Ietalurus punetaus dnmt1 gene knockout RNAi target sequence, sex regulation method and application

Abstract

The invention provides an ictalurus punctatus dnmt1 gene knockout RNAi target sequence, a sex regulation and control method and application, and belongs to the technical field of sex regulation and control. An RNAi sequence is designed based on the dnmt1 gene, and gonad differentiation intervention independent of exogenous hormones is realized in the species by inhibiting expression of the dnmt1 gene in the channel catfish. Experimental results show that dnmt1 expression down-regulation can effectively induce partial sex reversal of genetic female individuals, typical testis-like structures appear in ovarian tissues of the female individuals, it is proved that dnmt1 plays a key role in maintaining female gonad development stability, and it is hopeful that dnmt1 regulation is combined with a genetic screening or gene editing technology, so that the development stability of the female gonad is improved. A stable and efficient all-male channel catfish breeding system is established, and a new path is developed for all-male offspring seed production.

Description

Technical Field

[0001] This invention relates to the field of sex regulation technology, and more particularly to a spotted catfish. dnmt1 Gene knockout RNAi target sequences, sex regulation methods and applications. Background Technology

[0002] Spotted catfish ( Channel catfish As an important freshwater economic fish species globally, the male fish (Pterodon spp.) possesses advantages such as rapid growth, strong disease resistance, and excellent meat quality, and is widely used in aquaculture. In actual production, male individuals typically exhibit significantly better growth rates and feed conversion efficiency than females, and achieve superior meat yield and size consistency within the same rearing cycle. Therefore, achieving all-male aquaculture can not only significantly increase yield per unit area and economic benefits, but also optimize feed resource utilization, shorten the rearing cycle, and reduce environmental impact.

[0003] However, the sex determination mechanism of the channel catfish is extremely complex: its genome lacks typical heteromorphic chromosomes (such as the XX / XY or ZW / ZZ system), and sex differentiation is highly dependent on the interaction between genetic background and environmental factors (such as temperature, pH, and population density), belonging to a typical "multi-factor regulated" sex determination model. This uncertainty results in a near 1:1 female-to-male ratio in natural populations, severely restricting the large-scale production of all-male fry. Although traditional exogenous hormone (such as methyltestosterone)-induced sex reversal technology can achieve sex control in some fish species, its effectiveness in the channel catfish is unstable, and it poses multiple problems such as drug residues, ecological risks, food safety hazards, and consumer resistance, and its use has been strictly restricted or banned in many countries.

[0004] Against this backdrop, developing novel strategies for precise sex regulation based on endogenous molecular mechanisms and independent of exogenous hormones has become a core research focus in the breeding of channel catfish. In recent years, epigenetic regulation, particularly DNA methylation, has received increasing attention for its role in vertebrate sex determination and gonadal differentiation. DNA methyltransferase 1 (DNMT1), as a core enzyme maintaining DNA methylation patterns, is responsible for accurately transmitting methylation information from the parent DNA strand to the daughter strand during cell division, thereby ensuring genomic methylation homeostasis. However, current understanding of… dnmt1 The specific function of genes in sex regulation in channel catfish remains a mystery. Although traditional hormone-induced sex reversal methods have been successful in some fish species, they pose risks of residue buildup, ecological safety hazards, and low public acceptance. There is an urgent need to develop safer and more precise molecular regulatory methods. Summary of the Invention

[0005] The purpose of this invention is to provide a spotted catfish. dnmt1 Gene knockout RNAi target sequences, sex regulation methods and applications revealdnmt1 The core regulatory role of mediated DNA methylation in sex determination in channel catfish provides a safe, controllable, and transformable new strategy for sex manipulation, which has significant scientific value and industrial application prospects.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a spotted catfish dnmt1 The gene knockout RNAi target sequence is shown in SEQ ID NO.4.

[0007] This invention also provides a method for sex regulation in spotted catfish, comprising the following steps: (1) Design of RNAi plasmids for lentiviral packaging For spotted catfish dnmt1 Gene design of RNAi target sequences, sense strands, and antisense strands; and preparation of LV3-shRNA vector based on RNAi target sequences, sense strands, antisense strands, and LV3-pGLV-h1-GFP-puro vector; transformation of LV3-shRNA vector into DH5α competent cells to obtain... dnmt1 Gene RNAi plasmid; dnmt1 The gene RNAi plasmid was packaged with lentivirus to obtain lentivirus-packaged RNAi plasmid; (2) Sex regulation in spotted catfish The RNAi plasmid packaged with lentivirus in step (1) was injected into the yolk near the animal pole of the spotted catfish embryo, and the hermaphroditic spotted catfish were obtained by hatching and identification.

[0008] Preferably, in step (1), the RNAi target sequence is as shown in SEQ ID NO.4; the loop structure in the sense strand uses the “TTCAAGAGA” sequence to avoid forming a termination signal, and the “GATCC” sequence is added to the 5' end of the sense strand; the “AATTC” sequence is added to the 5' end of the antisense strand.

[0009] Preferably, the sense chain is as shown in SEQ ID NO.5; the antisense chain is as shown in SEQ ID NO.6.

[0010] Preferably, in step (2), the titer of the RNAi plasmid packaged with the lentivirus is 10. 7~8 The injection volume is 50 nL.

[0011] The present invention also provides the above-mentioned spotted catfish. dnmt1 Application of gene knockout RNAi target sequences in constructing a hermaphroditic spotted catfish model.

[0012] The present invention also provides the above-mentioneddnmt1 A hermaphroditic catfish model constructed using gene knockdown sex regulation methods.

[0013] Preferably, the hermaphroditic spotted catfish model is a hermaphroditic spotted catfish with female genes that has been reversed to have male traits.

[0014] This invention also provides an application of the above-mentioned hermaphroditic spotted catfish model in the study of sex determination mechanisms.

[0015] The beneficial effects of this invention compared to the prior art are as follows: This invention is based on dnmt1 The gene was designed with RNAi sequences to suppress the activity of spotted catfish. dnmt1 Gene expression in this species achieved gonadal intervention independent of exogenous hormones. Experimental results showed that... dnmt1 Downregulation of expression effectively induces partial sex reversal in genetically modified female individuals, resulting in the appearance of typical testis-like structures in their ovarian tissue, confirming... dnmt1 Its crucial role in maintaining the stability of female gonadal development is expected to be realized through... dnmt1 By combining regulation with genetic screening or gene editing technologies, a stable and efficient all-male channel catfish breeding system can be established, opening up a new path for the production of all-male seedlings. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 As described in the embodiments of the present invention dnmt1 Results of a gene knockdown sex regulation method in spotted catfish, where A represents... dnmt1 Gene knockout RNAi target sequence design; B is dnmt1 Flowchart of gene knockdown sex regulation method in channel catfish; C represents 4 groups of channel catfish. dnmt1 Gene expression levels; D represents the proportions of XY, XX, and XX ovarian abnormalities in the four groups of spotted catfish; Figure 2 The images show the sex identification and gene testing results of sex-reversed channel catfish individuals in this embodiment of the invention. A represents the anatomical, H&E section, and sex identification of sex-reversed channel catfish individuals; B represents the gene testing results. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1 Embodiment 1 of the present invention is based on Figure 1 The process shown provides a dnmt1 The specific steps for constructing a gene knockdown hermaphroditic spotted catfish model are as follows: (1) Sequence design 1) Targeting the spotted catfish dnmt1 Multiple RNAi target sequences were designed, and three optimal target sequences and one control target were selected for subsequent experimental procedures. The sequences are as follows: dnmt1-Icpu-NC (SEQ ID NO.1): 5'-GTTCTCCGAACGTGTCACGT-3'; dnmt1-Icpu-1026 (SEQ ID NO.2): 5'-TAAAAAGCTGGGCCCAATCA-3'; dnmt1-Icpu-2953 (SEQ ID NO.3): 5'-TTCGAAGACCCACCAAACCA-3'; dnmt1-Icpu-3789 (SEQ ID NO. 4): 5'-GTCTGACCTCCCAGAGATCC-3'.

[0024] 2) Design of the justice chain and anti-justice chain When designing the sense and antisense strands, the loop structure in the shRNA DNA template uses the "TTCAAGAGA" sequence to avoid forming a termination signal.

[0025] The 5' end of the sense strand template is augmented with the "GATCC" sequence, which is complementary to the sticky end formed by the LV3-pGLV-h1-GFP-puro vector after BamHI digestion; the 5' end of the antisense strand template is augmented with the "AATTC" sequence, which is complementary to the sticky end formed after EcoRI digestion. The specific base sequence of the template is shown below: Chain of Justice - (SEQ ID NO.5): 5'-GATCC-(GN20)-(TTCAAGAGA)-(N20C)-TTTTTTG-3'; Antisense strand - (SEQ ID NO.6): nnnnnnnnnnnnnnnnnnnn 3'-G(CN20)-(AAGTTCCT)-(N20G)-AAAAAAACTTAA-5'.

[0026] Wherein, N20C is the reverse complementary sequence of GN20 in 1); CN20 is the complementary sequence of GN20; and N20G is the complementary sequence of N20C.

[0027] Taking dnmt1-Icpu-1026 as an example, its justice chain is (SEQ ID NO.7): 5'-GATCCGTAAAAAGCTGGGCCCAATCATTCAAGAGATGATTGGG CCCAGCTTTTTACTTTTTTG-3'; The antisense chain is (SEQ ID NO.8): 3'-GCATTTTTCGACCCGGGTTAGTAAGTTCTCTACTAACCCGGGT CGAAAAATGAAAAAACTTAA-5'.

[0028] Referring to SEQ ID NO.7 and SEQ ID NO.8, four sets of sense and antisense strands were artificially synthesized based on sequences SEQ ID NO.1 to SEQ ID NO.4, respectively.

[0029] (2) dnmt1 Gene RNAi plasmid construction The four sets of sense and antisense strand sequences artificially synthesized in step (1) were dissolved and annealed using conventional methods to prepare shRNA DNA, which were then ligated into linearized LV3-pGLV-h1-GFP-puro vectors to construct four sets of LV3-shRNA vectors.

[0030] Four groups of LV3-shRNA vectors were transformed and cultured in DH5α competent cells (Thermofisher, Waltham, USA). After screening for positive clones by sequencing, four types of DNA were extracted using the SanPrep column-based plasmid DNA mini-extraction kit (Sangon Biotech (Shanghai) Co., Ltd.). dnmt1 Gene RNAi plasmids LV-dnmt1-shRNA, dnmt1-Icpu-1026, dnmt1-Icpu-2953, dnmt1-Icpu-3789.

[0031] (3) Using a fourth-generation lentiviral packaging system (GenePharma, Shanghai), RNAi plasmids packaged with lentiviral agents were prepared, and high-titer viral concentrate (10 μL) was prepared. 9 (units / mL), carrying green fluorescent protein (GFP) as a reporter gene in the vector.

[0032] (4) Sex reversal in spotted catfish Spread a thin layer of vegetable shortening on a 10 cm sterile culture dish, transfer 100 spotted catfish embryos that are 3 days post-fertilization (3 dpf) into the culture dish, adjust the embryos to maintain a monolayer arrangement, and immerse the embryos in a pre-prepared Holtfreter's solution (100%).

[0033] The culture dish was placed under a microscope, and the four lentivirus-packaged RNAi plasmids prepared in step (4) were delivered into the yolk near the animal pole using a Nanoject III microinjection system (Drummond, Broomall, USA). The injection dose of each lentivirus-packaged RNAi plasmid was 50 nL, and the titer was 10. 8Units / mL, 200 fertilized eggs per group were injected. The microinjected fertilized eggs were returned to Holtfreter's solution (100%), and the external environment was kept consistent across groups for continuous incubation. Larvae from the RNAi group and the control group were collected on days 3, 7, 14, 19, and 21 post-injection, rapidly cryopreserved in liquid nitrogen, and then stored at -80°C. Subsequently, qPCR was used for detection. dnmt1 The efficiency of gene silencing, the results are as follows Figure 1 As shown in C.

[0034] Figure 1 The data from the C-cell diagram shows that at 3, 7, 14, 19, and 21 days (dah) post-hatching, dnmt1 The expression levels all showed a downward trend, with the dnmt1-Icpu-3789 group showing the most significant expression inhibition.

[0035] (5) Gender reversal assessment Take 150 dah of gonadal tissue from the four groups in step (4), wrap it in aluminum foil, and freeze it directly on dry ice. Control the low-temperature chamber temperature at -15~-20℃. The frozen samples are continuously longitudinally sectioned into thin sections to 5~10 µm using a cryostat (Leica, Bensheim, Germany), with complete darkness protection. After sectioning, observe each phenotype and sex directly under a fluorescence microscope (results are shown in Figure 1). Figure 1 (As shown in D). Detection of male-related genes in the dnmt1-Icpu-3789 group ( dmrt1 , amh ) and female-related genes ( cyp19a1a , foxl2 ) Express the situation, the result is as follows Figure 2 As shown.

[0036] Figure 1 The results showed that all three interference groups exhibited varying degrees of sex reversal, with the dnmt1-Icpu-3789 group showing the highest reversal rate. Figure 2 The study revealed no individuals with complete reversal of ovarian masculinization; instead, partial ovarian masculinization was observed, manifested as testis-like tissue regions within the ovarian tissue. Male-related genes (GVGs) were also detected in these abnormal gonads. dmrt1 , amh ) and female-related genes ( cyp19a1a , foxl2 The common expression of ).

[0037] The above results indicate that inhibiting dnmt1 Expression of this substance can induce partial sex reversal in channel catfish, with some ovarian tissue transforming into testis-like structures. This is speculated to be... dnmt1Its main function is to maintain the methylation level of DNA throughout the genome. When it is suppressed, the overall methylation level will decrease, especially the demethylation of the X chromosome and sex-determining regions, thereby activating key genes related to male differentiation and causing changes in the direction of gonadal development.

[0038] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of spotted catfish dnmt1 Gene knockout RNAi target sequence, characterized in that, The RNAi target sequence is shown in SEQ ID NO.

4.

2. A method for sex control in channel catfish, characterized in that, Includes the following steps: (1) Design of RNAi plasmids for lentiviral packaging For spotted catfish dnmt1 Gene design of RNAi target sequences, sense strands, and antisense strands; and preparation of LV3-shRNA vector based on RNAi target sequences, sense strands, antisense strands, and LV3-pGLV-h1-GFP-puro vector; transformation of LV3-shRNA vector into DH5α competent cells to obtain... dnmt1 Gene RNAi plasmid; dnmt1 The gene RNAi plasmid was packaged with lentivirus to obtain lentivirus-packaged RNAi plasmid; (2) Sex regulation in spotted catfish The RNAi plasmid packaged with lentivirus in step (1) was injected into the yolk near the animal pole of the spotted catfish embryo, and the hermaphroditic spotted catfish were obtained by hatching and identification.

3. The method for sex regulation of spotted catfish according to claim 2, characterized in that, In step (1), the RNAi target sequence is shown in SEQ ID NO.4; the loop structure in the sense strand uses the "TTCAAGAGA" sequence to avoid forming a termination signal, and the "GATCC" sequence is added to the 5' end of the sense strand; the "AATTC" sequence is added to the 5' end of the antisense strand.

4. The method for sex regulation of spotted catfish according to claim 2, characterized in that, The justice chain is shown in SEQ ID NO.5; the antisense chain is shown in SEQ ID NO.

6.

5. The method for sex regulation of spotted catfish according to claim 2, characterized in that, In step (2), the titer of the RNAi plasmid packaged with the lentivirus is 10. 7~8 The injection volume is 50 nL.

6. A spotted catfish as described in claim 1 dnmt1 Application of gene knockout RNAi target sequences in constructing a hermaphroditic spotted catfish model.

7. A hermaphroditic channel catfish model constructed using the sex control method described in any one of claims 2 to 5.

8. The hermaphroditic spotted catfish model according to claim 7, characterized in that, The hermaphroditic spotted catfish model is a hermaphroditic spotted catfish with female genes that has been reversed to have male traits.

9. The application of the hermaphroditic spotted catfish model according to any one of claims 7 to 8 in the study of sex determination mechanisms.

Images