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Paralichthys olivaceus male related Dmrt1 recombinant protein and application thereof

A technology of recombinant protein and flounder, applied in the field of molecular biology, can solve the problems of not allowed to use, unsuitable for application, high cost of use, etc.

Pending Publication Date: 2022-07-15
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Sex hormones and their receptor inhibitors are not allowed to be used in aquaculture because they will pollute the water environment; aromatase inhibitors are not allowed due to the relatively high cost of use or the potential biological impact caused by the accumulation of exogenous inducers in the body. Suitable for application in farming production

Method used

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  • Paralichthys olivaceus male related Dmrt1 recombinant protein and application thereof
  • Paralichthys olivaceus male related Dmrt1 recombinant protein and application thereof
  • Paralichthys olivaceus male related Dmrt1 recombinant protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Prokaryotic recombinant expression of flounder Dmrt1 protein

[0031] 1. Construction of recombinant plasmid Blunt E2-Dmrt1 and recombinant strain

[0032] Extracting the RNA of the flounder testis tissue, and reverse transcribing it into cDNA, using PCR to amplify the entire coding region of the flounder Dmrt1, the encoded amino acid sequence is shown in SEQ ID NO: 1, and then using the designed specific primers, its The sequence is:

[0033] Dmrt1-F: 5'-ATGAACAAGGACAAGCAGAG-3';

[0034] Dmrt1-R: 5'-ATTGATGCCATCCACGTCGA-3'

[0035] PCR amplification was carried out. After the amplified fragments were verified by gel recovery, purification and sequencing, they were connected to the linearized Blunt E2 vector. The recombinant plasmids were transformed into E. coli DH5α competent cells. PCR amplification detection, and handed over to Ruibo Xingke Biotechnology (Beijing) Co., Ltd. for sequencing identification.

[0036] Amplification conditions are:

[0037] P...

Embodiment 2

[0046] 1. Construction of recombinant plasmid pET-30a-EGFP-Dmrt1 and recombinant strain

[0047] Extracting the RNA of the flounder testis tissue, and reverse transcribing it into cDNA, using PCR to amplify the entire coding region of the flounder Dmrt1, the encoded amino acid sequence is shown in SEQ ID NO: 1, and then using the designed specific primers, its The sequence is:

[0048] Dmrt1-F: 5'-AAGGCCATGGCTGATATGAACAAGGACAAGCAGAG-3';

[0049] Dmrt1-R: 5'-TGAATCGATACGCATATTGATGCCATCCACGTCGA-3'

[0050] PCR amplification was performed. After the amplified fragment was verified by gel recovery, purification and sequencing, it underwent homologous recombination with the linearized pET-30a-EGFP vector. The recombinant plasmid was transformed into E. coli DH5α competent. The cloned colonies were detected by PCR amplification of the bacterial solution, and were handed over to Ruibo Xingke Biotechnology (Beijing) Co., Ltd. for sequencing and identification.

[0051] Amplificatio...

Embodiment 3D

[0062] Example 3 In vitro activity verification of Dmrt1 recombinant protein

[0063] 1. Tissue incubation of Dmrt1 recombinant protein

[0064] The recombinant and purified flounder Dmrt1 protein in Example 2 was diluted to 1000 μg / mL in the aforementioned PBS.

[0065] Take the gonadal tissues of four healthy wild-type male and female adult fish, wash them repeatedly with the aforementioned PBS containing 1% penicillin (10kU / mL)-streptomycin (10mg / mL) mixed antibiotics, and cut them into about 1.0mm 3 The small pieces were placed in a 24-well plate, pre-incubated with L15 medium containing 1% antibiotics at 25 °C for 6 h, followed by Dmrt1 recombinant protein (100 μg) and Lipofectamine containing recombinant and purified Dmrt1 in Example 2. TM The fresh medium of 3000 transfection reagent was incubated at 25°C for 24h, and the incubated samples were stored at -80°C for later use.

[0066] 2. WB and qPCR analysis

[0067] A part of the samples were extracted with total pro...

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Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to paralichthys olivaceus Dmrt1 recombinant protein and application. The paralichthys olivaceus Dmrt1 recombinant protein is obtained for the first time by utilizing an in-vitro recombinant expression technology, a preparation method of the recombinant protein is provided, and the paralichthys olivaceus gonad tissue is incubated by utilizing the recombinant protein, so that the paralichthys olivaceus Dmrt1 recombinant protein has biological activity. The recombinant Dmrt1 protein is injected into an abdominal cavity of a female adult paralichthys olivaceus, so that cell nucleus chromatin of an oocyte in an ovarian tissue of the female adult paralichthys olivaceus is condensed. Further injecting the vaccine into the abdominal cavity of the fry with undifferentiated gonad to respectively cause the obvious decrease and increase (plt) of the estrogen and androgen levels in the juvenile fish body; 0.05), and the male rate is increased from 0% to 82%. The recombinant protein can be used for sex control of fishes such as paralichthys olivaceus, and a new method and way can be provided for sex control and unisexual production in fish culture.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a flounder Dmrt1 recombinant protein and application. [0002] technical background [0003] Dmrt1 (Doublesex and mab-3-related transcription factor 1), as a functionally conserved factor, has been shown to activate testis-specific genes and reduce estrogen levels in vivo, suggesting that it plays a key role in sex determination and testis differentiation in fish. The dmrt1 gene sequence has been obtained in a variety of fish, and the mRNA transcription and expression studies in the process of gonadal differentiation and development have been carried out. The results show that the gene starts to be expressed in the early stage of testis differentiation and continues to be highly expressed during the differentiation process. The researchers It is speculated that dmrt1 may be involved in the formation of male sex phenotype. Further gene knockout and overexpres...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/70A01K61/10C12R1/19
CPCC07K14/461C12N15/70A01K61/10
Inventor 舒畅王丽娟尤锋邹聪聪谭训刚邹玉霞吴志昊
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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