CN116855614A - miRNA marker for early pregnancy diagnosis of cattle and application thereof - Google Patents
miRNA marker for early pregnancy diagnosis of cattle and application thereof Download PDFInfo
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Abstract
The invention provides a miRNA marker for yellow Niu Zaoqi pregnancy diagnosis and application thereof, and belongs to the technical field of biology, wherein the miRNA marker is selected from any one or two of bta-miR-133a and bta-miR-19b, and the early pregnancy of a cattle is rapidly diagnosed by detecting the expression level of the miRNA in the blood of the cattle tested, and when the expression level of the bta-miR-133a in the blood of the cattle tested is obviously reduced or the expression level of the bta-miR-19b is obviously increased, or the expression level of the bta-miR-133a is obviously reduced and the expression level of the bta-miR-19b is obviously increased, the cattle tested is prompted to be in the early pregnancy. Therefore, any one or two of the bta-miR-133a and the bta-miR-19b can be used as a new marker for early pregnancy diagnosis of the cattle and used for early pregnancy diagnosis of the cattle.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a miRNA marker for early pregnancy diagnosis of cattle and application thereof.
Background
mirnas are a class of small RNA molecules that are expressed in both animal and plant cells, which are evolutionarily conserved. Lee et al 1999 first found a known first miRNA in caenorhabditis elegans and named Lin-4, thus opening up a study on miRNA. mirnas are small endogenous non-coding RNAs that are processed from intergenic regions or introns of protein-coding RNAs, approximately 20-24 nucleotides in length. Studies have shown that mirnas can target post-transcriptional messenger RNAs (mrnas) through epigenetic modifications, and that in mammalian cells, the seed regions of mirnas can complementarily bind to sequences on the 3' utr of a target gene, thereby inhibiting expression and translation of the target gene. In mammals, mirnas can be involved in a number of biological processes such as immunity, cell proliferation, differentiation, organogenesis, and embryogenesis by modulating the expression of target mRNA.
mirnas have been demonstrated to regulate ovarian function, endometrial receptivity, embryonic development, and placental function, and have recently become biomarkers for early pregnancy diagnosis with high clinical potential. Jason et al determined that miR-26a is a novel candidate biomarker for early pregnancy in cattle by analyzing a plasma miRNA population associated with early pregnancy in cattle. In sheep, miR-30c, miR-132, miR-379, miR-199a-3p and mi R-320 expressed a difference in serum from gestation 30d or 60 d. Circulating miR-XX1 and mi R-XX2 in plasma can be potential biomarkers for early pregnancy detection in buffalo. There is increasing evidence that mi RNA can be considered as a potential biomarker of cow pregnancy.
However, so far, miRNA has been less studied as a marker of early pregnancy detection molecules of cattle, and pregnancy recognition and pregnancy establishment at the early stage of cattle have a decisive role for successful pregnancy and calving. Therefore, the specific miRNA biomarker related to early pregnancy of cattle is provided, and has important significance for improving the fertility of cattle groups.
Disclosure of Invention
Therefore, the invention aims to provide a miRNA marker for yellow Niu Zaoqi pregnancy diagnosis and application thereof, and provides an effective diagnostic tool for early pregnancy diagnosis of cattle.
The technical scheme adopted for solving the technical problems is as follows:
a miRNA marker for early pregnancy diagnosis of cattle, the miRNA marker selected from any one or two of bt a-miR-133a and bta-miR-19 b; the sequence of the bta-miR-133a is shown as SEQ ID NO.1, and the sequence of the bta-miR-19b is shown as SEQ ID NO. 2.
Use of a miRNA marker for early pregnancy diagnosis of cattle in the preparation of a kit for early pregnancy diagnosis of cattle, wherein the miRNA marker is selected from any one or two of bta-miR-133a and bta-miR-19 b; the sequence of the bta-miR-133a is shown as SEQ ID NO.1, and the sequence of the bta-mi R-19b is shown as SEQ ID NO. 2.
Preferably, the kit is a kit for detecting the expression level of the miRNA biomarker in blood by adopting a Real-time RT-PCR method; the expression level of the bta-miR-133a in the pregnant cattle blood is obviously reduced, and the expression level of the bta-miR-19b in the pregnant cattle blood is obviously increased.
Preferably, the kit contains primers that can specifically amplify the miRNA markers; the primer sequence of the bta-miR-133a is shown in SEQ ID NO. 3-4, and the primer sequence of the bta-miR-19b is shown in SEQ ID NO. 5-6.
Preferably, the kit contains primers for detecting the expression level of the miRNA marker, wherein the reference genes are bta-GAPDH and bta-RPS18, the primer sequences of the reference genes bta-GAPDH are shown in SEQ ID NO. 7-8, and the primer sequences of the reference genes bta-RPS18 are shown in SEQ ID NO. 9-10.
According to the technical scheme, compared with the prior art, the invention has the beneficial effects that: the invention provides a miRNA marker for early pregnancy diagnosis of a cattle and application thereof, which are based on the results of small RNA sequencing screening and qRT-RCR verification, and show that bta-miR-133a and bta-miR-19b are differentially expressed between a 0d blood sample and a 21d blood sample of the pregnant cattle, and bta-miR-133a and bta-mi R-19b are differentially expressed between the pregnant cattle blood sample and a non-pregnant cattle control group, so that the miRN A can be used as a molecular diagnostic marker for early pregnancy diagnosis of the cattle, the early pregnancy of the cattle is rapidly diagnosed by detecting the expression quantity of the miRNA in the blood of the tested cattle, and when the expression quantity of bta-mi R-133a in the blood of the tested cattle is remarkably reduced or the expression quantity of bta-miR-19b is remarkably increased, or the expression quantity of bta-miR-133a is remarkably reduced and the expression quantity of bta-miR-19b is remarkably increased, the tested cattle is prompted to be in the pregnant cattle in the early pregnancy, and an effective diagnostic tool is provided for early pregnancy diagnosis of the cattle.
Drawings
FIG. 1 shows the early pregnancy diagnosis of cattle.
Fig. 2 is a volcanic plot of differentially expressed mirnas between sample groups.
FIG. 3 is a KEGG functional assay for differentially expressing miRNA target genes.
FIG. 4 is a bar graph of miR-133a expression in pregnant Niu Renshen blood at 0 and 21 d.
FIG. 5 is a bar graph of miR-19b expression in pregnant Niu Renshen blood at 0 and 21 d.
FIG. 6 is a bar graph of miR-133a expression in pregnant cattle and non-pregnant Niu Renshen, 21d blood.
FIG. 7 is a bar graph of miR-19b expression in pregnant cattle and non-pregnant Niu Renshen, 21d blood.
Detailed Description
The technical scheme and technical effects of the embodiments of the present invention are further described in detail below with reference to the accompanying drawings.
The instrument used in this example is as follows:
CFX 96Touch fluorescence quantitative PCR instrument (Bio-Rad Co.), multifunctional enzyme-labeled instrument (Bio-Rad Co.), electrophoresis gel imager (Bio-Rad Co.), and nucleic acid gel electrophoresis instrument (Bio-Rad Co.).
Example 1 screening of miRNA-specific markers
miRNA sequencing is carried out on blood samples of cattle gestation Nos. 0, 14 and 21d, and miRNAs related to early gestation of cattle are screened out by utilizing a bioinformatic technology, and the specific steps are as follows:
1. collection of samples
1.1 synchronous estrus management
In the early stage of the experiment, firstly, 9 nonpregnant fixed original cattle are screened by utilizing B-ultrasonic detection and rectal holding technology, and after 7d, the B-ultrasonic detection is used for rechecking, and finally 9 nonpregnant experimental cattle are screened, and synchronous estrus treatment is carried out.
(1) Medicament: gonadorelin (GnRH, 100 μg/count) for veterinary injection and sodium chloroprostanol (PG, 0.5 mg/count) for veterinary injection, with lot numbers 110912326 and 110917059, respectively; physiological saline; disposable syringes, and the like.
(2) 1-7-9 synchronous estrus method for cattle: two goserelin (200 mug) are intramuscular injected into each nonpregnant experimental cow in the morning of 1d, the oestrus state of the cattle is observed at any time, and the copulation is carried out when oestrus occurs; if the cattle is not in estrus, two branches of chlorprostinil sodium injection (1 mg) are intramuscular injected in the morning of 7d, the cattle state is continuously observed, and if the cattle is not in estrus, two branches of gonadorelin (200 mug) are intramuscular injected in the afternoon of 9d, and the breeding is carried out in the morning of 10 d.
1.2 blood collection
The experimental flocks were bled at jugular vein at 0, 7, 14, 21 and 28d after mating, respectively. The method comprises the following specific steps:
(1) Taking 1 15mL centrifuge tube, adding 6mL TRizol and 2mL fresh blood (TRizol: blood=3:1), and repeatedly blowing with a pipette to completely lyse blood cells;
(2) Placing the centrifuge tube on a vortex oscillator, and violently oscillating for 1-2min to dissolve floccules completely;
(3) Standing at room temperature for 5min to decompose nucleoprotein completely;
(4) And subpackaging the obtained mixed solution into 2mL numbered freezing pipes, sealing with sealing films, and storing in a refrigerator at-80 ℃.
1.3 pregnancy diagnosis
At 28d after the experimental cattle is bred, the pregnancy diagnosis is carried out on the experimental cattle by adopting a cattle early pregnancy rapid detection card (colloidal gold method) to detect the content of the protein PAG related to the cattle pregnancy. The specific operation method is that 3 drops of serum obtained by centrifugation are placed in a small hole of a detection card, after standing for five minutes, observation is carried out, a C line represents that a serum sample is pollution-free, the result is accurate, if the T line is darker than the B line, the pregnancy of the cattle is indicated, and otherwise, the cattle is not pregnant. The results are shown in FIG. 1. And re-diagnosis was performed by a cattle farm technician through the rectum at about 60 d. And judging the gestation state of the experimental cattle by combining the two gestation detection methods.
2. Extraction of blood Total RNA
According to the result of the pregnancy diagnosis, blood samples of 3 pregnant yellow cattle in the 0 th, 14 th and 21d th periods are finally selected and sent to companies for subsequent RNA extraction and miRNA sequencing.
The total RNA extraction steps of blood are as follows:
(1) A certain amount of blood sample is sucked, and three times of TRizol is added (after TRizol is added, the sample is gently beaten by a pipetting gun without being carried out on ice);
(2) Adding 200 mu L of chloroform, covering a centrifugal tube cover, shaking vigorously for 15s, and standing at room temperature for 5min;
(3) Transferring to a refrigerated centrifuge, and centrifuging at 12000rpm and 4deg.C for 15min;
(4) After centrifugation, the mixture is divided into three layers, wherein the upper layer contains RNA, the upper layer water phase is carefully sucked into a new centrifuge tube, and the precooled isopropanol with equal volume is added;
(5) Standing at room temperature for 10min after mixing, transferring to a centrifuge, and centrifuging at 12,000rpm and 4deg.C for 15min;
(6) Carefully discarding the supernatant, adding 1mL of 75% ethanol pre-cooled by DEPC water, fully washing the tube cover and the tube wall, flicking the tube bottom, and suspending the sediment;
(7) Transferring to a centrifuge, centrifuging at 12,000rpm and 4deg.C for 5min, discarding supernatant, and centrifuging at 7500rpm for 5min;
(8) Carefully sucking off excessive ice ethanol, horizontally placing, standing at room temperature for 5min, and air drying;
(9) Adding 20 mu L RNase free water, shaking after complete dissolution, and centrifuging;
(10) Taking 2 mu L of concentration measurement, 3 mu L of running electrophoresis analysis RNA quality (gel of 0.8% -1%, electrophoresis of about 110V for 15 min), and storing the rest in a refrigerator at-80 ℃.
The integrity of the extracted RNA was checked using agarose gel electrophoresis and an Agilent 2100 bioanalyzer.
Small RNA sequencing and screening of early pregnancy detection markers
The sequencing samples included 3 samples from pregnant cattle at gestation 0d, 3 samples from gestation 14d and 3 samples from gestation 21d, totaling 9 samples using the illumine high throughput sequencing technique. Results referring to figure 2, a in figure 2 is a volcanic plot of miRNAs differentially expressed between 14d and 0d of pregnancy, analyzed to identify a total of 7 differentially expressed miRNAs between 14d and 0d of pregnancy, 1 upregulated, 6 downregulated. In FIG. 2, B is a volcanic plot of miRNAs differentially expressed at gestation 21d and gestation 0d, where 10 differentially expressed miRNAs were detected at gestation 21d, with 1 up-regulated and 9 down-regulated, compared to gestation 0 d. In all downregulated differentially expressed mirnas, the expression of bta-miR-133a was significantly downregulated (20-fold change) at gestation 21d and the expression of bta-miR-19b was significantly upregulated (2-fold) at gestation 21 d. Further, referring to fig. 3, fig. 3a shows the result of KEGG analysis of the miRNA target gene differentially expressed by 14d and 0d of pregnancy, and B shows the result of KEGG analysis of the miRNA target gene differentially expressed by 21d and 0d of pregnancy. Through KEGG analysis on the target genes of the differential expression miRNA, the target genes of the differential expression miRNA are mainly enriched on the signal paths related to early pregnancy of cattle such as actin cytoskeleton regulation, ras, PI3K-Akt signal paths, focal adhesion and the like.
In conclusion, bta-miR-133a and bta-miR-19b are screened out to be possibly used as early pregnancy detection markers of cattle.
Example 2 effect verification of miRNA markers bta-miR-133a and bta-miR-19b for early pregnancy diagnosis of cattle
2.1 design of primers
According to the miRBase database, inquiring mature sequences of differential expression miRNAs (bta-miR-133 a and bta-miR-19 b), designing a specific stem-loop structure Primer for differential expression miRNA reverse transcription by a stem-loop method, designing a qRT-RCR Primer for differential expression miRNA by using Primer Premier 5.0 software, and entrusting general biology (Anhui) stock company to synthesize the Primer. The following sequence directions are all 5 '-3'.
Mature sequences of the bta-miR-133a and the bta-miR-19b are shown in SEQ ID NO. 1-2,
SEQ ID NO.1(bta-miR-133a)：UUUGGUCCCCUUCAACCAGCUG
SEQ ID NO.2(bta-miR-19b)：UGUGCAAAUCCAUGCAAAACUGA；
the primer sequence of the miRNA marker bta-miR-133a is shown in SEQ ID NO. 3-4,
SEQ ID NO.3(bta-miR-133a-F)：CTCAGATTTGGTCCCCTTCA
SEQ ID NO.4(bta-miR-133a-R)：AGTGCAGGGTCCGAGGTATT；
the primer sequence of the miRNA marker bta-miR-19b is shown in SEQ ID NO. 5-6,
SEQ ID NO.5(bta-miR-19b-F)：GGACTCTGTGCAAATCCATGC
SEQ ID NO.6(bta-miR-19b-R)：AGTGCAGGGTCCGAGGTATT。
2.2qRT-RCR
(1) removing genomic DNA from total RNA extracted in example 1, (2) obtaining a cDNA sample by RNA reverse transcription, and (3) performing Real-time PCR using cDNA as a template. The reaction system of each step is shown in the following tables 1 to 3:
TABLE 1 genomic DNA removal reaction System
Reaction conditions: 42 ℃ for 2min.
Table 2miRNA reverse transcription reaction system
Reaction conditions: 42 ℃,15min,85 ℃,5s,4 ℃.
TABLE 3qRT-PCR reaction System
Reaction conditions: pre-denaturation at 95℃for 30s, (95℃for 5s;60℃for 30 s) 39 cycles, 95℃for 10s,65℃for 5s.
The bta-GAPDH and the bta-RPS18 are simultaneously taken as internal reference genes, and the relative expression level of differential expression miRNA is 2 -ΔΔCt And (5) calculating a method. The primer sequence of the internal reference gene bta-GAPDH is shown as SEQ ID NO. 7-8, and the primer sequence of the internal reference gene bta-RPS18 is shown as SEQ ID NO. 9-10;
SEQ ID NO.7(bta-GAPDH-F)：GGCATCGTGGAGGGACTTATG；
SEQ ID NO.8(bta-GAPDH-R)：GCCAGTGAGCTTCCCGTTGAG；
SEQ ID NO.9(bta-RPS18-F)：GTGGTGTTGAGGAAAGCAGACA；
SEQ ID NO.10(bta-RPS18-R)：TGATCACACGTTCCACCTCATC。
GAPDH and RPS18 were tested for significance of group-to-group differentially expressed mirnas by t-test analysis of GraphPad 8.3 software, with P <0.05 indicating significant differences and P <0.01 indicating very significant differences.
Results referring to fig. 4, the results of miR-133a expression in the blood samples of pregnancy Niu Renshen, 0 and 21d, show that compared with the 0d pregnancy, the expression level of bta-miR-133a in pregnant cattle, 21d pregnancy, is extremely significantly reduced (P < 0.01), indicating that the expression difference of bta-miR-133a in early pregnancy of cattle is significant at different periods, and can be used as a marker for detecting early pregnancy of cattle. Referring to fig. 5, the results of expression of miR-19b in pregnant Niu Renshen blood samples at 0 and 21d show that compared with pregnant cow blood at 21d, the expression level of bta-miR-19b in pregnant cow blood at 21d is extremely significantly up-regulated (P < 0.01), which indicates that the expression difference of bta-miR-19b in early gestation of cattle is significant at different periods, and can be used as a marker for detecting early gestation of cattle.
Further, the expression of bta-miR-133a and bta-miR-19b in blood samples of pregnant cattle and non-pregnant cattle of the 21d gestation of the cattle is detected by adopting Real-time PCR, and the results are shown in FIGS. 6-7. It can be seen that the expression of bt a-miR-133a and bta-miR-19b in the blood sample of a pregnant cow, 21d, niu Yufei, is different. Bta-miR-133a showed very significant differences in expression levels in the blood of pregnant cows with pregnancy 21d and pregnancy Niu Yufei (P < 0.01), indicating that bta-miR-133a can be used as a marker for distinguishing pregnant cows from non-pregnant cows with cattle with pregnancy 21d (FIG. 6). Likewise, the difference in expression levels of bta-miR-19b in pregnant cow blood samples of pregnancy 21d and pregnancy Niu Yufei was very significant (P < 0.01), indicating that bta-miR-19b could be used as a marker for distinguishing pregnant and non-pregnant cows at cow pregnancy 21d (FIG. 7).
In conclusion, the invention provides any one or two of bta-miR-133a and bta-miR-19b in blood for the first time as a molecular marker for detecting early pregnancy of cattle, and can be applied to the technical field of early pregnancy diagnosis of cattle. For example, the application of the miRNA marker for early pregnancy diagnosis of cattle in preparation of a kit for early pregnancy diagnosis of cattle, wherein the kit is a kit for detecting the expression level of the miRNA biomarker in blood by adopting a Real-time RT-PCR method; when the expression level of bta-miR-133a in the blood of the tested cattle is obviously reduced or the expression level of bta-miR-19b is obviously increased, or the expression level of bta-miR-133a is obviously reduced and the expression level of bta-miR-19b is obviously increased, the tested cattle is indicated to be in early gestation. The kit contains the primer of the miRNA biomarker, the internal reference primer, namely the primers of the bta-miR-133a and bta-miR-19b, and the primers of the internal reference genes bta-GAPDH and bta-RPS18, and can also select common enzymes and/or reagents required by corresponding PCR reaction according to a specific adopted experimental method, wherein the common enzymes and/or reagents are well known to a person skilled in the art. The kit has the value that the variation trend of miRNA of the tested cattle blood sample can be detected by a Real-time RT-PCR method, and then the trend is used for assisting in pregnancy diagnosis of yellow Niu Zaoqi, so that the detection is convenient.
The foregoing disclosure is illustrative of the preferred embodiments of the present invention, and is not to be construed as limiting the scope of the invention, as it is understood by those skilled in the art that all or part of the above-described embodiments may be practiced with equivalents thereof, which fall within the scope of the invention as defined by the appended claims.
Claims (5)
1. A miRNA marker for early pregnancy diagnosis of cattle, characterized in that: the miRNA marker is selected from any one or two of bta-miR-133a and bta-miR-19 b; the sequence of the bta-miR-133a is shown as SEQ ID NO.1, and the sequence of the bta-miR-19b is shown as SEQ ID NO. 2.
2. The application of miRNA markers for early pregnancy diagnosis of cattle in preparing a kit for early pregnancy diagnosis of cattle is characterized in that: the miRNA marker is selected from any one or two of bta-miR-133a and bta-miR-19 b; the sequence of the bta-miR-133a is shown as SEQ ID NO.1, and the sequence of the bta-miR-19b is shown as SEQ ID NO. 2.
3. Use of the miRNA marker for early pregnancy diagnosis of cattle according to claim 2, for preparing a kit for early pregnancy diagnosis of cattle, characterized in that: the kit is used for detecting the expression quantity of the miRNA biomarker in blood by adopting a Real-time RT-PCR method; the expression level of the bta-miR-133a in the pregnant cattle blood is obviously reduced, and the expression level of the bta-miR-19b in the pregnant cattle blood is obviously increased.
4. Use of the miRNA marker for early pregnancy diagnosis of cattle according to claim 2, for preparing a kit for early pregnancy diagnosis of cattle, characterized in that: the kit contains a primer capable of specifically amplifying the miRNA marker; the primer sequence of the bta-miR-133a is shown in SEQ ID NO. 3-4, and the primer sequence of the bta-miR-19b is shown in SEQ ID NO. 5-6.
5. Use of the miRNA marker for early pregnancy diagnosis of cattle according to claim 2, for preparing a kit for early pregnancy diagnosis of cattle, characterized in that: the kit contains primers for detecting the internal reference genes of the miRNA marker expression quantity, wherein the internal reference genes are bta-GAPDH and bta-RPS18, the primer sequences of the internal reference genes bta-GAPDH are shown as SEQ ID NO. 7-8, and the primer sequences of the internal reference genes bta-RPS18 are shown as SEQ ID NO. 9-10.
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