CN112626236A

CN112626236A - CircRNA marker related to cow mammary epithelial cell apoptosis and application thereof

Info

Publication number: CN112626236A
Application number: CN202110049883.5A
Authority: CN
Inventors: 孙加节; 汪东阳; 张永亮; 习欠云; 陈婷; 罗君谊; 赵正江
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-01-14
Filing date: 2021-01-14
Publication date: 2021-04-09

Abstract

The invention discloses a circRNA marker related to milk cow mammary epithelial cell apoptosis and application thereof. The circRNA marker is circbEZH2, and the sequence of the circRNA marker is shown in SEQ ID NO: 1 is shown. The invention verifies whether circbEZH2 is looped and the over-expression vector plays a role in detecting the anti-apoptosis process of the mammary gland cells of the dairy cow, and discovers that the over-expression of circbEZH2 can inhibit the apoptosis of the mammary gland epithelial cells of the dairy cow by transfecting the over-expression vector plasmid of circbEZH2 into the mammary gland epithelial cells of the dairy cow, thereby providing a new thought for further analyzing the genetic mechanism of the growth and development of the mammary gland cells of the dairy cow, bringing deeper cognition for the molecular mechanism of the cyclic RNA for regulating and controlling the lactation traits of the dairy cow, being applied to the research of the molecular regulation and control mechanism of the heat stress resistance of livestock and having better application prospect; has important significance for researching milk secretion regulation and controlling of dairy cows and improving the quality of the dairy cows.

Description

CircRNA marker related to cow mammary epithelial cell apoptosis and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a circRNA marker related to milk cow mammary epithelial cell apoptosis and application thereof.

Background

With the generation of greenhouse effect and global warming, heat stress becomes one of the most important problems in the process of producing and breeding dairy cows. At present, the dairy cows in China are mainly Holstein dairy cows from the Netherlands, and belong to typical cold-resistant and heat-resistant animals. The heat-resistant milk cow is not heat-resistant in high-temperature weather, and is very easy to generate heat stress in summer, especially in the south of China. According to research, the lactation yield of the dairy cows is reduced by 20-45% under the condition of heat stress, which causes great economic loss to the dairy cows in China.

Milk, one of the best foods for humans, has a very high nutritional value, and contains not only most nutrients, but also a good source of many essential amino acids in the human body. However, as the heat stress of the cow occurs, the milk quality (milk protein, milk fat, lactose) is significantly reduced. With the increasing demand of consumers for milk quality, there is a strong need to improve the influence of heat stress on milk. Therefore, on the level of the mammary tissue cells of the dairy cow, the relationship between the heat stress and the lactation related characters is analyzed, and the mechanism of controlling the lactation by the excellent character related genes is discovered, so that the method is necessary and important for supplementing and perfecting the current dairy cow lactation theory and production practice.

With the advent of the "genome era", analysis of expression of trait-related genes at the gene level is one of the important research directions in dairy cow lactation at present. Circular RNA (circRNA) is one of non-coding RNAs, and plays an important role in the growth and development process of a living body as a hot spot for further research on an RNA family following micro RNA (miRNA) and long-chain non-coding RNA (lncRNA). Circular RNA (circRNA) can be used as a sponge of miRNA to contact the inhibition effect of miRNA on target genes. In recent years, there is increasing evidence that circRNA plays a key role in many physiological and pathological processes, including cell differentiation and apoptosis, growth and development, etc., where heat stress has been found to affect the biosynthesis of circRNA.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide a circRNA marker related to the apoptosis of the mammary epithelial cells of the dairy cow.

The invention also aims to provide application of the circRNA marker.

The invention provides a cyclic RNA of a dairy cow, which is named as circbEZH2 and provides a detection method and functional application of circbEZH2, and provides a method for identifying and constructing an over-expression vector of the cyclic RNA circbEZH2 of the dairy cow, which thoroughly verifies whether the cyclic RNA circbEZH2 of the dairy cow forms a ring or not and the function of the over-expression vector in the process of detecting the anti-apoptosis of the mammary gland cells of the dairy cow, and finds that the over-expression vector plasmid of the cyclic RNA circbEZH2 of the dairy cow transfects the mammary gland epithelial cells of the dairy cow and compares the over-expression vector plasmid with a blank control group to find that the over-expression cyclic RNA circbEZH2 of the dairy cow inhibits the apoptosis of the mammary gland epithelial cells of the dairy cow, so that the newly discovered cyclic RNA of the dairy cow can further analyze the genetic mechanism of the growth and development of the mammary gland cells of the dairy cow and provide a new thought, and has important significance for researching the genetic essence of the dairy cow during the heat stress lactation.

The purpose of the invention is realized by the following technical scheme:

the invention provides a circRNA marker related to milk cow mammary epithelial cell apoptosis, wherein the circRNA marker is circbEZH2, and the nucleotide sequence of the circRNA marker is shown as SEQ ID NO: 1 is shown. The circRNA marker is formed by circularizing 3 rd, 4 th, 5 th, 6 th, 7 th and 8 th exons of a cow EZH2 gene. The circbEZH2 has high expression in mammary gland tissue of a warm-fitting group milk cow, can influence milk secretion of the milk cow, and is applied to improve the milk secretion process of the milk cow under the condition of heat stress.

The further improvement lies in that: the primers for detecting the circRNA marker comprise an upstream primer (PF1) and a downstream primer (PR1), the primers for detecting the EZH2mRNA of the dairy cow comprise an upstream primer (PF2) and a downstream primer (PR2), and the nucleotide sequence of the upstream primer (PF1) is shown as SEQ ID NO:2, the nucleotide sequence of the downstream primer (PR1) is shown as SEQ ID NO: 3, the nucleotide sequence of the upstream primer (PF2) is shown as SEQ ID NO: 4, the nucleotide sequence of the downstream primer (PR2) is shown as SEQ ID NO: 5, the nucleotide sequence of the linker sequence of the circRNA marker is shown as SEQ ID NO: and 6.

The invention also provides an application of the circRNA marker in a dairy cow heat stress genetic marker and a dairy cow lactation process under the condition of improving dairy cow heat stress. In particular to application in preparing a heat stress genetic marker of a milk cow and a kit for improving the lactation process of the milk cow under the heat stress condition.

Further, the circRNA marker is applied to regulation and control of milk cow mammary epithelial cell apoptosis.

Preferably, the primer of the circRNA marker is applied to the genetic marker of heat stress of the dairy cow and the improvement of the lactation process of the dairy cow under the condition of heat stress of the dairy cow. In particular to application in preparing a heat stress genetic marker of a milk cow and a kit for improving the lactation process of the milk cow under the heat stress condition.

The experiment of the invention proves that the over-expression of the circRNA marker can effectively inhibit the apoptosis of the mammary epithelial cells of the dairy cow.

An overexpression vector of a circRNA marker, comprising SEQ ID NO: 1.

Further, SEQ ID NO: 1 is inserted between KpnI and BamHI.

The starting vector of the over-expression vector is pCD2.1-ciR and the like.

After the overexpression vector of the circRNA marker is transfected into mammary epithelial cells of a dairy cow, the apoptosis of the mammary epithelial cells can be inhibited, and the expression vector can be used for genetic marking of the dairy cow under heat stress and improving the lactation process of the dairy cow under the heat stress condition.

Further, the overexpression vector of the circRNA marker is applied to inhibiting the apoptosis of the mammary epithelial cells of the dairy cattle.

The invention also provides a kit for detecting the circRNA marker, which contains a primer of the circRNA marker.

The kit is applied to the quantitative detection of circbEZH2 in the mammary tissue of the milk cow.

The kit is suitable for all types of fluorescent quantitative gene amplification instruments on the market at present, has high sensitivity, quick and accurate quantification and good stability, and has good application prospect.

The invention also provides a method for detecting the circRNA marker, which comprises the following steps:

1) extracting total RNA of the mammary tissue of the milk cow;

2) digesting the total RNA by RNase R enzyme and obtaining cDNA through reverse transcription;

3) carrying out amplification detection on the cDNA obtained in the step 2) on a fluorescent real-time quantitative PCR instrument;

4) analysis by dissolution Curve, 2^-ΔΔCtThe method is used for relative quantification.

Compared with the prior art, the invention has the following advantages and effects:

(1) according to the full-length sequence of the circular RNA circbEZH2 of the dairy cow, the designed specific primers (shown as SEQ ID NO:2 and 3) are used for identifying whether the circular RNA is correctly cyclized or not and analyzing the relative expression quantity, the regulation and control effect of the circbEZH2 on the anti-apoptosis of the mammary epithelial cells of the dairy cow is researched, the PCR conditions required for quantitative analysis of the circular RNA circbEZH2 of the dairy cow are provided, and the primers and the method can be used for accurately detecting whether the circbEZH2 is cyclized or not and the expression level thereof.

(2) The overexpression vector of the circular RNA circbEZH2 of the dairy cow can stably express circbEZH2, and by transfecting mammary gland epithelial cells of the dairy cow, circbEZH2 is found for the first time to inhibit apoptosis of the mammary gland epithelial cells, so that a new thought is provided for further analyzing the genetic mechanism of growth and development of the mammary gland cells of the dairy cow, deeper cognition is brought to the molecular mechanism of the circular RNA for regulating and controlling the lactation character of the dairy cow, the overexpression vector is applied to the research of the molecular regulation and control mechanism of the heat stress resistance of livestock, and the application prospect is good; has important significance for researching cow lactation regulation and control.

Drawings

FIG. 1 is a schematic structural diagram of a circular RNA circbEZH2 of a cow in an embodiment of the invention.

FIG. 2 is an agarose gel electrophoresis of the expression product of the circular RNA circbEZH2 of the cow in the example of the present invention; wherein the content of the first and second substances,

shows the results of the upstream primer (PF1) and the downstream primer (PR1) of circbEZH 2;

results for the upstream primer (PF2) and the downstream primer (PR2) of the EZH2mRNA are shown.

FIG. 3 is a sequence peak diagram of the cyclization sites in the linker sequence of the circular RNA circbEZH2 of dairy cows in the example of the invention.

FIG. 4 is a diagram showing the relative expression levels of the cyclic RNA circbEZH2 of the cow after digestion with RNase R enzyme in the example of the present invention.

FIG. 5 is a structural map of circular RNA overexpression vector Pcd 2.1-ciR.

FIG. 6 is a graph showing the relative expression levels of circular RNA circbEZH2 after overexpression.

FIG. 7 is a western blot of proteins for inhibiting the apoptosis of the mammary gland cells of a cow after the overexpression of the circular RNA circbEZH2 of the cow in the embodiment of the invention.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

The cows used in the examples were Holstein cows, which are disclosed in the literature "determination of enzymatic activity in serums of Areca Jiang buffalo, Mora buffalo and Holstein cows [ J ]. Heilongjiang animal veterinarian 2016(5): 212-.

Example 1

A method for identifying the circular RNA of a cow comprises the following steps:

the method comprises the following steps: selecting healthy cows with moderate temperature in winter (12 months in 2018) and heat stress in summer (8 months in 2019) as study objects in a certain large dairy farm in Guangdong; mammary tissue of 3 cows is respectively selected in two periods, the tissue specimen is quickly frozen in liquid nitrogen for 30 seconds after being taken out, then the tissue specimen is stored in a refrigerator at the temperature of-80 ℃, one part of the tissue specimen is used for extracting gDNA (whole genome DNA) and total RNA, and the other part of the tissue specimen is attached with dry ice and sent to a sequencing company for circRNA sequencing.

Step two: extracting gDNA of the mammary tissue of the milk cow by using the Shanghai biological tissue gDNA extraction kit, specifically operating according to the kit specification, and storing the extracted gDNA in a refrigerator at the temperature of-80 ℃ for later use; extracting total RNA of the mammary tissue of the milk cow by using an RNA extraction kit (Trizol method), specifically operating according to the instruction of an Invitrogen kit, storing the extracted RNA in a refrigerator at-80 ℃ for later use, and preliminarily detecting the integrity of the total RNA by using 1.2% agarose gel electrophoresis; agilent 2100(Agilent Technologies, Santa Clara, Calif., USA) further detects the quality and solubility of total RNA; after the total RNA is qualified, the RNA is digested by RNase R enzyme and then is immediately subjected to reverse transcription to obtain cDNA, and a reverse transcription Kit (PrimeScript RT reagent Kit with gDNA Eraser) of Dalibao biology company is used for the reverse transcription. The operation steps are carried out according to the kit instructions, and the cDNA after reverse transcription is placed in a refrigerator at the temperature of minus 80 ℃ for later use.

Step three: according to the sequencing result of the milk cow mammary tissue, firstly, FastQC (http:// www.bioi informatics, babraham, ac, uk/projects/FastQC /) software is used for integrally evaluating the sequencing data quality, wherein the sequencing data quality comprises the mass value distribution of basic groups, the position distribution of mass values, GC content, PCR amplification content and the like; secondly, performing circRNA identification analysis on the circRNA-seq sequencing data by adopting the international recognized algorithms CIRC explorer2 (http:// circexplorer2. readthetdocs. io/en/late /), circRNA _ finder (https:// omictols. com/circrnawick-tool), CIRI (https:// sourceform. net/project /), find _ circle (https:// githu. com/orzechoj/circRNA _ finder) and pSnet (http:// w. lab. uky. edu/p/bioinfofo/pSPnice 2); then adopting an internationally recognized algorithm edgeR (h ttp:// www.bioconductor.org/packages/release/bioc/html/edgeR. html) to carry out differential screening, and finally selecting circbEZH2 with extremely obvious differential expression through analysis; finally, according to the transcriptome sequencing result and the cow gene information in the NCBI database, the full-length sequence of the circular RNA circbEZH2 of the cow is found to be formed by cyclization of

exons

3, 4, 5, 6, 7 and 8 on the cow EZH2 gene, as shown in figure 1, the nucleotide sequence is shown as SEQ ID NO: 1, the nucleotide sequence of the joint sequence of the circular RNA circbEZH2 of the dairy cow is shown as SEQ ID NO: and 6. The circbEZH2 is highly expressed in the breast tissue of the moderate-temperature dairy cow in winter.

The full-length sequence of circbEZH2 is used as a template, Primer sequences capable of identifying circular RNAs circbEZH2 and EZH2mRNA of the dairy cow are designed by using Primer5 software, and the sequences are as follows:

sequence of the upstream primer (PF 1): 5'-caacatagatggaccaaacgcc-3' (SEQ ID NO: 2)

Sequence of the downstream primer (PR 1): 5'-cgcatgtactctgactttacgc-3' (SEQ ID NO: 3)

Sequence of the upstream primer (PF 2): 5'-aatgctcttggtcagtacaatg-3' (SEQ ID NO: 4)

Sequence of the downstream primer (PR 2): 5'-tcgactctcctctagatctttct-3' (SEQ ID NO: 5)

Quantitative analysis is carried out on the circular RNA circbEZH2 and EZH2mRNA of the dairy cow by using an upstream primer (PF1) and a downstream primer (PR1) of circbEZH2, an upstream primer (PF2) and a downstream primer (PR2) of EZH2mRNA, and gDNA and cDNA obtained in the step two as amplification templates and adopting a qRT-PCR strategy, wherein the lengths of the products are 188bp and 96bp respectively;

the qRT-PCR reaction system is as follows: 10 mu L of Genstar SYBR GreenSupermix, 0.5 mu L of upstream primer, 0.5 mu L of downstream primer, 2 mu L of DNA template to be detected,ddH₂o7 uL, 20 μ L overall;

the qRT-PCR reaction conditions were: 94 ℃ for 3 min; 94 ℃, 10s, 60 ℃, 10s, 72 ℃, 30s, 40 cycles; 95 ℃, 10s, 62 ℃, 60s, 97 ℃, 1s, 1 cycle.

Taking 10 mu L of amplification products in the qRT-PCR reaction, adding 1 mu L of 10 loading buffer, mixing uniformly, spotting and carrying out electrophoresis on 2% agarose gel, taking 7 mu L of DNA standard molecular weight DL 2000Marker as reference, carrying out electrophoresis at 15V/cm, observing results in a gel imaging system after the electrophoresis is finished, taking pictures for storage, sending 10 mu L of amplification products in the remaining qRT-PCR reaction to Shanghai biological engineering Co., Ltd for sequencing, obtaining real base information of a linker sequence by a Sanger sequencing technology, carrying out comparative analysis on the sequencing result and a linker region of the cyclic RNA circeZH 2, finding that head and tail bases of the cyclic RNA circeZH 2 of the dairy cow are connected together to form a cyclization site, indicating that the cyclic RNA circeZH 2 of the dairy cow is correct, and indicating that the cyclization result is shown in figure 3.

Step four, exporting data after the qRT-PCR reaction in the step three is finished, and utilizing 2^-ΔΔCtThe method was analyzed and the results of the analysis were plotted using Graphad Prism software, as shown in FIG. 4, which shows that RNase R enzyme can digest linear RNA but circbEZH2 is not digested, further verifying the circularity of circbEZH 2.

Example 2

The construction process of the overexpression vector of the circular RNA circbEZH2 of the dairy cow is as follows:

the vector Pcd2.1-ciR empty vector (commercially available) is an engineering vector specially used for circular RNA expression, is marked as empty vector, contains a circular RNA expression framework as shown in figure 5, and is subjected to amplification by escherichia coli DH5 alpha, and then an endotoxin plasmid miniprep kit is used for extracting vector DNA, wherein the specific steps are shown in the kit specification.

The amplification process of the full-length sequence of the CircbEZH2 is as follows: primers capable of carrying out full-length amplification are designed by using a Primer5 software by taking a full-length sequence of circbEZH2 as a template, wherein KpnI and BamH I enzyme cutting sites and protective bases are respectively introduced to the 5' end, and the sequences are shown as follows:

sequence of the upstream primer (PF 3): 5'-ggggtacctgaaatatgctatcttacagaataatcatgggccagact gg-3' (SEQ ID NO: 7)

Sequence of the downstream primer (PR 3): 5'-cgggatcctcaagaaaaaatatattcacgatgtaggaagcagtcata ct-3' (SEQ ID NO: 8)

A primer pair is formed by using a designed upstream primer (PF3) and a designed downstream primer (PR3), and the full-length sequence of the circbEZH2 is amplified by using the cDNA of the dairy cow as a template.

The PCR reaction system is shown in the following table:

components	Volume (μ L)
		PCR Mix	25
10 μ M upstream primer (PF3)	1
		10 μ M downstream primer (PR3)	1
DNA template	50～200ng
		DEPC water	Supplement to 50

Double enzyme digestion process of vector and amplification product: pcd2.1-ciR empty vector plasmid and circbEZH2 full-length sequence amplification products were double-digested with restriction enzymes KpnI and BamHI, respectively.

The double digestion reaction system is shown in the following table:

components	Volume (μ L)
		KpnI enzyme	1
BamH I enzyme	1
		Buffer	2.5
DNA template	1μg
		DEPC water	Supplement to 50

Performing enzyme digestion on the system at 37 ℃ for two hours, performing electrophoresis on the enzyme digestion product in 2% agarose gel, and recovering the enzyme digestion product by adopting a column-passing centrifugation mode;

the PCR product of the empty vector plasmid pCD2.1-ciR and the full-length sequence amplification product circbEZH2, which were purified after digestion, were ligated with T4 ligase at 16 ℃ for 12 hours.

Adding 10 mu L of the ligation product into 50 mu L of DH5a competent cells under aseptic conditions, uniformly mixing, standing in an ice bath for 30min, thermally shocking at 42 ℃ for 90s, immediately placing on ice for 3min, adding 400 mu L of LB liquid culture medium without Amp antibiotics, placing on a constant-temperature shaking table at 37 ℃ for 45min, centrifuging at low speed in an environment of <5000g, reserving 150 mu L of supernatant, discarding the rest, blowing the rest supernatant, uniformly mixing, uniformly coating on a flat plate containing 100mg/L of Amp, flatly placing in a constant-temperature incubator at 37 ℃ for 1h, and then carrying out inverted culture.

Picking single colony with normal shape on the plate by using a small gun head, placing the single colony in a 1.5mL Ependorffi tube containing 400 mu L LB liquid culture medium, carrying out shaking table culture at the constant temperature of 37 ℃ for 8h, taking 1 mu L of bacterial liquid as a template for PCR amplification, detecting the PCR amplification product by agarose gel electrophoresis with the concentration of 2%, and taking a picture and recording in a gel imaging system.

And (3) carrying out PCR detection on the bacterial liquid, wherein the result is positive, the bacterial liquid containing the recombinant plasmid is sent to Shanghai biological engineering Co., Ltd for sequencing, carrying out amplification culture on the bacterial liquid with correct sequencing, and then extracting the vector DNA by using an endotoxin-removing plasmid small-amount extraction kit. The recombinant plasmid was designated as OE-circbEZH 2.

Example 3

The mammary epithelial cells of the dairy cattle are MAC-T cell lines, and the cell transfection process comprises the following steps:

the method comprises the following steps: 1 day before transfection, 5 x 10⁴Inoculating each cell into a 12-hole plate, and continuously culturing until the cell density reaches 80%;

step two: mu.g of plasmid (recombinant plasmid OE-circbEZH2 or empty vector in example 2) transfected per well was added to 125. mu.L of Opti-MEM medium and mixed, 2. mu.L of Lipofectamine 2000 was added to 125. mu.L of Opti-MEM medium and mixed, and the mixture was allowed to stand at room temperature for 5 min;

step three: uniformly mixing the two mixed solutions in the step two, and standing for 20min at room temperature;

step four: absorbing the original cell culture solution in each hole, and washing twice by using an Opti-MEM culture medium;

step five: adding 250 mu L of the mixed solution obtained in the third step into each hole of cells, and supplementing the mixed solution to 1000 mu L by using Opti-MEM culture medium;

step six: standing at 37 deg.C for 5% CO₂And (5) culturing in a cell culture box, and waiting for subsequent treatment.

After 48h, the cells are collected and subjected to qRT-PCR detection and western blot detection, the result of the circbEZH2 overexpression qRT-PCR is shown in figure 6, and the circbEZH2 is obviously overexpressed. Protein results show that after circbEZH2 is over-expressed, the expression level of apoptosis proteins (Bcl-2 and Bax) is remarkably reduced, and the expression level of anti-apoptosis protein Bcl-2 is remarkably increased, as shown in figure 7; wherein, Tubulin is an internal reference protein; therefore, the overexpression of circbEZH2 can inhibit the apoptosis of the mammary epithelial cells of the dairy cows. In conclusion, the circular RNA circbEZH2 vector of the dairy cow can stably over-express circbEZH2, and can also be used for detecting the apoptosis condition in mammary epithelial cells of the dairy cow.

According to the full-length sequence of the circular RNA circbEZH2 of the dairy cow, the designed specific primer is used for identifying whether the circular RNA is correctly looped or not, analyzing the relative expression quantity, researching the regulation and control effect of the circbEZH2 on the apoptosis of mammary epithelial cells of the dairy cow, providing PCR conditions required by quantitative analysis of the circular RNA of the dairy cow, and by using the primer and the method, whether the circbEZH2 in mammary tissues is looped or not and the expression condition of the circbEZH can be accurately detected.

The foregoing illustrates and describes the principles, essential features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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<120> circRNA marker related to dairy cow mammary epithelial cell apoptosis and application thereof

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Claims

1. A circRNA marker associated with apoptosis of mammary epithelial cells in a dairy cow, wherein:

the circRNA marker is circbEZH2, and the nucleotide sequence of the circRNA marker is shown in SEQ ID NO: 1 is shown.

2. A detection primer for the circRNA marker as claimed in claim 1.

3. The detection primer according to claim 2, wherein: the detection primer is shown as SEQ ID NO:2 to 3.

4. Use of the circRNA marker of claim 1 or the detection primer of any one of claims 2 to 3, characterized in that: the application is any one of the following:

the use of the circRNA marker of claim 1 or the detection primer of any one of claims 2 to 3 for genetic markers of heat stress in dairy cows and for improving the lactation process of dairy cows under the condition of heat stress in dairy cows;

use of the circRNA marker of claim 1 or the detection primer of any one of claims 2 to 3 in the preparation of a genetic marker for heat stress in dairy cows and a kit for improving the lactation process of dairy cows under heat stress conditions.

5. The use of the circRNA marker of claim 1 for modulating apoptosis in mammary epithelial cells of a dairy cow.

6. An overexpression vector of a circRNA marker, characterized in that: comprises the amino acid sequence shown in SEQ ID NO: 1.

7. Use of the overexpression vector of the circRNA marker according to claim 6, characterized in that: the application is any one of the following:

the over-expression vector of the circRNA marker is applied to inhibiting the milk cow mammary epithelial cell apoptosis;

the overexpression vector of the circRNA marker is applied to the genetic marker of heat stress of the dairy cow and the improvement of the lactation process of the dairy cow under the condition of heat stress of the dairy cow.

8. A kit for detecting the circRNA marker of claim 1, characterized by comprising a detection primer for the circRNA marker of claim 2 or 3.

9. Use of the kit according to claim 8 for the quantitative detection of circbEZH2 in milk cow breast tissue.

10. A method of detecting the circRNA marker of claim 1, characterized in that: the method comprises the following steps:

1) extracting total RNA of the mammary tissue of the milk cow;