CN116656841A - Cynoglossus semilaevis oocyte specific molecular marker circEPH6, primer, vector and application - Google Patents

Abstract

The invention discloses a cynoglossus semilaevis oocyte specific molecular marker circEPH6, a primer, a vector and application thereof, and belongs to the technical field of molecular biology. The nucleotide sequence of the molecular marker circEPH6 is shown as SEQ ID NO. 1. The invention also provides a detection primer and a probe for detecting the molecular marker circEPHA 6; the invention also provides an over-expression vector expressed by the molecular marker circEPHA6, and the over-expression vector is transfected in a cell line of cynoglossus semilaevis testis cells, so that the ovum can be promoted to generate the expression change of the related gene Attesk 1. The invention provides the specific molecular marker of the oocyte for researching the sex reversal mechanism of the cynoglossus semilaevis, and simultaneously the transfected over-expression vector can obviously promote the expression quantity of the circEPHA6, and the over-expression efficiency of an experimental group can be improved by 500 percent, so that the differentiation of ovarian cells can be promoted.

Description

Cynoglossus semilaevis oocyte specific molecular marker circEPH6, primer, vector and application

Technical Field

The invention belongs to the technical field of molecular biology, and particularly designs a cynoglossus semilaevis oocyte specific molecular marker circEPHA6, a primer, a vector and application.

Background

Functional molecular markers for oocyte development are helpful for researching reproductive mechanism and fertility of experimental objects, have been widely applied in research in the fields of mammal and clinical medicine research, in recent years, oocyte molecular markers which are identified and analyzed in fish are increasingly increased, including slbp2, figla and the like, but the molecular markers are more genes and are easily degraded by ribonuclease in external environment, so that more stable molecular markers are necessary to be explored for researching fish reproductive process.

Circular RNA (circRNA) is a covalently closed circular RNA molecule, and has the characteristics of universality, conservation, tissue specificity and high stable expression. The circRNA can play a role in regulation and control at the aspects of transcription, post-transcription, translation, epigenetic and the like, and is involved in physiological processes such as cell proliferation, cell differentiation, cell migration, apoptosis and the like. In recent years, the research finds that the circRNA has close relation with the biological ovum generation process and plays an important role in the oocyte maturation process. In view of the nature of circRNA and its relevance to egg production, it is expected to be a marker in the egg production process.

Cynoglossus semilaevis (Cynoglossus semilaevis) is an important sea water economic fish in China, and has delicious meat quality, rich nutrition and higher economic value. The breeding of the cynoglossus semilaevis and the occurrence process of the ovum thereof are inseparable, and the quantity and quality of the ovum can directly influence the quality of artificial breeding and the breeding yield, so that the development of the cynoglossus semilaevis oocyte specific molecular marker is of great significance to the promotion of the breeding of the cynoglossus semilaevis and the research of the development and maturation process of the ovary. In addition, the cynoglossus semilaevis has obvious sex two-state, female individuals grow fast, the meat quality is good, the economic value is higher than that of male fishes, and the economic benefit of full female cultivation can be greatly improved. Therefore, the specific molecular marker of the ovarian cells is mined, and the method has important significance for sex control of fishes, research on gonad development and maturation mechanism and cultivation application.

Disclosure of Invention

In order to achieve the above purpose, the invention adopts the technical scheme that:

the cynoglossus semilaevis oocyte specific molecular marker circEPHA6 gene has the nucleotide sequence shown in SEQ ID No. 1.

The invention also provides a primer for detecting the molecular marker circEPHA6 gene, wherein the primer sequences are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively.

The invention provides a method for detecting a molecular marker circEPH6 gene by using the primer, wherein the detection method adopts reverse transcription PCR and a real-time quantitative method for detection; the primers in the real-time quantitative PCR are back-to-back primers SEQ ID NO.2 and SEQ ID NO.3; the reverse transcription PCR primers are linear amplification primers SEQ ID NO.4 and SEQ ID NO.5.

A cynoglossus semilaevis oocyte specific molecular marker circEPHA6 gene probe has the nucleotide sequence shown in SEQ ID No. 6. Which is used for specific labeling and identification of oocytes.

The invention also provides an application of the probe in identifying oocytes, wherein the application method is to carry out fluorescence in situ hybridization by using the probe.

An over-expression vector for promoting cynoglossus semilaevis oocyte specific molecular marker circEPHA6 gene, wherein the vector contains a nucleotide sequence shown as SEQ ID NO. 1.

The invention also provides a preparation method of the over-expression vector, which comprises the following specific steps:

(1) Extracting total RNA of the ovary of cynoglossus semilaevis, and performing reverse transcription to obtain cDNA;

(2) The cDNA is used as a template, specific upstream and downstream primers SEQ ID NO.7 and SEQ ID NO.8 are used for carrying out PCR amplification on the circEPHA6 gene sequence, the restriction enzymes BamH1 and Not1 are utilized for carrying out double enzyme digestion on the target fragment and pcDNA3.1 (+) CircRNAmini Vector carrier, and T4 ligase is utilized for connecting the target fragment and the carrier after enzyme digestion.

Nucleotide sequence of forward primer SEQ ID NO. 7: 5' -cgggatcccgtgcattccaggcttctac-3’；

Nucleotide sequence of reverse primer SEQ ID NO. 8: 5' -gccggccgcgccctgatcagttctgacg-3', the BamH1 and Not1 cleavage site sequences are underlined, respectively.

The invention also provides an application of the over-expression vector in promoting the differentiation of the cynoglossus semilaevis oocyte, and the application method comprises the following steps: the over-expression vector is transfected in a cell line of cynoglossus semilaevis testis cells.

The invention also provides a detection method for over-expression of the molecular marker circEPHA6 gene, after the over-expression vector expressed by the circEPHA6 is transfected into a cell line of the cynoglossus semilaevis testis cell for 48 hours, the cynoglossus semilaevis testis cell is collected, RNA in the cynoglossus semilaevis testis cell is extracted, after the RNA is inverted into cDNA, fluorescence quantitative PCR is adopted to detect the expression change of the related gene Attesk1 of the ovum, and an Attesk1 fluorescence quantitative PCR upstream primer and an Attesk1 downstream primer are SEQ ID NO.9 and SEQ ID NO.10 respectively.

Compared with the prior art, the invention has the beneficial effects that: according to the invention, the Cynoglossus semilaevis ovary is cloned to obtain the circEPHA6 for the first time, the specific probe is obtained according to the cDNA sequence of the Cynoglossus semilaevis ovary, and the oocyte can be specifically marked by analyzing through a fluorescence in situ hybridization method, so that the Cynoglossus semilaevis ovary can be used for identifying the oocyte. The invention further discloses an application method for promoting the expression of the annular RNA and directly influencing the development of the oocyte by over-expressing the circEPHA6, and the method has the effects that after the cell is transfected, the expression quantity of the circEPHA6 gene can be obviously promoted by the over-expression vector transfected by the invention through quantitative gene expression change evaluation, the over-expression efficiency of an experimental group can be improved by 500%, and the differentiation of the ovarian cell can be further promoted;

the invention provides a specific molecular marker of oocyte for researching a sex reversal mechanism of cynoglossus semilaevis, and lays a foundation for subsequent research.

Drawings

FIG. 1 is a diagram showing a method for identifying circEPHA6 according to example 1 of the present invention. A is the position analysis of the circeph6 in the epha6 gene; b is a gel electrophoresis chart of PCR amplified products of the linear amplification primers on the ovary cDNA and DNA templates;

FIG. 2 is a graph showing comparison of the expression of the circEPHA6 of example 1 in the ovaries of various tissues and different developmental stages of Cynoglossus semilaevis; wherein A is the expression condition of cynoglossus semilaevis circEPHA6 in different tissues; b is the expression condition of cynoglossus semilaevis circlepha 6 in ovaries at different development stages;

FIG. 3 is a fluorescence in situ hybridization chart of the circEPHA6 gene provided in example 2 of the present invention in the ovary; bar,50 μm;

FIG. 4 is a schematic diagram of the construction of the circEPHA6 overexpression and the efficiency after the overexpression, which are provided in example 3 of the present invention;

FIG. 5 is an expression cassette of the gene Attesk1 related to ovum development after the overexpression of circEPHA6 provided in example 3 of the present invention.

Detailed Description

The following description of specific embodiments of the present invention is further illustrated in the accompanying drawings and examples, it being understood that the description of the embodiments herein is presented for purposes of illustration and explanation only, and is not intended to limit the scope of the invention.

Example 1 identification and expression of Cynoglossus semilaevis circreph 6 sequence: the embodiment is a cDNA of a cynoglossus semilaevis oocyte specific molecular marker circEPHA6 and a probe thereof, which are used for specific marking and identifying oocytes; the cynoglossus semilaevis oocyte specific gene circEPHA6 is overexpressed, so that the expression of genes related to ovum generation can be promoted, the oocyte development is affected, and further, the evaluation index after the circular RNA overexpression is promoted is obtained. The method comprises the following specific steps:

1. extracting total RNA of 10 tissues of cynoglossus semilaevis: taking female and male cynoglossus semilaevis of 6 months old, separating out 10 tissues of testis, ovary, brain, heart, kidney, liver, muscle, spleen, intestine and stomach, quick-freezing with liquid nitrogen, and storing in a refrigerator at-80 ℃ for later use. Extracting total RNA of 10 tissues of cynoglossus semilaevis by adopting a Trizol (Invitrogen) reagent method, detecting the quality of the RNA by ultraviolet and agarose electrophoresis, and storing in a refrigerator at-80 ℃ for standby.

2. Extracting the cynoglossus semilaevis ovary tissue DNA: the method comprises the steps of taking the ovary tissue of cynoglossus semilaevis of 6 months old, extracting the ovary tissue DNA of cynoglossus semilaevis by using a root plant genome DNA extraction kit (DP 305), detecting the DNA quality by agarose electrophoresis, and storing in a refrigerator at-20 ℃ for standby.

3. Identifying the Cynoglossus semilaevis circreph 6 sequence: the cynoglossus semilaevis circle EPHA6 sequence is SEQ ID NO.1, and the specific sequence is as follows. Then designing a pair of back-to-back primers and a linear amplification primer according to the sequences, wherein the upstream and downstream sequences of the back-to-back primers are SEQ ID NO.2 (cctacgccggaaacatcaag), SEQ ID NO.3 (ggcaggtgtcattcaggttg) and the upstream and downstream sequences of the linear amplification primers are SEQ ID NO.4 (cgtgcattccaggcttctac) and SEQ ID NO.5 (cgccctgatcagttctgacg), respectively;

SEQ ID NO.1 sequence:

cgtgcattccaggcttctacaaggcctacgccggaaacatcaagtgttccaaatgccctcctcacagcttcagttacgctgaaggatcgtccatctgccactgtgagaaaggcttctacagggcaaggaaagatccgcccaccatggcctgtacacgccctccgtctccacctcgtaacctgatgttcaacctgaatgacacctgcctgatgctggagtggtcgccacccagcgacaccggggggcgccgggacctcacttacaacgtgcagtgcaagcgctgcggtcctgagcccaaccactgccagctgtgcgaggaggacctgcgcttcctgccacggccactgggcctaaccaacgccacggtcaccgtcacagacttcacagcgcacgccaactacaccttcgagatcgagtcgctcaacggcgtgtcgaacttaagctccttccctcgccaggtggctgccatcaccgtcagaactgatcagggcg。

4. obtaining a reverse transcription PCR cDNA template: (1) The above ovarian RNAs were split in two, one of which was digested with RNase R (Epicentre) and the other served as a control. RNase R digested group was added with 2. Mu.L of 10X RNase R Reaction Buffer, 1. Mu.L of RNase R (20U/. Mu.L), (undigested group was added with 1. Mu.L of DEPC water), and digested at 37℃for 30min. After digestion, the mixture was purified and recovered using an RNA purification column kit (Qiangen);

(2) Reverse transcription reactions were performed using a reverse transcription reaction kit (Takara), all reactions were performed on ice; cDNA was synthesized using PrimeScript RT regent kit (TAKARA) reverse transcription kit, reverse transcriptionThe system is as follows: 1 μg total RNA,4 μl 5× PrimeScript Buffer 2,4 μl Random6 primer,1 μ LPrimeScript RT Enzyme Mix I, ddH ₂ 0 was added to 20. Mu.L. The reverse transcription procedure is: storing at 37℃for 15min,85℃for 5s and 4 ℃. Diluting the obtained cDNA in a ratio of 1:10, and storing at-20 ℃ for later use;

the obtained ovary cDNA and DNA template are amplified by PCR with the back-to-back primers and the linear amplification primers, and the back-to-back primers can amplify bands by cDNA but not bands by gDNA. Whereas for linear amplification primers, both cDNA and gDNA can be amplified to give bands, see B in FIG. 1. The obtained product is detected by 1% agarose gel electrophoresis, and the target band is cut and recovered and sent to a sequencing company for sequencing. The cDNA and trans-circular RNA primer cleavage site sequences of full-length circEPH6 were obtained as shown in SEQ ID NO.1 and A in FIG. 1.

5. Expression of Cynoglossus semilaevis circreph 6 in 10 tissues: inverting the RNA of 10 tissues of cynoglossus semilaevis in the step 1 into cDNA, detecting the expression condition of each tissue by using the circumflex 6 back-to-back primer, wherein the detection shows that the expression quantity in the ovary is highest, and then, some tissues in the brain, the kidney and the liver are expressed, and other tissues are expressed in a low way, such as A in figure 2.

Expression of cynoglossus semilaevis circephala 6 in ovaries at different developmental stages: the method is characterized in that the Trizol (Invitrogen) reagent method is used for extracting the tongue sole ovary RNA in different periods, the fluorescent quantitative PCR method is used for detecting the expression condition of the cireph 6 in the ovary in different development stages, and the result shows that the expression quantity of the cireph 6 is continuously increased in the ovarian maturation process, as shown in figure 2B.

Example 2 preparation of Cynoglossus semilaevis Cynoglossus cirrhosa 6 fluorescent probe in situ hybridization with ovary tissue according to the full length sequence of Cynoglossus semilaevis Cynoglossus, the sequences of 50bp each at the front and rear ends of the sheared sequence of Cynoglossus semilaevis is intercepted respectively to obtain a cyclized template SEQ ID NO.11, the specific sequence is as follows:

ctccttccctcgccaggtggctgccatcaccgtcagaactgatcagggcgcgtgcattccaggcttctacaaggcc tacgccggaaacatcaagtgttcc；

selecting a sequence of 20-25bp of the cyclized site, reversely complementing the sequence with the template, marking cy3 at the 5 'end with GC content of 40-60%, and obtaining a probe sequence of 5' cy3-tggaatgcacgcgccctgatcagtt. Oligonucleotide probes were synthesized by Shanghai Biotechnology Inc.

Treatment of cynoglossus semilaevis ovary tissue: fresh ovarian tissue 1cm was taken ³ Immediately left and right, the mixture was put into 4% paraformaldehyde and fixed at 4℃overnight. Paraffin sections are carried out on the fixed ovaries, and continuous transverse sections are carried out through dehydration of ethanol, transparency of xylene and paraffin embedding, wherein the thickness of the sections is 5-6 mu m.

Fluorescent in situ hybridization buffer was purchased from Shanghai Ji Ma pharmaceutical technologies, inc., the hybridization procedure was as follows:

1. dewaxing

1) Preheating paraffin sections in a 60 ℃ oven for 30min until paraffin melts;

2) Immersing the slices in xylene I, II for 10min each (recommended to be carried out in a dye vat);

3) Incubation in gradient alcohol (100%, 95%, 90%, 80%, 70%) for 10min each at room temperature (recommended in the dye vat);

4) The sections were washed twice with PBS for 2min each.

2. Proteinase K digestion

1) The proteinase K working solution is preheated to 37 ℃;

2) Dropwise adding 100 mu L of proteinase K working solution into each slice, and incubating for 20min at 37 ℃;

3) 100 μl 2 XSSC was added dropwise to each slice, and the slices were rinsed 3 times at room temperature for 1min each time;

4) Gradient alcohol 70%, 80%, 90%, 100% dehydrated for 2min each time, and air dried.

3. Denaturation (denaturation)

1) Preheating the denatured liquid at 78 ℃;

2) Each slice is dripped with 100 mu L of preheated denaturation liquid and incubated for 8min at 78 ℃;

3) Gradient alcohol 70%, 80%, 90%, 100% dehydrated for 2min each time, and air dried.

4. Hybridization

1) Incubating the hybridization buffer solution in a water bath kettle at 73 ℃ for 30min in advance until the hybridization buffer solution is clear and transparent;

2) Diluting the probe with DEPC water;

3) Preparing 8 mu M probe and hybridization mixture, and denaturing at 73 ℃ for 5min;

4) Preparing a wet box, horizontally placing slices, dropwise adding 100 mu L of denatured probe mixed solution into each slice, covering a cover glass, and sealing with sealing glue;

5) The mixture was placed in an in situ hybridization apparatus and incubated at 37℃for 12-16h, taking care to keep the humidity so as to prevent the plate from drying.

5. Washing after hybridization

1) Preheating the washing liquid at 43 ℃;

2) Gently removing the cover glass, removing the hybridization solution, and dripping 100 mu l of preheated washing solution into each slice for 15min;

3) Each slice was added dropwise with 2 XSSC 100. Mu.L (preheated to 37 ℃), washed 2 times for 10min each time;

4) The sections were washed 1 time with PBS for 10min.

6. Nuclear staining

1) 100 mu L of diluted DAPI working solution is added to each slice, and the slices are incubated for 20min at room temperature in a dark place;

2) Sucking DAPI working solution, washing the slices with PBS for 2 times, and 2min each time;

3) Dropping anti-quenching agent, covering with cover glass, sealing with sealing glue, and observing under fluorescent microscope.

FIG. 3 shows the localization of cynoglossus semilaevis circEPHA6 at different developmental stages of the oocyte.

Example 3 Cynoglossus semilaevis circreph 6 overexpression vector construction and functional study: the preparation method of the overexpression vector for the expression of the circEPH6 comprises the following steps: (1) Extracting total RNA of the ovary of cynoglossus semilaevis, and performing reverse transcription to obtain cDNA; (2) The cDNA is used as a template, and a specific upstream primer and a specific downstream primer are used as SEQ ID NO.7 (5' -cgggatcccgtgcattccaggcttctac-3 ') and SEQ ID NO.8 (5'-gccggccgcgccctgatcagttctgacg-3') the PCR amplification of the circEPH6 sequence SEQ ID NO.1 of example 1 was performed using restriction enzymes BamH1 and Not1 (NEB) to double cleave the fragment of interest and pcDNA3.1 (+) CircRNAmini vector vector (FIG. 4), and the cleaved fragment of interest and vector were ligated using T4 ligase.

Cynoglossus semilaevis testis cells are cultured by adopting a complete culture medium containing 20% FBS and 1% double antibody Leibovitz's L-15, and the cell culture is carried out at 24 ℃.

When the cell confluence reaches 90%, cell transfection experiments are carried out, and the cell transfection flow is as follows:

(1) 24h before transfection, 5X 10 cells were inoculated per well in 6-well plates ⁵ The cell density of each cell before transfection reaches 80 to 90 percent.

(2) Medium replacement: after 24h, the medium was replaced with replacement medium (Opti-MEMI medium).

(3) Preparing a transfection solution: A. diluting 3.0. Mu.g of the circEPHA6 over-expression plasmid with 250. Mu.l of Opti-MEMI low serum medium, and gently mixing; B. lipofectamine 2000 (Invitrogen) was gently shaken before use, and then 8. Mu.l of transfection reagent was diluted in 250. Mu.l of Opti-MEMI medium and incubated for 5min at room temperature; C. mixing the DNA diluted in the first two steps with transfection reagent (total volume 500. Mu.l), gently mixing, and standing at room temperature for 20min (turbidity of the solution can occur);

(4) Transfection: adding 100 mu l of transfection solution into each hole of cells, gently shaking, and culturing in a 24 ℃ incubator;

(5) Liquid replacement: cell liquid exchange is carried out after 4-6 h of transfection, and 2ml of complete culture medium is used for each hole;

(6) And (3) detection: cells were collected 48h after transfection to detect expression of the gene of interest. Total RNA was extracted from the cells collected from the 6-well plate by TRIzol method, reverse transcribed, and then qRT-PCR was performed to examine the expression level of circEPHA6, thereby verifying the overexpression efficiency.

FIG. 4 is a graph of the provided circEPHA6 overexpression construct and the efficiency after overexpression, and the circEPHA6 expression was significantly up-regulated after transfection of the circEPHA6 overexpression vector.

The functional research method of the CircEPH6 comprises the steps of transfecting a Cynoglossus semilaevis Gunther testis cell line with a circEPHA6 over-expression vector, collecting cells after 48h transfection, extracting RNA in the testis cells, reversing into cDNA, detecting the expression change of a related gene Attesk1 generated by ovum by adopting fluorescent quantitative PCR, determining the action of the annular RNA on oocyte development, wherein an Attesk1 fluorescent quantitative upstream primer and an Attesk1 downstream primer are respectively SEQ ID NO.9 (cgggatcccgtgcattccaggcttctac-3 ') and SEQ ID NO.10 (5' -gccggccgcgccct)gatcagttctgacg-3’)。

The above examples demonstrate that overexpression of circEPHA6 significantly promotes expression of the ovum-genesis related gene Attesk1, as shown in fig. 5.

The invention is not limited to the specific embodiments described above, which are intended only to illustrate the use of the invention in detail, but also to the production method and the technical details of the same. Indeed, those skilled in the art will be able to find different adjustment schemes as needed from the foregoing description, and such adjustments are within the scope of the claims appended hereto.

Claims (8)

1. The cynoglossus semilaevis oocyte specific molecular marker circEPHA6 is characterized in that the nucleotide sequence of the molecular marker circEPH6 is shown as SEQ ID NO. 1.

2. The primer for amplifying the cynoglossus semilaevis oocyte specific molecular marker circEPHA6 as set forth in claim 1, wherein the primer sequences are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively.

3. The method for detecting the cynoglossus semilaevis oocyte specific molecular marker circEPH6 by using the primer of claim 2, which is characterized by adopting a reverse transcription PCR and real-time quantitative method; the primers in the real-time quantitative PCR are back-to-back primers SEQ ID NO.2 and SEQ ID NO.3; the reverse transcription PCR primers are linear amplification primers SEQ ID NO.4 and SEQ ID NO.5.

4. The probe for detecting the cynoglossus semilaevis oocyte specific molecular marker circEPHA6 is characterized in that the nucleotide sequence of the probe is shown as SEQ ID NO.6, and the probe can detect the cynoglossus semilaevis oocyte specific molecular marker circEPHA6 through fluorescence in situ hybridization.

5. An over-expression vector for promoting the expression of a cynoglossus semilaevis oocyte specific molecular marker circEPHA6, which is characterized in that the vector contains a nucleotide sequence shown as SEQ ID NO. 1.

6. The method for preparing the over-expression vector according to claim 5, which comprises the following specific steps:

(1) Extracting total RNA of the ovary of cynoglossus semilaevis, and performing reverse transcription to obtain cDNA;

(2) PCR amplification of the circumEPHA 6 sequence is carried out by taking cDNA as a template and specific upstream and downstream primers SEQ ID NO.7 and SEQ ID NO.8, the target fragment and pcDNA3.1 (+) circumRNAminivector are cut by using restriction enzymes BamH1 and Not1, and the target fragment and the vector after enzyme cutting are connected by using T4 ligase.

7. The use of the over-expression vector of claim 5 for promoting the differentiation of the oocyte of cynoglossus semilaevis, wherein the application method is as follows: transfection of the over-expression vector of claim 5 in a cell line of cynoglossus semilaevis testis cells.

8. The method for detecting the over-expression of the molecular marker CircEPHA6 as claimed in claim 1, which is characterized in that after the over-expression vector expressed by the CircEPHA6 as claimed in claim 5 is transfected in a Cynoglossus semilaevis testis cell line for 48 hours, cynoglossus semilaevis testis cells are collected, RNA in the testis cells is extracted and inverted into cDNA, and then fluorescence quantitative PCR is adopted to detect the expression change of the ovum generation related gene Attesk1, wherein the Attesk1 fluorescence quantitative PCR upstream primer and the Attesk1 downstream primer are SEQ ID NO.9 and SEQ ID NO.9 respectively

NO.10。