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Cynoglossus semilaevis female specificity CSW3 protein as well as gene and application thereof

A semi-smooth tongue sole, protein technology, applied in application, genetic engineering, plant genetic improvement and other directions, to achieve the effect of increasing the proportion of female fish

Active Publication Date: 2014-04-23
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the screening and cloning of fish female-specific or determining genes, especially the screening and cloning of female-specific genes in tongue sole, has not been reported at home and abroad.

Method used

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  • Cynoglossus semilaevis female specificity CSW3 protein as well as gene and application thereof
  • Cynoglossus semilaevis female specificity CSW3 protein as well as gene and application thereof
  • Cynoglossus semilaevis female specificity CSW3 protein as well as gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Cloning and sequence of the female-specific CSW3 gene of half-smooth tongue sole

[0024] Whole-genome sequencing was carried out on the female and male fishes of half-smooth tongue sole, and through bioinformatics analysis, it was found that there was a specific CSW3 gene in the genome of ZW female individuals, and the female was cloned from the ovary of half-smooth tongue sole by rapid amplification of cDNA ends (RACE) technology. The full-length sequence of the cDNA of the specific gene CSW3 gene is SEQ ID NO:2, and the encoded amino acid sequence is SEQ ID NO:1.

[0025] The cDNA sequence of the female-specific gene CSW3 of half-smooth tongue sole is 1674bp in length, including an open reading frame (ORF region) of 414bp, encoding 137 amino acids, 173bp in the 5′ non-coding region, 1090bp in the 3′ non-coding region, and polyadenosine Acid tailing signal and polyA tail.

[0026] Specific steps are as follows:

[0027] 1. Screening and cloning of female-...

Embodiment 2

[0056] Example 2 Expression patterns of female-specific gene CSW3 in different tissues and developmental stages of half-smooth tongue sole

[0057] 1. Expression patterns of female-specific CSW3 gene in different tissues of tongue sole: semi-quantitative and real-time quantitative PCR techniques were used to analyze the expression patterns of CSW3 gene in different tissues of tongue sole (heart, liver, gill, skin, blood, kidney, intestine, brain, spleen, muscle). , pituitary, ovary, testis and testis of pseudo-male fish), it was found that the expression level of CSW3 gene was the highest in ovary, liver and pituitary of female fish, followed by gills, brain and spleen, heart, blood, intestine and pseudo-male fish. The expression level of the fish testis was the lowest, and there was almost no expression in the skin, muscle and testis of male fish ( figure 2 ). The genetic sex identification of female fish and pseudo-male fish was carried out according to the reported method...

Embodiment 3

[0085] Example 3. Construction of recombinant expression vector for female-specific CSW3 gene of half-smooth tongue sole and its recombinant expression in Escherichia coli and isolation and purification of expression products

[0086] Using conventional PCR technology, the open reading frame ORF region of CSW3 gene was amplified, its sequence is SEQ ID NO: 3, and it was cloned into the pET32a (+) expression vector to construct the CSW3 gene prokaryotic expression vector pET32-CSW3. The expression vector was transformed into Escherichia coli BL21, and the CSW3 gene was recombinantly expressed in vitro, and the recombinant expression product was obtained. After the recombinant expression product was purified by GE's His-tag affinity chromatography column, the purified recombinant CSW3 protein was obtained. , the recombinant CSW3 protein molecular weight is about 33kD. The specific plan to achieve the above goals is as follows:

[0087] 1. Construction method of CSW3 gene recomb...

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Abstract

The invention relates to a cynoglossus semilaevis female specificity CSW3 protein as well as a gene and an application thereof, which belong to the technical field of functional gene selection and application in an aquatic biological technology. The amino acid sequence of the cynoglossus semilaevis female specificity CSW3 protein is SEQ ID NO. 1; a nucleotide sequence for encoding the CSW3 gene of the CSW3 protein is SEQ ID NO.2. By adopting the in-vitro recombination cynoglossus semilaevis female specificity CSW3 protein of the CSW3 gene, the expression level of female relevant gene foxl2 can be obviously increased, and the biological activity for stimulating the female relevant gene expression can be realized; meanwhile, the biological activity for reducing the male relevant gene sox9 and amh expression level can be realized. After a CSW3 gene recombination product is used as a feed additive, the development of the female gonad can be stimulated, and the ratio of the female fish can be increased, so that the cynoglossus semilaevis female specificity CSW3 protein has the application potential on controlling the gender of the cynoglossus semilaevis and increasing the ratio of the female fish fries.

Description

technical field [0001] The invention belongs to the technical field of functional gene screening and application in aquatic biology technology, and in particular relates to a female-specific CSW3 protein of half-smooth tongue sole and its gene and application. Background technique [0002] In view of the significance of fish sex-determining genes in the basic and applied research of fish sex-determining mechanisms, scientists from the United States, Japan, Germany, France, Canada and other countries took salmon, tilapia and medaka as examples. Materials Screening of fish sex-specific genes and research on sex-determining mechanism were carried out. The most successful of these is the discovery of the first sex-determining gene DMY in fish by Japanese scientists using medaka as a material (Matsuda et al., Nature, 2002, 417:559-563). However, follow-up studies found that DMY was only found in medaka and medaka, while no DMY male-determining gene was found in other fishes (Mat...

Claims

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Application Information

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IPC IPC(8): C07K14/46C12N15/12C12N15/70A23K1/16A23K1/18A61K38/17A61P15/00
CPCA23K20/147A23K50/80C07K14/461
Inventor 王娜胡乔木王天姿陈松林
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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