Method for identifying lncRNA

Abstract

The invention discloses a method for identifying lncRNA, and belongs to the technical field of lncRNA identification. The invention aims to provide a simple, rapid and high-accuracy method for identifying the lncRNA of the mammal. The invention provides a method for identifying lncRNA. The method comprises the following steps: selecting a forward open reading frame of non-circular RNA to be identified, wherein the length of the non-circular RNA is greater than 200nt; connecting the obtained gene sequence of the open reading frame with a gene for coding fluorescent protein to obtain a recombinant sequence, and connecting the recombinant sequence with an expression vector to obtain a recombinant vector; carrying out in-vitro transcription on the obtained recombinant vector to obtain an RNA sequence; the obtained RNA is injected into a pig embryo, fluorescence intensity is detected by using a fluorescence microscope, if fluorescence expression exists, the RNA is not lncRNA, and if fluorescence expression does not exist, the RNA is lncRNA. And accurate and rapid screening of the lncRNA is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of identifying lncRNA, and in particular relates to a method for identifying lncRNA. Background Art

[0002] Long noncoding RNA (lncRNA) is a type of RNA molecule with a length of more than 200 nt that cannot encode proteins or can only encode short peptides. It is involved in the regulation of various biological processes. The following is a method for identifying pig lncRNA: Sample collection: extract total RNA from pig tissues or cells; RNA sequencing: use second-generation sequencing technology to sequence total RNA to obtain a large amount of sequencing data; data preprocessing: perform quality control and filtering on sequencing data to remove low-quality reads and adapter sequences; transcript assembly: use transcript assembly software such as StringTie or Cufflinks to assemble sequencing reads into transcripts; lncRNA identification: identify lncRNA from assembled transcripts based on the characteristics of lncRNA, such as length, coding potential and expression level; function prediction: predict the function of the identified lncRNA, which can be performed by association analysis with genes with known functions, or by using bioinformatics tools to predict its possible biological function; experimental verification: select some lncRNAs for experimental verification, such as through RT-PCR, Northern blot or RNA Interference and other technologies are used to verify its expression and function in pig tissues or cells. The above operation steps are cumbersome, time-consuming and costly, and a simple and convenient method is urgently needed. Summary of the invention

[0003] The purpose of the present invention is to provide a simple, rapid and highly accurate method for identifying pig lncRNA.

[0004] The present invention provides 1. a method for identifying lncRNA, wherein the specific steps of the method are as follows: Step 1: Select the positive open reading frame of the non-circular RNA to be identified with a length greater than 200 nt; Step 2: Connect the gene sequence of the positive open reading frame obtained in step 1 to the gene encoding the fluorescent protein to obtain a recombinant sequence, and connect the recombinant sequence to an expression vector to obtain a recombinant vector; Step 3: Perform in vitro transcription on the recombinant vector obtained in step 2 to obtain an RNA sequence; Step 4: Inject the RNA obtained in step 3 into pig embryos, and use a fluorescence microscope to detect the fluorescence intensity. If there is fluorescence expression, this RNA is not lncRNA; if there is no fluorescence expression, this RNA is lncRNA.

[0005] Further defined, the method for selecting the positive open reading frame in step 1: using the Open Reading FrameFinder tool, input the GI number, Accession number or fasta format of the cDNA of the RNA to be detected into the OpenReading Frame Finder tool, select Minimal ORF length as 75, Genetic code as standard, ORF start codon to use as "ATG" only, and after submitting, select all ORFs with standard as +.

[0006] It is further defined that the fluorescent protein is red fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein or green fluorescent protein.

[0007] It is further defined that the fluorescent protein is EGFP.

[0008] It is further defined that the RNA in step 1 has a sequence as shown in SEQ ID NO.5 or SEQ ID NO.3.

[0009] It is further defined that the gene sequence encoding the fluorescent protein in step 2 is as shown in SEQ ID NO.1.

[0010] It is further defined that the starting vector of the recombinant vector in step 2 is the pEASY-T1 vector.

[0011] Further defined, the method in step 3: capping the DNA sequence, then removing the DNA sequence, and finally tailing, and obtaining the RNA sequence after purification.

[0012] It is further defined that the pig embryo in step 4 is a pig parthenogenetic embryo at the 1-cell stage.

[0013] It is further defined that in step 4, the fluorescence intensity of the embryo is observed when the embryo develops to the 2-cell stage.

[0014] Beneficial effect: Compared with the traditional in vitro method for verifying whether RNA is lncRNA, the in vivo verification experiment of the present invention is more accurate, with an accuracy rate of 100%, and is more convenient to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This figure shows the result of observing green fluorescent protein expression in 2-cell stage embryos after in vitro transcribed RNA was injected into embryos. DETAILED DESCRIPTION

[0016] Example 1. A method for identifying pig lncRNA Step 1: Select the positive open reading frame of the lnRNA to be identified (LncFKBPL; Gas5 is a confirmed mouse lncRNA; GAPDH is a control gene and is a known non-lncRNA): enter the cDNA sequence of the RNA to be detected (RNA length is greater than 200nt and non-circular) into the Open Reading Frame Finder (URL: https: / / www.ncbi.nlm.nih.gov / orffinder / ), enter the GI number, Accession number or fasta format of the sequence of the RNA to be detected into the Open Reading Frame Finder tool, select Minimal ORF length as 75, Genetic code as standard, ORF start codon to use as "ATG" only, after submitting, select all ORFs with standard as +, the result is as follows Figure 1 shown.

[0017] Step 2: Connect the gene sequence of the open reading frame obtained in step 1 with the gene encoding the fluorescent protein to obtain a recombinant sequence, submit the recombinant sequence information to the company, and connect the recombinant sequence with the expression vector (pEASY-T1) to obtain a recombinant vector; EGFP sequence and inserted EGFP cDNA sequence: (SEQ ID NO.1) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA; EGFP cDNA sequence with the stop codon sequence removed: (SEQ ID NO.2) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAG; Gas5 cDNA sequence, SEQ ID NO.3: Ctttcggagctgtgcggcattctgagcaggaatggcagtgtggacctctgtgatgggacatcttgtgggatctcacagccagttctgtggcaaaggaggatgaaggcttacgaggactcgtcaggaagctggataacagagcgagcgcaatgtgctagaatagaagaccagaaaatgaaatggtggagtttgaggctggatagacagtttgaaagttaactggttgcatgcttgttcatttggctggcttgcttgggtacaaataatggtttgaataaagaaaggtattaatgggtcacctcaagtgaaggcactgcaaacacaatgattggtcattctgaatttccggtccttcattctgaatttcaaaggctcctgtgacaagtggacatgcagtgactgcacctttgtttctgaggtgcctggatggaggctcaaatagaagatggtgtcagatatattgtgttaaaattttaccattaaagtgtattataacatgaa; GAPDH cDNA sequence SEQ ID NO.4: CTCTGAGCCTCCTCCAATTCAACCCTTAAGAGGGATGCTGCCCTTACCCCGGGGTCCCAGCTTAGGTTCATCAGGTAAACTCAGGAGAGTGTTTCCTCGTCCCGTAGACAAAATGGTGAAGGTCGGTGTGAACGGATTTGGCCGTATTGGGCGCCTGGTCACCAGGGCTGCCATTTGCAGTGGCAAAGTGGAGATTGTTGCCATCAACGACCCCTTCATTGACCTCAACTACATGGTCTACATGTTCCAGTATGACTCCACTCACGGCAAATTCAACGGCACAGTCAAGGCCGAGAATGGGAAGCTTGTCATCAACGGGAAGCCCATCACCATCTTCCAGGAGCGAGACC CCACTAACATCAAATGGGGTGAGGCCGGTGCTGAGTATGTCGTGGAGTCTACTGGTGTCTTCACCACCATGGAGAAGGCCGGGGCCCACTTGAAGGGTGGAGCCAAAAGGGTCATCATCTCCGCCCCTTCTGCCGATGCCCCCATGTTTGTGATGGGTGTGAACCACGAGAAATATGACAACTCACTCAAGATTGTCAGCAATGCATCCTGCACCACCAACTGCTTAGCCCCCCTGGCCAAGGTCATCCATGACAACTTTGGCATTGTGGAAGGGCTCATGACCACAGTCCATGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCTGGAAAGCTGTGGCGTGAT GGCCGTGGGGCTGCCCAGAACATCATCCCTGCATCCACTGGTGCTGCCAAGGCTGTGGGCAAGGTCATCCCAGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTTCCTACCCCCAATGTGTCCGTCGTGGATCTGACGTGCCGCCTGGAGAAACCTGCCAAGTATGATGACATCAAGAAGGTGGTGAAGCAGGCATCTGAGGGCCCACTGAAGGGCATCTTGGGCTACACTGAGGACCAGGTTGTCTCCTGCGACTTCAACAGCAACTCCCACTCTTCCACCTTCGATGCCGGGGCTGGCATTGCTCTCAATGACAACTTTGTCAAGCTCATTTCCTGGTATGA CAATGAATACGGCTACAGCAACAGGGTGGTGGACCTCATGGCCTACATGGCCTCCAAGGAGTAAGAAACCCTGGACCACCCACCCCAGCAAGGACACTGAGCAAGAGAGGCCCTATCCCAACTCGGCCCCCAACACTGAGCATCTCCCTCACAATTTCCATCCCAGACCCCCATAATAACAGGAGGGGCCTAGGGAGCCCTCCCTACTCTCTTGAATACCATCAATAAAGTTCGCTGCACCCACCTC; LncFKBPL cDNA sequence: SEQ ID NO.5

[0018] GADPH is a known internal reference gene that can encode proteins. It has four positive open reading frames as shown in Table 1. One of them is 1080 nt long and can encode proteins, so the positive open reading frame ORF1 was selected as a control. Table 1 GPADH positive open reading frame

[0019] musGas5 has only one open reading frame, and the results are shown in Table 2 ; Table 2 Gas5 positive open reading frame

[0020] lncFKBPL has 7 open reading frames, and the results are shown in Table 3 ; Table 3 lncFKBPL positive open reading frame

[0021] Positive open reading frame of GAPDH: GAPDH ORF1 cDNA sequence: SEQ ID NO.6 GAPDH ORF4 cDNA sequence: SEQ ID NO.7 ATGCCGGGGCTGGCATTGCTCTCAATGACAACTTTGTCAAGCTCATTTCCTGGTATGACAATGAATACGGCTACAGCAACAGGGTGGTGGACCTCATGGCCTACATGGCCTCCAAGGAGTAAGAAACCCTGGACCACCCACCCCAGCAAGGACACTGAGCAAGAGAGGCCCTATCCCAACTCGGCCCCCAACACTGAGCATCTCCCTCACAATTTCCATCCCAGACCCCCATAA; GAPDH ORF2 cDNA sequence: SEQ ID NO.8 ATGACTCCACTCACGGCAAATTCAACGGCACAGTCAAGGCCGAGAATGGGAAGCTTGTCATCAACGGGAAGCCCATCACCATCTTCCAGGAGCGAGACCCCACTAACATCAAATGGGGTGAGGCCGGTGCTGAGTATGTCGTGGAGTCTACTGGTGTCTTCACCACCATGGAGAAGGCCGGGGCCCACTTGA; GAPDH ORF3 cDNA sequence: SEQ ID NO.9 ATGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCTGGAAAGCTGTGGCGTGATGGCCGTGGGGCTGCCCAGAACATCATCCCTGCATCCACTGGTGCTGCCAAGGCTGTGGGCAAGGTCATCCCAGAGCTGA; Forward open reading frame of Gas5: ORF1 cDNA sequence: SEQ ID NO.10 ATGAAGGCTTACGAGGACTCGTCAGGAAGCTGGATAACAGAGCGAGCGCAATGTGCTAGAATAGAAGACCAGAAAATGAAATGGTGGAGTTTGAGGCTGGATAGACAGTTTGAAAGTTAA; Forward open reading frame of lncFKBPL: lncFKBPL ORF6 cDNA sequence: SEQ ID NO.11 TGCTTCCCTACCTCTGTGGGTGGCGCCATTTCCCCAATGCCCTTATCCAAAAACTTGATGCCTCCTCCATCTCCTCCCCACCTTTATTTTTGGTGAGCTATCAAGCCCAGTCAATACTACCATCGCGGAGTTCCCATGGTGGCACAGCTGAAATGAATCCGACCAGGAACCATGAGGTTGCAGGTTCGATCCCTGGCCTCGCTCAGTGGGTTAAAGATCTGGCATTGCCGTGAG; lncFKBPL ORF2 cDNA sequence: SEQ ID NO.12 ATGAGGTTGCAGGTTCGATCCCTGGCCTCGCTCAGTGGGTTAAAGATCTGGCATTGCCGTGAGTTGTGGTGTAGGTTGAAGACGTGGCTGGAATCTGGCATTGCTTTGGCTGTGGTGTAG; lncFKBPL ORF1 cDNA sequence: SEQ ID NO.13 ATGACACTGAAAACATGCTTCCCTACCTCTGTGGGTGGCGCCATTTCCCCAATGCCCTTATCCAAAAACTTGATGCCTCCTCCATCTCCTCCCCACCTTTATTTTTGGTGA; lncFKBPL ORF3 cDNA sequence: SEQ ID NO.14 ATGGACCTGCTGTATAGCACAGGGAACTCTACCCAGTGTTCTGTGATAATCCATATGGAAAAAGAATTTGAAAGAGAATGGATGTGTGTATTTGTGTAA; lncFKBPL ORF5 cDNA sequence: SEQ ID NO.15 ATGTATTTACAGGGAGACATTTTTCTGGTTTCTGTGGAAAATGGCCACATGGGCAATACTGCTGGTCCTCTAAACGTAGCATGTTCCAAAGCAGACTGA; lncFKBPL ORF7 cDNA sequence: SEQ ID NO.16 ATGGATGTGTGTATTTGTGTAACTGAATCACTTTGTTGTACAGCAGAAATGATCACAACTTGTAAATCAACTCTACTTCAATAA; lncFKBPL ORF4 cDNA sequence: SEQ ID NO.17 ATGAGGATGGGTTGGAGTATGGAAATCATGGGTTCTCCTTTGGACGTTTCAGTGTCTTGTTTTTTTTCTCCCTTGCCTTAA; Step 3: Perform in vitro transcription on the recombinant vector obtained in step 2 to obtain RNA: 1. Use commercial M13F and M13R primers to perform PCR amplification on the recombinant plasmid. Purify the PCR product and use it as a DNA template for in vitro transcription.

[0022] 2. Capping & transcription, the system is shown in Table 4: Table 4

[0023] Incubate in PCR instrument at 37℃ for 30min. In 20μL system, 8μL is left except T7 enzyme and NTP-Mix, so the minimum X concentration cannot be lower than 125ng / μL. 30min for larger than 500bp and 1h or more to overnight (16h) for smaller than 500bp.

[0024] Remove template: Add 2 μL DNase I, mix well, and incubate at 37°C for 15 min.

[0025] Add enough ATP to the tailing-capping mix without adding additional tailing substrate. The system is shown in Table 5: Table 5

[0026] Incubate at 37°C for 45 minutes. A 30-minute tailing reaction will provide a poly(A) tail of 150 nt or longer for most mRNAs. Due to the nature of the tailing reaction, the tail length will vary depending on the RNA sequence, structure, yield, length, etc. For very short RNAs (<300 nt), the tailing time can be extended to 1 hour to achieve adequate tailing.

[0027] mRNA purification To 50 μl of the tailing reaction, add 25 μl of lithium chloride solution and mix well. Incubate at -20°C for 30 minutes. 3. Centrifuge at top speed at 4°C for 15 minutes to precipitate the RNA. Carefully remove the supernatant. Resuspend in 500 μl of cold 70% ethanol and centrifuge at 4°C for 10 minutes. Carefully aspirate the ethanol. Carefully remove any remaining liquid. Allow to dry for 5 min and resuspend the mRNA in 50 μl of 0.1 mM EDTA or a suitable RNA storage solution. Heat the RNA at 65°C for 5-10 minutes to completely dissolve the RNA and mix well. Store the RNA at -20°C or below. Step 4: Inject the RNA obtained in step 3 into pig embryos: (1) Acquisition of parthenogenetic embryos GV stage oocytes were obtained from pig ovaries. When the oocytes developed to the MII stage, hyaluronidase (0.1%) was used to remove the cumulus cells on the cumulus-oocyte complex, and mature oocytes with polar bodies were selected under a microscope for use.

[0028] Acquisition of parthenogenetic embryos: The electro-activation method was used, and the specific operation was as follows: the fusion tank was placed under the microscope, the positive and negative electrodes were installed, and the preheated fusion solution was added. The mature oocyte was then placed in the center of the fusion tank, and the whole was in a single straight line. Direct current was used for electric shock twice, 1.2 kV / cm, and the pulse time was 30 μs. The embryos were then moved to the PZM-3 wash drop and washed four times. After that, every 50 embryos were moved into a four-well plate with 500 μL PZM-3 culture solution in each well and cultured in an incubator at 39°C, 5% carbon dioxide, and saturated humidity.

[0029] (2) RNA injection 1) Preparation of fixation needle and injection needle A thin-walled capillary with a specification of 1.0 mm OD and 0.78 mm ID was used to pull a micromanipulator on a P-97 needle puller. The thin-walled capillary was fixed on the needle puller, and the appropriate parameters were selected to break the thin-walled capillary, which was used to prepare fixed needles and injection needles respectively. The parameters for the fixed needle were: temperature 565℃, power 200 W, tension 50, time 50 seconds, and then placed on the needle calcining instrument. The needle was broken at the 140 μm ruler, and the broken needle mouth was kept flat. The broken mouth was made smooth using high heat, and the inner diameter was about 30 μm. The needle tip was placed near the glass ball and continuously heated to bend the fixed needle to the optimal angle to complete the preparation of the fixed needle. The injection needle parameters were set as follows: temperature 600℃, power 200 W, tension 150, time 130 seconds, and polished at a 45° angle with a needle grinder. Then the thin-walled capillary was fixed to the needle calcining instrument. The heating point was located 2 cm away from the needle tip, and the needle tip was burned and bent to a 15° angle.

[0030] 2) Microinjection The RNA obtained by in vitro transcription was dissolved in RNase-free H2O to a final concentration of 10 mM, and stored in a -80°C refrigerator after aliquoting. A Nikon inverted microscope (TS-100), Nikon microinjection system (IM-9A) and Eppendorf Femtojet Express quantitative injection system were used as micromanipulation systems. The aliquoted siRNA was centrifuged at 13000 rpm at 4°C and the supernatant was aspirated for standby use. The supernatant was aspirated through a micropipette tip and added to the tip of the injection tube, which was placed on the operating arm. After the injection system was opened, the injection parameters were injection pressure 100 hPa, injection time 0.2 seconds and compensation pressure 40 hPa. The injection volume per embryo was about 20 pL.

[0031] The fluorescence intensity was detected by fluorescence microscopy. If there was fluorescence expression, the RNA was not lncRNA. If there was no fluorescence expression, the RNA was lncRNA. If the embryo did not express green fluorescent protein, it meant that the ORF did not have the ability to encode protein, indicating that the RNA was lncRNA; if the embryo expressed green fluorescent protein, it meant that the ORF had the ability to encode protein, indicating that the RNA was not lncRNA.

[0032] Figure 1 Mouse lncRNA Gas5 and porcine lncRNA lncFKBPL were used as positive controls, and mouse internal reference gene GAPDH was used as a negative control. The results are as follows Figure 1 As shown, LncFKBPL and Gas5 verified by the present invention are confirmed mouse lncRNA and GAPDH control genes, and the 7 forward open reading frames of LncFKBPL have no expression function and are determined to be lncRNA; 1 open reading frame of Gas5 also has no expression ability and is determined to be lncRNA.

Claims

1. A method for identifying lncRNA, characterized in that: The specific steps of the method are as follows: Step 1: Select the positive open reading frame of the non-circular RNA to be identified with a length greater than 200 nt; Step 2: Connect the gene sequence of the positive open reading frame obtained in step 1 to the gene encoding the fluorescent protein to obtain a recombinant sequence, and connect the recombinant sequence to an expression vector to obtain a recombinant vector; Step 3: Perform in vitro transcription on the recombinant vector obtained in step 2 to obtain an RNA sequence; Step 4: Inject the RNA obtained in step 3 into pig embryos, and use a fluorescence microscope to detect the fluorescence intensity. If there is fluorescence expression, this RNA is not lncRNA; if there is no fluorescence expression, this RNA is lncRNA.

2. The method according to claim 1, characterized in that Method for selecting the positive open reading frame in step 1: Use the Open Reading Frame Finder tool to input the GI number, Accession number or fasta format of the cDNA of the RNA to be detected into the Open Reading Frame Finder tool, select Minimal ORF length as 75, Genetic code as standard, ORF start codon to use as "ATG" only, and after submitting, select all ORFs with standard as +.

3. The method according to claim 1, characterized in that The fluorescent protein is red fluorescent protein, blue fluorescent protein, cyan fluorescent protein, yellow fluorescent protein or green fluorescent protein.

4. The method according to claim 3, characterized in that The fluorescent protein is EGFP.

5. The method according to claim 1, characterized in that The sequence of RNA in step 1 is shown as SEQ ID NO.5 or SEQ ID NO.

3.

6. The method according to claim 1, characterized in that The gene sequence encoding the fluorescent protein in step 2 is shown as SEQ ID NO.

1.

7. The method according to claim 1, characterized in that The starting vector of the recombinant vector in step 2 is pEASY-T1 vector.

8. The method according to claim 1, characterized in that The method in step 3: using the DNA sequence as a template to perform in vitro transcription to obtain capped RNA, then removing the DNA sequence, and finally performing tailing, and obtaining the RNA sequence after purification.

9. The method according to claim 1, characterized in that: The pig embryo in step 4 is a pig parthenogenetic embryo at the 1-cell stage.

10. The method according to claim 1, characterized in that In step 4, the fluorescence intensity of the embryo is observed when the embryo develops to the 2-cell stage.

Images