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Cyclin B gene related to procambarus clarkii cell cycle and encoding protein identification method thereof

A technology of Crayfish and cyclin, applied in genetic engineering, plant genetic improvement, animal/human peptides, etc., can solve the problems of uncontrollable temperature control and light control, shortening of parental molting cycle, and decreased egg quality.

Inactive Publication Date: 2016-01-06
FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

However, there will be unavoidable side effects in practice, such as the uncontrollable process of temperature control and light control; cumbersome hormone treatment process; surgical intervention, such as removal of eye stalks, can easily lead to shortened molting cycle, increased mortality, and decreased egg quality. as a result of

Method used

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  • Cyclin B gene related to procambarus clarkii cell cycle and encoding protein identification method thereof
  • Cyclin B gene related to procambarus clarkii cell cycle and encoding protein identification method thereof

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Embodiment 1

[0012] Example 1. Isolation and Identification of Procambarus clarkii cyclinB Gene cDNA Sequence and Its Coding Sequence

[0013] Use the gene sequence in the NCBI public database to search for EST fragments homologous to the cDNA of Procambarus clarkii cycle proteins, design merger primers based on the homologous fragments, and then use the RACE method to amplify the sequences at the first and last ends respectively after sequencing. Sequence assembly was performed to form the cDNA sequence of the complete ORF, and specific PCR primers were designed according to these sequences, and the complete cyclinB gene was obtained by RT-PCR. The cDNA sequence of Procambarus clarkii cyclinB gene is shown in SEQ ID No.1, and the full-length sequence of the protein encoded by the open reading frame is shown in SEQ ID No.2.

Embodiment 2

[0014] Example 2. Quantitative RT-PCR analysis of the expression of the cyclinB gene in Procambarus clarkii

[0015] Total RNA was extracted from the ovary tissue of Procambarus clarkii, and the gene expression was studied by real-time fluorescent quantitative RT-PCR technology. First, the total RNA (2μg / sample) of different tissues (ovary, testis, heart, muscle, liver, gill, eye, brain, intestine and blood cells) was reversed using reverse transcriptase (M-MLVRNaseH-ReverseTranscriptase, Promega) cDNA was recorded; then, using cDNA as a template, quantitative PCR was carried out with gene-specific primers (5'-GGATGTGGAGGAAGTGGC-3' and 5'-AATTGCCAGACTGCAATTTATTG-3') and Real-time PCR MasterMix (TOYOBO, Japan). The 18SrRNA gene of Procambarus clarkii (GenBankaccessionno.AF436001) was used as the reaction internal standard (the primer sequences were 5'-TGGTGCATGGCCGTTCTTA-3' and 5'-AATTGCTGGAGATCCGTCGAC-3'), and the amplification of each cycle of the target gene was amplified by...

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Abstract

The invention provides a cyclin B gene related to the procambarus clarkii cell cycle and an encoding protein identification method thereof, and discloses a procambarus clarkii cycle protein cyclin B gene full-length sequence. The full length of cDNA of the gene is 1295 bp, the length of a 5' end noncoding region is 86 bp, and the length of an open reading frame is 1209 bp; 402 amino acid residues are encoded, the average molecular mass of encoding protein is 45745.5, and the pI is 8.93. The cyclin B gene encoding protein includes 2 cyclin superfamily structural domains. It is discovered after BLAST comparison that homology of the amino acid sequence of procambarus clarkii and the amino acid sequence of penaeus monodon reaches about 90%. Semiquantitative RT-PCR results show that the cyclin B gene has obvious tissue specificity in different tissue expressions, and a high expression quantity is achieved in ovaries and hearts. It is speculated that the difference is mainly related to the function of regulating and controlling cell division in the cell cycle by the cyclin B, and the gene can be used for gonad development regulating research of procambarus clarkii artificial breeding.

Description

Technical field: [0001] The invention relates to the gene of Procambarus clarkii and belongs to the field of aquatic genetic engineering. Specifically, it is the cloning and identification of a cell cycle-related cyclin B gene and its encoded protein, which is used for the research on the regulation of gonad synchronization in the artificial culture of Procambarus clarkii. Background technique: [0002] Procambarus clarkii (Procambarus clarkii), commonly known as lobster and crayfish, belongs to Arthropoda, Crustacea, and Decapod, and is one of the aquaculture species with high economic value; meanwhile, Procambarus clarkii also has low cost, easy It has the advantages of breeding, large size and easy artificial breeding. Therefore, in recent decades, the domestic artificial breeding scale and breeding technology of crayfish have developed rapidly. However, with the strong advancement of development, many problems have emerged, such as: artificially cultured female shrimp o...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/435
Inventor 水燕徐增洪沈怀舜周鑫
Owner FRESHWATER FISHERIES RES CENT OF CHINESE ACAD OF FISHERY SCI
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