CN112831572A - Fluorescent quantitative PCR kit for auxiliary prediction of pork quality - Google Patents
Fluorescent quantitative PCR kit for auxiliary prediction of pork quality Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR (polymerase chain reaction) kit for assisting in predicting pork quality, belonging to the technical field of livestock breeding. The kit provided by the invention comprises a reagent for detecting the expression quantity of LncRNA TCONS _00654335, wherein the gene sequence of LncRNA TCONS _00654335 is shown as SEQ ID NO.1, and the reagent for detecting the expression quantity of LncRNA TCONS _00654335 is a primer pair of LncRNA TCONS _ 00654335. By the kit, the fat content in pork can be known, so that the quality of the pork can be identified, and the kit can be used in breeding to breed high-quality live pigs with required meat quality.
Description
Technical Field
The invention belongs to the technical field of livestock breeding, and particularly relates to a fluorescent quantitative PCR kit for assisting in predicting pork quality.
Background
China is the first major country for pork production and consumption, and pork is the most important source of meat consumption for residents. With the continuous improvement of the national economic level and the living standard of people, the consumption of pork by people is greatly converted from the simple requirement in the past into the interest of the inherent quality of the pork, which puts higher requirements on pork producers, processing enterprises and retailers.
From the nutrition point of view, pork is a good source of protein, fat, vitamins and minerals of human bodies, and eating a proper amount of pork is beneficial to obtaining nutrient substances required by the human bodies, but excessive meat intake is not beneficial to health. The fat character of the pig directly influences the pork quality, such as the intramuscular fat content, the carcass lean meat percentage and the like. When a human body eats the pork with high fat content, various diseases such as obesity, cardiovascular and cerebrovascular diseases, diabetes and the like are easily caused. Therefore, the research on molecular markers and molecular mechanisms related to the fat deposition of pork has important significance for predicting and improving the pork quality.
Disclosure of Invention
The invention aims to provide a fluorescent quantitative PCR kit capable of assisting in predicting pork quality.
In order to achieve the purpose, the invention provides the following technical scheme:
first, the invention provides a fluorescence quantitative RCR kit for assisting in the prediction of pork quality, which comprises a reagent for detecting the expression level of LncRNA TCONS _ 00654335.
Preferably, the gene sequence of the LncRNA TCONS _00654335 is shown as SEQ ID NO. 1.
Preferably, the reagent for detecting the expression level of the LncRNA TCONS _00654335 is a primer pair of the LncRNA TCONS _ 00654335.
Preferably, the sequence of the upstream primer of the primer pair is shown as SEQ ID NO.2,
the upstream primer sequence of the primer pair is CAAAGCCTCTCACTTCTTTGGC, SEQ ID NO. 2;
the downstream primer sequence of the primer pair is TTATTGTAGCAGCCTCACCTGG, SEQ ID NO. 3.
Preferably, the kit further comprises SYBR Green fluorescent dye, GAPDH primer pair, ddH2O。
Secondly, the invention provides application of a reagent for detecting the expression level of LncRNA TCONS _00654335 in preparation of a fluorescent quantitative PCR kit for assisting in predicting pork quality.
Preferably, the reagent for detecting the expression level of the LncRNA TCONS _00654335 is a primer pair of the LncRNA TCONS _ 00654335.
Preferably, the sequence of the upstream primer of the primer pair is shown as SEQ ID NO.2,
the upstream primer sequence of the primer pair is CAAAGCCTCTCACTTCTTTGGC, SEQ ID NO. 2;
the downstream primer sequence of the primer pair is TTATTGTAGCAGCCTCACCTGG, SEQ ID NO. 3.
Finally, the invention provides application of the LncRNA TCONS _00654335 inhibitor in preparing a precursor adipocyte differentiation inhibitor.
Preferably, the LncRNA TCONS _00654335 inhibitor is siRNA of LncRNA TCONS _00654335,
sense strand of the siRNA: GGAUAGAUUUCAACCUCAAGC, SEQ ID NO. 6;
the antisense strand of the siRNA: UUGAGGUUGAAAUCUAUCCCU, SEQ ID NO. 7.
The invention has the beneficial effects that:
1. the invention provides a fluorescent quantitative PCR kit for auxiliary prediction of pork quality, which indicates that the fat content of pork is higher when the expression level of LncRNA TCONS _00654335 is higher, and indicates that the lean meat content of pork is higher when the expression level of LncRNA TCONS _00654335 is lower, so that the kit can be used for identifying the pork quality, and can be used in breeding to breed high-quality pigs with required meat quality. Compared with the traditional method, the fluorescence quantitative PCR has higher sensitivity and specificity.
2. Secondly, the present invention provides specific siRNA of LncRNA TCONS _00654335, and the present invention found through experiments that transfection of si-LncRNA TCONS _00654335 in porcine preadipocytes can effectively inhibit expression of lipo-transformation associated proteins PPAR- γ and C/ebpa and can inhibit differentiation of porcine preadipocytes, so that the inhibitor of LncRNA TCONS _00654335 can be used in pig breeding to improve meat quality.
Drawings
FIG. 1 ROC curves of the difference and the difference in expression of LncRNA TCONS _00654335 in subcutaneous adipose tissues of Changbai and Laiwu pigs;
FIG. 2 detection of the inhibitory effect of si-LncRNA TCONS-00654335;
FIG. 3 effect of si-LncRNA TCONS-00654335 on differentiation of porcine preadipocytes;
FIG. 4 shows the effect of si-LncRNA TCONS-00654335 on the transformation-related proteins PPAR-gamma and C/EBP alpha of porcine preadipocytes into fat.
Detailed Description
In order to clearly illustrate the technical features of the present solution, the present solution is explained below by way of specific embodiments.
Example 1 expression difference of LncRNA TCONS _00654335 in subcutaneous adipose tissue of Long white pig and Laiwu pig
RNA sample extraction
(1) Collecting subcutaneous adipose tissues of 32 Changbai pigs and 32 Laiwu pigs of 150 days old, and placing in a refrigerator at-80 ℃ for later use;
(2) taking about 100mg of tissues into a precooled mortar, adding a small amount of liquid nitrogen, quickly grinding into powder, and transferring the powder into a 1.5ml EP tube after grinding;
(3) adding 1ml Trizol reagent into each tube, carrying out vortex oscillation to fully and uniformly mix the tissue and the reagent, and standing for 15 minutes at room temperature;
(4) centrifuging at 12000rpm at 4 ℃ for 10min, and transferring the supernatant to a new EP tube;
(5) adding 200ul chloroform, shaking, mixing, standing at room temperature for 15min, centrifuging at 4 deg.C and 12000rpm for 15min, and transferring the supernatant to a new EP tube;
(6) adding 0.5ml of isopropanol, mixing uniformly, standing at room temperature for 10 minutes, centrifuging at 4 ℃ and 12000g for 10min, and removing supernatant to obtain RNA precipitate;
(7) adding 1ml of 75% ethanol, gently shaking an EP tube, suspending and precipitating, centrifuging at room temperature of 8000g for 5min, and removing a supernatant;
(8) after air-drying at room temperature, the precipitate was dissolved in 40ul DEPC water.
2. Reverse transcription reaction and fluorescent quantitative PCR reaction
(1) And (2) carrying out reverse transcription reaction by taking the total RNA obtained in the step (1) as a template to obtain cDNA, wherein the reverse transcription reaction system is as follows: 5.5. mu.L of RNA, 8. mu.L of dNTPs, 1.5. mu.L of Random primer, 4. mu.L of 5 XM-MLV Buffer, 0.5. mu.L of RRI, 0.5. mu.L of L M-MLV;
the reverse transcription reaction conditions are as follows: 10min at 30 ℃, 1h at 42 ℃ and 5min at 70 ℃;
(2) designing a primer according to the gene sequence of LncRNA TCONS _00654335, wherein the LncRNA TCONS _00654335 is positioned on the pig No.7 chromosome 117789046-117791862, and the sequence is shown as SEQ ID NO. 1;
the primer sequences of LncRNA TCONS _00654335 are as follows:
an upstream primer: CAAAGCCTCTCACTTCTTTGGC, SEQ ID NO. 2;
a downstream primer: TTATTGTAGCAGCCTCACCTGG, SEQ ID NO. 3;
the primer sequences of GAPDH are as follows, taking GAPDH as an internal reference:
an upstream primer: TTCCAGTATGATTCCACCCACG, SEQ ID NO. 4;
a downstream primer: GGACTCCACAACATACGTAGCA, SEQ ID NO. 5;
(3) fluorescent quantitative PCR detection
The fluorescent quantitative PCR reaction was carried out according to the following reaction system and reaction conditions
Reaction system: 10 μ l SYBR Green Premix Ex Taq (2X), 0.4 μ l forward primer, 0.4 μ l reverse primer, 2 μ l cDNA template, 7.2 μ l ddH 2O.
Reaction conditions are as follows: 5min at 95 ℃; at 95 ℃ for 10s, at 60 ℃ for 30s, at 72 ℃ for 25s, for 35 cycles; 7min at 72 DEG C
By using 2-△△CtThe method calculates the relative expression amount.
Results of the experiment
The experimental results obtained from example 1, as shown in fig. 1, show that the relative expression amount of LncRNA TCONS _00654335 was 3.769 ± 1.569 in the subcutaneous adipose tissues of leuw pigs, indicating that the expression amount of LncRNA TCONS _00654335 was significantly higher in the subcutaneous adipose tissues of leuw pigs with high fat content than in the subcutaneous adipose tissues of long white pigs with low fat content. Meanwhile, the ROC curve shows that the AUC value between the two is 0.9521, which indicates that the differential expression has very excellent specificity and sensitivity. Therefore, the expression level of LncRNA TCONS _00654335 in subcutaneous adipose tissues of the pigs can be detected to assist in diagnosing the fat content of the pork, and breeding pigs with different fat contents is assisted, so that compared with the traditional method, the fluorescent quantitative PCR detection method has higher sensitivity and specificity.
Example 2
Obtaining Laiwu pig precursor adipocytes
(1) Directly inserting a 1-day-old Laiwu pig into a heart by using a scalpel for killing, putting the Laiwu pig into an alcohol basin for disinfection, performing debridement in an ultra-clean workbench to obtain subcutaneous adipose tissues, and putting the subcutaneous adipose tissues into a prepared streptomycin mixed solution;
(2) cutting tissue pieces to 1mm using an ophthalmic scissors3Transferring the small particles into a 15ml centrifuge tube, and adding 10 times of volume of collagenase I;
(3) placing the centrifuge tube in an incubator at 37 deg.C for 100min, and shaking the centrifuge tube 1 time every 10 min;
(4) filtering the liquid in the centrifugal tube by using a 100-micron filter screen membrane, centrifuging for 10min at 200g, removing the supernatant, adding an equal volume of erythrocyte lysate, and standing for 20min at room temperature;
(5) centrifuging at 200g for 10min, removing supernatant, counting with dolol blue at 1 × 105Cell density of one/ml cells were seeded to 25cm2The cell culture flask of (1);
(6) and (3) placing the cell culture bottle in a cell culture box, culturing for 24h, and then replacing the culture medium to obtain the pig precursor fat cells.
Example 3
Design and verification of siRNA of LncRNA TCONS _00654335
(1) According to the gene sequence of LncRNA TCONS _00654335, siRNA is designed, and the designed siRNA is as follows:
sense strand: GGAUAGAUUUCAACCUCAAGC, SEQ ID NO. 6;
antisense strand: UUGAGGUUGAAAUCUAUCCCU, SEQ ID NO. 7;
(2) the preadipocytes obtained in example 2 were seeded on a cell culture plate and transfected into groups of an empty transfer group (empty liposome lipofectamine 2000), a si-NC group (transfected siNC), and a si-LncRNA TCONS _00654335 group (transfected LncRNA TCONS _00654335 siRNA), each group being set to 5 replicates;
(3) after transfection, RNA was extracted according to the procedure of example, reverse transcription reaction was performed and the relative expression amount of LncRNA TCONS _00654335 was detected by fluorescent quantitative PCR.
Results of the experiment
The experimental results obtained in example 3 are shown in fig. 2, and it can be seen from the graph that there is no significant difference in the expression amount of LncRNA TCONS _00654335 between the siNC group and the idling group, while the relative expression amount of LncRNA TCONS _00654335 of the si-LncRNA TCONS _00654335 group is significantly reduced to 0.278 ± 0.089, which indicates that the si-LncRNA TCONS _00654335 designed by the present invention can effectively inhibit the expression of LncRNA TCONS _00654335 in porcine precursor adipocytes.
Example 4
Effect of transfection of si-LncRNA TCONS _00654335 on differentiation of porcine preadipocytes
1. Pig precursor fat cell induction differentiation
(1) Inoculating the pig precursor adipocytes transfected with si-NC and si-LncRNA TCONS _00654335 into a cell culture plate, and when the cell density reaches 100%, performing contact inhibition for 48h, wherein 3 repeats are set in each group;
(2) changing the culture medium into inducing solution I (10% DMEM/F12 culture medium containing 1M dexamethasone, 5g/ml insulin and 0.5mM IBMX), culturing for 2 days, changing into inducing solution II, and continuously culturing for 6 days, and changing the solution every 2 days;
2. oil red O dyeing
(1) Adding 2g of oil red O powder into 200ml of isopropanol to be fully dissolved, and mixing the oil red O powder and the isopropanol according to the proportion of 2 parts of oil red O saturated liquid to 3 parts of distilled water for use;
(2) gently washing the cells in the step 1 by using PBS (phosphate buffer solution) for 3 times, adding 10% formaldehyde, and fixing at room temperature for 15 min;
(3) removing methanol fixing solution, washing with deionized water for 2 times, and then washing with 60% isopropanol for 2 times;
(4) discarding the cleaning solution, adding oil red O staining solution to completely cover the cells, and standing in the dark for 20 min;
(5) absorbing staining solution, washing for 2 times by using deionized water, observing by using an inverted microscope, and taking a picture;
(6) after the photographing is finished, 500 mul of isopropanol is added to extract the oil red O staining solution combined in the cells;
(7) after 20 minutes, the extracts were added to a 96-well plate and the OD was measured at 490 nm.
Results of the experiment
The experimental results obtained in example 4 are shown in FIG. 3, which shows that the fat droplet content in the pig preadipocytes is significantly reduced after si-LncRNA TCONS-00654335 transfection, wherein the OD value of the si-LncRNA TCONS-00654335 group is 0.643 + -0.041 and the OD value of the si-NC group is 1.344 + -0.078, and the results show that the differentiation of the pig preadipocytes into adipocytes can be effectively inhibited by inhibiting LncRNA TCONS-00654335.
Example 5
Effect of si-LncRNA transfection TCONS _00654335 on lipotropy transformation related proteins PPAR-gamma and C/EBP alpha in the process of transforming pig precursor adipocytes into adipocytes
1. Protein extraction
(1) Removing the culture medium of the porcine preadipocytes induced according to step 1 of example 4, washing 2 times with PBS, adding 150. mu.l of cell lysate per well, scraping the cells completely off the culture plate using a cell scraper and adding to a 1.5ml sterile centrifuge tube;
(2) centrifuging at 12000g for 5min, carefully sucking supernatant, determining protein concentration by BCA protein quantification method, adding sample buffer solution, and heating with boiling water for 5min to obtain protein sample;
2. electrophoresis
(1) Preparing 5% of upper layer glue and 10% of lower layer glue, and adding a protein marker and a protein sample into glue holes according to an electrophoresis frame;
(2) the voltage of the upper layer glue is set to be constant voltage of 80V, the voltage of the lower layer glue is set to be constant voltage of 120V, and the bromophenol blue runs out of the lower layer glue;
3. transfer film and sealing
(1) Cutting a PVDF membrane, putting the PVDF membrane in methanol for activation for 30s, removing the upper layer of glue, and putting the cut gel in a membrane transferring buffer solution for soaking for about 5 min;
(2) placing the rotating membrane clamp cathode with the sponge in a membrane transferring solution, placing 3 pieces of soaked filter paper, then placing the gel on the filter paper, placing the PVDF membrane on the gel, and slightly extruding the PVDF membrane by using a plastic plate to remove bubbles;
(3) covering 3 pieces of soaked filter paper on the PVDF membrane, covering the soaked sponge, covering the anode and the cathode of the membrane-rotating clamp together, fixing, placing in an electric rotating tank, and rotating at 250mA for 1.5 h;
(4) after the electrotransformation is finished, taking out the PVDF membrane, placing the PVDF membrane in a plate, and adding TBST to wash for 10 min;
(5) after washing, adding a sealing liquid to completely cover the membrane, and sealing for 1 h;
4. incubating antibodies and developing
(1) After the sealing is finished, adding TBST to wash the membrane for three times, adding diluted PPAR-gamma, C/EBP alpha and beta-actin primary antibody into an incubation box, and placing the incubation box in a refrigerator at 4 ℃ for overnight incubation;
(2) after the incubation is finished, recovering the primary antibody, adding TBST for washing three times, adding the diluted secondary antibody, and incubating for 1h at room temperature;
(3) in a dark room, development exposure was performed.
Results of the experiment
The experimental result is shown in fig. 4, and it can be seen from the figure that after si-LncRNA TCONS _00654335 is transfected, the protein expression of PPAR-gamma and C/ebpa in the adipogenic-induced porcine precursor adipocytes is significantly reduced, which indicates that in the process of transforming the porcine precursor adipocytes into the adipocytes, the LncRNA TCONS _00654335 can effectively inhibit the expression of PPAR-gamma and C/ebpa proteins, thereby effectively inhibiting the transformation of the porcine precursor adipocytes into the adipocytes. Therefore, the LncRNA TCONS _00654335 inhibitor can be used for preparing the pig precursor adipocyte differentiation inhibitor, thereby providing possibility for further and effectively breeding pigs with high quality and meat quality.
Sequence listing
<110> Jiang Zhi Biotechnology GmbH of Qingdao
SHANDONG NEW HOPE LIUHE GROUP Co.,Ltd.
<120> fluorescent quantitative PCR kit for auxiliary prediction of pork quality
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2817
<212> DNA
<213> pig (Susscrofa domistic)
<400> 1
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gaagcaccgt ctccatggag acagggatgg caacagctca gtgcgtcctt caccagaaca 360
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agggcgaccg gtacggacta aatctgcata ctgcagaacc aagcaccaaa gccgggcatg 2580
ctctgggcag cgggggtccg cccctagaac cctgtgcctc tgggtgagac cacccttctg 2640
atccacctga aggagccagg gggaggccca gggcccacgt gcaaccctaa cacttcggcc 2700
aggagattat ctcagctctg ggaaccccgt ggggcttttg ttttcatcga ggtggctttc 2760
ggggtggggc cggttaattt ttcagggaat aaaaatatga caaagcgatg aagggaa 2817
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
caaagcctct cacttctttg gc 22
<210> 3
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ttattgtagc agcctcacct gg 22
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttccagtatg attccaccca cg 22
<210> 5
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ggactccaca acatacgtag ca 22
<210> 6
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggauagauuu caaccucaag c 21
<210> 7
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
uugagguuga aaucuauccc u 21
Claims (10)
1. The fluorescent quantitative RCR kit for assisting in the prediction of pork quality is characterized by comprising a reagent for detecting the expression level of LncRNA TCONS _ 00654335.
2. The kit of claim 1, wherein the LncRNA TCONS _00654335 has the gene sequence shown in SEQ ID No. 1.
3. The kit of claim 1, wherein the reagent for detecting the expression level of LncRNA TCONS _00654335 is a primer set of LncRNA TCONS _ 00654335.
4. The kit according to claim 3,
the upstream primer sequence of the primer pair is CAAAGCCTCTCACTTCTTTGGC, SEQ ID NO. 2;
the downstream primer sequence of the primer pair is TTATTGTAGCAGCCTCACCTGG, SEQ ID NO. 3.
5. The kit according to claim 4, wherein said kit further comprises,characterized in that the kit also comprises SYBR Green fluorescent dye, GAPDH primer pair and ddH2O。
6. Application of a reagent for detecting the expression level of LncRNA TCONS _00654335 in preparation of a fluorescent quantitative PCR kit for assisting in predicting pork quality.
7. The use as claimed in claim 6, wherein the reagent for detecting the expression level of LncRNA TCONS _00654335 is a primer set of LncRNA TCONS _ 00654335.
8. The use according to claim 7,
the upstream primer sequence of the primer pair is CAAAGCCTCTCACTTCTTTGGC, SEQ ID NO. 2;
the downstream primer sequence of the primer pair is TTATTGTAGCAGCCTCACCTGG, SEQ ID NO. 3.
9. Application of LncRNA TCONS _00654335 inhibitor in preparation of precursor adipocyte differentiation inhibitor.
10. The use of claim 9, wherein the LncRNA TCONS _00654335 inhibitor is siRNA of LncRNA TCONS _00654335,
sense strand of the siRNA: GGAUAGAUUUCAACCUCAAGC, SEQ ID NO. 6;
the antisense strand of the siRNA: UUGAGGUUGAAAUCUAUCCCU, SEQ ID NO. 7.
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| CN202110284400.XA CN112831572B (en) | 2021-03-17 | 2021-03-17 | Fluorescent quantitative PCR kit for auxiliary prediction of pork quality |
| CN202210731963.3A CN115058521A (en) | 2021-03-17 | 2021-03-17 | Biological agent for improving pork quality |
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| CN202110284400.XA CN112831572B (en) | 2021-03-17 | 2021-03-17 | Fluorescent quantitative PCR kit for auxiliary prediction of pork quality |
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| CN112831572B CN112831572B (en) | 2022-07-05 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881249A (en) * | 2017-12-18 | 2018-04-06 | 中国农业科学院北京畜牧兽医研究所 | LncRNA and its target gene are applied in seed selection high-quality livestock and poultry species |
| CN108085399A (en) * | 2017-12-28 | 2018-05-29 | 中国农业科学院北京畜牧兽医研究所 | The new application of lncRNA and its trans controlling gene WNT11 |
| CN108103206A (en) * | 2017-12-18 | 2018-06-01 | 中国农业科学院北京畜牧兽医研究所 | A kind of relevant lncRNA of intramuscular fat and its application |
-
2021
- 2021-03-17 CN CN202110284400.XA patent/CN112831572B/en active Active
- 2021-03-17 CN CN202210731963.3A patent/CN115058521A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881249A (en) * | 2017-12-18 | 2018-04-06 | 中国农业科学院北京畜牧兽医研究所 | LncRNA and its target gene are applied in seed selection high-quality livestock and poultry species |
| CN108103206A (en) * | 2017-12-18 | 2018-06-01 | 中国农业科学院北京畜牧兽医研究所 | A kind of relevant lncRNA of intramuscular fat and its application |
| CN108085399A (en) * | 2017-12-28 | 2018-05-29 | 中国农业科学院北京畜牧兽医研究所 | The new application of lncRNA and its trans controlling gene WNT11 |
Non-Patent Citations (1)
| Title |
|---|
| PIG LNCRNANET: "TCONS_00654335", 《PIG LNCRNANET》 * |
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| CN115058521A (en) | 2022-09-16 |
| CN112831572B (en) | 2022-07-05 |
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