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Sepiella japonica neuropeptide sCAP and application thereof

A Mann's needle-free squid and neuropeptide technology, applied in the field of neuropeptides, can solve problems such as affecting breeding efficiency and unfavorable artificial breeding of squid, and achieve the effects of enhancing hindgut contraction, reducing precocious puberty of cultured individuals, and stimulating fallopian tube contraction.

Inactive Publication Date: 2019-01-04
ZHEJIANG OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Zheng Xiaodong et al. also reported this phenomenon. They found that generally in the natural environment, it takes at least six to eight months for Sepiella japonica to reach sexual maturity. However, the squid under artificial breeding conditions, It only takes less than 4 months to reach sexual maturity. Due to the shortening of the growth cycle, the cuttlefish under artificial breeding are much smaller than wild squid. This phenomenon is not conducive to the artificial breeding of squid and seriously affects the breeding efficiency.

Method used

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  • Sepiella japonica neuropeptide sCAP and application thereof
  • Sepiella japonica neuropeptide sCAP and application thereof
  • Sepiella japonica neuropeptide sCAP and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Squid neuropeptide sCAP is obtained by cloning the sCAP gene of Squid by RT-PCR and RACE technology, and the specific steps are as follows:

[0035] 1) Sampling was taken from the cephalopod seed breeding base of Cangnan Base in Wenzhou, Zhejiang Province, and healthy and vigorous squid were selected. After the base was dissected, the tissue was taken out and stored in RNA preservation solution, and transported back with dry ice, and the samples were Place in a -80 degree refrigerator;

[0036] 2) Extraction of total RNA:

[0037] ①Take a 2ml EP tube without RNase, add 500ul Trizol to the tip of the used pipette, use tweezers soaked in alcohol to pick up the brain tissue sample and dissolve it in the Trizol liquid, grind it with an electric homogenizer until the tissue is completely dissolved in the liquid, and fill it up Trizol to 1mL, 4°C, 12000rpm, centrifuge for 10min;

[0038] ②Take the supernatant and put it into a 1.5mL EP tube, so that no impurities can be abs...

Embodiment 2

[0137] Squid Squid Neuropeptide sCAP, Squid Squid Neuropeptide sCAP was obtained by cloning the sCAP gene of Squid Squid by RT-PCR and RACE technology; the full-length cDNA of Squid Squid Neuropeptide sCAP gene is 696bp in length. Manson's needleless cuttlefish cardiokinin sCAP has the most expression in the optic lobe, followed by the brain tissue, other tissues have a small amount of expression, and the heart tissue has the least expression. It controls squid activity through the central nervous system and has the ability to stimulate heart movement. , control muscle contraction, stimulate fallopian tube contraction, participate in feeding behavior, enhance hindgut contraction, regulate various nervous system functions including vision and smell.

[0138] The sCAP gene includes a 111bp 5' non-coding region sequence and a 324bp 3' non-coding region sequence; the predicted open reading frame of the sCAP gene is 261bp, and it is predicted to encode 86 amino acids.

[0139] The ...

Embodiment 3

[0148] Tissue-specific expression analysis of the neuropeptide sCAP in Manson's needleless squid:

[0149] a. Extraction of total RNA from different tissues: Tissues of male squid: brain, optic lobe, heart, gills, liver, stomach, pancreas, intestine, muscle, testis; tissues of female squid: brain, optic lobe, heart, gills, liver , stomach, pancreas, intestine, muscle, ovary, ovary gland, paravertebral gland tissue, the total RNA extraction is the same as the brain tissue total RNA extraction, the specific steps refer to Example 1 total RNA extraction;

[0150] b. cDNA template synthesis:

[0151] (1) Reverse transcribe the total RNA extracted in the previous step from different tissues to prepare the cDNA template required for the real-time PCR experiment. For the specific method, refer to the instruction manual of the M-MLV Reverse Transcriptase reagent. Before reverse transcription, it is necessary to remove the interference of genomic DNA in the extracted total RNA on the ...

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Abstract

The invention discloses a sepiella japonica neuropeptide sCAP and application thereof. Sepiella japonica sCAP genes are cloned by the aid of RT-PCR (reverse transcription-polymerase chain reaction) and RACE [rapid amplification of cDNA (complementary deoxyribonucleic acid) ends] technologies to obtain cDNA sequences with the total lengths of 696 bp and include 5' non-coding region sequences with the lengths of 111 bp and 3' non-coding region sequences with the lengths of 324 bp, the total lengths of predicted open reading frames are 261 bp, 86 parts of amino acid are predicted and coded, the relative molecular weights are 9.331 kDa, the isoelectric points are 8.52, and the sepiella japonica neuropeptide sCAP contains obvious signal peptide sequences and obvious transmembrane regions and isa hydrophilic protein; the sepiella japonica neuropeptide sCAP can be applied to carrying out reproductive regulation in cephalopoda culture procedures; the sepiella japonica neuropeptide sCAP is a protein secreted in cells, and can be secreted out of the cells from the insides of the cells, so that effects can be realized; activities of cuttlefishes can be controlled by central nervous systems,accordingly, cephalopoda reproduction can be regulated, and the sepiella japonica neuropeptide sCAP can contribute to cephalopoda artificial culture and multiplication and releasing.

Description

technical field [0001] The invention relates to the technical field of neuropeptides, in particular to the neuropeptide sCAP and its application. technical background [0002] Cephalopods belong to molluscs, which are an important part of the marine ecosystem and play an indispensable role in the balance of marine ecology. Neuropeptides are small molecular polypeptides secreted by endocrine tissues of the nervous system, which exist in various animals and play an indispensable role in physiological activities such as neuromodulation, development, and reproduction in organisms. With the continuous maturity of experimental techniques such as molecular cloning and immunohistochemistry, research on the mechanism of action of neuropeptides has also continued to deepen. Up to now, seven neuropeptides have been found in cephalopods, and the central kinin is of great significance to the reproductive regulation of cephalopods. The cardiokinin sCAP was first extracted from the brain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12
CPCC07K14/43504
Inventor 迟长凤肖燃单雨菡黄伟刘慧慧吕振明
Owner ZHEJIANG OCEAN UNIV
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