CN114854745A - MiRNA analogue related to development of portunus trituberculatus ovary and application thereof - Google Patents
MiRNA analogue related to development of portunus trituberculatus ovary and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of aquaculture biotechnology, and discloses a miRNA analogue related to the development of portunus trituberculatus ovary and application thereof. The miRNA analogue is one or more of let-7-5p and miR-141-3 p. After synthesizing let-7-5p agomir and miR-141 agomir, the two are injected into female portunus trituberculatus through the base of the third step, namely, key enzyme genes in the decomposition pathway of methyl farnesyl ester can be reducedJHE‑like CXE‑1AndJHE‑like CXE‑2expression and upregulation of the vitellogenin GenevtgExpression, realizes the purposes of promoting the exogenous yolk synthesis of the portunus trituberculatus and accelerating the ovarian development and maturation of the portunus trituberculatus, has small side effect on the parent, is efficient and reliable, and is suitable for a large number of parentsPromoting the maturity of the body.
Description
Technical Field
The invention belongs to the field of aquaculture biotechnology, and particularly relates to a miRNA analogue related to the development of portunus trituberculatus ovary and application thereof.
Background
Portunus trituberculatus (blue crab)Portunus trituberculatus) Belonging to Crustacea, Decapoda, Paralithodes, and Paralithodes (genus)Portunus) Is an important mariculture variety in China. In recent years, the swimming crab breeding industry develops rapidly. However, due to the lack of effective reproductive control technology in the industry, the yield of high-quality seedlings cannot meet the demand, and the development of the breeding industry is severely limited. Therefore, the establishment of an efficient reproductive regulation and control technology has important significance for the sustainable development of the Portunus trituberculatus breeding industry.
At present, the method for stimulating the maturation of crustacean ovaries is commonly used in production, but the removal of the eyestalk damages parents and causes the problems of egg quality reduction, hatchability reduction and the like. microRNA (miRNA) can be specifically combined with 3' UTR of a target gene to carry out negative regulation on the expression of the target gene, and has an important effect on regulation and control of animal reproduction. By injecting the miRNA analogue artificially synthesized, the regulation and control effect of endogenous miRNA on the target gene can be simulated, and the control on the development process of ovaries can be realized.
Disclosure of Invention
The invention aims to provide a miRNA analogue related to the development of portunus trituberculatus ovary and application thereof. The miRNA analogs comprise let-7-5p and miR-141-3p, and the two have good inhibition targeting effects on methyl farnesyl ester (MF) decomposition pathway key enzymes, so that the mature development of the portunus trituberculatus ovary is promoted.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides a miRNA analogue related to the development of portunus trituberculatus ovary, wherein the miRNA analogue is one or more of let-7-5p and miR-141-3 p.
The invention also provides application of the miRNA analogue in preparation of a portunus trituberculatus ovary development promoter.
Further, the ovarian development promoter simultaneously contains let-7-5p agomir and miR-141 agomir.
Further, the sense strand sequence of the let-7-5p agomir is as follows: AACUAUACAACCUACUACCUCAGG, the antisense strand sequence is: UGAGGUAGUAGGUUGUAUAGUUUU is added.
Further, the sense strand sequence of the miR-141 agomir is as follows: UAACACUGUCUGGUAAAGAUGGGG, the antisense strand sequence is: CCAUCUUUACCAGACAGUGUUAUU are provided.
Further, the preparation and use method of the ovarian development promoter comprises the following steps: synthesizing let-7-5p agomir and miR-141 agomir, mixing the two to prepare the portunus trituberculatus ovary development promoter, and directly injecting the ovary development promoter into the portunus trituberculatus female crab through the basal part of the third step.
Further, the miRNA analogs are regulated by down regulationJHE-like CXE-1AndJHE-like CXE-2gene expression and upregulationvtgGene expression to accelerate the development and maturation of the portunus trituberculatus ovary.
The invention also provides application of the miRNA analogue in preparing a key enzyme inhibitor of a methyl farnesyl ester decomposition pathway in crustaceans.
Further, the key enzymes of the methylfarnesyl ester decomposition pathway are JHE-like CXE-1 and JHE-like CXE-2.
The invention also provides application of the miRNA analogue in preparing a yolk synthesis promoter in crustaceans.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the experimental verification of the invention firstly obtains two miRNA analogues related to the development of the portunus trituberculatus ovary, namely let-7-5p and miR-141-3p, which can be regulated downJHE-like CXE-1AndJHE-like CXE-2gene expression and upregulationvtgThe gene expression can promote the synthesis of exogenous yolk and accelerate the development and maturation of the portunus trituberculatus ovary.
2. Compared with the common method for stimulating the maturation of crustacean ovaries to cut off eye handles in production, the method for promoting the development of the ovary of the Sanyou swimming crab by using the miRNA analogs has the advantages that the side effect on parents is small, the tissue damage or death of individuals cannot be caused by injecting a trace non-toxic solution at the foot basement membrane through the needle with the diameter of 0.5 mm, the method is efficient and reliable, the accumulation of vitelline of the ovary tissues of the parents can be obviously improved in a short time, and the development and maturation of the ovaries are accelerated. In addition, the method has the advantages of low cost, simple and quick operation and suitability for accelerating the maturation of a large number of parents. Therefore, the method has wide application prospect.
Drawings
FIG. 1 shows let-7-5p and miR-141-3p withJHE-like CXE-1AndJHE-like CXE-2prediction of gene 3' UTR binding.
FIG. 2 shows the relative activities of firefly luciferase and Renilla luciferase after co-transfection of different sets of miRNA mimics and plasmids; wherein a is transfection let-7-5p mimics; b is transfected miR-141-3p mimics.
FIG. 3 shows the control and treated groups after injection of different agomirsJHE-like CXE-1AndJHE-like CXE-2the expression profile of the gene.
Figure 4 shows that after injection of different agomirs,vtgthe expression profile of the gene.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to the following specific examples.
In the following examples, unless otherwise specified, the experimental methods used were all conventional methods, and materials, reagents and the like used were all available from biological or chemical reagents companies.
Example 1
Methyl farnesyl ester (MF) is an important reproductive hormone of crustaceans, and can promote the development and maturation of ovaries. In the Portunus trituberculatus, key enzymes of MF decomposition pathway juvenile hormone esterase-like carboxylesterase-1 (JHE-like CXE-1) and juvenile hormone esterase-like carboxylesterase-2 (JHE-like CXE-2) play important roles in regulating and controlling MF abundance. By inhibiting the expression of the two enzymes, MF decomposition can be slowed down, so that the exogenous yolk synthesis (in liver pancreas) of the blue crab is promoted, and the ovary maturation is accelerated.
Prediction of targeting by bioinformatics methodsJHE-like CXE-1AndJHE-like CXE-2miRNA of gene 3' UTR, the screening standard is as follows:
(1) in the presence of miRNAJHE-like CXE-1AndJHE-like CXE-2the 3 'UTR of the gene is not mismatched with a seed region (2 nt to 8 nt at the 5' end);
(2) MiRNA andJHE-like CXE-1andJHE-like CXE-2the binding free energy of the 3' UTR of the gene is less than or equal to-20 kcal/mol.
The prediction results are shown in figure 1, and let-7-5p and miR-141-3p can be respectively related to blue crabJHE-like CXE-1AndJHE-like CXE-2the 3' UTR of the gene binds.
Example 2
Verification of let-7-5p pairs at the cellular level by a Dual luciferase reporter assayJHE-like CXE-1And miR-141-3p pairJHE-like CXE-2Regulating and controlling effect.
Each validation experiment sets different miRNA mix combinations with plasmids.
1. let-7-5p pairsJHE-like CXE-1The regulation and control verification experiment group is as follows:
(1) miRNA mimics negative control and pmirGLO-JHE-like CXE-1-3' UTR-wild type;
(2) let-7-5p mimics and pmirGLO- JHE-like CXE-1-3' UTR-mutant; wherein, the sense chain sequence of the let-7-5p mimics is as follows: AACUAUACAACCUACUACCUCA (SEQ ID No. 1), transThe sense chain sequence is: UGAGGUAGUAGGUUGUAUAGUU (SEQ ID No. 2);
(3) let-7-5p mimics and pmirGLO-JHE-like CXE-1-3' UTR-wild type;
(4) miRNA mimics negative control and pmirGLO-JHE-like CXE-1-3' UTR-mutant.
2. miR-141-3p pair JHE-like CXE-2The regulation and control verification experiment group is as follows:
(1) miRNA mimics negative control and pmirGLO-JHE-like CXE-2-3' UTR-wild type;
(2) miR-141-3p mimics and pmirGLO- JHE-like CXE-2-3' UTR-mutant; wherein, the sense chain sequence of the miR-141-3p mimics is as follows: UAACACUGUCUGGUAAAGAUGG (SEQ ID No. 3), the antisense strand sequence being: CCAUCUUUACCAGACAGUGUUA (SEQ ID No. 4);
(3) miR-141-3p and pmirGLO- JHE-like CXE-2-3' UTR-wild type;
(4) miRNA mimics negative control and pmirGLO-JHE-like CXE-2-3' UTR-mutant.
Combinations of different miRNA mix and plasmids were transfected into 293T cells separately using transfection reagent extract 2000. After 48 h of transfection, adherent cells were collected, the activities of firefly Luciferase and Renilla Luciferase were determined using the Dual-Luciferase Reporter Assay System, and the relative activities of firefly Luciferase and Renilla Luciferase were calculated.
The results are shown in FIG. 2, let-7-5p mimics and pmirGLO- JHE-like CXE-13' UTR-wild type (a in FIG. 2), and miR-141-3p mimics and pmirGLO- JHE-like CXE-2The relative luciferase activity of-3' UTR-wild type (b in FIG. 2) was significantly lower than that of the other groups, and it was preliminarily confirmed that let-7-5p and miR-141-3p were directed to separatelyJHE-like CXE-1AndJHE-like CXE-2has inhibitory effect.
Example 3
Through injection experiments, artificially synthesized let-7-5p agomir (double strand, sense strand sequence: AACUAUACAACCUACUACCUCAGG (SEQ ID No. 5), antisense strand sequence: UGAGGUAGUAGGUUGUAUAGUUUU (SEQ ID No. 6)) was simultaneously injected from in vivo level verificationCholesterol modification is carried out, two thio skeletons at the 5 'end are modified, and four thio skeletons at the 3' end are modified to be full-chain methoxy modification), and miR-141-3p agomir (double chains, wherein the sequence of a sense chain is as follows: UAACACUGUCUGGUAAAGAUGGGG (SEQ ID No. 7), the antisense strand sequence being: CCAUCUUUACCAGACAGUGUUAUU (SEQ ID No. 8); cholesterol modification is carried out at the 3 ' end of the antisense chain, two thio skeletons at the 5 ' end and four thio skeletons at the 3 ' end are modified to modify the whole chain methoxy group) on the blue crabJHE-like CXE-1AndJHE- like CXE-2regulating and controlling function of the compound and promoting function of ovary development.
Two groups are set in the experiment, and an agomir negative individual is taken as a control group, and a let-7-5p agomir and a miR-141 agomir individual are simultaneously taken as a treatment group. Using a 1ml syringe (needle diameter of 0.5 mm), agomir was injected into female portunus trituberculatus through the basal part of the third step. After 48 h of injection, the crabs were dissected to collect the hepatopancreas tissues. Extracting total RNA of liver pancreas tissue of blue crab by using Trizol reagent, carrying out reverse transcription by adopting Evo M-MLV RT Kit with gDNA Clean for qPCR II Kit, and carrying out reverse transcription by adopting AG SYBR Green Pro Taq HS qPCR KitJHE-like CXE-1、JHE-like CXE-2、vtgAnd (4) analyzing gene expression.
The reaction system for qPCR (20 μ L) was: 10 μ L SYBR Green Pro Taq HS Premix II (2 ×), 0.4 μ L ROX Reference Dye II, 0.4 μ L10 μmol/L forward primer, 0.4 μ L10 μmol/L reverse primer, 3.0 μ L DEPC water and 1.0 μ L cDNA dilution.
The sequences of the amplification primers for qPCR were as follows:
JHE-like CXE-1a forward primer: TGTTCGAAGGAGGACCAATC (SEQ ID No. 9);
JHE-like CXE-1reverse primer: CAGTCTGAAGGGCGAGGTAG (SEQ ID No. 10).
JHE-like CXE-2A forward primer: AGCCTGTCCGACTGTTATGG (SEQ ID No. 11);
JHE-like CXE-2reverse primer: GGCGAAGTTTGTCCACATCT (SEQ ID No. 12).
vtgA forward primer: AGCGGCATCATCTCTTCAGT (SE)Q ID No.13）；
vtgReverse primer: AATGCGGGAGATGGTATCTG (SEQ ID No. 14).
The reaction program for qPCR was: 30 s at 95 ℃; 95 ℃ for 5 s, 60 ℃ for 34 s, 40 cycles.
let-7-5p、miR-141-3p、JHE-like CXE-1、JHE-like CXE-2、vtgRelative expression amounts are 2 –ΔΔCt The method is used for calculation, statistical analysis is carried out through SPSS 20.0 software, and whether the different groups of data have significant differences or not is determined, so thatP<A significant difference was found to be 0.05.
As shown in FIG. 3, let-7-5p agomir and miR-141 agomir were injected in comparison with the control groupJHE- like CXE-1(a in FIG. 3) andJHE-like CXE-2(b in FIG. 3) the gene expression was significantly down-regulated,vtgthe expression level was significantly up-regulated (fig. 4). During the generation of the egg yolk, the blue crab secretes estrogen to induce liver cells to generate a large amount of Vitellogenin (VTG), which is transported to the ovary through blood and is converted into egg yolk protein after being absorbed and modified for the development of blue crab ovary. Therefore, the experimental result shows that the simultaneous injection of let-7-5p agomir and miR-141 agomir can obviously inhibitJHE-like CXE-1AndJHE-like CXE-2the gene expression effectively promotes the synthesis of exogenous yolk, thereby accelerating the development and maturation of the portunus trituberculatus ovary.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Sequence listing
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<120> miRNA analogue related to development of portunus trituberculatus ovary and application thereof
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Claims (10)
1. The miRNA analogue related to the development of the ovary of the blue crab is characterized in that the miRNA analogue is one or more of let-7-5p and miR-141-3 p.
2. The miRNA analogue of claim 1, wherein the miRNA analogue is used for preparing a portunus trituberculatus ovary development promoter.
3. The application of the miRNA analogue in preparing the blue crab ovary development promoter according to claim 2, wherein the ovary development promoter simultaneously comprises let-7-5p agomir and miR-141 agomir.
4. The application of the miRNA analogue in preparing a portunus trituberculatus ovary development promoter according to claim 3, wherein the sequence of the sense strand of let-7-5p agomir is as follows: AACUAUACAACCUACUACCUCAGG, the antisense chain sequence is: UGAGGUAGUAGGUUGUAUAGUUUU are provided.
5. The application of the miRNA analogue in preparation of the blue crab ovary development promoter according to claim 3, wherein the sense strand sequence of the miR-141 agomir is as follows: UAACACUGUCUGGUAAAGAUGGGG, the antisense strand sequence is: CCAUCUUUACCAGACAGUGUUAUU are provided.
6. The application of the miRNA analogue in preparing the portunus trituberculatus ovary development promoter according to claim 3, wherein the ovary development promoter is prepared and used by the following steps: synthesizing let-7-5p agomir and miR-141 agomir, mixing the two to prepare the portunus trituberculatus ovary development promoter, and directly injecting the ovary development promoter into the portunus trituberculatus female crab through the basal part of the third step.
7. The application of the miRNA analogue in preparation of the blue crab ovary development promoter according to claim 3, wherein the miRNA analogue is used for reducing the regulation of the blue crab ovary developmentJHE-like CXE-1AndJHE-like CXE-2gene expression and upregulationvtgGene expression to accelerate the development and maturation of the portunus trituberculatus ovary.
8. Use of the miRNA analog of claim 1 for the preparation of inhibitors of methyl farnesyl ester decomposition pathway key enzymes in crustaceans.
9. The use of an miRNA analogue of claim 8 for preparing an inhibitor of a methyl farnesyl ester decomposition pathway key enzyme in a crustacean, wherein the methyl farnesyl ester decomposition pathway key enzyme is JHE-like CXE-1 and JHE-like CXE-2.
10. Use of the miRNA analog of claim 1 for producing a yolk synthesis promoter in crustaceans.
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| XIANLIANG MENG等: "Integrative Proteomic and MicroRNA Analysis: Insights Into Mechanisms of Eyestalk Ablation-Induced Ovarian Maturation in the Swimming Crab Portunus trituberculatus", 《FRONT ENDOCRINOL》 * |
| 史莉莉: "中华绒螯蟹miRNA的分离及在卵母细胞成熟过程中的表达分析", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
| 张小辉: "MiRNA及其合成通路相关基因在三疣梭子蟹性腺发育过程中的功能分析", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
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