CN114107301A - Application of miR-9 in preparation of portunus trituberculatus ovary development promoter - Google Patents
Application of miR-9 in preparation of portunus trituberculatus ovary development promoter Download PDFInfo
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Abstract
The invention discloses application of miR-9 in preparation of a portunus trituberculatus ovary development promoter. The ovarian development promoter comprises miR-9 agomir. After the miR-9 agomir is synthesized, the miR-9 agomir is injected into female portunus trituberculatus through the basal part of a third step, namely, the gene of the forkhead box protein L2 can be reducedfoxl2Expression and upregulation of ovarian maturation marker genesvtgThe method and the portunus trituberculatus ovary development promoter have small side effect on parents, are efficient and reliable, and are suitable for accelerating the maturity of a large number of parents.
Description
Technical Field
The invention belongs to the field of aquaculture biotechnology, and particularly relates to application of miR-9 in preparation of a portunus trituberculatus ovary development promoter.
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 provides application of miR-9 in preparation of a portunus trituberculatus ovary development promoter. Proved by experiments, the miR-9 pairfoxl2The gene has good inhibition targeting effect, and further promotes the maturity of the portunus trituberculatus ovary.
In order to realize the purpose of the invention, the invention adopts the following technical scheme to realize:
the invention provides application of miR-9 in preparation of a portunus trituberculatus ovary development promoter.
Further, the ovarian development promoter comprises miR-9 agomir.
Further, the sense strand sequence of the miR-9 agomir is as follows: UCUUUGGUUAUCUAGCUGUAGG, the antisense strand sequence is: UACAGCUAGAUAACCAAAGAUU are provided.
Further, the preparation and use method of the ovarian development promoter comprises the following steps: after the miR-9 agomir is synthesized, the miR-9 agomir is directly injected into the female portunus trituberculatus through the basal part of the third step.
Further, the miR-9 is regulated downfoxl2Gene expression level and Up-regulationvtgThe gene expression level to achieve the effect of promoting the development of the portunus trituberculatus ovary.
The invention also provides application of the miR-9 in preparation of a forkhead box protein L2 inhibitor in crustaceans.
Further, the forkhead box protein L2 inhibitor comprises miR-9 agomir.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the experimental verification of the invention firstly proves that the miR-9 can be combined with Portunus trituberculatusfoxl23' UTR binding and by downregulationfoxl2Up-regulation of gene expression level to ovarian maturation marker genevtgThe expression quantity achieves the effect of promoting the development and maturity of the portunus trituberculatus ovary.
2. Compared with the common method for stimulating the maturation of crustacean ovaries to cut eye stems in production, the method for promoting the development of the blue crab ovaries by using miR-9 agomir has small side effect on parents, and can not cause tissue damage or death of the individuals by only injecting a trace non-toxic solution at the foot basement membrane through a needle head with the diameter of 0.5 mm. 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 miR-9 andfoxl2prediction of 3' UTR binding.
FIG. 2 shows the relative activities of firefly luciferase and Renilla luciferase after co-transfection of different sets of miRNA mics and plasmids.
FIG. 3 shows the expression of miR-9 in the ovary of Portunus trituberculatus after injection of different agomirs.
FIG. 4 shows that after miR-9 agomir is injected,foxl2expression in the ovary of Portunus trituberculatus.
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
The forkhead box protein L2 (Foxl 2) plays an important role in the development of crustacean ovaries and can obviously inhibit the development of the ovaries.
Prediction of targeting by bioinformatics methodsfoxl2miRNA of gene 3' UTR, the screening standard is as follows:
(1) in the presence of miRNAfoxl2The 3 'UTR of the gene is not mismatched with a seed region (2 nt to 8 nt at the 5' end);
(2) MiRNA andfoxl2the 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 miR-9 can be combined with Portunus trituberculatusfoxl2The 3' UTR was bound with a free energy of-23.7 kcal/mol.
Example 2
By means of double-luciferase reporter gene experiment, miR-9 pairs of portunus trituberculatus are verified at cell levelfoxl2The regulation and control function of (1).
The experiment sets the combination of four miRNA mimics and plasmids, which are respectively:
(1) miRNA mimics negative control and pmirGLO-foxl2-3' UTR-wild type;
(2) miR-9 mimics and pmirGLO-foxl2-3' UTR-mutant; wherein the sense strand of the miR-9 mimics is as follows: UCUUUGGUUAUCUAGCUGUA (SEQ ID No. 1); the antisense strand is: UACAGCUAGAUAACCAAAGA (SEQ ID No. 2);
(3) miR-9 mimics and pmirGLO-foxl2-3' UTR-wild type;
(4) miRNA mimics negative control and pmirGLO-foxl2-3' 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, wherein miR-9 mimics and pmirGLO-foxl2The relative activity of the firefly luciferase and the renilla luciferase in the-3' UTR-wild type co-transfection group is obviously lower than that of other groups, and the primary confirmation of miR-9 mimics on Portunus trituberculatus is thatfoxl2Has inhibitory effect.
Example 3
Through injection experiments, the in vivo level verification of the synthetic miR-9 agomir (double-chain, positive-sense chain sequence is UCUUUGGUUAUCUAGCUGUAGG (SEQ ID No. 3), antisense chain sequence is UACAGCUAGAUAACCAAAGAUU (SEQ ID No. 4), cholesterol modification is carried out on the 3 'end of the antisense chain, two thio-skeletons are modified on the 5' end, four thio-skeletons are modified on the 3 'end, and full-chain methoxy modification is carried out on the 3' end) on the blue crabfoxl2Regulating and controlling function of the compound and promoting function of ovary development.
The miR-9 agomir is injected into the female portunus trituberculatus through the base of the third step by using a 1ml syringe (the diameter of a needle is 0.5 mm). After 48 h of injection, the crabs were dissected to collect ovarian tissues. Extracting miRNA of ovarian tissues by using a miRcute miRNA extraction and separation kit, carrying out reverse transcription by using a miRcute enhanced miRNA cDNA first strand synthesis kit, and analyzing the expression quantity of miR-9 by using a miRcute enhanced miRNA fluorescent quantitative detection kit.
The reaction system (20. mu.L) was: 10 uL of 2 × miRcute Plus miRNA PreMix, 0.4 uL of forward primer (sequence: GCCTCTTTGGTTATCTAGCTGTAT, SEQ ID No. 5), 0.4 uL of reverse primer (kit belt), 1 uL of miRNA first strand cDNA, and 8.2 uL of DEPC water.
The reaction procedure is as follows: 15 s at 95 ℃; 95 ℃ 20 s, 60 ℃ 34 s, 40 cycles.
Extracting total RNA of the portunus trituberculatus ovary tissue by using Trizol reagent, carrying out reverse transcription by adopting Evo M-MLV RT Kit with gDNA Clean for qPCR II Kit, and using AG SYBR Green Pro Taq HS qPCR reagentCassette to carry outfoxl2Andvtgand (4) analyzing gene expression.
The qPCR reaction system (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.
qPCR amplification primers:
foxl2the forward primer sequence is: CGTTGTCCTGATCTCACTGC (SEQ ID No. 6), the reverse primer sequence is: CGTCTTTCATCGTGCCGTAG (SEQ ID No. 7).
vtgThe forward primer sequence is: AGCGGCATCATCTCTTCAGT (SEQ ID No. 8), the reverse primer sequence is: AATGCGGGAGATGGTATCTG (SEQ ID No. 9).
The qPCR reaction procedure was: 30 s at 95 ℃; 95 ℃ for 5 s, 60 ℃ for 34 s, 40 cycles.
miR-9、foxl2、vtgRelative expression amounts are 2–ΔΔCtThe method is used for calculation, independent sample t test is carried out through SPSS 20.0 statistical analysis software, and whether different groups of data have significant difference or not is determined so as toPA significant difference was found to be < 0.05.
The results are shown in figures 3 and 4, compared with the control group, the abundance of miR-9 in the injection agomir group is obviously increased,foxl2marked gene with obviously down-regulated gene expression and ovary maturityvtgThe gene expression level is obviously up-regulated. Therefore, the miR-9 agomir injection can remarkably improve the abundance of the miRNA in the blue crab body and inhibitfoxl2The gene is expressed, thereby promoting the ovary to develop and mature. The results prove that the injected miR-9 can effectively promote the development and maturity 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.
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Claims (7)
1. Application of miR-9 in preparation of portunus trituberculatus ovary development promoter.
2. The application of the miR-9 in preparation of the portunus trituberculatus ovary development promoter according to claim 1, wherein the ovary development promoter comprises miR-9 agomir.
3. The application of the miR-9 in preparation of the portunus trituberculatus ovary development promoter according to claim 2, wherein the sense chain sequence of the miR-9 agomir is as follows: UCUUUGGUUAUCUAGCUGUAGG, the antisense strand sequence is: UACAGCUAGAUAACCAAAGAUU are provided.
4. The application of the miR-9 in the preparation of the portunus trituberculatus ovary development promoter according to claim 2, wherein the preparation and use methods of the ovary development promoter are as follows: after the miR-9 agomir is synthesized, the miR-9 agomir is directly injected into the female portunus trituberculatus through the basal part of the third step.
5. The application of miR-9 in preparation of portunus trituberculatus ovary development promoter according to claim 1, wherein miR-9 is used for reducing the ovarian development promoter of portunus trituberculatus by down-regulatingfoxl2Gene expression level and Up-regulationvtgThe gene expression level to achieve the effect of promoting the development of the portunus trituberculatus ovary.
Application of miR-9 in preparation of a forkhead box protein L2 inhibitor in crustaceans.
7. The use of miR-9 in the preparation of an inhibitor of forkhead box protein L2 in crustaceans according to claim 6, wherein miR-9 agomir is comprised in said inhibitor of forkhead box protein L2.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110130435A1 (en) * | 2009-12-02 | 2011-06-02 | Ying-Nan Chen | Time-released medication for applying to crustacean ovarian development |
| CN110760545A (en) * | 2019-10-31 | 2020-02-07 | 福建农林大学 | miRNA-based shrimp and crab ovary development promoter |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110130435A1 (en) * | 2009-12-02 | 2011-06-02 | Ying-Nan Chen | Time-released medication for applying to crustacean ovarian development |
| CN110760545A (en) * | 2019-10-31 | 2020-02-07 | 福建农林大学 | miRNA-based shrimp and crab ovary development promoter |
Non-Patent Citations (5)
| Title |
|---|
| MINGCAN ZHOU ET AL.: "miR-9 and miR-263 Regulate the Key Genes of the ERK Pathway in the Ovary of Mud Crab Scylla paramamosain", 《MARINE BIOTECHNOLOGY》, vol. 22, pages 594 - 606 * |
| XIANLIANG MENG ET AL.: "Identification and comparative profiling of ovarian and testicular microRNAs in the swimming crab Portunus trituberculatus", 《GENE》, vol. 640, pages 6 - 13 * |
| 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", 《中国知网硕士电子期刊》, no. 2, pages 1 - 68 * |
| 张梦倩等: "三疣梭子蟹（Portunus trituberculatus）foxl2 基因功能 初探及相关miRNA 分析", 《海洋学报》, vol. 44, no. 4, pages 85 - 94 * |
| 郭梁等: "水产虾蟹microRNA 组学研究进展", 《生物资源》, vol. 42, no. 1, pages 67 - 76 * |
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