Method for inducing eclamys farreri feminization through miRNA
By injecting the miRNA Agomir with a specific sequence to reduce the expression of the DMRT1 gene in the scallop, males were converted into females, solving the problem of low breeding efficiency in the scallop and providing a new method for sex control.
Patent Information
- Application Number
- CN202511153386.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-18
- Publication Date
- 2025-11-07
AI Technical Summary
Current technologies have not yet overcome the key bottlenecks in sex-controlled breeding, resulting in low breeding efficiency for scallops and an inability to efficiently regulate sex to obtain more female individuals and prevent inbreeding depression.
By injecting a specific sequence of miRNA Agomir, the expression level of the DMRT1 gene in the gonads of the scallop was reduced. The conversion from male to female was achieved by micro-injection of miRNA mimics and Agomir into the adductor muscle.
Successfully reducing DMRT1 gene expression, inducing testicular development arrest and sex reversal, and achieving feminization of the scallop is a new method for sex control that is simple to operate, low in cost, and has few side effects.
Smart Images
Figure CN120905303A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of aquaculture biological genetic breeding technology, specifically relating to a method for inducing feminization of scallops through miRNA. Background Technology
[0002] Comb scallop ( Chlamys farreri Scallops, belonging to the phylum Mollusca and class Bivalvia, are one of my country's important native marine aquaculture species. They possess the biological characteristics of dioecy and stable sex characteristics, making them ideal model organisms for studying the molecular mechanisms of sex determination and differentiation in shellfish. In recent years, although artificial breeding technology has provided a core driving force for the sustainable development of shellfish aquaculture, current technologies have not yet overcome the key bottleneck of sex-controlled breeding, resulting in low efficiency in the selection of high-yield and high-quality new varieties. If the sex of scallops can be controlled through specific technical means, the following advantages can be obtained: more female individuals can be obtained and more offspring can be produced in a short period, significantly improving breeding efficiency; the staggered development of sexes in hermaphroditic scallops can be controlled to prevent self-pollination depression; and single-sex individuals can be obtained, thus protecting the intellectual property rights of important varieties. Therefore, establishing efficient sex control technology is of great significance for promoting the breeding of high-yield and high-quality new shellfish varieties and the sustainable development of the shellfish aquaculture industry.
[0003] miRNAs, as endogenous gene expression regulators, specifically bind to target mRNAs in the seed region (bases 2-8), negatively regulating key developmental genes and playing important roles in organogenesis, gonadal development, immunity, and disease defense. Studies have shown that miRNAs play a central role in sex determination pathways in vertebrates and model organisms, but their functional mechanisms and applications in mollusks' sex differentiation remain a blank.
[0004] Agomir, a synthetically produced double-stranded RNA molecule, mimics the function of endogenous miRNAs and enhances their activity. This technology leverages the advantages of in vitro synthesized short-chain RNAs, achieving gain-of-function studies of target genes through specific binding to RISC complexes. Currently, microinjection of Agomir has been successfully applied in mammals and bony fishes, achieving long-term regulation in various tissues such as nerves, muscles, epidermis, and vascularized organs. However, successful cases of sex induction in lower invertebrates (especially mollusks) have yet to be seen. Summary of the Invention
[0005] This invention provides a method for controlling the sex differentiation of male scallops through miRNA, that is, a sex control method for inducing male scallops to transform into females, thereby overcoming the shortcomings of the prior art.
[0006] The application provides a sex control method for inducing the sex transformation of male chlamys farreri into female chlamys farreri. DMRT1 The application reduces the expression of a male sex differentiation gene in the gonad of chlamys farreri, realizes the sex control of male into female, and achieves the sex transformation of male chlamys farreri into female chlamys farreri. Further, the method is injecting miRNA, miRNA mimics or miRNA Agomir which can reduce the expression of the male sex differentiation gene. DMRT1 Further, the method is injecting miRNA, miRNA mimics or miRNA Agomir which can reduce the expression of the male sex differentiation gene.
[0007] Further, the miRNA has the sequence as follows: 5'-UUUGUUCGUUCGGCUCGCGUUAU-3' (SEQ ID NO: 1), The sequence information of the miRNA mimics is as follows: The sequence of the sense strand is 5'-UUUGUUCGUUCGGCUCGCGUUAU-3' (SEQ ID NO: 2); The sequence of the antisense strand is 5'-ACGCGAGCCGAACGAACAAAUUG-3' (SEQ ID NO: 3); The miRNA Agomir is that the nucleic acid fragments of the miRNA mimics are modified with sulfur skeleton at the 1st, 2nd and 19th-22nd nucleotides of the antisense strand in the direction from 5' to 3'; the 3' end of the antisense strand is modified with cholesterol; and the whole chain is modified with 2' methoxy; Further, the method is injecting through the adductor muscle.
[0008] The application further provides a miRNA which can reduce the expression of a male sex differentiation gene in chlamys farreri. DMRT1 The sequence of the miRNA is as follows: 5'-UUUGUUCGUUCGGCUCGCGUUAU-3' (SEQ ID NO: 1).
[0009] The miRNA of the application can target the male sex differentiation gene and down-regulate the expression of the male sex differentiation gene, mediates the participation of the male sex differentiation gene in spermatogenesis and testis function maintenance; the application first uses the miRNA overexpression technology to perform function verification in shellfish, and builds a technical platform for the function research of miRNAs. DMRT1 The application first uses the miRNA overexpression technology to perform function verification in shellfish, and builds a technical platform for the function research of miRNAs. In addition, the method has low invasiveness, small side effects, and only needs to use a 32G needle to inject a small amount of non-toxic solution into the adductor muscle, so that the Agomir can be efficiently and reliably delivered to the target organ. The method is simple, fast and low in cost, and has a wide application prospect in the reproductive regulation of chlamys farreri. BRIEF DESCRIPTION OF DRAWINGS
[0010] Figure 1Target binding site map of miRNA in CDS sequence of Chlamys farreri DMRT1 Figure 2 Figure of relative expression of miRNA and DMRT1 in testis after injection of miRNA Agomir P < 0.01 Figure 3 Figure of relative expression of miRNA and DMRT1 in testis after injection of miRNA Agomir P < 0.0001 Figure 4 Figure of morphological characteristics of testis of Chlamys farreri after overexpression of miRNA Figure 5 Figure of gonadal index of Chlamys farreri after overexpression of miRNA Figure 6 Figure of histological observation of testis of Chlamys farreri after overexpression of miRNA Figure 7 Figure of histological observation of testis of Chlamys farreri after overexpression of miRNA DETAILED DESCRIPTION
[0011] The present application is based on the existing miRNA transcriptome data of Chlamys farreri, and a miRNA with high female-specific expression characteristics is screened; by injecting the artificially synthesized miRNA agonist (Agomir), the key gene DMRT1 for male development is specifically inhibited, the testis development is induced to be blocked, and the male phenotype is transformed into female, thereby providing a new means and idea for establishing the application of miRNA Agomir technology in sex control breeding of shellfish.
[0012] To achieve the above object, the present application adopts the following technical scheme: 1. By bioinformatics analysis of the miRNA transcriptome data of the gonad of Chlamys farreri, we found a miRNA (5'-UUUGUUCGUUCGGCUCGCGUUAU-3') that can specifically bind to the key gene DMRT1 for male development of Chlamys farreri.
[0013] 2. By transfecting artificially synthesized miRNA mimics (sense strand sequence: 5'-UUUGUUCGUUCGGCUCGCGUUAU-3'; antisense strand sequence: 5'-ACGCGAGCCGAACGAACAAAUUG-3') and negative control (NC) mimics (sense strand sequence: 5'-UUGUACUACACAAAAGUACUG-3'; antisense strand sequence: 5'-GUACUUUUGUGUAGUACAAUU-3') into HEK293T cells, dual-luciferase reporter gene assay results showed that the relative luciferase activity of the co-transfected miRNA-mimics group was significantly lower than that of the negative control group. Preliminary in vitro validation confirmed that miRNA and... DMRT1 The expression of has a negative regulatory relationship.
[0014] 3. Chemically synthesized double-stranded miRNA Agomir (sense strand sequence: 5'-UUUGUUCGUUCGGCUCGCGUUAU-3'; antisense strand sequence: 5'-mA(*)mC(*)mGmCmGmAmGmCmCmGmAmAmCmGmAmAmCmAmA(*)mA(*)mU(*)mU(*)-Chl3'; nucleotides at positions 1, 2, and 19-22 of the antisense strand are thiocarbamate-modified nucleotides along the 5' to 3' direction; cholesterol modification at the 3' end of the antisense strand; 2' methoxy modification throughout the strand) was injected into the adductor muscle of *Scallop scallop*, and the overexpressed miRNA was analyzed. DMRT1 The relative expression levels of miRNAs were analyzed, and the results showed that the relative expression levels of miRNAs in the testes were significantly increased after injection of miRNA Agomir. DMRT1 The relative expression level was significantly reduced; histological observation showed a significant decrease in the number of male gametes overexpressing miRNA, and individuals exhibiting positive sex reversal (testis to ovary conversion) were observed; based on this, it is proposed that miRNA downregulates the expression level of *Scallop schizocarp*. DMRT1 Gene expression, mediating DMRT1 This study successfully established a novel method for regulating male sex differentiation pathways in the scallop using miRNAs to control spermatogenesis and maintain testicular function.
[0015] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.
[0016] Example 1 When performing in vitro validation at the cellular level, miRNAs and DMRT1 The binding sequence ( Figure 1 HEK293T and its upstream and downstream 100bp DNA segments were inserted into the pmirGLO vector to construct a dual-luciferase reporter gene vector; HEK293T was then inserted at a ratio of 2×102...5 Seeds were placed into 24-well plates, and 0.5 mL of prepared DMEM complete medium (containing 10% fetal bovine serum from Gibco, USA) was added. The plates were then incubated at 37°C in a 5% CO2 incubator. When cells reached 90% confluence, the synthesized miRNA-mimics / NC mimics (40 pmol / well) and pmirGLO- liposome transfection reagent (purchased from Invitrogen) were transfected using Lipofectamine® 2000 reagent. DMRT1- WT / HEK293T cells were co-transfected with pmirGLO-DMRT1-MT (500 ng / well). 48 h after transfection, adherent cells were collected and centrifuged at low speed with cell lysis buffer. The supernatant was then used for luciferase activity assays. Luciferase Assay Reagent II (LARII) was added to the supernatant, and the activity (RLU1) of firefly luciferase was read using a Promega GLOMAX luminescence analyzer. Then, Stop&GLO reagent was added, and the activity (RLU2) of Renilla luciferase was read. The ratio of the former to the latter activity values represents the relative activity of the dual-luciferase reporter gene. The dual-luciferase assay results showed that the relative luciferase activity of the miRNA-mimics group was significantly different from that of the negative control group (NC mimics). P < 0.01, its relative luciferase activity decreased by 37%. However, the luciferase activity in the miRNA-mimics mutant sequence group (with a 6-nt mutation introduced at the binding site) was not significantly different from the negative control group. Figure 2 Therefore, it was determined that miRNA and DMRT1 There is a targeting relationship between them, and they can be negatively regulated in vitro. DMRT1 The expression.
[0017] pmirGLO- DMRT1 Using the pmirGLO dual-luciferase miRNA target expression vector (purchased from Promega) as the plasmid backbone and *Scallop scallop* testis cDNA as a template, a designed... DMRT1 The target fragment was amplified using F / R specific primers, ligated into the pmirGLO vector, and wild-type pmirGLO-1 was obtained. DMRT1- WT reporter vector; following the instructions of the FastMutagenesis System kit (purchased from TransGen Biotech), a mutation site was designed by selecting the miRNA seed region and the 6-nt binding region of the target gene. MT-F / R primers containing the mutation site and wild-type pmirGLO- were used. DMRT1-WT report plasmid as template for constructing pmirGLO- DMRT1- MT mutant plasmid.
[0018] DMRT1-F: 5'-GAGCTCGCTTATCCACCGTTATCCTCAT-3'; DMRT1-R: 5'-CTCGAGGGCTTTATTCATTGCCTCGTCT-3'; MT-F: 5'-GAAAATAGACGAGGCAAGACCGCCAGCCCTTGAC-3'; MT-R: 5'-GGCGGTCTTGCCTCGTCTATTTTCCCTATGGCCTTT-3'.
[0019] Example 2 At the overall level, the effects of miRNA overexpression technology on key target genes in the gonads were validated in vivo. DMRT1 The regulatory effect of miRNA was investigated. A blank control group, a negative control group (NC Agomir), and an experimental group (miRNA Agomir) were established. Each bivalfalfa in the experimental and negative control groups received 30 μg miRNA Agomir (dissolved in 80 μL PBS) or NCAgomir per injection. The blank control group received no treatment. Multiple injections were performed into the adductor muscle using a 32G needle (0.23 mm diameter). The first injection was recorded as day 0, and injections were performed every 7 days, specifically on days 7, 14, and 21, for a total of 4 injections. The injection dosage and method were the same as the first injection. The overexpression effectiveness was assessed 72 h (3 days) after the first injection. Total RNA was extracted from testicular tissue using the Trizol method (purchased from Tiangen Biotech). The Mir-X miRNA First-Strand Synthesis Kit (purchased from TaKaRa) and PrimeScript were used for the extraction. TM The RT reagent kit (purchased from TaKaRa) was used to reverse transcribe total RNA into cDNA. RT-qPCR was then performed using the cDNA as a template. The reaction mixture consisted of: 1.0 μL cDNA, 1.0 μL each of 2 μM forward and reverse primers, 5 μL of 2×SYBR Green I Real-time PCR Master Mix (purchased from TaKaRa), and 2.0 μL sterile water in a 10 μL volume. The reaction program was: 95℃ pre-denaturation for 10 s; 95℃ denaturation for 5 s; 60℃ annealing and extension for 20 s; 40 cycles. 2... -ΔΔCtRelative quantification was performed for expression, all data were expressed as mean ± standard deviation, SPSS Statistic 22.0 software was used and one-way ANOVA and t Significance analysis was performed for the test, setting P <0.05 as significant difference.
[0020] The upstream and downstream primers of RT-qPCR were designed according to the differential miRNAs sequences obtained in the Chlamys farreri gonad samples, 5S rRNA was used as the miRNA internal reference gene, and the internal reference primer was designed as 5S-F / R to detect the relative expression of miRNA in each gonad; according to the CDS sequence of Chlamys farreri, the specific primer RT-F / R was designed, and the elongation factor 1 alpha (EF1a) of Chlamys farreri was used as the mRNA internal reference gene, and the internal reference primer was designed as EF1a-F / R to detect the relative expression of mRNA in each gonad. DMRT1 ef-1α ef-1α- DMRT1
[0021] 375-F: 5'-TTTGTTCGTTCGGCTCGCGTTAT-3'; 375-R: 5'-GTGCAGGGTCCGAGCT 24 -3'; 5S-F: 5'-TATCACGTTGAAAACACCGGTTCTCG-3'; 5S-R: 5'-AGTCTACGACACCTGGTATTCCCAGG-3'; RT-F: 5'-GACGGAGATACACAGAAGGC-3'; RT-R: 5'-CCAATGAGCCCCATACCGAC-3'; ef-1α- F: 5'-ATCCTTCCTCCATCTCGTCCT-3'; ef-1α- R: 5'-GGCACAGTTCCAATACCTCCA-3'.
[0022] The results showed that the miRNA level in the testis of Chlamys farreri injected with Agomir was extremely significantly higher than that of the control group (P < 0.001), P DMRT1 The mRNA expression level was extremely significantly decreased (P < 0.001). P Figure 3 The testis tissue of the experimental group was smaller and translucent. Figure 4 ); comparing the GSI of testis among groups, it can be seen that the GSI of testis in the first and second injection samples in the experimental group has no significant difference with the blank control group and the negative control group; after the third injection and the fourth injection, the GSI of testis in the experimental group is extremely significantly lower than that in the blank control group and the negative control group P < 0.001) Figure 5 ).
[0023] Comparing the histology of different injection groups, the arrangement of germ cells in the testis of the experimental group is loose, most of the germinal follicles have obvious cavities in the center, only a small amount of spermatogonia and spermatocytes are scattered in the follicles, and the number of germ cells is significantly less than that in the blank control group and the negative control group Figure 6 , and the individual of sex reversal appears in the fourth overexpressed genetic sex male (testis turns to ovary, and female germ cells appear in the testis) Figure 7 ) of the scallop, which indicates that the miRNA successfully induces the feminization of the scallop.
[0024] The above only describes the preferred embodiments of the present application, and any equivalent changes and modifications made according to the scope of the patent application of the present application shall be included in the scope of the present application.
Claims
1. A sex control method for inducing the transformation of Chlamys hirata males into females, characterized by, The method is to reduce the expression of the gene in the sex gland of Chlamys variegata DMRT1 and achieve sex control of male to female transformation.
2. The method of claim 1, wherein, The method is injecting into the subject a miRNA, miRNA mimics, or miRNA Agomir that reduces the expression of the gene. DMRT1 The method is injecting into the subject a miRNA, miRNA mimics, or miRNA Agomir that reduces the expression of the gene.
3. The method of claim 2, wherein, The miRNA has the sequence of 5'-UUUGUUCGUUCGGCUCGCGUUAU-3'.
4. The method of claim 2, wherein, The miRNA mimics has the sequence information as follows: The sequence of the sense strand is 5'-UUUGUUCGUUCGGCUCGCGUUAU-3'; The sequence of the antisense strand is 5'-ACGCGAGCCGAACGAACAAAUUG-3'.
5. The method of claim 2, wherein, The miRNA Agomir is that the nucleic acid fragment of the miRNA mimics is modified according to the 5' to 3' direction, the 1st, 2nd and 19th-22nd nucleotides of the antisense strand are sulfur skeleton modified nucleotides, the 3' end of the antisense strand is cholesterol modified, and the whole chain is 2' methoxy modified.
6. The method of claim 2, wherein, The method is to inject through the obturator muscle.
7. A method to reduce the concentration of [unclear text - possibly related to scallops] in [unclear text - possibly DMRT1 miRNAs that express gene levels are characterized by, The sequence of the miRNA is 5'-UUUGUUCGUUCGGCUCGCGUUAU-3'.