zGrad plasmid sequence information used to express zGrad mRNA is as follows:

[0065]

[0066]

[0067]

[0068] in, The sequence is Sp6 promoter. The green part is the F-box sequence, the orange part is the GFP nanobody sequence, the pink part is the SV40 poly(A)signal sequence, the green underlined part is the AmpR sequence, the blue part following it is the AmpR promoter sequence, and the gold underlined part is the CMV IE94 promoter.

[0069] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit the present application. Although the present application is explained in detail with reference to the examples, the technical solutions of the present application can be modified or equivalently replaced by those skilled in the art according to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for controlling the fertility of fish, characterized by, The control method comprises inducing degradation of maternal Rbm24a protein of fish by plant hormone treatment; specifically, the control method comprises: In rbm24a knocking in a modified rbm24a fourth exon, fusing the endogenous Rbm24a protein with green fluorescent protein and degron sequence; and simultaneously rbm24a flanking the gene with an E3 ubiquitin ligase gene responsive to plant auxin TIR1 , said E3 ubiquitin ligase gene TIR1 driven by a promoter, such that the TIR1 protein is expressed in large amounts in the egg and early embryo; by passaging, a rbm24a-gfp-degron KI homozygote line is selected; then the early embryo is treated with auxin.

2. The control method according to claim 1, characterized by, The sgRNA sequence for performing gene knock-in is shown as SEQ ID NO. 1; the knock-in plasmid is shown as SEQ ID NO. 2; The plant hormone is potassium 1-naphthaleneacetate, the concentration is 1-5 μM, and the treatment time is 1-10 hours.

3. A method for controlling the fertility of fish, characterized by, The method is specifically: introducing the mRNA of zGrad into the zygote produced by the homozygous KI line to produce sterile offspring fish. rbm24a-gfp-degron The method is specifically: introducing the mRNA of zGrad into the zygote produced by the homozygous KI line to produce sterile offspring fish. The rbm24a-gfp-degron A specific method for constructing a KI homozygous line includes: In rbm24a knock-in of a modified rbm24a fourth exon, fusing the endogenous Rbm24a protein to a green fluorescent protein and a degron sequence; and simultaneously rbm24a flanking the gene with an E3 ubiquitin ligase gene responsive to plant auxin TIR1 , said E3 ubiquitin ligase gene TIR1 driven by a promoter, thereby causing a high expression of TIR1 protein in the egg and early embryo; by passaging, a KI homozygous line is selected rbm24a-gfp-degron ​ Alternatively, construct Tg( ef1α : zGrad Genetically modified fish, making the homozygous males of this strain... rbm24a-gfp-degron When KI homozygous female fish mate, they produce sterile offspring.

4. The control method according to claim 3, characterized by, Expression zGrad The plasmid for mRNA is shown in SEQ ID NO.

3.

5. The control method according to any one of claims 1-4 is applied in any one or more of the following: (a) mass production of sterile fish; (b) breeding of excellent lines of fish and controlling fertility.

6. The use according to claim 5, wherein the compound is ###0002### In the application (a), the sterile fish comprises sterile fish embryos; In the application (b), the controlling fertility specifically means controlling sterility of fish.

7. The use according to claim 5, wherein the compound is ###0002### The fish comprises economic fish.

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