CN108676801B - cDNA of oocyte marker gene slbp2 of Epinephelus coioides and application thereof - Google Patents
cDNA of oocyte marker gene slbp2 of Epinephelus coioides and application thereof Download PDFInfo
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Abstract
The invention discloses a cDNA of an oocyte marker gene slbp2 of garrupa, the nucleotide sequence of which is shown as SEQ ID NO: 1. The invention also discloses a preparation method of the cDNA of the oocyte marker gene slbp2 of the garrupa, and the coded protein of the oocyte marker gene slbp2 of the garrupa, the amino acid sequence of which is shown as SEQ ID NO: 2. The invention further discloses a specific probe of the oocyte marker gene slbp2 of the garrupa, a preparation method thereof and application of the specific probe of the oocyte marker gene slbp2 of the garrupa in specific marking of oocytes and identification of oocyte types.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to cDNA of an oocyte marker gene slbp2 of garrupa and application thereof.
Background
Only a small portion of the mRNA in eukaryotes is phosphorylated and carries a poly (a) tail; whereas most mRNAs are replication-dependent mRNAs that are not phosphorylated and do not have a poly (A) tail, the 3' -non-coding region of such mRNAs comprises a highly conserved stem-loop structure. This structure interacts with a protein that specifically binds to RNA, which is called the stem-loop binding protein (SLBP or HBP). Previous studies have shown that stem-loop binding proteins are involved in a variety of important processes such as synthesis of mRNA precursors, embryonic development, cell proliferation, and the like. There are generally two subtypes of SLBP, and most of the current researches are type 2, namely SLBP2, and the gene encoding SLBP2 is SLbp2.
The genus Epinephelus (Perciformes), the family of bass (Serratidae), the subfamily of Epinephelus (Epinepheline), and the genus Epinephelus (Epinephelus). The grouper has delicious quality, rich nutrition and high economic value, and is a main sea water economic cultured fish in the coastal area of south China. The groupers are hermaphrodite fishes, and have sex reversal phenomenon in the individual development process of the groupers. The phenomenon causes the problem that the number of the male fishes is insufficient in the artificial propagation process, and severely restricts the large-scale production of the artificial propagation technology of the groupers and the development of the breeding industry. Therefore, the exploration of a specific mechanism of the sex reversal of the groupers is necessary, the sex marker gene with specific, obvious and credible signals is an effective tool in the research process, and the development of the groupers marker gene is important for the subsequent experimental research.
Disclosure of Invention
The invention aims at providing cDNA of an oocyte marker gene slbp2 of garrupa, a preparation method and a coding protein thereof.
The invention also aims to provide a specific probe of the oocyte marker gene slbp2 of the garrupa, and a preparation method and application thereof.
The first object of the present invention is achieved by the following technical solutions: the cDNA of the oocyte marker gene slbp2 of the garrupa has a nucleotide sequence shown in SEQ ID NO: 1.
The coded protein of the oocyte marker gene slbp2 of the garrupa has an amino acid sequence shown in SEQ ID NO: 2.
The preparation method of the cDNA of the oocyte marker gene slbp2 of the garrupa comprises the following steps:
(1) Extracting total RNA of gonads of the garrupa;
(2) Subjecting the total RNA extracted to SMART TM Reverse transcription of the PCR cDNA synthesis kit to form a template for RACE;
(3) Designing two pairs of specific upstream and downstream primers, and obtaining cDNA of the oocyte marker gene slbp2 of the garrupa by RACE amplification.
Two pairs of specific upstream and downstream primers described in the step (3), wherein the nucleotide sequences of one pair of specific upstream and downstream primers are respectively shown in SEQ ID NO: 3-SEQ ID NO:4, the nucleotide sequences of the other pair of specific upstream and downstream primers are respectively shown in SEQ ID NO: 5-SEQ ID NO:6.
the second object of the present invention is achieved by the following technical solutions: a specific probe of an oocyte marker gene slbp2 of garrupa has a nucleotide sequence shown in SEQ ID NO: shown at 7.
The preparation method of the specific probe of the oocyte marker gene slbp2 of the garrupa comprises the following steps:
(1) Extracting total RNA of gonads of the garrupa;
(2) Subjecting the total RNA extracted to SMART TM Reverse transcription of the PCR cDNA synthesis kit to form a template for RACE;
(3) Designing two pairs of specific upstream and downstream primers, and obtaining cDNA of an oocyte marker gene slbp2 of the garrupa through RACE amplification;
(4) Selecting a relatively conserved fragment in cDNA of slbp2, designing an upstream primer and a downstream primer, carrying out PCR amplification, carrying out electrophoresis on the obtained product, recovering a target product, carrying out recombinant plasmid construction on the target fragment, screening positive clones, extracting plasmids, cutting the plasmids by using restriction endonucleases (ApaI, pstI), obtaining the target fragment after agarose gel electrophoresis, then recovering the cut adhesive tape, and preparing a specific probe of the oocyte marker gene slbp2 of the garrupa by using a DIG probe kit from the product recovered by the adhesive tape.
The nucleotide sequence of the upstream primer and the downstream primer in the step (4) is shown as SEQ ID NO: 8-SEQ ID NO: shown at 9.
The specific probe of the oocyte marker gene slbp2 of the garrupa is applied to the specific marking of oocytes and the identification of oocyte types.
The probe in the invention can specifically mark the oocyte, define the development period of the oocyte, and identify the cell type in the research reversal molecular mechanism.
The invention has the following advantages:
(1) The invention designs a specific primer by utilizing a grouper genome database combined with molecular biology method, and obtains the full-length sequence of slbp2 gene by RACE amplification cloning;
(2) According to the invention, a specific fragment is selected from an ORF sequence, a probe for in-situ hybridization is reversely transcribed, and through inspection, the slbp2 gene is only expressed in female germ cells of the garrupa, and the expression quantity and the expression position in oocytes in different development stages are different, so that the formation and degradation processes of the papilionaceous body are clearly reflected in the whole expression process;
(3) The invention provides a specific marker gene of oocyte for researching the sex reversal mechanism of garrupa, and lays a foundation for subsequent experimental research.
Drawings
FIG. 1 shows the expression of the slbp2 of Epinephelus coioides in 16 tissues in example 1, wherein M is DS 2000DNA marker, and 1-16 represent 16 tissues of olfactory bulb, telencephalon, metaencephalon, cerebellum, brain extension, hypothalamus, pituitary, heart, head kidney, stomach, gill, gonad, kidney, liver, spleen and intestine, respectively;
FIG. 2 is a diagram showing the analysis of homology between the SLBP2 of Epinephelus coioides and SLBP of other species in example 1;
FIG. 3 is an electrophoretogram of the slbp2 probe of Epinephelus coioides in example 2, M is DS 2000 DNAMaroker, and P is slbp2 probe;
FIG. 4 shows the expression of slbp2 from Epinephelus coioides in example 3 at different developmental stages of the oocyte, a representing the gonad of the oogenic stage, b representing the gonad of the oocyte containing a large number of primary growth stages (PO), c representing the gonad of the oocyte containing a large number of cortical vesicles (PVO) and d representing the gonad of the vitelline production stage (VO);
FIG. 5 shows the localization of the slbp2mRNA of Epinephelus coioides at different developmental stages of the oocyte in example 3, A representing the gonad of the oogenic stage, B representing the gonad of the oocyte containing a large number of primary growth stages (PO), C representing the gonad of the oocyte containing a large number of cortical small vesicles (PVO) stages, D representing the gonad of the vitelline stage (VO), a-D representing the slbp2 probe negative patterns, respectively, as negative controls;
FIG. 6 is a graph showing the expression pattern of the slbp2 of Epinephelus coioides in example 3 in oocytes at various developmental stages;
FIG. 7 shows the expression of slbp2mRNA of Epinephelus coioides in example 3 in the artificial induction reversal process, wherein a, c and e respectively represent the early, middle and end gonads, g is the natural male gonad, and b, d, g, h is the DAPI (nuclear specific dye) nuclear staining chart corresponding to a, c, e, g.
Detailed Description
The invention is described in further detail below with reference to the drawings and the specific examples. Unless specifically indicated, the apparatus and methods employed in the present invention are those conventionally commercially available in the art.
EXAMPLE 1 Gene cloning of slbp2 from Epinephelus coioides
1. Extraction of total RNA of 16 tissues of Epinephelus coioides
Taking Epinephelus coioides (Epinephelus coioides), taking the Epinephelus coioides with the body length of 30-40 cm and the body weight of 1-1.5 kg, carrying out ice bath anesthesia for about 2min, killing fish, sampling, separating 16 tissues of olfactory bulb, end brain, middle brain, cerebellum, brain extension, hypothalamus, pituitary gland, heart, head kidney, stomach, gill, gonad, kidney, liver, spleen and intestine, quick-freezing in liquid nitrogen, and storing in a refrigerator at-80 ℃ for standby. Extracting total RNA of 16 tissues of Epinephelus coioides by Trizol reagent method, and OD thereof 260/280 ＝1.90。
2. Distribution of slbp2 of Epinephelus coioides in 16 tissues
And (3) reversely transcribing RNA of 16 tissues of the garrupa in the step (1) into cDNA, and detecting the expression condition of slbp2 in each tissue by PCR. The detection shows that the expression level in the gonad is highest, the kidney has a small amount of expression, and other tissues have no expression (figure 1).
3. Cloning of the complete cDNA sequence of the slbp2 Gene of Epinephelus coioides
construction of cDNA library
Using SMART TM Construction of PCR cDNA amplification kit (Clontech)The cDNA library was subjected to experimental procedures according to the instructions, and all centrifuge tubes and tips used were subjected to enzyme-free treatment. The template is synthesized by the corresponding steps of the kit, and corresponding buffer solution is added according to the concentration of the final product, and the template is preserved for three months at minus 20 ℃.
Cloning of the complete cDNA sequence of the slbp2 Gene of Epinephelus coioides
Designing two specific pairs of upstream and downstream primers SEQ ID NO according to slbp2 gene fragments of other species in NCBI database: 3 (CGGCGACAGAAACAGATA), SEQ ID NO:4 (GTACAGTCGTCGCTCTTG), SEQ ID NO:5 (ACACCACCACCTTCTTCC), SEQ ID NO:6 (TCTCATCATCTTCCACCTG), the DNA polymerase used for PCR amplification is a high fidelity Taq DNA polymerase, using SEQ ID NO:3 and SEQ ID NO:4, diluting the PCR reaction product of the first round 100 times after the completion of the first round of PCR reaction as a template for the second round of PCR reaction, and using SEQ ID NO:5 and SEQ ID NO:6 performing a second round of PCR amplification. The PCR reaction products of the second round are loaded to carry out 2% agarose gel electrophoresis, and if the target strip exists, the gel is cut and recovered.
The excised gel was purified and recovered to obtain the desired product, which was then ligated into pGEM-easy vector, transformed into DH 5. Alpha. E.coli, and selected positive clones for sequencing by the company. Blast homology comparison analysis shows that the non-coding sequences at the 3 'and 5' ends of the slbp2 gene are obtained, and the cDNA complete sequence of the slbp2 gene can be obtained by splicing overlapping parts through software. ATG is located at 27-30 nucleotides, stop codon is located at 795-798 nucleotides, open reading frame is 798 nucleotides, presumably encoding a 225 amino acid protein. The 5' UTR is 27bp, the 3' UTR is 798bp, and the 3' UTR is provided with a typical tailing signal AATAAA. RACE amplification to obtain the complete sequence SEQ ID NO of slbp2 gene cDNA: 1 and the corresponding amino acid sequence SEQ ID NO. 2 is predicted, the bold black region being the probe sequence in example 2 (SEQ ID NO. 7).
4. Homology analysis of Epinephelus coioides SLBP2 with other species
The amino acid sequence predicted by the garrupa is subjected to homology comparison with SLBP of other species by using MEGA 6.06 software, and the comparison result is shown in figure 2. The results showed that the garrupa SLBP2 and xenopus are clustered together, and that likely both SLBP2 are relatively close in evolutionary distance.
Example 2 preparation of Epinephelus coioides slbp2 Gene-specific Probe
Synthesis of first strand cDNA
DNAase I treatment is carried out on 10 mu g of total RNA sample of the garrupa gonads to remove the pollution of genome DNA, the DNA sample is mixed with RNA Oligo dT, reverse transcription PCR is carried out, and the obtained product is preserved at the temperature of minus 20 ℃ for standby.
Cloning of slbp2 Gene Probe sequence of Epinephelus coioides
According to the full-length slbp2 sequence in example 1, a section of SEQ ID NO:7, designing primers SEQ ID NO at the 3 'and 5' ends of the fragment respectively: 8 (CAGCAACAAACCTCGAAGGC), SEQ ID NO:9 (CGGAGCCAGTCAGTCATGTT), performing PCR amplification, loading the obtained PCR product, performing 1.5% agarose gel electrophoresis, separating DNA fragments by low-voltage electrophoresis, and purifying and recovering the target product from the gel. And connecting the purified target product to pGEM-easy vector to transform DH5 alpha escherichia coli, and selecting positive clone for sequencing. Blast homology comparison analysis shows that the target product is an slbp2 probe, and the sequence is shown as SEQ ID NO: shown at 7.
Construction of the slbp2 recombinant plasmid of Epinephelus coioides
And constructing the obtained target fragment, screening positive clones, extracting plasmids, cutting the plasmids by using restriction endonucleases (ApaI, pstI) to obtain the target fragment (666 bp), then recovering the target fragment by using glue, and preparing a probe from the recovered DNA fragment by using a DIG probe kit. The whole process ensures no RNase pollution and is operated on ice. The purified probe was used in the RNA area, the RNA fragment was separated by electrophoresis on a 2% agarose gel at low voltage, the fragment of the target size was present and the band was single and clear (FIG. 3). The probe is stored at-80deg.C, and diluted probe can be placed at-20deg.C for use.
Example 3 in situ hybridization of Epinephelus coioides slbp2 probe to gonadal tissues at different times
Treatment of gonad tissue of Epinephelus coioides
Fresh gonadal tissue block 1cm was taken 3 Immediately left and right, put into 4% paraformaldehyde at 4℃overnight. Samples were immersed in 30% sucrose and shaken overnight at 4 ℃. After 30% sucrose and OCT embedding agent (sakura) are treated for 2-4 hours according to the proportion of 1:1, the sample is embedded by the OCT embedding agent on dry ice, and the solidified sample is preserved at-80 ℃.
Frozen sections of gonad tissue of Epinephelus coioides
The frozen microtome (Lecia) is pre-cooled to-20 ℃ in advance, and the embedded tissue blocks are placed into the cavity of the frozen microtome (Lecia) for 30-60 minutes for balancing, and then are sliced. The immature ovarian tissue section has a thickness of 7 μm and the gonadal tissue section of oocyte containing a large amount of yolk particles has a thickness of 8-10 μm. Slicing, and storing at-80deg.C.
In situ hybridization
The ovarian gonad sections in different periods are baked for 45 minutes at 42 ℃, and then in-situ hybridization can be performed for two days. The concentration of the probe is 0.5-1 ng/. Mu.L, the hybridization temperature is 55-65 ℃, and specific conditions need to be adjusted according to actual conditions. The whole process ensures no RNase pollution.
The first day flow: rehydration for 30min in 1 XDenPBS (DEPC water formulation), prehybridization (50% deionized formamide, 5 XSSC, 0.5mg/ml salmon sperm RNA,1 XDenhard's, 5% dextran sulfate) at 65℃for 1h, then prehybridization with addition of probes at 65℃overnight.
The first day flow: 50% formamide: 2×ssct=1:1 elution for 30min, 2 washes; eluting with 2 XSSCT for 15min; eluting with 0.2 XSSCT for 30min, and washing for 2 times. The elution process was carried out in an oven at 65 ℃. After 1h of blocking at room temperature, incubation with Anti-DIG-AP was carried out for 2h and finally color development with NBT/BCIP Stock Solution.
FIG. 4 shows the expression of slbp2 from Epinephelus coioides at different developmental stages of the oocyte, a representing the gonad of the oogenic stage, b representing the gonad of the oocyte containing a large number of primary growth stages (PO), c representing the gonad of the oocyte containing a large number of cortical vesicles (PVO) and d representing the gonad of the vitelline stage (VO). The result shows that the slbp2 expression quantity of the garrupa is extremely low in the oogenic cells, and is accumulated in a large quantity in the oocyte in the primary growth stage, and then the slbp2 expression quantity is gradually reduced.
FIG. 5 shows the localization of slbp2 of Epinephelus coioides at different developmental stages of the oocyte, A representing the gonad of the oogenic stage, B representing the gonad of the oocyte containing a large number of primary growth stages (PO), C representing the gonad of the oocyte containing a large number of cortical vesicles (PVO) and D representing the gonad of the vitelline stage (VO).
FIG. 5 shows in situ hybridization results that slbp2mRNA pools in perinuclear papules to form a dense sphere when the oocyte is in the early stage of the primary growth phase. When the oocyte is in the middle of the primary growth stage, this compact body gradually moves away from the nucleus and migrates to the cytoplasm; when the oocyte is at the end of the primary growth phase, the pasteurizer disappears and the slbp2mRNA is dispersed in the cytoplasm. In cortical bleb oocytes, the signal of slbp2mRNA in the cytoplasm is greatly reduced, mainly converged at one side edge of the cell; in egg yolk-producing oocytes, slbp2mRNA showed little or no signal in the cytoplasm or only a small signal at one side edge of the cell.
FIG. 6 is a graph showing the expression pattern of slbp2 from Epinephelus coioides in oocytes at various developmental stages.
FIG. 7 shows the expression of the slbp2mRNA from Epinephelus coioides in the artificial induction of sex reversal. The results show that the expression localization of slbp2 in oocytes is consistent with FIG. 5, and that slbp2 is not expressed in male germ cells, again demonstrating that slbp2 is an oocyte-specific marker gene.
The invention is not limited to the specific embodiments described above, which are only intended to be able to describe in detail the course of use of the invention, and also the production methods and technical details with equivalent functions are part of the present disclosure. Indeed, those skilled in the art will be able to find different adjustment schemes according to the needs of each, and these adjustments are within the scope of the claims appended hereto.
Sequence listing
<110> university of Zhongshan
HAINAN CHENHAI AQUATIC Co.,Ltd.
Yangjiang vocational and Technical College
<120> cDNA of oocyte marker gene slbp2 of Epinephelus coioides and application thereof
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Ser Ala Ala Met Trp Asn Gly Leu Pro Asp Pro Leu Leu Thr Ala Ala
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Asn Ser Ser Arg Cys Ser Ala Pro Glu Pro Trp Leu Leu Pro Gly Cys
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Ser Ser Val Phe Asp Ser Leu Val Ser Asn Thr Ser Pro Thr Pro Ser
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Pro Pro Ala Ala Ser Gly Leu Arg Gly Arg Glu Arg Ala Val Ser Asn
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Lys Pro Arg Arg Pro Ser Ile Leu Glu Arg Cys Ile Leu Lys Val Ser
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Thr Ser Ser Val Ala Val Gly Thr Glu Asp Leu Asp Asn Lys Arg Pro
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Pro Ile Gly Arg Cys Tyr Pro Arg Leu Pro Asp Pro Ala Asn Thr Glu
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Thr Asn Gly Ala Val Leu Lys Arg Arg Gln Lys Gln Ile Gln Tyr Gly
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Met Arg Asp Ala Lys Leu His Pro Ser Thr Pro Asn Lys Tyr Arg Lys
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Tyr Ser Arg Arg Ser Trp Asp Met Gln Val Arg Leu Trp Arg Arg Ala
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Leu His Leu Trp Asp Pro Pro Ser Glu Ser Gln Pro Asp Ala Thr Asp
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Thr His Asp Pro Val Glu Gln Leu Gln Ser Gln Leu Ala Lys Met Thr
225 230 235 240
Ser Asp Leu Cys Glu Asp Gly Glu Asp Lys Gln Arg Glu Lys Glu Thr
245 250 255
Pro Glu Ala Ser Lys Ala Ser Ser Val Ser Pro Leu Ser Ala Val Met
260 265 270
Ser Leu Glu Leu Pro Gly Ser Trp Asn Val Pro Leu Ser Pro Glu Ala
275 280 285
Glu Met Asn Leu Arg Thr Leu Arg Ser Pro Pro Gly Leu Ser Gln Leu
290 295 300
Thr Asn Asp Asn Asn Asn Met Thr Asp Trp Leu Arg Leu Leu Leu Glu
305 310 315 320
Ala Asp Asn Asp Gln Asp Phe Gly Ser Asp Asp Gln Gln Val Pro Val
325 330 335
Phe Ser Asp Gln Leu Phe Trp Asn Pro Tyr
340 345
<210> 3
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
cggcgacaga aacagata 18
<210> 4
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
gtacagtcgt cgctcttg 18
<210> 5
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
acaccaccac cttcttcc 18
<210> 6
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
tctcatcatc ttccacctg 19
<210> 7
<211> 666
<212> DNA
<213> stem-loop binding protein (stem loop binding protein)
<400> 7
cagcaacaaa cctcgaaggc cgtccatcct ggagcgatgt atcctcaaag tgtccaccag 60
cagtgtcgct gtggggacag aagatctgga caacaagcgg ccccctatcg gccgctgcta 120
cccccgcctc ccggacccgg ccaacaccga gaccaacggt gcggttctga agcggcggca 180
gaaacagatc cagtacggta aaaacaccag cggctaccag aactacctgc agcaagtccc 240
caaacatatg agggacgcca aactccaccc gtccaccccc aacaagtaca ggaagtacag 300
ccggcgctcc tgggacatgc aggtgcgtct gtggaggcga gcgctgcacc tttgggaccc 360
tccatctgag agccagccgg acgccaccga cacacatgac ccagtggagc aactgcagag 420
ccagctggcg aagatgacct ccgacctgtg tgaggacgga gaagacaagc agagagagaa 480
ggagacacca gaagcctcta aagcctcctc tgtgtctcct ctgtcggctg tgatgtcgct 540
ggagctgcct ggatcctgga acgtcccgct gtcaccggag gctgagatga acctcaggac 600
tctgcgctct cctccaggcc taagccagct gaccaacgac aacaacaaca tgactgactg 660
gctccg 666
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
cagcaacaaa cctcgaaggc 20
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
cggagccagt cagtcatgtt 20
Claims (6)
1. Oocyte marker gene of garrupaslbp2Is characterized in that: the nucleotide sequence of the polypeptide is shown in SEQ ID NO: 1.
2. The oocyte marker gene of Epinephelus coioides of claim 1slbp2Is characterized in that: the amino acid sequence of the polypeptide is shown in SEQ ID NO: 2.
3. The oocyte marker gene of Epinephelus coioides of claim 1slbp2The preparation method of cDNA is characterized by comprising the following steps:
(1) Extracting total RNA of gonads of the garrupa;
(2) Subjecting the total RNA extracted to SMART TM Reverse transcription of the PCR cDNA synthesis kit to form a template for RACE;
(3) Designing two pairs of specific upstream and downstream primers, and obtaining the oocyte marker gene of the garrupa by RACE amplificationslbp2Is a cDNA of (C);
two pairs of specific upstream and downstream primers described in the step (3), wherein the nucleotide sequences of one pair of specific upstream and downstream primers are respectively shown in SEQ ID NO: 3-SEQ ID NO:4, the nucleotide sequences of the other pair of specific upstream and downstream primers are respectively shown in SEQ ID NO: 5-SEQ ID NO:6.
4. oocyte marker gene of garrupaslbp2Is characterized in that: the nucleotide sequence of the polypeptide is shown in SEQ ID NO: shown at 7.
5. The oocyte marker gene of Epinephelus coioides as defined in claim 4slbp2The preparation method of the specific probe is characterized by comprising the following steps:
(1) Extracting total RNA of gonads of the garrupa;
(2) Subjecting the total RNA extracted to SMART TM Reverse transcription of the PCR cDNA synthesis kit to form a template for RACE;
(3) Designing two pairs of specific upstream and downstream primers, and obtaining the oocyte marker gene of the garrupa by RACE amplificationslbp2Is a cDNA of (C);
(4) Selectingslbp2The cDNA of the sequence is designed with upstream and downstream primers, PCR amplification is carried out, the obtained product is electrophoresed, the target product is recovered, the target fragment is constructed by recombinant plasmid, positive clone is screened, plasmid is extracted, the plasmid is cut by endonuclease to obtain the target fragment, then the gel is recovered, and the gel is recoveredPreparation of oocyte marker gene of Epinephelus coioides by DIG probe kitslbp2Is a specific probe of (a);
the nucleotide sequence of the upstream primer and the downstream primer in the step (4) is shown as SEQ ID NO: 8-SEQ ID NO: shown at 9.
6. The oocyte marker gene of Epinephelus coioides as defined in claim 4slbp2The specific probe of the (2) is applied to the specific marked oocyte of the garrupa and the identification of the garrupa oocyte type.
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| WO2013056252A1 (en) * | 2011-10-14 | 2013-04-18 | Gema Diagnostics, Inc. | Assessment of oocyte competence by detecting spsb2 and/or tp53i3 gene expression |
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| CN107267536A (en) * | 2017-07-10 | 2017-10-20 | 山西大学 | Sinopotamon henanense reproduction marker gene vasa and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US7572601B2 (en) * | 2004-10-15 | 2009-08-11 | Northwestern University | Compositions and methods for determining oocyte development potential |
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| WO2013056252A1 (en) * | 2011-10-14 | 2013-04-18 | Gema Diagnostics, Inc. | Assessment of oocyte competence by detecting spsb2 and/or tp53i3 gene expression |
| CN103342742A (en) * | 2013-06-09 | 2013-10-09 | 北京市水产科学研究所 | Sturgeon oocyte maturation associated proteins and correlation biomaterials thereof |
| CN103724415A (en) * | 2013-12-31 | 2014-04-16 | 中山大学 | Epinephelus coioides sex control gene Rspo1 and preparation method and application thereof |
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