Long-chain non-coding rna molecule lnc46198 related to sex gland development of hemigynous duck embryo and application thereof
By studying the long non-coding RNA molecule lnc46198, which regulates the proliferation and apoptosis of duck embryonic fibroblasts, the problem of gonadal development defects in Muscovy ducks has been solved, the reproductive capacity of Muscovy ducks has been improved, and the development of the Muscovy duck industry has been promoted.
Patent Information
- Application Number
- CN202310418997.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-04-19
- Publication Date
- 2025-10-24
- Estimated Expiration
- 2043-04-19
AI Technical Summary
The incomplete reproductive system of the Muscovy duck results in low fertility. Current technology has failed to effectively explain the regulatory mechanism of its gonadal development defects, which limits the development of the Muscovy duck industry.
By studying lnc46198, a long non-coding RNA molecule associated with gonadal development in Muscovy ducks during the embryonic period, primer pairs and recombinant plasmids were designed to overexpress lnc46198 to regulate the proliferation and apoptosis of duck embryonic fibroblasts and promote gonadal development.
The scientific explanation of the molecular genetic mechanism of sterility in Muscovy ducks provides a new way to promote gonadal development and improve the reproductive performance of Muscovy ducks.
Smart Images
Figure CN116463346B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of genetic engineering, and particularly relates to a long-chain non-coding RNA molecule lnc46198 related to embryonic period half-bantai gonad development and application thereof. BACKGROUND
[0002] Half-bantai production is an advantageous and characteristic industry of Fujian duck production. Half-bantai has strong hybrid vigor and combines the advantages of parents, and has great development potential. However, there is still a serious problem in half-bantai production, that is, half-bantai has no actual breeding value. In order to obtain half-bantai ducklings, the parent ducks must be fed and artificial insemination technology is used, which increases the production cost and greatly limits the development of half-bantai industry. Compared with the parent ducks of Beijing duck or bantai duck with normal reproductive performance, the reproductive system of half-bantai is incomplete, and the gonad development is often defective, which directly leads to low fertility of half-bantai. However, the reason for the sterility of half-bantai and the regulation mechanism affecting the gonad development defect of half-bantai are still unclear. The present application aims to explore the function of the key transcript lnc46198 of half-bantai and its parent ducks in the process of gonad development and cell differentiation at the sexual maturation period and embryonic period, so as to promote the normal development of half-bantai gonad by gene editing technology, exogenous injection or chimera in the later stage, and then explore the possibility of half-bantai breeding, and provide a scientific basis for half-bantai germplasm controllable technology. SUMMARY
[0003] The present application aims to provide a long-chain non-coding RNA molecule lnc46198 related to embryonic period half-bantai gonad development and application thereof.
[0004] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:
[0005] A long-chain non-coding RNA molecule lnc46198 related to embryonic period half-bantai gonad development, the base sequence of which is shown in SEQ ID NO. 1.
[0006] A primer pair for detecting the expression level of the long-chain non-coding RNA molecule lnc46198, the sequence of which is:
[0007] F: 5'-CGGCTGATTCCGTTATGGGTAT-3',
[0008] R: 5'-CAGGGCTTCCTCCACCTTT-3';
[0009] or
[0010] F2: 5'-CAAATCTAGGCAACACCACA-3',
[0011] R2: 5'-CAGCAACAATCATAGGAGGC-3'.
[0012] A recombinant plasmid lnc46198-pcDNA3.1 containing a long-chain non-coding RNA molecule lnc46198.
[0013] The application of a long-chain non-coding RNA molecule lnc46198 in the preparation of a product for regulating the development of gonads of semi-muscovy ducks in the embryonic period.
[0014] A method for inhibiting the proliferation of duck embryo fibroblasts and promoting the apoptosis of duck embryo fibroblasts by overexpressing a long-chain non-coding RNA molecule lnc46198.
[0015] Further, the recombinant plasmid lnc46198-pcDNA3.1 is used to overexpress the long-chain non-coding RNA molecule lnc46198.
[0016] A product for regulating the proliferation and apoptosis of duck embryo fibroblasts, comprising a substance for overexpressing a long-chain non-coding RNA molecule lnc46198.
[0017] The application has the advantages that:
[0018] The research on the sterile molecular mechanism of semi-muscovy ducks and the differences in gonad development between semi-muscovy ducks and paternal muscovy ducks and maternal Beijing ducks is still very insufficient. In the present research, the semi-muscovy ducks, a characteristic and dominant hybrid breed in Fujian Province, are used as experimental objects, and the influence of lnc46198 on the embryonic and cellular differentiation and development of semi-muscovy ducks is verified for the first time. The molecular genetic mechanism of the sterility of semi-muscovy ducks after intergeneric crossbreeding is scientifically explained from the molecular and cellular levels, and a reference basis and a new research approach are provided for the sterility of semi-muscovy ducks. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 : Enrichment level of 17beta-HSD3 protein to lnc46198.
[0020] Figure 2 : Expression of lnc46198 in different gonads of semi-muscovy ducks at different embryonic ages.
[0021] Figure 3 : Specific expression of lnc46198 in different tissues of semi-muscovy ducks in the embryonic period.
[0022] Figure 4 : CDS PCR amplification result of lnc46198.
[0023] Figure 5 : Morphology of duck embryo fibroblasts.
[0024] Figure 6 : Overexpression efficiency of lnc46198 detected by qPCR.
[0025] Figure 7 : Effect of overexpression of lnc46198 on the viability of duck embryo fibroblasts.
[0026] Figure 8 :Flow cytometric analysis of apoptosis in duck embryo fibroblasts after lnc46198 overexpression.
[0027] Figure 9 :Effect of lnc46198 overexpression on apoptosis of duck embryo fibroblasts.
[0028] Figure 10 : TUNEL staining results of apoptosis in duck embryo fibroblasts after lnc46198 overexpression (×100).
[0029] Figure 11 : The TUNEL method was used to detect the effect of overexpression of lnc46198 on apoptosis of duck embryo fibroblasts. DETAILED DESCRIPTION
[0030] The present invention will be further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.
[0031] Those skilled in the art will appreciate that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer.
[0032] The base sequence corresponding to the long non-coding RNA molecule TCONS_00246198 (hereinafter referred to as lnc46198) described in the present application is shown in SEQ ID NO.1. lnc46198 is a long non-coding RNA molecule obtained by the applicant using the embryonic male gonad tissue of Muscovy duck (Minhou Qianliyang Poultry Breeding Co., Ltd.) and Muscovy duck (Zhangzhou Changlong Agriculture and Animal Husbandry Co., Ltd.) as the research objects, extracting total RNA, treating it with DNase I to remove the residual genomic DNA, and then performing ribosomal chain-specific library sequencing. After that, edgeR software was used to perform expression difference significance analysis, and Padj (corrected P value) <0.05 was used as the difference significance standard for screening. The applicant further predicted its target gene through the positional relationship (co-location) and expression correlation (co-expression) of lnc46198 with protein-coding genes, and found genes known to be related to reproduction. 17β-HSD Targeting lnc46198, it was speculated that lnc46198 may be involved in the gonadal development of duck embryos.
[0033] Example 1: 17β-HSD3-Flag RNA Immunoprecipitation Detection
[0034] To study the relationship between lnc46198 and 17β-HSD3 The present invention cultivates duck embryo fibroblasts and transfects 17β-HSD3 -pcDNA3.1-Flag overexpression plasmid, and RNA binding protein immunoprecipitation (RIP) detection was performed using Flag antibody. Transfection was performed according to the instructions of LipofectamineTM 3000. RNA binding protein immunoprecipitation (RIP) experiment was performed according to the instructions of EZMagna RIP Kit (Millipore). -ΔΔCt Quantitative analysis of lnc46198 and 17β-HSD3 Expression. Among them, 17β-HSD3 -The construction of pcDNA3.1-Flag overexpression plasmid is as follows:
[0035] Total RNA was extracted from male gonadal tissue of Mule ducks and reverse-transcribed into cDNA as a template for PCR amplification using primers 17β-HSD3-F and 17β-HSD3-R. The total PCR reaction volume was 50 μL: deionized water (1.0 μL), 17β-HSD3-F (10 μmol / L) (1.0 μL), 17β-HSD3-R (10 μmol / L) (1.0 μL), cDNA (2.0 μL), and Platinμm® PCR SμperMix (High Fidelity) (45 μL). PCR amplification conditions included initial denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 60°C for 30 s, and 68°C for 1 min. PCR products were recovered from the gel using the QIAqμick PCR & Gel Cleanμp Kit (refer to the kit instructions for details). The obtained PCR product was double-digested with endonucleases BamHI and EcoRI, and then the digested product was purified and recovered. The pcDNA3.1 plasmid (purchased from Thermo Fisher, USA) was double-digested with endonucleases BamHI and EcoRI. The double-digested pcDNA3.1 plasmid was ligated with the PCR fragment using GeneArt® Seamless Cloning and Assembly Enzyme Mix to obtain 17β-HSD3 -pcDNA3.1-Flag overexpression plasmid was transformed into DH5α competent cells. The sequences of 17β-HSD3-F and 17β-HSD3-R are as follows:
[0036] 17β-HSD3-F: 5'-TTGGTACCGAGCTCGGATCCGCCACCATGCATGCTAGCTTGCTGCCTT-3',
[0037] 17β-HSD3-R: 5'-CTGTGCTGGATATCTGCAGAATTCGGGCTTCCTCCACCTTTTCT-3'.
[0038] The results show that ( Figure 1 ), compared with the IgG group, 17β-HSD3 The expression level of lnc46198 in the -pcDNA3.1-Flag overexpression plasmid group was enriched 71.51 times, indicating that lnc46198 interacted directly or indirectly with 17β-HSD3 protein, and lnc46198 could be precipitated after incubation with 17β-HSD3 antibody protein, indicating that there may be a targeting relationship between the two.
[0039] Example 2: qPCR technology to determine the specific expression of lnc46198
[0040] Mule duck embryos were collected at embryonic age 12.5, and the brain, heart, liver, lungs, muscles, kidneys, and male gonads were collected under a stereomicroscope. Male gonadal tissues were also collected at different embryonic ages: E10, E15, E20, and E25. Total RNA was extracted, and cDNA was stored at -20°C for use after reverse transcription. qPCR was used to determine the relative expression levels of lnc4619 in different tissues and at different embryonic ages of Mule ducks. GAPDH Quantitative PCR primers were designed using PrimerPremier 6.0 and Beacon designer 7.8 software and synthesized by Shanghai Bioengineering. PCR reaction system: Power SYBR® Green Master Mix 10.0 μL, cDNA 1.0 μL, SDW 8.0 μL, forward primer (10 μmol / L) 0.5 μL, reverse primer (10 μmol / L) 0.5 μL. PCR reaction conditions: 95°C for 1 min; 95°C for 15 s, 63°C for 25 s, 40 cycles. Each sample was repeated three times, and the relative expression level was expressed as 2. -ΔΔCt Statistical analysis was performed. The primer sequences are as follows:
[0041] GAPDH-F: 5'-GGAGCTGCCCAGAACATTATC-3',
[0042] GAPDH-R: 5'-GCAGGTCAGGTCCACGACA-3';
[0043] lnc46198-F1: 5'-CGGCTGATTCCGTTATGGGTAT-3',
[0044] lnc46198-R1: 5'-CAGGGCTTCCTCCACCTTT-3'.
[0045] As shown in Figure 2 , lnc46198 was specifically expressed in the gonad of different embryonic stages of semi-muscovy duck, and the relative expression of lnc46198 in the gonad gradually increased with the increase of embryonic age, indicating that lnc46198 had high spatiotemporal specificity. As shown in Figure 3 , the expression of lnc46198 in the gonad of semi-muscovy duck was significantly higher than that in other tissues (P < 0.01), suggesting that lnc46198 might play a role in the gonad.
[0046] Example 3: Construction of lnc46198 overexpression vector
[0047] Semi-muscovy duck embryos were collected, total RNA was extracted, and cDNA was reverse transcribed as a template, and the CDS sequence of lnc46198 was obtained by PCR amplification. The total volume of the PCR amplification reaction system was 50 μL: deionized water 1.0 μL, lncRNA-F (10 μM) 1.0 μL, lncRNA-R (10 μM) 1.0 μL, cDNA 2.0 μL, Platin PCR SμperMix, High Fidelity 45 μL. The PCR amplification conditions were: pre-denaturation 94℃ for 2 min, 94℃ for 30 sec, 60℃ for 30 sec, 68℃ for 3.5 min, 35 cycles. The PCR amplification product was recovered by gel cutting using QIAqμick PCR & Gel Cleanμp Kit. As shown in Figure 4 , the lnc46198 PCR amplification product was about 3525 bp. Among them, the lncRNA-F and lncRNA-R sequences are as follows:
[0048] lncRNA-F: 5'-TTGGTACCGAGCTCGGATCCGTTAGATGATGTTTAGTTAGTTTAA-3',
[0049] lncRNA-R: 5'-CTGTGCTGGATATCTGCAGAATTCCAGAAATAAGAGCCTGTTTAA-3'.
[0050] The obtained PCR product was double-digested with endonucleases BamHI and EcoRI, and then the digested product was recovered after purification. The pcDNA3.1 plasmid (purchased from Thermo Fisher, USA) was double-digested with the two endonucleases BamHI and EcoRI. The double-digested pcDNA3.1 plasmid was ligated with the PCR fragment using GeneArt® Seamless Cloning and Assembly Enzyme Mix, to obtain the lnc46198-pcDNA3.1 overexpression plasmid.
[0051] Example 4: Duck embryo fibroblast cell culture and transfection experiment
[0052] The duck embryo fibroblast cells (DEF) were resuscitated using MEM a culture solution (containing 10% FBS and double-antibiotics) and cultured at 37°C, 5% CO2 to a confluence of about 90%; Trypsin-EDTA (0.25%) was added for digestion, and the cells were passaged at a cell density of 5×10 5 The duck embryo fibroblast cells were observed under an inverted microscope, and it was found that the cells were all adherent cells and grew in a fibroblast-like or spindle shape. Figure 5
[0053] The duck embryo fibroblast cells were seeded in a 6-well plate at a density of 5×10 5 The cells were incubated at 37°C, 5% CO2 overnight, and then fresh complete culture solution was replaced when the cell confluence was about 70%; according to the LipofectamineTM 3000 instructions, the lnc46198-pcDNA3.1 overexpression plasmid and the pcDNA3.1 control plasmid were transfected, respectively, and fresh complete culture solution was replaced 8h later; the cells were collected 48h after transfection to extract total RNA for subsequent qPCR analysis of overexpression efficiency.
[0054] The overexpressed DEF cells and control cells were collected, total RNA was extracted using TRIzol® Plus RNA Purification Kit, residual genomic DNA was removed by DNase I, and then cDNA was synthesized according to the SuperScript™ III First-Strand Synthesis SuperMix instructions. The overexpression efficiency of lnc46198 was analyzed by qPCR using the following primers: GAPDH For internal reference, Real-time PCR amplification was performed using PowerUp SYBR™ Green Master Mix kit. The PCR reaction system was as follows: 10 μL PowerUp SYBR™ Green Master Mix, 4 μL cDNA (the original cDNA solution was diluted 1:10), 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), and H2O was added to 20 μL. The reaction conditions were as follows: 95 °C for 1 min; 95 °C for 15 sec, 60 °C for 25 sec, for a total of 40 cycles. Each experiment was repeated 3 times, and 3 replicate wells were set for each sample. The relative expression was calculated using 2 -ΔΔCt The results showed that Figure 6 Compared with the transfection control group pcDNA3.1, the lnc46198 level in the lnc46198-pcDNA3.1 overexpression plasmid group was up-regulated by 29.28 times, indicating that the duck embryo fibroblast lnc46198 overexpression strain was successfully constructed. The primer sequences used are as follows:
[0055] lnc46198-F2: 5'-CAAATCTAGGCAACACCACA-3',
[0056] lnc46198-R2: 5'-CAGCAACAATCATAGGAGGC-3',
[0057] GAPDH-F: 5'-GGAGCTGCCCAGAACATTATC-3',
[0058] GAPDH-R: 5'-GCAGGTCAGGTCCACGACA-3'.
[0059] The duck embryo fibroblasts transfected with pcDNA3.1 control plasmid and lnc46198-pcDNA3.1 overexpression plasmid for 24 h were collected by digestion, and the cell density was adjusted. The 96-well plate was inoculated with 2x10 3 / 100 μL cell density per well, and after the cells adhered, it was recorded as 0h, and 10 μL CCK8 was added after 48h of continuous culture. After incubation at 37 °C, 5% CO2 for 2h, the absorbance value at 450nm was detected by microplate reader. Each group was repeated 6 replicate wells. The results showed that Figure 7 Compared with the blank group and the pcDNA3.1 group, the duck embryo fibroblast activity was extremely significantly reduced after overexpression of lnc46198 (P<0.01) for 48h, indicating that overexpression of lnc46198 can inhibit the proliferation activity of duck embryo fibroblasts.
[0060] The duck embryo fibroblasts transfected with pcDNA3.1 control plasmid and lnc46198-pcDNA3.1 overexpression plasmid for 48 h were collected by digestion, and the cells were rinsed with pre-cooled PBS twice. 300 μL of preheated non-EDTA trypsin was added for digestion for 3-5 min, and then 200 μL of preheated serum-containing medium was added to terminate the reaction; at the same time, 500 μL of PBS was added to wash the residual cells into a 2 mL centrifuge tube. Centrifugation (1500 g, 5 min) was performed, and the supernatant was discarded; 1 mL of preheated PBS was added to resuspend the cells by gentle blowing, and centrifugation was performed as above; 1 mL of PBS was added to resuspend the cells again, and centrifugation was performed as above. 500 μL of Binding Buffer was added to each tube, and the cells were resuspended gently. 5.0 μL of Annexin V-FITC and 10 μL of PI were added, mixed by inversion, and incubated at room temperature for 5-15 min in the dark, followed by flow cytometry analysis. The results showed that Figure 8 to Figure 9 compared with the pcDNA3.1 control group, the overexpression of lnc46198 increased the apoptosis of duck embryo fibroblasts; the apoptosis of duck embryo fibroblasts in the overexpression group was 7.30 times that of the pcDNA3.1 control group (P < 0.01), indicating that overexpression of lnc46198 can promote the apoptosis of duck embryo fibroblasts.
[0061] The TUNEL experiment was performed according to the TUNEL Bright Red Apoptosis Detection Kit instructions. Three parallel samples were set up for each group, and three fields (x100) were randomly selected for observation and photography using an upright fluorescence microscope. Image-Pro Plus software was used to analyze the images and calculate the apoptotic index (AI). Among them, DAPI (blue) labeled the nuclei of all cells, and TUNEL (purple) labeled the nuclei of apoptotic cells. The results showed that Figure 10 to Figure 11 the pcDNA3.1 group had a small number of TUNEL-labeled conjunctival cells, while the proportion of TUNEL-labeled cell apoptosis in the overexpression group increased significantly (P < 0.01), suggesting that overexpression of lnc46198 can promote the apoptosis of duck embryo fibroblasts.
Claims
1. A long-chain non-coding RNA molecule Inc46198 associated with the development of gonads in semi-muscovy duck embryos, characterized in that: The base sequence is shown in SEQ ID NO.
1.
2. Use of the long non-coding RNA molecule lnc46198 according to claim 1 in the preparation of a product for regulating gonadal development in embryonic mule ducks.