CN115281151B - Application of miR-34b/c gene inhibitor in regulation and control of early sexual differentiation of Acipenser schrenki - Google Patents
Application of miR-34b/c gene inhibitor in regulation and control of early sexual differentiation of Acipenser schrenki Download PDFInfo
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Abstract
The invention discloses application of a miR-34b/c gene inhibitor in regulation and control of early sexual differentiation of Acipenser sinensis. The invention discloses a novel method for regulating and controlling early sex differentiation of sturgeon for the first time, namely, a method of injecting miR-34b/c inhibitor into multiple-site gonads in early sex differentiation sensitive period of sturgeon is adopted, the proportion of early sex differentiation of sturgeon to male direction is increased, and male fish with relatively high growth speed is provided for sturgeon culture industry.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a miR-34b/c gene inhibitor in regulation and control of early sexual differentiation of Acipenser schrenki.
Background
Sturgeon is an ancient fish, living on the earth for 2 hundred million years in the same time as dinosaur, and has high economic value and scientific research value. The sturgeon fish is nutritious and superior, is precious throughout the body, and is a 'good' in fish; the most important economic added value of sturgeon is represented on caviar made of sturgeon roe, which is rich in essential amino acids, various unsaturated fatty acids, vitamins and other substances of human body, has extremely high nutritive value and enjoys the reputation of black gold.
Acipenser sinensis (Acipenser schrenckii) is a special rare and precious large-scale economic fish of the black longriver system in China. The artificial breeding of the Acipenser schrenki starts in the 30 th 19 th century, and has the advantages of high growth speed, strong disease resistance, strong adaptability and the like, and has become one of important breeding varieties in the sturgeon breeding industry for producing hybrid parents and caviar in the sturgeon breeding in the 90 th century.
In the cultivation of Acipenser schrenki, if sex screening can be carried out in the early stage of sex differentiation, male fish with higher growth speed can be made into commercial fish to be marketed in advance, and excellent female Acipenser schrenki is selected to be remained as caviar for production, so that higher economic benefit can be obtained. Therefore, a new breeding technology for regulating and controlling the sex differentiation direction of sturgeons becomes a key attention field for sustainable development of the current sturgeon culture industry. The gonad development of the sturgeon at 5 months after hatching enters an active period, the sturgeon at 6 months is in a sex differentiation sensitive period, and then the initial female-male different reproductive epithelium characteristics begin to appear at 7 months, the female is in a rugosity shape, the male is in a smooth reproductive epithelium shape, and according to statistics, 75.86% of individuals in the sturgeon population appear in the initial sex differentiation characteristics at 8 months. The intervention from the sex differentiation sensitive period of sturgeons provides theoretical possibility for regulating and controlling early sex differentiation of sturgeons.
Disclosure of Invention
The invention aims to invent a new breeding technology and a method for regulating and controlling early sexual differentiation of Acipenser sinensis.
The first object of the invention is to provide the application of miR-34b/c gene inhibitor in regulation of early sexual differentiation of Acipenser sinensis.
Preferably, the application of the miR-34b/c gene inhibitor in regulating the differentiation of early-stage sex of Acipenser sinensis towards the male direction is provided.
Preferably, the sequence of miR-34b is as follows: UAGGCAGUGUUGUUAGCUGAUUG; the sequence of miR-34c is as follows: AGGCAGUGCAGUUAGUUGAUUAC.
Preferably, the gene inhibitor is at least one of siRNA, shRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular compounds, peptides or antibodies.
A second object of the present invention is to provide a method for controlling early sexual differentiation of sturgeon, comprising the steps of: the miR-34b/c gene inhibitor is injected into the Acipenser sinensis in the sex differentiation sensitive period.
Preferably, the Acipenser sinensis in the sex differentiation sensitive period refers to Acipenser sinensis 6 months after hatching.
Preferably, the injection is from between the caudal fin plates 2-3 and between 5-6 using a two-sided multi-site injection.
Preferably, the miR-34b/c gene inhibitor is injected, specifically: the miR-34b gene inhibitor and the miR-34c gene inhibitor are alternately injected, 8nmol of each time is injected, the injection is carried out twice a week, the interval between the two injections is 3-4 days, and the injection is carried out for three weeks.
A third object of the present invention is to provide the use of the above method for controlling the early sex differentiation of Acipenser sinensis into the male direction.
The miR-34b/c is remarkably and highly expressed in the early sexual differentiation process of the Acipenser sinensis, is specifically expressed in gonad germ cells and participates in germ cell fate regulation, and further, in the early sexual differentiation sensitive period (6 months old) of the Acipenser sinensis, a method for injecting miR-34b/c inhibitor into multiple loci of gonads is established, so that the proportion of the gonads of the Acipenser sinensis to the male direction is remarkably increased, the operation method is simple and easy to operate, the effect is remarkable, a novel target gene and a breeding method are provided for the early sexual differentiation regulation technology of the Acipenser sinensis, and the sustainable and green healthy development of the Acipenser sinensis cultivation industry can be promoted and supported.
The invention provides a novel target gene and a breeding method for sturgeon early sexual differentiation regulation technology. Compared with the prior art, the invention firstly discloses a novel method for regulating and controlling early sex differentiation of sturgeon, namely, in the early sex differentiation sensitive period of sturgeon, a multi-site gonad injection miR-34b/c inhibitor method is adopted, the proportion of early sex differentiation of sturgeon towards the male direction is increased, and male fish with relatively high growth speed is provided for the sturgeon culture industry.
Drawings
FIG. 1 is a graph showing the expression changes of miR-34b and miR-34c in the early sexual differentiation course of Acipenser sinensis.
FIG. 2 is an in situ hybridization technique to detect miR-34c expression in 36 month old ovaries and spermary.
FIG. 3 is a graph showing the detection of the proliferation of the germ cells of Acipenser schrenki by the overexpression or the inhibition of miR-34 b/c.
FIG. 4 is an apoptosis assay of germline cells of Acipenser schrenki by miR-34b and miR-34c overexpression.
FIG. 5 is a miR-34b/c agonist or inhibitor gonadal injection site.
FIG. 6 shows the female (F) and male (M) differentiation of Acipenser schrenki in the miR-34 b/c-excited group (J) or in the inhibited group (Y).
FIG. 7 is the change in estradiol and 11K-testosterone in serum before and after gonadal injection of miR-34b/c agonists and inhibitors.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
Example 1
1. Expression characteristics of miR-34b/c in early sexual differentiation process of Acipenser schrenki.
The Real-time RT-PCR method was used to detect the changes in the spatial and temporal expression of miR-34b (SEQ ID NO: 5'-UAGGCAGUGUUGUUAGCUGAU UG-3') and miR-34c (SEQ ID NO: 5'-AGGCAGUGCAGUUAGUUGAUUAC-3') in the early sexual differentiation process of Acipenser sinensis (comprising four stages, namely, 5 month old undifferentiated gonad UGs (before sexual differentiation) and differentiated (after sexual differentiation) 12 month old, 24 month old and 36 month old gonads). Extracting total RNA of gonadal tissue of four development stages of sex differentiation process of Acipenser sinensis by RNAiso reagent (TaKaRa, tokyo, japan), detecting total RNA concentration and purity by 1.5% gel electrophoresis and ultraviolet spectrometer, and collecting qualified total RNA sample (clear 28S and 18S rRNA bands and OD 260/280)>1.8 Further used in Real-time PCR experiments. 200ng of total RNA per sample was used for reverse transcription first strand cDNA synthesis with specific miR-34b and miR-34c specific stem-loop primers, respectively. The reaction procedure: 42 ℃,60 min and 70 ℃ for 15min. Real-time PCR at the Applied Biosystems Quant-Studio TM 5 platform (Thermo Fisher Scientific) execution, 20 μl reaction system comprising: 0.5. Mu.L cDNA, 0.5. Mu.L upstream and downstream primer (10. Mu.M), 10. Mu.LGreen Master Mix (Applied Biosystems, carlsbad, USA), 8.5. Mu.L ultrapure water make-up system. The reaction procedure: 50 ℃, activated-able universal-DNA glycylase (UDG) for 2min followed by 95 ℃ for 2min; after that (95 ℃,15s,60 ℃,30s and 72 ℃,30 s). Times.40 cycles. The data analysis adopts a relative quantitative analysis method, namely 2 -△△CT U6 snRNA is used as an internal reference gene, and undifferentiated gonad is used as a UGs reference sample.
The results show that the expression patterns of miR-34b and miR-34c are similar, the expression level is relatively very low at UGs, and then the relative expression level of gonads (testis and ovary) after sex differentiation is increased, wherein the gonad expression level is highest at 12 months of age; ovary was significantly relatively highly expressed at 12 and 24 month old testis, whereas ovary tissue was significantly highly expressed at 36 month old ovary tissue (fig. 1).
2. miR-34c is specifically expressed in gonad germ cells of Acipenser sinensis
The localization expression characteristics of miR-34c in the 36 month old testis and ovary of Acipenser sinensis are detected by utilizing a miR-34c specific digoxin probe (5'-GTAATCAACTAACTGCACTGCCT-3', 5 'and 3' end marked digoxin, marked and synthesized by Guangzhou exon biotechnology limited company) and an in situ hybridization technology. The results indicate that miR-34c is expressed predominantly in the cytoplasm of ovarian oogonial clusters (FIG. 2A), and in the nuclei of spermatids (FIG. 2B).
3. miR-34b/c promotes proliferation and reduces apoptosis of gonadal germ cells of sturgeon
miR-34b/c mimics (mimic) and inhibitors (inhibitor) are synthesized in vitro by Guangzhou Ruibo biological company, and the synthesis information is as follows: miR-34b mimic (double stranded RNA sequence, sense strand 5'-UAGGCAGUGUUGUUAGCUGAUUG-3' and antisense strand 5'-CAAUCAGCUAACAACACUGCCUA-3') and inhibitor (single stranded RNA sequence, 5'-CAAUCA GCUAACAACACUGCCUA-3'). miR-34c mic (double stranded RNA sequence, sense strand 5'-AGGCAGUGCAGUUAGUUGAUUAC-3' and antisense strand 5'-GUAAUCAACUAACUGCACUGCCU-3') and inhibitor (single stranded RNA sequence, 5'-GUAAUCAACUAACUGCACUGCCU-3').
For isolation of primary germ cells from gonads of Acipenser schrenki in sex differentiation sensitive period (5-7 months of age) and establishment of culture system, miR-34b and miR-34b are performed at cellular levelmiR-34c over-expression or interference experiments. The transfection method is mainly (1) respectively transfecting miR-34b and miR-34c mimics/inhibitors into sturgeon gonadal cells cultured in vitro on day 5, and simultaneously setting miRNA NC control, wherein the transfection experiments are divided into three groups: (1) miR-34b mimetic/inhibitor transfection; (2) miR-34c mimetic/inhibitor transfection; (3) miR-34b and miR-34c mimics/inhibitors were co-transfected while miRNA NC controls (treatment with MEM alpha broth) were set up. (4) In 96-well plates, the transfection concentration of miRNA mic or miRNA inhibitor per well was 50nM. (5) At 25 ℃,5% CO 2 After culturing in an incubator for 4-6 hours, the medium was replaced with complete medium containing serum and antibiotics (medium composition mainly based on MEM. Alpha. (Gibco Co., LOTC12571500 BT) and 10% FCS (Gibco Co., ltd., LOT 10270-106)), while adding 4 growth factors required for the renewal and maintenance of undifferentiated germ stem cells, including 10ng/mL LIF (Peprotech Co., ltd., LOT 300-05-5), 100ng/mL EGF (Peprotech Co., LOT AF-100-15-100), 20ng/mL GDNF (Peprotech Co., LOT 450-10-10), 10ng/mL bFGF (Peprotech Co., LOT 100-18C-10)), and 5% CO at 25 ℃ 2 Culturing in incubator for 48-72 hr. (6) According to cell proliferation kit CellTiterAQUEUS Non-Radioactive cell proliferation assay (MTS) (Promega Co., G5421) protocol, 2-3 h were processed and absorbance at 490nm was measured using a microplate reader (Thermofischericitic Co., model 1510-04524).
The result shows that compared with a miRNA NC control group, the miR-34b over-expression or interference has a decreasing trend on the number and activity of the gonadal germ cells of the Acipenser sinensis, but the influence is not obvious, the miR-34c over-expression obviously promotes the proliferation of the gonadal germ cells of the Acipenser sinensis, and the miR-34b and the miR-34c are over-expressed simultaneously to have a combined promotion effect; and simultaneously, after the expression of miR-34b and miR-34c is respectively inhibited, the miR-34b and miR-34c are found to have obvious inhibition effect on the in-vitro proliferation of the gonadal germ cells of the sturgeon (figure 3).
Meanwhile, at the end of miR-34b/c overexpression, the experimental cells of the overexpression and control group are digested and collected, and the apoptosis of the cells is detected according to an apoptosis detection kit Bioscience Annexin V-FITC Apoptosis kit (Invitrogen corporation, LOT BMS500 FI-100).
The results show that miR-34b, miR-34c and control groups are negative in Annexin V staining, so that miR-34b, miR-34c and control groups are indicated to be free of early apoptotic cells, PI staining shows that over-expression of miR-34c can effectively prevent apoptosis of gonadal germ cells relative to the control group (12.97% +/-3.48%) and miR-34b (11.22% +/-3.49%), and fluorescence results show that miR-34c (5.59% +/-2.40%) is significant in reducing cell numbers (P < 0.05) in middle and late stages and normal death (FIG. 4).
4. Administration of miR-34b/c agonist and inhibitor gonadal injections during sex differentiation sensitivity phase
Custom miR-34b/c agomir (miRNA agonist) and antagomir (miRNA antagonist) were synthesized by Guangzhou Ruibo biological company with the following synthesis information: miR-34b agamir (double-stranded RNA sequence, sense strand 5'-UAGGCAGUGUUGUUA GCUGAUUG-3' and antisense strand 5'-CAAUCAGCUAACAACACUGCCUA-3') and antagomir (single-stranded RNA sequence, 5'-CAAUCAGCUAACAACACUGCCUA-3'). miR-34c agomir (double-stranded RNA sequence, sense strand 5'-AGGCAGUGCAGUUAGUUGAUUAC-3' and antisense strand 5'-GUAAUCAACUAACUGCACUGCCU-3') and antagomir (single-stranded RNA sequence, 5'-GUAAUCAACUAACUGCACUGCCU-3'). The method takes the Acipenser schrenki 6 months after the sex differentiation sensitive period hatching as an experimental animal object, and the experiments are divided into three groups:
(1) miR-34b/c activation group, 10 tails;
(2) miR-34b/c inhibition group, 10 tails;
(3) saline control group, 30 tails;
gonadal injection site: because of the longer length of sturgeon gonads, a double-sided multi-site injection mode is adopted, and injection from between 2-3 and 5-6 of the caudal fin bone plates is confirmed after pre-experiments (see figure 5);
injection dose: the miR-34b agonir and miR-34c agonir are used in a crossing manner, and each time of injection is 4nmol; inhibiting the cross use of the miR-34b antagomir and miR-34c antagomir, and injecting 8nmol each time; and the control group was injected with physiological saline;
injection frequency: the injection is twice a week, and the interval between the two injections is 3-4 days, and the injection is three weeks. And (5) continuing to culture normally for five weeks after the experiment, namely, sampling and deeply analyzing the experimental fish of the Acipenser sinensis after eight weeks until the experimental fish of the Acipenser sinensis develop to the age of 8 months.
5. miR-34b/c combined inhibition significantly increases the proportion of male differentiation of Acipenser schrenki
Sex differentiation of each group was observed and counted by using the gonadal tissue HE staining method.
In the normal saline control group, 30 experimental fish: tail 11 developed into potential females (i.e., early ovarian differentiation), tail 11 developed into potential males (i.e., early testis differentiation), and 7 were still in an undifferentiated state, with 1 sample lost.
miR-34b/c agonism group: tail 5 developed into potential females (i.e., early ovarian directional differentiation) (JF in fig. 6 1 -JF 5 ) The 4 tail developed into potential male (i.e., early testis-directed differentiation) (JM in FIG. 6) 1 -JM 4 ) Also, 1 tail is still in an undifferentiated state.
miR-34b/c inhibition group: tail 2 develops into a potential female (i.e., early ovarian directional differentiation) (YF in fig. 6 1 -YF 2 ) Tail 6 develops into potential males (i.e., early testis-directed differentiation) (YM in FIG. 6 1 -YM 6 ) There are also 2 tails that are still in an undifferentiated state.
The statistics of the male and female differentiation ratios of all three groups show (Table 1), compared with the physiological saline control group (1:1) and the miR-34b/c activation group (1.25:1), the miR-34b/c inhibition group significantly increases sex differentiation in the early male direction, and the male and female sex differentiation ratio is 1:3.
TABLE 1 influence of MiR-34b/c treatment on early sexual differentiation ratio of Acipenser sinensis
| Group of | Normal control group | miR-34b/c agonism group | miR-34b/c inhibition group |
| ♀:♂ | 1:1 | 1.25:1 | 1:3 |
6. Effects of miR-34b/c treatment on concentration levels of estradiol and 11K-testosterone in serum
The serum was tested for changes in estradiol and 11K-testosterone before and after miR-34b/c agomir and antagomir gonadal injection treatment (6 months to 8 months of age). Estradiol (cat No. 501890) and 11K-testosterone (cat No. 582751) were tested using ELISA test kits available from Cayman corporation.
The results show that the estradiol levels of the control group after sexual differentiation (8 months of age), all 6 groups differentiated in ovarian direction and in spermary direction show an increasing trend compared to UGs before sexual differentiation, wherein the miR-34b/c agonistic group (miR-34 b/c agonir) and the miR-34b/c inhibitory group (miR-34 b/c antagonir) differentiated in spermary direction are significantly increased (P < 0.05) compared to UGs; in addition, miR-34b/c inhibition group (miR-34 b/c antagomir) differentiated in the testis direction was found to be in a significantly elevated mode (P < 0.05) compared with the control group, as shown in FIG. 7A.
Whereas the 11K-testosterone profile exhibited the following characteristics, see FIG. 7B: (1) before and after sex differentiation of ovarian differentiation, 11K-testosterone exhibited a pattern of decline, especially 11K-testosterone was significantly reduced (P < 0.05) in miR-34b/c inhibition group (miR-34 b/c antagomir) compared to UGs; (2) before and after sex differentiation, the control group had a significantly elevated pattern of 11K-testosterone (P < 0.05) compared to UGs; (3) after sex differentiation with testis-directed differentiation (8 months of age), 11K-testosterone of both the miR-34 b/c-excited group (miR-34 b/c agonir) and the miR-34 b/c-inhibited group (miR-34 b/c antagonir) was in a significantly reduced pattern (P < 0.05) compared to the control group, although 11K-testosterone of the miR-34 b/c-inhibited group (miR-34 b/c antagonir) was relatively higher than that of the miR-34 b/c-excited group (miR-34 b/c agonir).
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (5)
1. A method for regulating early sex differentiation of sturgeon into male direction, comprising the steps of: injecting miR-34b/c gene inhibitor into the Acipenser schrenki in the sex differentiation sensitive period; the injection adopts a double-side multi-site injection mode, namely the injection is carried out between the tail fin bone plates 2-3 and 5-6, and specifically comprises the following steps: the miR-34b gene inhibitor and the miR-34c gene inhibitor are alternately injected, 8nmol of each time is injected, the injection is carried out twice a week, the interval between the two injections is 3-4 days, and the injection is carried out for three weeks.
2. The method according to claim 1, wherein the sexually sensitive phase of sturgeon is 6 months after hatching.
3. The method of claim 1, wherein the sequence of miR-34b is:
UAGGCAGUGUUGUUAGCUGAUUG; the sequence of miR-34c is as follows: AGGCAGUGCAGUUAGUUGAUUAC.
4. The method of claim 1, wherein the gene inhibitor is at least one of siRNA, shRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, a low molecular compound, a peptide, or an antibody.
5. Use of the method according to any one of claims 1-4 for regulating early sex differentiation of sturgeon towards males.
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