Application of tex11 gene in sex-controlled fish breeding

Abstract

The present invention belongs to the field of molecular biology and discloses tex11 The application of genes in fish sex control breeding, the applicant found that the knockout fish tex11 By incorporating a gene into a male brooding line, all fish can be produced as fertile males. This method is easy to implement, simple to operate, hormone-free, and environmentally friendly. Using gene mutation to create an all-male fish strain addresses the difficulties of traditional sex-controlled breeding, including the difficulty of producing seeds with super-male parents and the environmental impacts of sex-changing production with hormones. By eliminating the need for extensive and tedious hormone-induced sex reversal procedures, all-male fish can be produced, demonstrating significant application value.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology, and specifically relates to the application of the tex11 gene in fish sex control breeding. By using the gene provided by the present invention as a target sequence and using conventional methods in the field to deprive it of its original function, fish that all develop into fertile males can be obtained. Background Art

[0002] Before sexual maturity, the nutrients and energy consumed by fish are primarily used for body growth. Many fish species exhibit sex-specific differences in the age of sexual maturity, resulting in significant sexual dimorphism in their growth characteristics. In some fish, such as common carp and tongue sole, females are larger than males due to delayed ovarian maturation. In other fish, such as yellow catfish, snakehead, and snakehead, males have a significant growth advantage. Therefore, developing sex-controlled breeding techniques to cultivate new all-male or all-female farmed fish varieties is of great practical value.

[0003] Current fish sex-control breeding techniques primarily involve artificially inducing sex reversal by adding exogenous hormones to recipient fish, resulting in fish with inconsistent genetic and biological sex. These fish are then hybridized to create all-male or all-female monosex fish populations. Fish, as the lowest vertebrates, exhibit a high degree of plasticity in gonadal differentiation. During the early stages of gonadal differentiation, sex steroid treatment can induce sex reversal, causing the fish's biological sex to be inconsistent with its genetic sex, leading to the development of sex-control breeding techniques. Currently, artificial sex reversal in fish is achieved through two main approaches. One is by directly adding sex hormones to the fish's feed, altering hormone levels and thereby reversing their sex. The other is by adding exogenous drugs to the feed, interfering with the binding of hormones to their corresponding receptors, rendering them ineffective and thus promoting biological sex reversal. For example, treatment with methyltestosterone, estradiol, and letrozole, respectively, has resulted in pseudo-male common carp (XX) and super-male yellow catfish (YY), establishing sex-control breeding techniques for common carp and yellow catfish, and fostering the development of new aquaculture varieties. By hybridizing pseudo-male common carp (XX) with control female common carp (XX), all-female common carp were obtained (Wu Qingjiang, Ye Yuzhen, Chen Rongde, et al. Generation and biological characteristics of the gynogenetic line Red Carp 8305. Oceanology and Limnology, 1991, 22:295–300.); and by hybridizing super-male yellow catfish (YY) with control yellow catfish (XX), all-male yellow catfish were obtained [Liu H, Guan B, Xu J, et al. Genetic manipulation of sex ratio for the large-scale breeding of YY super-male and XY all-male yellow catfish (Pelteobagrus fulvidraco (Richardson)). Mar Biotechnol (NY), 2013, 15:321–328.]. In addition, in a few fish species such as tilapia, interspecific hybridization and sex reversal technology have been combined to establish new sex-controlled breeding technologies, thereby cultivating new all-male aquaculture varieties. Sex reversal technology was used to obtain YY supermale Nile tilapia (sex determination type is XX / XY), and WY females were obtained through interspecific hybridization between Nile tilapia and Aurelia tilapia (sex determination type is ZZ / ZW). The YY supermale maintainer line was obtained by hybridizing WY females with YY supermale Nile tilapia. The YY supermale Nile tilapia were then hybridized with control female Nile tilapia to cultivate a new all-male tilapia variety "Yuemin No. 1" [Liu Zhigang, Lu Maixin, Cao Jianmeng, Gao Fengying. Analysis of genetic diversity and genetic relationships of tilapia "Yuemin No. 1" and its breeding population. Progress in Fisheries Science, 2018, 39(06): 31-41.].

[0004] While these sex-controlled breeding techniques have yielded new fish species, they require the use of hormones to induce sex reversal in recipient fish. This leads to issues such as low efficiency and potential environmental impacts. Furthermore, after the exogenous hormones are removed, some sex-reversed fish may revert to their natural sex, making it difficult to obtain stable sex-controlled breeding parents. Therefore, there is an urgent need to develop new, precise, efficient, environmentally friendly, and safe sex-controlled breeding technologies.

[0005] As an important model organism, zebrafish are widely used in studies of vertebrate growth, development, reproduction, sex, physiology, toxicology, and disease. Existing studies suggest that zebrafish, after long-term domestication, have lost their sex-determining genes (Wilson CA, High SK, McCluskey BM, et al. Wild sex in zebrafish: loss of the natural sex determinant in domesticated strains. Genetics, 2014, 198:1291-1308.). The sex of laboratory-reared zebrafish is regulated by multiple genetic factors and influenced by environmental factors (Hosseini S, Ha NT, Simianer H, et al. Genetic mechanism underlying sexual plasticity and its association with color patterning in zebrafish (Danio rerio). BMC Genomics, 2019, 20:341). Male-associated factors such as dmrt1, amh, and gsdf, as well as female-associated factors such as cyp19a1a and foxl2, play a role in zebrafish sex differentiation. In addition, the sex differentiation of zebrafish is also related to the number of early primordial germ cells (PGCs). Knocking down or knocking out the dead end (dnd) gene, which plays an important role in the migration and survival of PGCs, will cause all zebrafish without PGCs to develop into infertile males [Slanchev K, Stebler J, de la Cueva-Mendez G, et al. Development without germ cells: the role of the germ line in zebrafish sex differentiation. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(11): 4074-4079. Tzung KW, GotoR, Jolly M, et al. Early Depletion of primordial germ cells in zebrafish promotes testis formation. Stem Cell Reports, 2015, 4, 61–73.].

[0006] Testis-expressed gene 11 (Tex11) was originally identified and processed in mouse spermatogonial-specific transcripts and is an X-linked germ cell-specifically expressed gene [Wang PJ, McCarrey JR, Yang F, et al. Anabundance of X-linked genes expressed in spermatogonia. Nature Genetics, 2001, 27(4): 422-426.]. The zebrafish tex11 gene is 19,158 bp long, and its mRNA is 3,128 bp long, encoding 913 amino acids. Domain prediction revealed that the Tex11 protein may have two tetratricopeptide repeat motifs (TPRs) that mediate protein-protein interactions. Currently, there are no reports on the function of the tex11 gene in fish. Summary of the Invention

[0007] The present invention aims to provide an application of the tex11 gene in fish sex control breeding. The NCBI number of the tex11 gene is: Gene Bank Accession Number: NC_007125. This gene is used as the target gene, and gene mutation or deletion is performed to make it lose its original function, so that male fertile fish can be quickly obtained.

[0008] In order to achieve the above object, the present invention has taken the following measures:

[0009] The application of the tex11 gene in fish sex control breeding can be achieved by deleting or mutating the fish tex11 gene as the target gene using conventional methods in the art, thereby losing its original function and obtaining a fish strain that develops entirely as males.

[0010] In the above application, preferably, the fish is zebrafish, yellow catfish or mandarin fish;

[0011] In the above applications, preferably, TALENs are used for gene editing;

[0012] In the above application, preferably, when the fish is zebrafish, the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 are used as target sites for gene editing;

[0013] In the above application, preferably, the obtained zebrafish contains a gene sequence encoding the protein shown in SEQ ID NO.6 or SEQ ID NO.7;

[0014] In the application described above, the tex11 mutant parents were obtained, and the F1 generation embryos were produced by hybridizing the F0 mutants with wild-type zebrafish. The selected juveniles were further cultured, and the F1 generation mutants were self-pollinated to screen out the tex11 homozygous mutation. All the tex11 homozygous mutants developed into stable genetic male zebrafish.

[0015] Compared with the prior art, the present invention has the following advantages:

[0016] The method of the present invention can be used to accurately develop all-male fish strains. The method is easy to implement, has the advantages of simple operation, no use of hormones, and is environmentally friendly. The use of gene mutation to obtain all-male fish strains solves the problems of difficulty in producing super-male parents in traditional sex-controlled breeding and the environmental impact of hormone treatment for sex-changing production. It does not require a large number of tedious hormone-induced sex reversal operations to obtain fish that have all developed into males, and has important application value. This method not only provides target genes with important breeding value and new technologies for accurate and efficient sex-controlled breeding for the cultivation of new sex-controlled fish varieties, but the established fertile all-male fish model can also be used for basic research on the mechanism of interaction between fish germ cells and somatic cells, and the regulatory mechanism of sex determination and differentiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the mutation design and sequence analysis of the F0 target site for the zebrafish tex11 gene;

[0018] The target site was determined to obtain sequence mutations.

[0019] Figure 2 Appearance and gonadal developmental characteristics of wild-type female zebrafish, wild-type male zebrafish, and tex11 homozygous mutant zebrafish. A is a wild-type female zebrafish (WT♀); B is a wild-type male zebrafish (WT♂); C is a stably inherited adult male zebrafish mutant obtained by target mutation of the tex11 gene using TALENs. SG: spermatogonia; SC: spermatocyte; ST: spermatid; I: stage I oocyte; II: stage II oocyte.

[0020] Figure 3 Gonadal indices of wild-type zebrafish males and tex11 homozygous mutant zebrafish males and their fertilization rates when crossed with wild-type females. Specific implementation methods

[0021] Unless otherwise specified, the technical solutions described in the present invention are all conventional methods in the art; the reagents or materials used, unless otherwise specified, are all from commercial channels.

[0022] The present invention uses zebrafish TEX11 gene editing as an example to conduct fish sex control experiments. Based on the zebrafish TEX11 gene sequence, the applicant simultaneously amplified the TEX11 genes of yellow catfish and mandarin fish, and simultaneously knocked out the genes, also obtaining fertile male offspring.

[0023] Example 1:

[0024] Application of tex11 gene in fish sex control breeding:

[0025] The present invention obtains an all-male fertile zebrafish strain by knocking out the zebrafish tex11 gene. The ORF frame of the zebrafish tex11 gene is shown in SEQ ID NO.1, and the encoded protein is shown in SEQ ID NO.2.

[0026] 1.1 Experimental Materials

[0027] The zebrafish used in the present invention are of the AB strain, cultured in a constant temperature circulating water at 28°C at the Institute of Hydrobiology, Chinese Academy of Sciences, with a 14-hour light / 10-hour dark regime per day. The embryos used for microinjection were obtained from naturally spawned zebrafish.

[0028] 1.2 Experimental methods

[0029] The TALENs target sites of the zebrafish tex11 gene were determined to be 5'-GTCAGAGAAACTCCTCCA-3' on the left, 5'-ACTTGGATGAAGTGATTG-3' on the right, and 5'-CAGACAGAGCCATTCAG-3' in the middle.

[0030] 1.2.1 Construction of tex11 gene knockout plasmid

[0031] The tex11 gene knockout plasmids tex11-TALEN-Left and tex11-TALEN-Right were constructed using the Golden Gate method. The construction process was based on the published literature [Liu Y, Luo D, Lei Y, Hu W, Zhao H, Cheng CH. (2014) A highly effective TALEN-mediated approach for targeted gene disruption in Xenopus tropicalis and zebrafish. Methods. 69(1): 58-66].

[0032] The specific steps are as follows:

[0033] (1) The single modular plasmid that recognizes the first 10 bases of tex11-TALEN-Left and tex11-TALEN-Right and pFUS_A were linearized with BsaI endonuclease (NEB, R0535S), and then ligated with T4 ligase (NEB, M0202T) to obtain tex11-Left-pFUS_A and tex11-right-pFUS_A. The single modular plasmid that recognizes the 7 bases from positions 11 to 17 of tex11-TALEN-Left and tex11-TALEN-Right and pFUS_B were linearized with BsaI endonuclease, and then ligated with T4 ligase to obtain tex11-Left-pFUS_B and tex11-Right-pFUS_B.

[0034] (2) The correctly sequenced tex11-Left-pFUS_A, tex11-Left-pFUS_B, the single modular plasmid that recognizes the 18th base, and pCS2-TALEN-ELD were linearized with Esp3 I endonuclease (Fermentas, ER0451) and ligated with T4 ligase to obtain tex11-TALEN-Left;

[0035] The correctly sequenced tex11-Right-pFUS_A, tex11-Right-pFUS_B, the single modular plasmid that recognizes the 18th base, and pCS2-TALEN-KKR were linearized with Esp3 I endonuclease and ligated with T4 ligase to obtain tex11-TALEN-Right.

[0036] 1.2.2 In vitro transcription of tex11-TALEN-Left and tex11-TALEN-Right mRNA

[0037] tex11-TALEN-Left and tex11-TALEN-Right were linearized with Not I endonuclease (NEB, R0189S) and transcribed using in vitro mMESSAGE mMACHINE TM In vitro transcription was performed using the SP6 KIT (Life Technologies, AM1340) kit. mRNA was recovered using the RNeasy mini Kit (Qiagen, 74104), dissolved in enzyme-free water, and the concentration was determined using a Nanodrop 2000 (Thermo Scientific, USA). The mRNA was then aliquoted and stored at -80°C.

[0038] 1.2.3 Microinjection

[0039] The day before injection, male and female zebrafish were separated by a partition in a spawning tank. Before injection, the partition was removed, and fertilization was allowed to occur naturally. Fertilized eggs were collected every 15 minutes. Before injection, tex11-TALEN-Left and tex11-TALEN-Right mRNAs were mixed at a 1:1 ratio to a final concentration of 300 ng / μL, and a small amount of phenol red was added as an indicator. Experimental samples were injected into the animal pole of zygotes before they reached the two-cell stage using a nitrogen-pressurized quantitative microinjection system (Warner, USA). Injected zygotes were then incubated at 28°C.

[0040] 1.2.4 Screening for homozygous tex11 mutants

[0041] After injection, 24 hpf embryos were collected for genotyping. DNA was extracted and the mutation status of the target site was detected by PCR.

[0042] The detection primers were F1: 5′-GAAGTAAAAGGTACGTTTGCGGTAA-3′ and R1: 5′-GACTAATACAAGGACAGACCTTTGG-3′.

[0043] The PCR system consisted of 10 μL of 2× Taq MasterMix (CWBIO, Jiangsu), 1 μL of upstream and downstream primers, 1 μL of genomic DNA template, and 7 μL of sterile water. Reaction conditions included pre-denaturation at 94°C for 3 minutes, 30 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 10 seconds, followed by an additional extension at 72°C for 5 minutes. PCR products were directly sequenced, and embryos showing overlapping peaks near the target site were raised to sexual maturity and designated the P0 generation.

[0044] The F1 generation was obtained by testcrossing individual zebrafish from the P0 generation group with WT. After the F1 generation reached sexual maturity, the tail fin was clipped and the test was performed as above to identify positive mutants. In this example, two positive mutants were identified: a (-18, +1) bp heterozygous mutant (the protein encoded by the mutated tex11 gene is shown in SEQ ID NO. 6) and a -10 bp heterozygous mutant (the protein encoded by the mutated tex11 gene is shown in SEQ ID NO. 7). The specific mutations are shown in FIG. Figure 1 shown.

[0045] The (-18, +1) bp heterozygous mutant and the -10 bp heterozygous mutant were self-pollinated to obtain the F2 generation. After the F2 generation reached sexual maturity, the tail fin was clipped and tested as above to identify the (-18, +1) bp homozygous mutant and the -10 bp homozygous mutant.

[0046] The obtained (-18, +1) bp tex11 homozygous mutant and -10 bp homozygous mutant all developed into fertile males. Figure 2 Shows the appearance and gonadal development characteristics of wild zebrafish females, wild zebrafish males, and tex11 homozygous mutant zebrafish. Figure 3 Shown are the gonadal indices of wild-type zebrafish males and tex11 homozygous mutant zebrafish males, as well as the fertilization rates of their hybrids with wild-type females.

[0047] Example 2:

[0048] The tex11 transgene can rescue the all-male phenotype of the tex11 homozygous mutant

[0049] Conventional methods were used to construct an overexpression zebrafish strain in which the β-actin promoter drives the expression of tex11. During the construction, the zebrafish β-actin promoter sequence, tex11 CDs and the self-cleavage sequence P2A were connected to the PSKD-RFP backbone plasmid by the in-fusion method to obtain the tex11 transgenic plasmid Tol2-β-actin Promoter-tex11 CDS-P2A-RFP-Tol2. The tex11 transgenic plasmid was injected into the homozygous mutant zebrafish 1-cell stage fertilized eggs prepared in Example 1 by microinjection to develop transgenic fish with overexpression of tex11. According to conventional methods for establishing transgenic fish families, a transgenic zebrafish family with overexpression of tex11 was bred. The overexpression strain was hybridized with the tex11 knockout strain and then self-pollinated to obtain Tg (β-actin:tex11); tex11 + / + 、Tg(β-actin:tex11);tex11 + / - and Tg(β-actin:tex11);tex11 - / - Three genotypes of strains.

[0050] By counting the number of males and females in each genotype, we knocked out the ectopic overexpression line Tg(β-actin: tex11); - / - Female zebrafish (53 / 145) were found in the 145 samples, indicating that ectopic overexpression can rescue the all-male phenotype caused by endogenous knockout, and also showing that the all-male phenotype of tex11 homozygous mutants is indeed caused by tex11 mutation.

Claims

1. tex11 The application of genes in fish sex control breeding is to use the conventional method in the field to tex11 The target gene is deleted or mutated to lose its original function, thereby obtaining a fish strain that develops entirely into males; the fish is zebrafish, and the tex11 The NCBI number of the gene is: GeneBank Accession Number: NC_007125.

2. The use according to claim 1, wherein TALENs are used to perform gene deletion or mutation.

3. The use according to claim 1, wherein gene editing is performed using the sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 as target sites.

4. The use according to claim 3, wherein the gene-edited zebrafish obtained comprises a gene sequence encoding the protein shown in SEQ ID NO. 6 or SEQ ID NO.

7.

5. The use according to claim 1, wherein the obtained tex11 The mutant parents were hybridized with wild-type zebrafish to produce F1 generation embryos. The selected juveniles were cultured and the F1 generation mutants were self-fertilized to screen out the tex11 Homozygous mutation, tex11 All homozygous mutants developed into stable male zebrafish.

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