CN114875030A

CN114875030A - dsRNA (double-stranded ribonucleic acid) of rainbow trout ccne1 gene and application thereof

Info

Publication number: CN114875030A
Application number: CN202210630641.XA
Authority: CN
Inventors: 黄天晴; 刘恩慧; 徐革锋; 谷伟; 王高超; 王炳谦
Original assignee: Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
Current assignee: Heilongjiang River Fisheries Research Institute of Chinese Academy of Fishery Sciences
Priority date: 2022-06-06
Filing date: 2022-06-06
Publication date: 2022-08-09

Abstract

The invention discloses dsRNA of rainbow trout ccne1 gene and application thereof, belonging to the technical field of biology. The invention provides an oncorhynchus mykiss ccne1 gene, an interference gene fragment and dsRNA thereof. Firstly, cloning to obtain a gene sequence of ccne1 by taking total RNA of gonad tissue of rainbow trout parr as a template, wherein the nucleotide sequence is SEQ ID NO.1, and the amino acid sequence is SEQ ID NO. 2. Designing a primer of an interference gene segment by taking a ccne1 gene sequence as a template to obtain the interference gene segment, wherein the nucleotide sequence is SEQ ID NO.3, and synthesizing the dsRNA of the rainbow trout ccne1 gene through in vitro transcription. The dsRNA of the rainbow trout ccne1 gene can efficiently silence the ccne1 gene, effectively reduce the expression of other meiosis related genes in the rainbow trout body, and provide a research idea for analyzing the meiosis pathway of the rainbow trout.

Description

dsRNA (double-stranded ribonucleic acid) of rainbow trout ccne1 gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to dsRNA of rainbow trout ccne1 gene and application thereof.

Background

Rainbow trout (Oncorhynchus mykiss) belonging to the order Salmoniformes, Salmonidae, and Oncorhynchus, is named for its body-side banded reddish streak. The rainbow trout as a main breeding variety of salmon and trout in China has fine and smooth meat quality and delicious taste, is deeply favored by consumers and has very high commercial value. However, adverse effects which disturb production and cultivation, such as reduction in product performance and reduction in stress tolerance due to gonad development, are observed during production. In recent years, the key scientific problem of rainbow trout gonad differentiation has become a research hotspot, and the analysis of the key regulation molecular mechanism of rainbow trout is explored.

RNAi (RNAi) technology, which specifically degrades or inhibits the expression of homologous mRNA by small double-stranded RNA to inhibit the expression of a specific gene, is currently widely studied in the field of gene function studies. Therefore, the RNAi technology is used for carrying out molecular mechanism regulation and control on the rainbow trout gene, particularly the gene related to meiosis, and has certain guiding significance for analyzing the gonad development mechanism of the rainbow trout.

Disclosure of Invention

The invention aims to provide dsRNA of rainbow trout ccne1 gene and application thereof, and provides a new gene resource for fish meiosis research.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides application of dsRNA of rainbow trout ccne1 gene in inhibiting meiosis or preparing meiosis inhibitor, wherein the dsRNA is synthesized by in vitro transcription by taking an interference gene segment as a template; the nucleotide sequence of the interference gene fragment is shown as SEQ ID NO. 3.

Further, the dsRNA inhibits the meiosis of the gonads of the rainbow trout by reducing the expression level of genes comprising a ccne1 gene, a SYCP1 gene, an MLH1 gene, a DMC1 gene, a Spindlin gene and a beta-Tubulin gene.

The invention also provides application of the interference gene segment or dsRNA of the rainbow trout ccne1 gene in silencing the ccne1 gene, which is characterized in that the dsRNA acts on the interference gene segment in the ccne1 gene so as to inhibit the expression of the ccne1 gene.

Further, the dsRNA takes an interference gene fragment as a template and is synthesized by in vitro transcription; the nucleotide sequence of the interference gene fragment is shown as SEQ ID NO. 3.

The invention also provides a method for inhibiting gonad meiosis, which is characterized in that dsRNA of the ccne1 gene of the rainbow trout is injected into the body of the rainbow trout.

The invention discloses the following technical effects:

(1) the dsRNA segment for the rainbow trout ccne1 gene can be efficiently combined with a target gene, the expression of the ccne1 gene is reduced, help is provided for analyzing a meiosis pathway of the rainbow trout through establishment of the model, clues and theoretical guidance are provided for analyzing an action mechanism of the rainbow trout gonad development, and the dsRNA segment has application significance.

(2) The rainbow trout ccne1 gene is knocked down at the body level by using an RNA interference technology, the silencing efficiency of the dsRNA after injection in the gonad tissue of the rainbow trout reaches more than 85 percent, and the expression of the rainbow trout ccne1 gene is effectively inhibited.

(3) According to the invention, the expression of the meiosis key gene of an individual with the knock-down rainbow trout ccne1 gene is investigated, and the injection of dsRNA can effectively reduce the expression of other meiosis related gene mRNA in the rainbow trout body, so that a research idea is provided for the meiosis pathway analysis of the rainbow trout.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a gel electrophoresis strip of dsRNA of rainbow trout ccne1 gene, wherein M is DL5000 DNA Marker, lanes 1-5 are rainbow trout ccne1 dsRNA segment;

FIG. 2 shows the survey result of the expression level of ccne1 gene in rainbow trout after dsRNA injection, wherein different letters represent that the difference is significant and P is less than 0.05;

FIG. 3 shows the results of the investigation of the expression level of meiosis-associated genes in rainbow trout after dsRNA injection. Indicates significant difference from control group (P < 0.05); indicates a very significant difference from the control group (P < 0.01).

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

The rainbow trout parr in the embodiment of the application is taken from a Bohai cold water fish test station (peony river) of the research institute of aquatic products in Heilongjiang, China aquatic products science institute, is aerated with water and transported back to a laboratory, and is raised in a circulating temperature control aquarium.

The reagents used in the examples of the present application are generally commercially available unless otherwise specified.

Example 1 cloning of rainbow trout ccne1 Gene fragment

1. Total RNA extraction

The experimental rainbow trout parr (weight 30 +/-5 g) is anesthetized by MS222, the whole gonadal tissue is collected on an ice tray, a sample is collected by liquid nitrogen, the total RNA of the gonadal tissue of the rainbow trout is extracted by a kit method, and the specific method refers to the instruction of a Simpoly P total RNA extraction kit (Bio Flux). The RNA concentration and purity were determined using a UV spectrophotometer (Analytikjena, Germany).

Reverse transcription of cDNA

Carrying out reverse transcription on the total RNA, and carrying out reverse transcription by utilizing a reverse transcription Kit PrimeScript RT Reagent Kit (TaKaRa, Dalian) to obtain cDNA, wherein a reverse transcription system is as follows: total RNA 1. mu.g, Oligo dT Primer 1. mu.L, dNTP mix 1. mu.L, RNAase-free dH ₂ O to 10. mu.L. After keeping the temperature at 65 ℃ for 5min, the mixture is rapidly cooled on ice. To the above system were added 4. mu.L of 5 XPrimeScript II Buffer, 0.5. mu.L of RNase Inhibitor, 1. mu.L of PrimeScript II RTase, and RNase Free dH ₂ O to 20. mu.L. Slowly mixing, cooling at 42 deg.C for 30-60min, 95 deg.C for 5min, and cooling on ice. And (3) packaging and storing the cDNA after reverse transcription in a refrigerator at the temperature of-20 ℃ to avoid repeated freeze thawing as much as possible.

3. Amplification of fragments of interest

The rainbow trout ccne1 sequence was obtained in NCBI data with sequence ID XM _ 021585952.1. Primers were designed using Primer5.0 software and the primers obtained are detailed in Table 1.

TABLE 1

Primer and method for producing the same	Sequence of
		Upstream primer Osccne1-F (SEQ ID NO.4)	ATGACCAACAGAGGCCACGAATCGA
Downstream primer Osccne1-R (SEQ ID NO.5)	ATGCCAAGCAAAGGGGAAAATGTGG

And (3) taking cDNA obtained by reverse transcription as a template, and carrying out PCR amplification to obtain a target product fragment. The PCR amplification system is 2 XTaq mix 25. mu.L, Forward Primer 1.25. mu.L, Reverse Primer 1.25. mu.L, cDNA 5. mu.L, plus ddH ₂ O to 50. mu.L. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 2 min; 94 ℃ 30s, 59.5 ℃ 30s, 72 ℃ 30s (30 cycles total); extension at 72 ℃ for 5min, 4 ℃ for forever.

The PCR product was detected by electrophoresis on a 1% agarose gel at 110V for 30 min. The target band was cut and recovered (the band size was about 1230bp), and the band was recovered and purified using Gel DNA Extraction kit 4.0(Takara) kit, and the specific procedures were referred to the kit instructions.

4. Target fragment transformation of Escherichia coli

The recovered product was ligated with pMD18-T vector in the following system: 1 mu L of pMD-18T vector, 4 mu L of gel recovery product, 5 mu L of Solution, slowly mixing uniformly, and connecting for 12-16h at 16 ℃.

And transforming the connecting product into escherichia coli DH5 alpha competent cells, spreading the cells in an Amp resistant solid culture medium, airing the cells, and performing inverted culture in a constant-temperature incubator at 37 ℃ for 12-24 h. After picking up a single colony in an Amp-resistant liquid LB culture medium and shaking the colony for 12 ℃ at 37 ℃, plasmids are extracted according to a plasmid extraction kit (Beijing Tiangen Biochemical technology Co., Ltd.), and the plasmids are sent to Hongxi Biotechnology Co., Suzhou for sequencing.

And comparing the sequencing result with an NCBI database to obtain a sequence fragment of the rainbow trout ccne1 gene, wherein the sequence fragment is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 2.

SEQ ID NO.1：

ATGCCAAGCAAAGGGGAAAATGTGGAATCCAGGACAATTGATCATGAGATGCCCAAAGAAACTGCAGTGAGGTCCAAGAAAAGAAAATCAGACGTTGCCGTTTATTTACAAGACCCAGATGAAGTGGCAGAGATGACGAAAAATAACCAGTGTGGAGCTTTGGTTTGCTGGAATCCTGAGTCGGTTCACACCATCCCGCACAGCCGGATCCCCACACCTGACAAAGTCGATCAACCAGTTGCTTTGAATGCCACTGGGTTTTCTACTCAGTACACCTTCAAAAACATATTTGTCACCCCCACCAGGTCTTCCCCTCTTCCTGTGCTATGCTGGGCAAGCAGAGACGACGTATGGAACAACCTGTTGAAGAAAGACGACACGTATTCTCGAGACATCCACGTCATGAAGAAACACCCACACCTCCAACCCAAGATGAGGGCTATTCTTCTGGACTGGCTAATGGAGGTGTGCGAGGTGTACAAGCTTCACAGAGAAACCTTCTACCTGGCTCAGGATTACTTTGATCGGTTCATGGCCACACAGAGCAATGTTTTCAAGTGCACACTACAACTCATCGGCATCTCTTCTCTGTTTATAGCTGCCAAGATGGAGGAAATCTACCCTCCAAAAGTGCATCAATTTGCCTATGTAACTGATGGTGCCTGCACTGAGGATGATATCCTATGTATGGAGCTAATCATCATGAAGGAGCTGAAGTGGCGTTTGAGTCCCCTGACACCTGTGTCCTGGCTAAACATCTACATGCAGGTTGCCTACCTGAAAGAGTCCGAAGAGGTTCTCATCCCACAGTACCCGCAAACTACATTTGTGCAGATTGTCGAGCTCCTGGACCTCTGTGTGTTGGATGTGAAATGTCTGGAGTTCTCCTACGGGGTGCTGGCTGCCTCTGCCTTGTTCCATTTCTCCTCGCTGGAGCTGGTGGAGAAAGTCTCAGCTCTGAAGTGGTCTGATGTTGAGGAGTGTGTGAGGTGGATGGTCCCCTTTGCCATGTCCATCAGGGAAGTGGGCAGCTCAGCCCTGAGGACCTTCAAAGGAATCCCGGCAGACGACATGCACAACATCCAGACCCATGCTGCCTATCTGGACTGGATGGCGAAGGCATGCGCCTACCCACAAGTGGATGTAGATCGCAGCCAGAGTTCACCTATACCATCCGGAGTGCTCACTCCGCCCCCTAGCAGCGAGAAGCCAGAGGGCACAGTCTCTTGA。

SEQ ID NO.2：

MPSKGENVESRTIDHEMPKETAVRSKKRKSDVAVYLQDPDEVAEMTKNNQCGALVCWNPESVHTIPHSRIPTPDKVDQPVALNATGFSTQYTFKNIFVTPTRSSPLPVLCWASRDDVWNNLLKKDDTYSRDIHVMKKHPHLQPKMRAILLDWLMEVCEVYKLHRETFYLAQDYFDRFMATQSNVFKCTLQLIGISSLFIAAKMEEIYPPKVHQFAYVTDGACTEDDILCMELIIMKELKWRLSPLTPVSWLNIYMQVAYLKESEEVLIPQYPQTTFVQIVELLDLCVLDVKCLEFSYGVLAASALFHFSSLELVEKVSALKWSDVEECVRWMVPFAMSIREVGSSALRTFKGIPADDMHNIQTHAAYLDWMAKACAYPQVDVDRSQSSPIPSGVLTPPPSSEKPEGTVS。

Example 2 Synthesis of interference Gene fragment of rainbow trout ccne1 Gene and dsRNA

Based on the rainbow trout ccne1 gene with the nucleotide sequence shown in SEQ ID NO.1, https:// www.dkfz.de/signaling/e-rnai 3/website is used for designing an interference gene fragment primer of the rainbow trout ccne1 gene, a T7 sequence is added before an upstream primer and a downstream primer, and the primer information is shown in Table 2.

An upstream primer SEQ ID NO.6 and a downstream primer SEQ ID NO.7 containing a T7 promoter are used for obtaining a PCR product through PCR amplification, the sequence is shown as SEQ ID NO.3, and the interference gene fragment of the rainbow trout ccne1 gene is obtained through purification and recovery by a PCR purification kit.

TABLE 2

The sequence of the interference gene fragment of the rainbow trout ccne1 gene is shown in SEQ ID NO. 3.

SEQ ID NO.3：

CCAGTGTGGAGCTTTGGTTTGCTGGAATCCTGAGTCGGTTCACACCATCCCGCACAGCCGGATCCCCACACCTGACAAAGTCGATCAACCAGTTGCTTTGAATGCCACTGGGTTTTCTACTCAGTACACCTTCAAAAACATATTTGTCACCCCCACCAGGTCTTCCCCTCTTCCTGTGCTATGCTGGGCAAGCAGAGACGACGTATGGAACAACCTGTTGAAGAAAGACGACACGTATTCTCGAGACATCCACGTCATGAAGAAACACCCACACCTCCAACCCAAGATGAGGGCTATTCTTCTGGACTGGCTAATGGAGGTGTGCGAGGTGTACAAGCTTCACAGAGAAACCTTCTACCTGGCTCAGGATTACTTTGATCGGTTCATGGCCACACAGAGCAATGTTTTCAAGTGCACACTACAACTCATCGGCATCTCTTCTCTGTTTATAGCTGCCAAGATGGAGGAAATCTACCCTCCAAAAGTGCATCAAT。

Synthesizing dsRNA by taking the PCR product obtained by purification and recovery as a template, and carrying out in-vitro transcription on the dsRNA by using MEGAscript ^TM The RNAi Kit transfer and RNAi prediction (Invitrogen by Thermo Fisher Scientific) Kit is operated and embodiedThe test steps are as follows:

(1) assembling a transcription reaction: 1 μ g of Linear template DNA, 2 μ L of 10 XT 7 Reaction Buffer, 2 μ L of ATP Solution, 2 μ L of CTP Solution, 2 μ L of GTP Solution, 2 μ L of UTP Solution, 2 μ L T7 Enzyme Mix, adding water to 20 μ L, mixing, instant centrifugation, and overnight incubation at 37 ℃ (12-16h) to obtain transcribed dsRNA;

(2) nuclease digestion removes DNA and ssRNA: taking 20 mu L of the transcription dsRNA obtained in the step (1), 21 mu L of nucleic-free Water, 5 mu L of 10 Xdigestion Buffer, 2 mu L of LDNase I and 2 mu L of RNase, and incubating for 1.5h at 37 ℃;

(3) purification of dsRNA: mu.L of dsRNA50, 50. mu.L of 10X Binding Buffer, 150. mu.L of nucleic-free Water, 250. mu.L of absolute ethanol, from which DNA and ssRNA were removed by Nuclease digestion in step (2), was taken, and the total volume was 500. mu.L. Mixing, transferring to adsorption column, and centrifuging for 2 min; wash with 2X 500. mu.L Wash Solution and recover dsRNA with 50. mu.L Solution (preheated at 95 ℃).

(4) Quantification and storage: the purity of the dsRNA was checked on a 1% agarose gel electrophoresis (FIG. 1), stored at-80 ℃ for further use.

The dsRNA acts on an interference gene segment in the ccne1 gene to degrade or inhibit the expression of mRNA, so that the expression of the ccne1 gene is inhibited, and the effect of silencing the ccne1 gene is achieved.

Example 3 detection of Gene silencing efficiency of rainbow trout ccne1

1. Rainbow trout dsRNA injection

50 μ l PBS containing 30 μ g dsRNA was injected into the abdominal cavity of rainbow trout using a 1mL syringe. The experimental fish are divided into two groups, namely an experimental group and a control group, wherein each group comprises 10 fish, 50 mu l of PBS solution containing dsRNA (the concentration is 0.6 mu g/mu l) is injected into the abdominal cavity of rainbow trout juvenile fish (the body mass is 20g +/-1.2 g) by using a 1mL injector in the experimental group, equal volume of 1 XPBS solution is injected into the control group as a control group, the injection is continuously carried out for three days, and the gonadal tissue is taken 72h after the last injection.

Change in expression level of ccne1 Gene

Extracting total RNA of gonadal tissue 72h after injection, carrying out reverse transcription to obtain cDNA, carrying out fluorescence quantification (Real Time PCR) by taking the cDNA as a template to detect the relative expression quantity of the ccne1 mRNA, wherein the internal reference gene is beta-actin, and the primers are shown in Table 3. The results show that after 3 days of continuous injection, the gene expression level of ccne1 is significantly reduced in the dsRNA experimental group compared with the control group without injection, and the silencing efficiency is calculated according to the gene expression level and is 85% (fig. 2).

TABLE 3

Primer and method for producing the same	Sequence of
		Upstream primer Osccne-qF (SEQ ID NO.8)	CCTACCTGAAAGAGTCCGAAGA
Downstream primer Osccne-qR (SEQ ID NO.9)	CCTACCTGAAAGAGTCCGAAGA
		Osβ-actin-qF(SEQ ID NO.10)	ATCCTGACGGAGCGCGGTTACAGC
Osβ-actin-qR(SEQ ID NO.11)	TGCCCATCTCCTGCTCAAAGTCCA

Example 4 Effect of ccne1 Gene silencing on expression levels of other meiosis Key genes

In order to investigate the influence of dsRNA injection on the meiosis of rainbow trout, the relative expressions of other meiosis key genes in the rainbow trout are compared, total RNA of gonad tissues 72h after the last dsRNA injection in example 3 is extracted, cDNA is obtained by reverse transcription, and then the cDNA is used as a template to perform fluorescence quantification (Real Time PCR) to detect the relative expression of other meiosis key genes in the rainbow trout, wherein the internal reference gene is beta-actin, and primers are shown in Table 4.

TABLE 4

Primer and method for producing the same	Sequence of
		Upstream primer OsSYCP1-qF (SEQ ID NO.12)	ACCGAAGCTCTCAGAACTCC
Downstream primer OsSYCP1-qR (SEQ ID NO.13)	TGTTCCGAGCTGTCAGACTT
		Upstream primer OsSYCP3-qF (SEQ ID NO.14)	AGCCATGCAAGCCAAGAGAA
Downstream primer OsSYCP3-qR (SEQ ID NO.15)	GACAGTGGCCATCTCTTGCT
		Upstream primer OsMLH1-qF (SEQ ID NO.16)	TAAAGACCCAACCCAAACCC
Downstream primer OsMLH1-qR (SEQ ID NO.17)	CTGCTCACCACCTCCACAAT
		Upstream primer Osdmc1-qF (SEQ ID NO.18)	CATCAGCAATTCCCCCTGGA
Downstream primer Osdmc1-qR (SEQ ID NO.19)	GTTGGATTTTGTGGCAGCCA
		Upstream primer OsSpindin-qF (SEQ ID NO.20)	CACCGTCAACACCTCCCTCTA
Downstream primer OsSpindin-qR (SEQ ID NO.21)	ACTCCACCTGCTTACCCACAA
		Upstream primer Os beta-Tubulin-qF (SEQ ID NO.22)	TTGGATGTGGTGAGGAAAGA
Downstream primer Os beta-Tubulin-qR (SEQ ID NO.23)	ATAGGTGGGCGTGGTAAGTT

As shown in FIG. 3, the synaptonemal complex (synaptonemal complex) SYCP1 gene is significantly lower than that of the control group, the expression level of the experimental group is 0.064 times that of the control group, while the expression level of the family SYCP3 is higher than that of the control group, and we speculate that the two genes may have functional complementation during meiosis of rainbow trout. MLH1(mutL homologous gene 1, MLH1) affects meiosis by making up the MLH1/MLH3 complex. The experimental result shows that the MLH1 gene expression level of the experimental group injected with dsRNA is 0.156 times of that of the control group. DNA meiotic recombinase 1(DNA meiotic recombination enzyme 1, DMC1) is a meiotic gene and is essential for homologous chromosome pairing and cross-recombination. Injection of dsRNA induced significantly lower expression of DMC1 gene, 0.233 times that of the control group. Similarly, Spindlin and β -Tubulin, which are key meiosis regulatory genes, were observed to have a tendency of decreasing expression levels in the dsRNA-injected group, which were 0.718-fold and 0.156-fold, respectively, compared to the control group. The results show that the dsRNA has a certain inhibiting effect on meiosis of the gonad of the rainbow trout.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> institute of aquatic products of Heilongjiang, China institute of aquatic science

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gcctgcactg aggatgatat cctatgtatg gagctaatca tcatgaagga gctgaagtgg 720

cgtttgagtc ccctgacacc tgtgtcctgg ctaaacatct acatgcaggt tgcctacctg 780

aaagagtccg aagaggttct catcccacag tacccgcaaa ctacatttgt gcagattgtc 840

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Ser Arg Asp Ile His Val Met Lys Lys His Pro His Leu Gln Pro Lys

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<213> Artificial Sequence (Artificial Sequence)

<400> 8

cctacctgaa agagtccgaa ga 22

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cctacctgaa agagtccgaa ga 22

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atcctgacgg agcgcggtta cagc 24

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tgcccatctc ctgctcaaag tcca 24

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

accgaagctc tcagaactcc 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgttccgagc tgtcagactt 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

agccatgcaa gccaagagaa 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gacagtggcc atctcttgct 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

taaagaccca acccaaaccc 20

<210> 17

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

ctgctcacca cctccacaat 20

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

catcagcaat tccccctgga 20

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gttggatttt gtggcagcca 20

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

caccgtcaac acctccctct a 21

<210> 21

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

actccacctg cttacccaca a 21

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ttggatgtgg tgaggaaaga 20

<210> 23

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ataggtgggc gtggtaagtt 20

Claims

1. The application of dsRNA of rainbow trout ccne1 gene in inhibiting meiosis or preparing meiosis inhibitor is characterized in that the dsRNA takes an interference gene segment as a template and is synthesized by in vitro transcription; the nucleotide sequence of the interference gene fragment is shown as SEQ ID NO. 3.

2. The use of claim 1, wherein the dsRNA inhibits meiosis of rainbow trout gonads by decreasing the expression level of genes comprising a ccne1 gene, a SYCP1 gene, an MLH1 gene, a DMC1 gene, a Spindin gene and a β -Tubulin gene.

3. An application of an interference gene segment or dsRNA of an oncorhynchus mykiss ccne1 gene in silencing of a ccne1 gene is characterized in that the dsRNA acts on the interference gene segment in the ccne1 gene so as to inhibit the expression of the ccne1 gene.

4. The use of claim 3, wherein the dsRNA is synthesized by in vitro transcription using an interfering gene fragment as a template; the nucleotide sequence of the interference gene fragment is shown as SEQ ID NO. 3.

5. The use of claim 3, wherein the dsRNA inhibits meiosis of rainbow trout gonads by decreasing the expression level of genes comprising a ccne1 gene, a SYCP1 gene, an MLH1 gene, a DMC1 gene, a Spindin gene and a β -Tubulin gene.

6. A method for inhibiting gonad meiosis is characterized in that dsRNA of the ccne1 gene of rainbow trout is injected into the body of the rainbow trout.