CN110760545B - miRNA-based shrimp and crab ovary development promoter - Google Patents
miRNA-based shrimp and crab ovary development promoter Download PDFInfo
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- 239000002679 microRNA Substances 0.000 title claims abstract description 22
- 108091070501 miRNA Proteins 0.000 title claims abstract description 20
- 241000238557 Decapoda Species 0.000 title abstract description 19
- 230000006408 female gonad development Effects 0.000 title abstract description 7
- 241001672730 Scylla paramamosain Species 0.000 claims abstract description 10
- 108091028689 miR-277 stem-loop Proteins 0.000 claims description 52
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 230000000694 effects Effects 0.000 abstract description 17
- 238000000034 method Methods 0.000 abstract description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- 210000001672 ovary Anatomy 0.000 abstract description 8
- 230000018109 developmental process Effects 0.000 abstract description 7
- 241000238102 Scylla Species 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 3
- 238000012404 In vitro experiment Methods 0.000 abstract description 2
- 108700011259 MicroRNAs Proteins 0.000 abstract description 2
- 238000009792 diffusion process Methods 0.000 abstract description 2
- 238000001727 in vivo Methods 0.000 abstract description 2
- 108020004999 messenger RNA Proteins 0.000 abstract 2
- 239000003112 inhibitor Substances 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 9
- 230000002401 inhibitory effect Effects 0.000 description 8
- 239000005089 Luciferase Substances 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 230000002710 gonadal effect Effects 0.000 description 5
- 108060001084 Luciferase Proteins 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 4
- 108091036066 Three prime untranslated region Proteins 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 210000000496 pancreas Anatomy 0.000 description 4
- 108010052090 Renilla Luciferases Proteins 0.000 description 3
- 108010090932 Vitellogenins Proteins 0.000 description 2
- 238000012761 co-transfection Methods 0.000 description 2
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- 241000238424 Crustacea Species 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
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- 102000004190 Enzymes Human genes 0.000 description 1
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- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 241000238095 Scylla serrata Species 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention relates to a method for promoting shrimp and crab ovary development based on microRNA (miRNA for short), which is characterized in that the method is used for injecting an eyestalk capable of specifically combining with scylla paramamosainvihTo inhibitvihThe expression of the gene can promote the development of the ovary of the shrimp and crab. Can be specifically combined with scylla paramamosain eyestalkvihThe miRNA sequence of (a) is GUUAUGGAGUGCGUGUCUGGUGAAG. In vitro experiments prove that the composition can effectively reduce Scylla paramamosainvihThe expression of mRNA, furthermore, in vivo experiments also prove that the miRNA is injected into the eye stem to inhibit the eye stem of the scylla paramamosainvihExpression of mRNA. Due to the specificity of the action of miRNA, the side effect is less than that of the method of cutting out the eyestalk; because the molecular weight of miRNA is less than that of antibody, the miRNA has strong diffusion capability in the eye handle and high efficiency.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a shrimp and crab ovary development promoter based on miRNA.
Background
Gonadal development of decapod crustaceans such as shrimps and crabs is negatively regulated by gonadal-inhibiting hormone (GIH or VIH) secreted from the eye stems thereof. Therefore, inhibiting the secretion of gonadal-inhibiting hormone is an effective means for promoting the gonadal development of shrimps and crabs. The development of gonads, particularly ovaries of shrimps and crabs is promoted by a method of cutting eye stalks of the shrimps and the crabs at the earliest. However, since the hormone secreted by the shrimp and crab eyestalk contains other hormones besides VIH, the method has more negative effects such as higher mortality rate, poorer ovum quality and the like. Attempts have been made to inhibit VIH by injecting antibodies to VIH into the eye stems to promote gonadal development in shrimp and crab. However, this method is inefficient, involves a low ability of the antibody to diffuse into the eye stem, and is expensive.
Disclosure of Invention
The invention aims to provide the miRNA-based shrimp and crab ovary development promoter which has stronger specificity and higher action efficiency.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for promoting development of shrimp and crab ovary based on microRNA (miRNA for short) is characterized in that the eyestalk is injected with an eyestalk capable of specifically combining with scylla paramamosainvihTo inhibitvihThe expression of the gene can promote the development of the ovary of the shrimp and crab.
1. By bioinformatics analysis of the miRNA group of scylla paramamosain eyestalk, we found a miRNA, namely miRNA-277 (hereinafter referred to as mir-277, the sequence of which is GUUAUGGAGUGCGUGUCUGGUGAAG), which can specifically bind to the scylla paramamosain eye vih.
2. Through cell transfection of artificially synthesized miR-277 mics (the sequence is GUUAUGGAGUGCGUGUCUGGUGAAG) and miR-277 mics NC (the sequence is AGGUUGCUUGACGUUGGGGGAGAUU) and miR-277 inhibitor (the sequence is CUUCACCAGACACGCACUCCAUAAC) and miR-277 inhibitor NC (the sequence is AAUCUCCCCCAACGUCAAGCAACCU), the miR-277 micromics enters HEK293T cells, and luciferase activity analysis shows that the activity of miR-277 micromics is remarkably reduced compared with that of a control group miR-277 micromics NC, and the activity of miR-277 inhibitor is remarkably increased compared with that of the control group miR-277 inhibitor NC; results preliminarily verify miR-277 pairsvihHas inhibitory effect.
3. Injecting chemically synthesized miR-277 agomir (sequence: GUUAUGGAGUGCGUGUCUGGUGAAG) and miR-277 agomir (AAUCUCUCCCCCCAACGUCAAGCAACCU) into the base of the eye stalk of the scylla paramamosain, and detecting the position of the eye stalkvihRelative expression amount with miR-277, the result shows that: after miR-277 agomir is injected, the expression level of miR-277 in the eye handle is obviously increased, andvihthe expression level of (a) is significantly reduced; the miR-277 expression quantity in the eye handle after miR-277 antagomir injection is obviously reduced,vihthe expression level of (B) is obviously increased. Further verifies the miR-277 pairvihThe inhibitory action of (1).
The invention has the advantages that: the miRNA-based method established by the invention has smaller side effect than the method for cutting out the eye handle due to the specificity of the miRNA action; since the molecular weight of miRNA is smaller than that of antibody, the miRNA has stronger diffusion capability in the eye handle and higher efficiency.
Drawings
FIG. 1 shows miR-277 and Scylla paramamosainvihA graph of the predicted results of the 3' -UTR action of (2);
FIG. 2 shows miR-277mimics NC andvih3' -UTR prediction result map of (2);
FIG. 3 shows miR-277 mimics/mimics NC and pRR-vihResults of Dual luciferase reporter Gene Activity after 3' -UTR Co-transfection (p<0.05）；
FIG. 4 is the miR-277 inhibitor/inhibitor NC and pRR-vihResults of Dual luciferase reporter Gene Activity after 3' -UTR Co-transfection (p<0.05）；
FIG. 5 is vitellogenin in ovaries after miR-277 agomir and miR-277 antagomir injectionvtgRelative expression amount of Gene: (p<0.05）；
FIG. 6 is in the liver pancreas after miR-277 agomir and miR-277 agomir injectionvtgRelative expression amount of Gene: (p<0.05）。
Detailed Description
Example 1
In vitro experiments, artificially synthesized miR-277mimics (with the sequence being GUUAUGGAGUGCGUGUCUGGUGAAG)/miR-277 mimics NC (with the sequence being AGGUUGCUUGACGUUGGGGGAGAUU) and miR-277 inhibitor NC (with the sequence being CUUCACCAGACACGCACUCCAUAAC)/miR-277 inhibitor NC (with the sequence being AAUCUCCCCCAACGUCAAGCAACCU) are respectively used for replacing an internal reference plasmid and a target plasmid pRR-vih-3’-UTR（Zhou M, Jia X, Wan H, et al. miR‐34 regulates reproduction by inhibiting the expression of MIH, CHH, EcR, and FAMeT genes in mud crab Scylla paramamosain [J]Molecular reduction and reduction, 2019, 86(2): 122-131.) was performed, and lipofectin reagent used in the experiment was Lipofectamine 2000; the ratio of the target plasmid to the reference plasmid is 3: 1; the ratio of transfection reagent to plasmid was 2.1: 1. After entering HEK293T cells after cell transfection, collecting adherent cells, adding cell lysate, centrifuging at low speed, taking supernatant, measuring enzyme activity, adding Luciferase Assay II (LARII) Reagent into the supernatant, and recording the activity value of firefly Luciferase(ii) a Then adding Stop&And the Glo reagent records the activity value of the renilla luciferase (the two reagents are both protected from light), and the ratio of the activity value of the renilla luciferase to the activity value of the renilla luciferase is the relative activity of the plasmid dual-luciferase reporter gene. By comparing the relative activities of the target plasmid and luciferase reporter genes of miR-277 mics/miR-277 mics NC and miR-277 inhibitor/miR-277 inhibitor NC respectively, the activities of miR-277 mics and control miR-277 micromics NC are remarkably reduced (figure 3), and the activities of miR-277 inhibitor and control miR-277 inhibitor NC are remarkably increased (figure 4); results preliminarily verify miR-277 pairsvihHas inhibitory effect.
Said pRR-vihThe-3' -UTR is obtained by using pmir-RB-REPORTTM (Ruibo Biotech, Guangzhou) as a plasmid framework and amplifying VIH-F/R from the genome of the Scylla paramamosainvihThe 3' -UTR gene was ligated to pmir-RB-REPORTTM vector to obtain pRR-vih-3' -UTR plasmid of interest;
VIH-F：5’-CCGCTCGAG GGCACTGAACCTCATTAAACCTTTC-3’；
VIH-R：5’-GAATGCGGCCGC ATTTATCGTTTGATGACTGT-3’。
example 2
In vivo experiment, chemically synthesized miR-277 agomir (sequence: GUUAUGGAGUGCGUGUCUGGUGAAG) and miR-277 agomir (AAUCUCCCCCCAACGUCAAGCAACCU) are injected into the base of the eye stalk of scylla paramamosain, and the eye stalk is detectedvihRelative expression with miR-277, relative expression (RQ) values of the genes to be tested were calculated from the Cp values of each sample as indicated by the LightCycler 480 real-time fluorescent quantitative PCR instrument, relative expression levels of all the genes were calculated using the comparative threshold cycle method (Δ Δ CT) (Δ CT = CT of the target gene-the reference gene, Δ Δ CT = Δ CT-the calibrator sample), and the data were analyzed using SPSS20.0 statistical software for one-way anorgasmy using SPSS20.0 statistical analysistChecking to determine the difference in mean values between data treatments top<The difference was significant at 0.05. The results show that: after miR-277 agomir is injected, the expression level of miR-277 in the eye handle is obviously increased, andvihthe expression level of (a) is significantly reduced; injection of miR-277 antagomirThe miR-277 expression level in the posterior eye handle is obviously reduced,vihthe expression level of (B) is obviously increased. Further verifies the miR-277 pairvihThe inhibitory action of (1).
On the basis that miR-277 agomir and miR-277 agomir are injected into the base of the eye stalk of Scylla paramamosain, vitellogenin in ovary, liver and pancreas is detectedvtgThe results show that: after miR-277 agomir injectionvtgThe expression level in ovary, liver and pancreas is obviously increased, and miR-277 antagomir is injectedvtgThe expression level in both ovary and liver pancreas was significantly reduced (fig. 5, fig. 6). The miR-277 can be used as the ovarian development promoter of the scylla paramamosain.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Fujian agriculture and forestry university, college university
<120> miRNA-based shrimp and crab ovary development promoter
<130> 6
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> Artificial sequence
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agguugcuug acguuggggg agauu 25
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cuucaccaga cacgcacucc auaac 25
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aaucuccccc aacgucaagc aaccu 25
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ccgctcgagg gcactgaacc tcattaaacc tttc 34
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gaatgcggcc gcatttatcg tttgatgact gt 32
Claims (1)
1. Specifically combined scylla paramamosain eyestalkvihThe miRNA is miR-277, and the sequence of the miRNA is GUUAUGGAGUGCGUGUCUGGUGAAG;
the preparation is used for injection of scylla paramamosain eyestalk.
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| CN114085839A (en) * | 2021-12-03 | 2022-02-25 | 中国水产科学研究院黄海水产研究所 | MiRNA analogue related to development of portunus trituberculatus ovary and application thereof |
| CN114107301B (en) * | 2021-12-03 | 2023-06-27 | 中国水产科学研究院黄海水产研究所 | Application of miR-9 in preparation of portunus trituberculatus ovarian development promoter |
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| WO2018084077A1 (en) * | 2016-11-01 | 2018-05-11 | 国立研究開発法人国際農林水産業研究センター | Method for releasing oocyte maturation regulation in useful shrimps |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2018084077A1 (en) * | 2016-11-01 | 2018-05-11 | 国立研究開発法人国際農林水産業研究センター | Method for releasing oocyte maturation regulation in useful shrimps |
| CN109715806A (en) * | 2016-11-01 | 2019-05-03 | 国立研究开发法人国际农林水产业研究中心 | Release the method that the maturation of ovum of useful shrimps inhibits |
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| Title |
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| 拟穴青蟹 miR-34/miR277/miR279 的表达特征及其对眼柄重要基因的调控;梁萌萌;《cnki中国优秀硕士论文数据库》;20170503;第4,5，24，32页，第38页第2段，第40页图4.1，第1.6.1，3.1.1，3.3.1节 * |
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