CN118638800A - Application of a cpeb1 gene in regulating animal reproductive ability - Google Patents

Abstract

The invention discloses an application of cpeb gene in regulating and controlling animal reproductive capacity, belonging to the technical field of genetic engineering. According to the invention, by constructing a tilapia cpeb gene homozygous mutant line and carrying out histological analysis through hematoxylin eosin staining and fluorescence immunohistochemistry, the quality of the tilapia heterozygote ovary ovum is obviously reduced, and the tilapia homozygous ovary germ cell is completely deleted and lacks reproductive capacity, so that the cpeb gene can be used as a target spot for cultivating high-fertility fish breeding materials in aquaculture, and a novel way is provided for artificial breeding and assisted reproduction of fishes and mammals to obtain high-quality ovum.

Description

Application of a cpeb1 gene in regulating animal reproductive ability

Technical Field

The invention belongs to the technical field of genetic engineering, and specifically relates to an application of a cpeb1 gene in regulating animal reproductive ability.

Background Art

The quality of animal eggs is determined by the collection and spatiotemporal distribution of all maternal substances stored in the eggs and the maturation of oocytes. Egg quality is one of the decisive factors affecting animal fertility and is crucial to the reproduction of animal populations. At present, due to the impact of environmental pollution on the ecology, the egg quality of many wild animals has declined significantly, seriously threatening the reproduction of animal populations. In recent years, human egg quality and fertility have declined sharply. Therefore, research aimed at maintaining animal egg quality and fertility has important research significance and application value for animal reproduction. At present, the research and mechanism between the cpeb1 gene and animal egg quality and fertility are still unclear, and the cpeb1 gene has not been used as a target for cultivating high-fertility fish breeding materials in aquaculture, so further research is needed.

Nile tilapia is an important farmed fish species. The availability of genetic all-female and all-male fish, mature CRISPR/Cas9 genome editing methods, available high-quality genome sequences, shorter spawning cycle (14 days) and convenience of blood sample collection make it an ideal model for functional studies of oogenesis. Therefore, it is urgent to study the defense mechanisms that affect the quality of tilapia eggs in order to create tilapia materials with strong reproductive performance, and also provide potential molecular targets for the study of ovarian function maintenance and anti-premature ovarian failure regulation in animals.

Summary of the invention

The technical problem to be solved by the present invention is to provide an application of a cpeb1 gene in regulating animal reproductive ability to solve the technical problem of decreased animal egg quality.

To achieve the above object, the technical solution adopted by the present invention is: to provide an application of a cpeb1 gene in regulating animal reproductive ability, the nucleotide sequence of the cpeb1 gene is shown in SEQ ID NO:1.

On the basis of the above technical solution, the present invention can also be improved as follows:

Further, the application is to reduce the reproductive capacity of animals by silencing the cpeb1 gene.

Furthermore, reducing the reproductive capacity of animals means reducing the quality of the eggs of animals and inhibiting the reproductive development of animals.

Furthermore, the animal is tilapia.

The invention also discloses a preparation for regulating animal reproductive ability, which contains the cpeb1 gene.

The beneficial effects of the present invention are as follows: by constructing a homozygous mutant line of the cpeb1 gene of tilapia, the present invention performs histological analysis through hematoxylin and eosin staining and fluorescent immunohistochemistry, and finds that the quality of ovarian eggs of heterozygous tilapia is significantly reduced, and the ovarian germ cells of homozygous tilapia are completely absent and lack reproductive ability, so the cpeb1 gene can be used as a target for cultivating high-fertility fish breeding materials in aquaculture, and at the same time provides a new way to obtain high-quality eggs for artificial breeding and assisted reproduction of fish and mammals.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram of the cpeb1 gRNA target design;

Figure 2 is the result of enzyme electrophoresis;

FIG3 shows the sequencing results of the cpeb1 target site with frameshift mutation;

FIG4 is a schematic diagram showing the prediction of the wild-type complete Cpeb1 protein and the truncated Cpeb1 protein of the homozygous mutant;

FIG5 is a schematic diagram of the construction of the cpeb1 homozygous knockout line;

Figure 6 is a Sanger sequencing peak diagram of the wild type and homozygous mutant;

Figure 7 is an image of an embryo at 24 hpf;

Figure 8 is a graph of relative egg diameters from cpeb1 ^+/+ and cpeb1 ^+/- XX fish;

FIG9 is a graph showing embryo survival rates from cpeb1 ^+/+ and cpeb1 ^+/- XX fish;

FIG10 is a histological analysis of the ovaries of control fish and cpeb1 homozygous mutant fish at 60 and 90 dah;

FIG11 shows the gonadal maturation coefficients of 60dah control fish and cpeb1 homozygous mutant fish;

FIG12 is a statistical diagram of the number of oogonia and phase I and II oocytes of 60 dah control fish and cpeb1 homozygous mutant fish;

FIG13 shows the gonadal maturation coefficients of 90 dah control fish and cpeb1 homozygous mutant fish;

FIG14 is a statistical diagram of the number of oogonia and phase I and II oocytes of 90 dah control fish and cpeb1 homozygous mutant fish;

FIG15 is a fluorescent immunohistochemical detection of ovaries of WT and cpeb1 ^-/- female fish at 60 dah and 90 dah;

Figure 16 is a statistical analysis of Vasa-positive cells at 60 dah;

FIG17 shows the relative expression level of vasa mRNA at 60 dah;

Figure 18 is a statistical analysis of Vasa-positive cells at 90 dah;

FIG. 19 shows the relative expression level of vasa mRNA at 90 dah.

DETAILED DESCRIPTION

The specific embodiments of the present invention are described below to facilitate the understanding of the present invention by those skilled in the art. If the specific conditions are not specified in the examples, the methods are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturers are not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the attached claims, these changes are obvious, and all inventions and creations using the concept of the present invention are protected.

The nucleotide sequence of the cpeb1 gene is shown below:

GGACTGAGGCCCTCGAGCACTGTCATTAGCTGGGCACACGTTCACTAAATTATGAGTAATTAGCTGTTAGCACGCCTCATAAATATGACCTTTTATTGCAGCCAGCGGTCGTTGTTGTGACATTTCTTTTAGGCTGTCTGTCCACATCATGGCGTTTTCCCTGGTGAGTTGAAACGGCATTAAATGGGTGAAATTAGTCAGTTTTGCGCCACGTCTAACGTTATTATTTGAACGTATACCGCCTCATTTGGCCCATTAGTTC CTCCCCGGAACAAAACAAACAAACAAACAAAAATTCACTTTGCAGCATGGTGCACAAGCTTGACCTTT AGCTTTTAGCATGAGTGCTGGGTTAATTGTGTCCCTCTCACGGCCTCCTTCTGCAGAGAAACGAAGCCCGCAGGCCTGAGAGCCCGGACACCGATTCTCCGGCACTGTCCACCTGCAGCAACGCAGACATCTTCCGCAGGATGAACACTATGCTGGGCAACGCTCTGGATTTCACCGGTGTGTGCACCACACCCAACACCAAGGGAAAACCAAACCTGATGTCCGGTAAACCCTCACTCGTCATTCACTCTGCAAGAG TTAAACATTGGTGGGCTTGAGTGCAAAGGCGAGAAACATTAGTATCCGACACTACTGCAGCCTAAACTTAT TTAAACGAGGCTGTAGCCATGAGGGTTGCAAGGGTTTTAGAAATGCTCAAATAGCTTAATACGTAGCAAAGTCCGCGCAAGTCTGTAGTACTTTTATTAAGGTCTAATAGTCAAACTGCATTTTCTGCTAACTTTATTTGCCTACACATCATGTGCTCATTGTGGTCACTTGCAGTCAGGGAAACGTGAAGGCCTTGATTTTGGGCCCCAGATGGTATCAACAGCTGTTTTCACTGTCTGATAAAAATGTTTTAATCTTG TAGACTACAGTTTCCCAAATGGGTTTCCTTTCCAGTGTTTTCATCTTAAAATGCAGTTTTTGACCAGGAA ACAGCCTGAGATGATATTTATAACAAAGCAGGAAATCCTTGTGCAAATCCTAGAAAAGGAAGTGTTTTAGCTATTCAAGCAGAATTTTCTTTCAGTAAACCTAAAGATTGGGCTATATTAGTAATACAGGTACAGTAGTATTGAGGGATATTACATAAGCAATACTTCAACAATTTGAGTCCCAAAGTGCTTGAATCTCTCCCAGTATTTCTCAGTTGTGTGAATTTTGATGCTTTTAGTTTCATGTACAGTGAAGTG TTAGAAAGCAGGTCACATGACCTCTAACCTCGGTGGGAGCTTCTCCTGCTGCTGTGGTTTGCATTTTCCTTAA AACCACAAAACCATGAAGGAACATGCATGCAGTGTGCAAAACCAATGTGGTACTCTGTAAGATGAGAGGCTGCATTGCCTATAATGGAAATCCCCCTTTGACTTTTTAAAGCTGTTGAACCTCCTTCACCTTGTCTTTTTATTCAGGTGCCAGCAGCTGATTCATACTGCATTGCTGGTCTGCAGCATTGCAGTAGTTCTAAGTGTCTTTTTTTTTTTTTTAAACCACAGGAGAGGTATTTGCCAAGATTTTGGCTCAGCTC AGTGCTGGAGTCCTTTCTTTCAGTTAGTTTGCTTAAGGCATGTCTGCTCTTGTGACTACTGGTCTTA TTTTTAGATGACAATACAATCCTTAGTTTGACATGAGCAGAGCATAAATGTTGCATAGCAAAGGTAAGCTGGATCTTGATGCACAGTGAGCTGAAGTTCTTCTTTGGTCTCTGTCCCATCCACTCCAGCTTGCTGAATCAGGGGTGTAACTGGCAGCTGGAAGCTTTCCAGGCCTGGAGAGGGAAGGGAGGAGCTGGTGGTGCAAATCACTCAGTCCTCAACATCTGATCAGTCAGCTGCTTCAGCTACA GCGAGACACTGCTTGCCCGTTTGTTCAGTGTTGGTGTGCTAAAATAAATTCACAAGCCAGTCTTGAAACTGAAATA CCTTGAGCATCCTCAAGTCTACAATACTTTAGTTGAAGTCTGCTGAGTCATTTGCTCCCATGCTGCCTCCCGCCATGCTTCTGTAGCTCTGCCCCTCTCTCCCCCCCCTCCCTCCTGCCTCTGTCAGCTTGCTCGGGGAAACAAGGCTAGACACAGTAAGCAGCAGTAACGGCAGCAGCTGTCTTCACACTGACTCGGAGACGTGCTGTGCCTACCAGAGGGGAGTACTTCTATCAACAAGCCAATAGCTCCAACAGGA ATTTTTCATTACACAGTTGTTAGGCAAGGAAGCGGGGAGGTGGGGGAAGCTGATGTCATTAGCAGGGGT GTGCGAGTGCATCAGCAGCCATGTCACATGCTGCTAGGTGGGTCAGTGATGCGGACGGAGATGCATGGCAGTTGCACCAGGCACAGAGGCAGCAGGTTAGCTCCGCCGTCAGCCAGGGTAGGGGAGATGTGTTTATTGAGGGAGTTTATTGCGGTTTCTTTTACCAAAAGGGGTTTTGACAAGTCTTGCTTTGATCTTTTCTGTATAATGATGCCGATGGGAGGTTGGAAAACGTTCTTTATGGCTGTCTA ACACTTATCTCCTTTCGCTCTTGCTTAATCTGTGAACTGGATTTCGGCAGAGTCGGACCTGGCTGCAGTGGGCAGCAGGAG GATGCTTTTCCCAAACTGCGATGCCATGCCTGGCTCTCAGGAGATGTCGTTGTCCAGGGACCCGTCGGGTGTCACAGACCTGGGCCTGGGTCTTCAGTCTCTCCGTCTGTCTGGCTGGGACAAACCTTGGAGCAGCCAGGAGGCAGATACACACACCTCCCCATCTCACGTTCAAACCAGCTCACCCTCCAGTAAGTGCCAGACCTTTAACTGTGTGTTATAGCATTCTGTTGTTTTGGAGGTACCAGTTTCCCCAAA AGTTTTTCTGGCTCTTACTTTTGAAGAGAAAATTGGAATGGATATAGTAAGGTTGTTTTTTATTGTGTAGA TCTTATTAATATCAAAATAAGATGGTTGATTAACAGCAGTCGGTGTGGGAACAGCCTGTAGCGTCACTACGGAGGATGATGGCAGTTTTGCTTTACTGCATCCCTAGCAAACACACGAACAGCCAGTGAAGCTACTTTTCTTTCCCAAGTTAAATAGAGAGTCAAGTTAAATATTTGTTACCTGTTGATCAAACTTCAGTCAGGGATGTCAGTCAGAAAACTGCACTAACCCTACATATCAGCCTAAGTCAAAGC TGAGTGACAGAGGTACGCCAAGTATGTGTAAATTTTGACATTCTTCAGACAGGTCTCGTTTGAGGCTGTTTTG CCATTTGAGCGGCACTGATAAGGATGCCCCATTTGTCCTGACCCATTTGTAGCACTATAGTTTTGCTGCATGTCCACACCAACCCAGTGTAACTTCATTTAAAGCTTTCCAAACTGGGCCTGCAACAAAGTGGGACAAAGCTTTGAATGCGGCCTACAGCTGAATAATCCCCCTCAGAGCCTCATGTTGACTTGCAGGCCACACCGTACATACCCATCACTGTTGCCAGCTGACTCACTTGGGCATCAAGAACAAGTGACTTGTTAATGCT TAAGGAATTGTTGCTTGGTATTTACTGGATTTGTGTTAGAAACACTGGCTTCCAATTGA TTTGACTTATAAAAAGGCATCTAGCATCTGAGGGGAGAAATTGTTTCATCAGCAGTCTGGTTAGAAAATAAAACCAATTGAATGACTCAACAACCTGAGCTTTAAGGCTCTGTTGGCAGCCGGCTTGCACATCTGGTATCTTGACTAAGAATATTTTTGGAAACCTCCGTTCACATGGCTACCTTTCAGTTCTGATCAATGTCTGTCTGTAGATTCAGCACAAAACTAAATTTAAGATGCCCTTGAAGATGCACCTTTT GAAACTCGAGTTTGCTGTGCAGCTTCATTCCTAACAAATCAATGCAGCTCTTTGGGCACAAATCCCATTTTC TTCAGTTTACTTGAAAAGCTGAAATGGCAAACAAGCTATCGAGTAACCTTACATGGCTGCTGGCACAGATCGCACTCTTTCATACTGATTGTAATTGTTTTTCAGTGTTGGGTCTACTGAACAGTCCCTTTCAGTGGCCTCCTTGGAAAGCCCCCAGGCTACCTGCCCCCCGATTCCATGAGCATGACGGCTGACTTTCTGGAGAAGTTTCCCGGTGTTGCTCGTATGGCAAACCGTCTAGACTCATGCTCCTTTTTGGACTC TCGCTCCAGCAGTCCTGAAGACTCGGAGACCAGTGGCTTCAGCTCTGGCTCAGATCACCTGTGTGAC ATGTTGGTAAGGCAGATGACTTTTATTCTCCAGCTTTGTGTTGGATTGGTGGAAGGTGAATATCTCAAACCTCAATCGACCCATTTACTGTTTTACCGTAGTATCCAAATCTACAGGGTGTGGCATGAAAAGATGGGCTTAGAGTGTGATTTGTGTGCATGAACTTCACTTTCATTTCTCTGCAAGAGTCTTGCACTTAAAGGTATGATAAAGTAATTTAAGGTTATTTAAAGGGGAAAAATTGTTGTTCTAATAAATG TACATATTAATGTGTAATTACATCTTCCAACAGTTGATGCATTGTCATAAATCTAAAATTAATCTATTAGAAAT GCACAAGATTAGACAGTTTGGGGGAAGCCACCATGTTGCCCTGCCATATTGTATACAGGGGCTGAGAGAGAGCGCCATTTCAACTATAACCTCTTCACCTCAAAGCTCATTACCTGAACTGAAGTCTCCTCTAACACAAACATGCTTATTTAGTCCCAATATTGTCACAGTTGTCAATGGTCTGGGTCACGTTTAATTCTACTAAATGGCAGCAGCTGGTTATAAGCTAACATTATGCCAGCTAATGTTAGGCTTTTGTGTC CTAAAGTCAGACTTTTTAGAAAGACAGTTTCACTAATACTGTAAAAAGCAGTGCTTACAACCAAAA CTGATATCTTCTGCTCATCGCAATATCTGTCATTGGACAGAAGTGAGCTTTACTGACTCACCAGCTTGTATTGGACGCCTTTCAAAGTTTTACAGCCACTTTCAGAGTAAATGGACACTCGGCATATTGACAGCTGATGAACAGTGACTGATGGCCAACAATCACTACAGATGAATAGTGGCCAGAGCTGTTTGTCCTCCAGCAGCCACTGTAGATAAGATTATGTACTTAAATCTGGAGGGGCACTAAATCGAATTTGGTTG ACTACAATCTGCAGTATACTGATATCTAGCGGTGAGATTTTACACTTTACCTTTTTAAGATTGTTACG TTCTCCTCAACTGACAGGAATCTACCTTAACCTTCCTTTGGCCTGCTTTTTAACTTTGAGGATTTAAATTGTTTGCTGTGTCCCCAATAGATTAATGTCAATCATCAGCTTCATTGGTCAGGCTTCATCACAGGATATTTTCAGTTTTAACTTTCTGGCTTTTTTTCTTGTAAAACATCAGAAATGATTTTGAGATGGATGTCAGAAATTAATCAGTTATATTAAGGCATGCAAAGAAAAGATTAATCCTGTAGGGT TTTTATTTTTTGTGCCACATTGGCATTTATTAACTTCGAGTTTGGCAGCAGACAGTGTGGACTGCAAATTTC TGATTATCAAGTCAGATACTGCACCCAGTCTAAACCTTTCAGTGAATTTCAGACTTTTTATTTTAATACACAGAATGGAGGAAAATATGAGGCCACTTTACACTAACGCATATCTTCTTCTCCAGTCCACTTTGCGGATTTCCCCTCCCCTGCCTTATCTCACGTCAAACGTGCAGAAGGACCTGCTGCGACTGGGCTCTCGCCTGGACCCTCAGGACACTAGCTCTCCTCTGACCCCTCCATCCAGTGCCAGCCCTTCC TCCTTGGCCATTTCCCGTCGCTGGCGTGGTGGGTCTTCGTGGCCAGGTGCAGATGTACTTGATCAGCCAG AGGATGCTTTCAGCATTGAGAGGGAAGCCCGCCTGCATAGACAGGCTGCAGGTAGTCACTACTCATTAATCATCTTGAAACTGTTCAAGCATGACAATCCACAAACCTTGTCTGTCTTGTTTTCAACTCAGCTGTGAATGAGGCGACATTCACCTGGAGTGGACAGCTGCCTCCCCGTAACAATAAAGATCCTCTTTACTCCTGCAAGGTCTTTCTTGGTGGTGTACCTTGGGACATCACAGAAGGTAAGTGTGGCTGTAAACGGTCT TTATTTTCAAACACGTTCTTCTGATTTTATGACGGGGGTTGTTTGGTTTTTGTTTTAAAGAT TTGAAAAGATTGTGTATTGAAATCCCAGATACAGACATGCTTAAATCTGTTTCTGTGGCTGCACATAGACTTTTATTCCCTTTGGGTTTTTCAGGTTTGGCCCTACTTAATGTTTTAACACATGTAGCCCTGATGGTTCTGAACAGTTCACTTAACAGTCAGCTATAGTGTGTTTTACTTGGTACATGCCAGCTTTTTTATTGTGGCTTTACAGAATTGATTAATGCTGAATTGTTTAGTTTGCTAACCAGTATCTTGGCAACA GCTGCAGATGTGTGGCTATAAATGGCAGCCACTGAAAACTAGAGCTTGGGACTAATGGCAGCATGA AATAAACATGCATTCTTGGATATCCATTCATTTTTAAAAATGACTATACTTAAACCCTTATTTACTGTAGTCTTTTTGGCAAGGATTACAGGCATTGGTTCAAACCTCACTATCTTGGTACATCTCACTGTCACACGTTGAAGGTGTGCATTGTTATTCCACCCTCACAAACTGATGCTTTATTTCAGCTGGCCTGATCAACACCTTCAGTGGTTATGGCCCGCTGACTGTGGAGTGGCCGGGTAAGGATGGAAAGCACCCTCGCTGTC CTCCCAAAGGTAATGTCGCCAAAGGTAACAAAGAGCTGGTAGGCTATTTTTTTTTTTCCT TCTGGTTTTAAGGTGGCACTGTGGTGAGCATGTAGAAAGGTGGTGAGTAACTGATGGAGATTCTGAGCATTGCTGGAGCATAAAACACGGTGTTAGAAGCAGGGGTGGGCAAATGCTTTCGGTAACACTTTGCTAATTAGTTTGTATAAATGAGTTTTGTGGACCTCCAGTGGTGACATACCCCCACAGGCCATTAAGTTGTAAACTGGTTTTATGACAGTTTTTCAAGTCATTATTTCTCAAATTTCTTAAATCTACATT TTATATGGACTTGGGCTAAATTTTGGTCAGCATTTCATCACTGATTTATCAGTCTAATTATGCATTACTT TACACTCCTAACTTTTTCGGTTGGACTTGGAACTTAGTGTTTCACTTTTGCAGTCGCATTAAAAGCATTAAACCTGGCTTGGTGTCAAGTACTCGGCACCTTTCAAAGCTTTCAGCAAAACGGCTACAGACAAGTTGTAAACTGTCAGGACCTTTTGGAAACTATATTAAATGCTAGCAAGTCTTACTGTAGTTGCTGTGTTACTCAGTCTTGGGTTGTTGTAGCTTGTAGTTCAAGAGGCAACATGCACTGTAGTACCCGTGG TATCGGTCTGCCCTGCAGAATGATGCAGCAAGTAAGCCTCTTTTAAAGTGTTTTGCTGTTTGT CTGTAGGCTACGTTTACCTGGTGTTTGAGAATGAGAAGTCTGTCCGGGCTTTGCTCCACGCCTGCTCCCAGGACCCGTTTCACCCTGAGGACAACAGAGAGTACTACTTCAAGATGTCCAGCCGCAGAATGCGATGCAAGGATGTAGGTGGAAACGAAGCATGCAAGGCATTATTTACTCACTGCATTTACAACTTACGAAATGCTGGATAATCAAATAGCTGTCATATTGTACTCGAGTATGTTTAAGAAATATAGGTGTAGT TTGAATTCTGTCTGTGATGTAACTCCAAGAAATGGGTTGTCCAGGATTAGATCCTGTAGAATCTGAT TTCACACACCTTGTTTGTCAGATTTCAACAATGACTAATTCAAGTGAGATCACCAGCACACACATCGATATATATGGACACATGTAGAAAGCTTCTACGTCTATGGATTCTTTGGTCAGACCTTCTTGTTGAGCACACTTATTACGAGACAGTTGGCTTTTGTTTAGTGAGCGATCCTATTGAAGGACAGGAAGCGAGGATGTCTGTCCTGAATGCTGTGGCTCTTGGCTGCTACAGGTGCAGGTGATACCCTGGGTGA TCTCTGACAGCAACTACGTCCGTTGCCCTGCTCAACGTCTGGACCCGAGTAAGACTGTGTTTGTTGGCGCG CTGCACGGCATGCTGAATGCTGAGGCGCTGGCCAGCATCATGAACGATCTCTTTGGAGGAGTCATGTATGCTGGCATCGACACGGACAAGCACAAGTACCCAATAGGTGAGACTGCTAGTCTCGAATTGTCAGCATCTACTGCAGTGTGGTTCTTGGTGACCACTGTAGGTGTGGTTTGACCTTTAAGAGCTGCTTTGAGTGACATGATTTGTCTTCTGTACAGGCTCTGGACGTGTCACTTTCAATAATCAGCGC AGTTACCTGAAGGCTGTGTGCGCAGCATTTGTGGAGATTAAAACACCCAAGTTTACAAAGAAGGTAAAGA CATTGTTTACTGCATGTGTTCTCCAGCAGCAGAGAAGTCTTTCAACAGTTTTGTGCTGCTTTTGGTTAAGTGTCAATTTTCAAAACTTGTCTGCTATAAATTGAGCGTGTTTCCCCTTCAGGTTCAGATTGACCCCTACCTGGAAGACTCCATGTGTCAAGTTTGCCTTCGCCAACCTGGACCTTTCTTCTGCAGAGACCAGGTCAGTGGAAACTCCTGCTCCCCTTTTTGTGAGCAGGTGGCTGGGTTCAGTC TAGCTTTCTGTCCAGTAGACTTCATGTCTTATGCTGAACCAGAATATTAAAATTTGGTAATTAAATCATGATCC TTTAAACTTGTTTGTAAATGGACAGAAAAAGGGAATTATGCAGCTTGAGCTTAACTGTAGTTCAACCTCATCTGTGAGAACAGTTGTACTTAAAGTTTCCTATTGAAATATGGGAGTGATGTTGTTATAGAAGTTTAGAACTTGTCTTGTTTTATTGGTAGTTTAATTAAAGTTTAAGTGATGATATTGTTAGACTGATTTTAAATCAGATGTGCCTCTATAATGCACTCTTAGGCCTGCTTCAAGTACTACTG CCGCTCCTGCTGGCACTGGCAGCACTCAATGGACATCTTAAGCAACCACCGGCCCCTCATGCGCAACCAGAAGA ACAGGGACTCCAGCTAGAGGAGATGGATTCAGAGTCCCTGAAGGGCAAGAGGAGGTGCCCGTCTGCACGGGGCAAGGCTGCCCGCCAAGGAGAGCACGTGTGCCAGACCTCCGGTACAAGGATACCATGGCACACCACCAGGCACTGGGGATATCACTATAGAAAATGTTCACTTTTGCACTTGCTACATTTTTGCTGTTCAGTGGCACTATGCACAGTGACCTTGTTGGGAAAGACAAGGGGCCCTTTCACTCTTATGCATTTTC CCAGACTTGTGGCCAGATGGAAAGATGGGAGTCAGCCTAAGGTTAAGAAGTGGATGAAAACACCA TTTCTTTAATTTTTTTTTTTCGTTTACATGTGTTGCATAGTGAAGAGCAATCACGCAAGTCTCATTGCCGTTCATTGGCAAACGCCATACAGTGCCTTTAAATTTTTTATTTCCCCCCCTCCCCCTACCCCCAGTTGGCATCAGTTTTTTCCTGTGCAAGCCAACTGGTGTTTCTTACTTTTGGGAGTTGAATGTTGGGCTTCATTTAACCAGTCGCATTTCAGACATTTTTACGCAGTTTTTTACTAATTTGTACACAAACGG TTTTAAGTCATGACATGAAAATTCTGAACAATTATACCCAGTGGCTGTTTATGGGAAAACCCCCTTT TATTTTTTTTTTCCTGGCTTTTTTTTTTTTTTTAATTTGCTCTCCCAAATTTCAACTGAATGGGCACAAATTGTTGGATGTACTACTTCGACTTTGTTGGTCCAGTTATATTAAATGTAACGGTAGTACTGGATAACTCAACTGGTAATCCATTTTCTTTTTCCAAGCAACTGATGGATCTTGATGAAAGTACAGTGAGCAAATCATTTGTACATAGTTTGATTTTTGTTTTTTGCACTTTTTAATTTGCACGTGTGGAGG TGGCAGTGGTTTTAATCACTTTTTGTACATCGGCAGGGGGACATGCCTATGTTAGAACAGCTGCT TGTTTCCTGTTGAATGTAAGAACTTCCACTTAGCTGATGATAAATTATCTATCCCAGAGCTTGAGTAGACCAGTACATGATTCAATGCTTTCTTATCCCATTTTGCCTTCCTGTTCCAGTTGGACATAAATGCATAATAAGCTGTGTGCCAAAGACGAAGTGTCTTTTCTTTATCCATTAGGTTTGTCATGCTGAGCTTGTTGTCCCTTAGCCCAGCTGCTTTTTAGAGTTTTGAAAGTCTACCTCATTGTGGTCTGC CAAGTTTGCTGCTACTTAACAGTTGTGGAGTATGTTCAGATTGGGCACAATAAAGGTGTTTAAGCATTA (SEQ ID NO: 1).

The amino acid sequence encoded by the cpeb1 gene is shown below:

MAFSLRNEARRPESPDTDSPALSTCSNADIFRRMNTMLGNALDFTGVCTTPNTKGKPNLMSESDLAAVGSRRMLFPNCDAMPGSQEMSLSRDPSGVTDLGLGLQSLRLSGWDKPWSSQEADTHTSPSHVQTSSPSMLGLLNSPFSGLLGKPPGYLPPDSMSMTADFLEKFPGVARMANRLDSCSFLDSRSSSPEDSETSGFSSGSDHLCDMLSTLRISPPLPYLTSNVQK DLLRLGSRLDPQDTSSPLTPPSSASPSSLAISRRWRGGSSWPGADVLDQPEDAFSIER EARLHRQAAAVNEATFTWSGQLPPRNNKDPLYSCKVFLGGVPWDITEAGLINTFSGYGPLTVEWPGKDGKHPRCPPKGYVYLVFENEKSVRALLHACSQDPFHPEDNREYYFKMSSRRMRCKDVQVIPWVISDSNYVRCPAQRLDPSKTVFVGALHGMLNAEALASIMNDLFGGVMYAGIDTDKHKYPIGSGRVTFNNQRSYLKAVCAAF VEIKTPKFTKKVQIDPYLEDSMCQVCLRQPGPFFCRDQACFKYYCRSCWHWQHSMDILSNHRPLMRNQKNRDSS (SEQ ID NO: 2).

Example 1 CRISPR/Cas9 system knocks out cpeb1 gene

(1) Selection of cpeb1 gene targets

By accessing the NCBI database, the Nile tilapia cpeb1 gene sequence was obtained, and then the start codon and stop codon of the gene were found, and the exon region was marked. Since the first exon sequence of the cpeb1 gene was short (the number of bases was 30bp), the target was selected on the second exon, and the second exon sequence of the gene was entered into the online target prediction website to obtain a target with a length of 20bp. The target sequence of the cpeb1 gene was 5'-GAGCCCGGACACCGATTCTC-3.

(2) Synthesis and extraction of gRNA

T7 promoter and homology arms were added to both ends of the gRNA, and the gRNA was sent to BGI for synthesis. The gRNA double-stranded template was transcribed in vitro using a T7 in vitro transcription kit. The sequence of the oligo fragment containing the cpeb1 gene knockout target was 5’-TAATACGACTCACTATAGGGCCCGGACACCGATTCTCGTTTTAGAGCTAGAA-3’.

(3) Co-injection of gRNA and Cas9 mRNA

Normal male and female wild broodstock were selected for mating, and fertilized eggs were taken for use. The synthesized gRNA and Cas9 mRNA were mixed with phenol red in a ratio of 1:1, and co-injected into the fertilized eggs of wild tilapia at the 1-cell stage using a microinjector, and then the fertilized eggs were placed in a shaking incubator at 37°C for incubation. After the fertilized eggs were hatched, they were transferred to a circulating aerated freshwater system with a natural light cycle (12h light/12h dark), a water temperature of 26°C, a pH of 7.5, and a dissolved oxygen of 6.8mg/L.

(4) Genotype identification of F0 generation tilapia

The F0 generation was raised, the tail of tilapia was cut for genome testing, and primers were designed to amplify the sequences near the target site of cpeb1 gene. The sequences were connected to the vector for sequencing and the gene mutation was identified.

(5) F0 generation tilapia breeding and F1 generation tilapia genotype identification

When the F0 generation male cpeb1 mutant tilapia grew to 6 months old, they were mated with wild female fish to obtain F1 generation tilapia; the genomic DNA from the tail of the F1 generation tilapia was extracted, the sequence near the cpeb1 gene target site was amplified by PCR, the specific mutation type of the F1 generation individuals was detected, and the mutation type with a deletion of 8bp was obtained by screening.

(6) F1 generation tilapia breeding and F2 generation tilapia genotype identification

The F1 generation male and female individuals carrying the same cpeb1 mutation type were mated to obtain the F2 generation tilapia. The F2 generation genome was extracted and PCR amplified to detect the mutation type of the F2 generation individuals. After screening, the cpeb1 gene homozygous knockout line was successfully obtained, as shown in Figures 1 to 6.

Figure 1 shows that Nile tilapia cpeb1 F0 positive fish were obtained using CRISPR/Cas9 technology. The tilapia cpeb1 genome contains 13 exons. A mutation target was designed on the second exon, which contains a recognition site for the restriction endonuclease Hinf I, which can be used to identify mutations.

After the F0 generation fertilized eggs were digested with Hinf I, the enzyme electrophoresis results (Figure 2) showed that the agarose gel electrophoresis results of the fertilized eggs injected with gRNA and Cas9 at the same time showed three bands, including the top 673bp band that could not be cut due to a mutation at the enzyme cutting site, and two bands (538bp and 109bp) that were completely cut by Hinf I. The electrophoresis results of the fertilized eggs that were not injected or only injected with gRNA or Cas9 showed two completely cut bands, indicating that cpeb1 was successfully mutated by CRISPR/Cas9.

The sexually mature mutant fish F0 generation XY was fertilized with the wild type XX to obtain the F1 generation. Five different mutation types were identified by PAGE detection and Sanger sequencing. The F1 generation heterozygote with a deletion of 8bp was selected for passage and establishment of the line. The mutant sequence and the PAM structure of Hinf I are shown in Figure 3. Figure 4 shows the protein structure of normal Cpeb1 and the truncated protein with deletion of RRM1, RRM2 and ZZ domains after the mutation of 8bp. F1 generation XX and XY fish with the same mutation type were mated to obtain F2, and the F2 homozygous mutant fish with a deletion of 8bp were screened by Sanger sequencing (Figure 5). Figure 6 is the Sanger sequencing peak diagram of wild type and homozygous mutant.

Example 2 Determination of the effect of cpeb1 gene on egg quality and fertility

(1) Embryos of F1 female fish with 8bp deletion mutation and F1 male fish were obtained 210 days after hatching, and the embryos of control fish were observed. The results showed that the egg diameter of the embryonic offspring of F1 female fish was significantly smaller than that of wild fish offspring.

(2) The embryos of the F1 generation female fish with the 8bp deletion mutation type and the F1 generation male fish were taken, the embryos of the F1 generation female fish with the 8bp deletion mutation type and the wild male fish were taken, and the embryos of the wild male and female fish were taken, and the embryo survival rate was recorded. The results showed that the embryonic mortality rate of the offspring of the F1 generation heterozygous mutant fish was high, and most of the embryos died before 3 days after fertilization (dpf), and their survival rate was significantly lower than that of wild fish embryos.

The above results indicate that haploinsufficiency of cpeb1 will lead to a significant decrease in the quality of eggs and impaired developmental capacity of fertilized eggs, as shown in Figures 7 to 9.

Smaller egg diameters were observed in embryonic offspring of cpeb1 ^+/- female tilapia compared to wild type (Figure 7). As can be seen in Figure 8, statistical data showed that the egg diameters of heterozygous mutant fish offspring were significantly smaller than those of wild fish offspring. As can be seen in Figure 9, embryonic mortality rates of cpeb1 ^+/- female fish were high, with most embryos dying before 3 days post fertilization (dpf), and their survival rates were significantly lower than those of wild fish embryos.

(3) Ovaries of cpeb1 knockout fish and wild-type fish were collected at 60 and 90 days after hatching for ovarian morphology analysis.

As shown in Figures 10 to 14 , obvious oogonia and phase I and phase II oocytes can be observed in wild female fish, however, no oogonia and oocytes of any phase were observed in the ovaries of cpeb1 knockout fish, and cavities appeared to varying degrees.

As shown in Figure 10, at 60dah and 90dah, obvious oogonia and phase I and phase II oocytes can be observed in wild female fish, however, no oogonia and oocytes of any phase were observed in the ovaries of cpeb1 ^-/- fish, and cavities appeared to varying degrees. As shown in Figure 11, at 60dah, there was no statistical difference in the GSI between wild fish and cpeb1 ^-/- fish. As shown in Figure 12, at 60dah, the statistics of the number of germ cells in the same visual field showed that there was a lack of germ cells in the ovaries of cpeb1 ^-/- fish. As shown in Figure 13, at 90dah, the GSI of cpeb1 ^-/- female fish was significantly lower than that of WT fish. As shown in Figure 14, at 90dah, the statistics of the number of germ cells in the same visual field showed that there was a lack of germ cells in the ovaries of cpeb1 ^-/- fish.

As shown in Figures 15 to 19 , no positive signal of the germ cell marker gene Vasa could be detected in the ovaries of the cpeb1 knockout fish.

Figure 15 detected the expression of the vasa gene in the wild-type ovaries at 60dah and 90dah by immunofluorescence staining, while no Vasa positive signal could be detected in the ovaries of cpeb1 ^-/- female fish. The number of germ cells was counted using the positive signal as a marker, and the results showed (Figure 16) that the number of Vasa positive cells in the ovaries of cpeb1 ^-/- female fish at 60dah was zero, significantly lower than that of the ovaries of WT female fish. The qRT-PCR results at 60dah showed (Figure 17) that vasa mRNA was not expressed in the ovaries of cpeb1 ^-/- female fish, and its expression level was significantly lower than that of WT female fish. As shown in Figure 18, the number of Vasa positive cells in the ovaries of cpeb1 ^-/- female fish at 90dah was zero, significantly lower than that of WT female fish ovaries. The qRT-PCR results at 90dah also showed (Figure 19) that vasa mRNA was not expressed in the ovaries of cpeb1 ^-/- female fish, and its expression level was significantly lower than that of WT female fish.

The above results indicate that the homozygous mutation of cpeb1 leads to a complete absence of germ cells in tilapia, which in turn causes the loss of its fertility.

Claims (5)

1. A use of a cpeb1 gene in regulating animal reproductive capacity, wherein the nucleotide sequence of the cpeb1 gene is as shown in SEQ ID NO: 1. 2. The use according to claim 1, characterized in that the use is to reduce the reproductive capacity of animals by silencing the cpeb1 gene. 3. The use according to claim 2, characterized in that reducing the reproductive capacity of animals is to reduce the quality of the eggs of animals and inhibit the reproductive development of animals. 4. The use according to any one of claims 1 to 3, characterized in that the animal is tilapia. 5. A preparation for regulating animal reproductive ability, characterized in that it comprises the cpeb1 gene according to claim 1.