KR102166717B1 - Compositions for promoting breeding of mollusks comprising cephalotocin - Google Patents

Abstract

The present invention relates to the use of cephalotocin to promote the breeding of mollusks and to confirm the state of incubation. Cephalotocin according to the present invention is overexpressed in female individuals who are incubating in mollusks, and when administered to mollusks, movement and sociality are increased. This means that cephalotocin can be used as a marker for checking the incubation status and for promoting the breeding, thereby being able to be variously applied in various ways in the field of mollusk breeding and farming.

Description

Compositions for promoting breeding of mollusks comprising cephalotocin}

The present invention relates to the use of Separotosine to promote the reproduction of mollusks and to confirm the state of incubation.

The octopus ( Octopus minor ) belongs to the octopus family ( Animalia Cephalopoda Octopoda Octopodidae ) taxonomically, and it mainly lives in the coast of Northeast Asia such as Korea, China and Japan. Octopus is a very popular fishery product in Korea (Korea).According to the Ministry of Maritime Affairs and Fisheries' Maritime and Fisheries Statistics System in 2018, it ranked 5th in the import performance of fishery products by major item (36,000 tons, $340 million), and Fisheries resource research institutes are also interested in ways to proliferate domestic octopus resources.

Meanwhile, sephalotocin (CPT) is a peptide that was first discovered in Octopus vulgaris and belongs to the oxytocin/vasopressin superfamily. In general, products made by homologous genes that are evolutionarily conserved in different species are all called the same name and are considered to have similar functions, but with exceptions, peptides belonging to the oxytocin/vasopressin superfamily function even with slight chemical structural differences. Because of this large difference, if there is any difference in amino acid sequence, a separate name is given and treated as a separate substance.

In previous studies, it has been known that injecting cephalotosin from the octopus into the European patterned cuttlefish ( Sepia officinalis ) belonging to the cephalopod, an invertebrate such as an octopus, increases long-term memory. In addition to cephalotosin, the octopus also expresses a peptide called octopressin, which belongs to the same vasopressin/oxytocin superfamily. It has been reported that octopressin contracts the peripheral tissues of octopus, and when octopressin is injected into octopus ocellatus belonging to the genus Octopus, the osmotic pressure of blood lymph is regulated. However, there is no research on the relationship of cephalotosin to social behaviors such as mating and incubation, and whether a gene similar to cephalotosin of true octopus exists in octopus, and whether it is related to mating and incubation has not been previously revealed.

Reich, Gerhard. "A new peptide of the oxytocin/vasopressin family isolated from nerves of the cephalopod Octopus vulgaris." Neuroscience letters 134.2 (1992): 191-194. Van Kesteren, R. E., et al. "A vasopressin-related peptide in the mollusc Lymnaea stagnalis: peptide structure, prohormone organization, evolutionary and functional aspects of Lymnaea conopressin." Progress in Brain Research. Vol. 92. Elsevier, 1992. 47-57.

Accordingly, the present inventors completed the present invention by confirming that as a result of studying social behaviors such as mating and incubation of octopus, separotocin is overexpressed in the incubating octopus and increases movement and sociality of the octopus.

Accordingly, it is an object of the present invention to provide a composition for promoting reproduction of mollusks, including cepharotosine represented by the amino acid sequence of SEQ ID NO: 1 or a gene encoding the same.

Another object of the present invention is to provide a method for promoting the reproduction of mollusks comprising the step of administering the composition for promoting the reproduction of mollusks to the mollusks.

Another object of the present invention is to provide a biomarker composition for confirming the incubation status of mollusks, including cepharotosine represented by the amino acid sequence of SEQ ID NO: 1 or a gene encoding the same.

Another object of the present invention is a composition for checking the incubation state of molluscs comprising an agent for measuring the expression of separotosine represented by the amino acid sequence of SEQ ID NO: 1 or a gene encoding the same, and a kit for checking incubation status comprising the same Is to provide.

Another object of the present invention is to provide a method for confirming the incubation state of mollusks, comprising measuring the expression of a gene encoding the separotocin represented by the amino acid sequence of SEQ ID NO: 1 in a biological sample.

In order to achieve the above object, the present invention provides a composition for promoting reproduction of mollusks comprising a gene encoding the separotosine represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides a method for promoting the reproduction of mollusks comprising administering the composition for promoting the reproduction of mollusks to the mollusks.

In addition, the present invention relates to a biomarker composition for confirming the incubation state of mollusks comprising cepharotosine represented by the amino acid sequence of SEQ ID NO: 1 or a gene encoding the same.

In addition, the present invention provides a composition for checking the incubation state of mollusks comprising an agent for measuring the expression of cepharotosine represented by the amino acid sequence of SEQ ID NO: 1 or a gene encoding the same.

In addition, the present invention provides a kit for checking the incubation status including the composition for checking the incubation status of the mollusk.

In addition, the present invention provides a method for confirming the incubation state of a mollusk, comprising measuring the expression of a gene encoding the separotocin represented by the amino acid sequence of SEQ ID NO: 1 in a biological sample.

It was confirmed that the separotocin according to the present invention is overexpressed in female individuals who are incubating in mollusks, and when administered to mollusks, movement and sociality are increased. This means that separotocin can be used as a marker for checking the incubation status and for promoting reproduction, and it can be applied in various ways in the field of mollusc breeding and aquaculture.

1 is a diagram confirming the results of predicting and verifying the presence of separotocin in octopus based on the result of transcript analysis.
Figure 2 is a diagram showing the sequence of the active peptide predicted from two types of Separotocin-expressing genes identified in octopus.
3 is a diagram showing the results of confirming the expression of separotosine isoform-1 and -2 in a female octopus in an incubation.
4 is a schematic diagram showing an experiment for observing changes in movement and sociality of an octopus according to administration of separotocin.
5 is a diagram showing a list of movements for observing octopus movements and social changes.
6 and 7 are diagrams showing the results of observing changes in movement and sociality of the octopus according to the continuous administration of separotocin (ASW: artificial seawater injection, CPT: separotocin injection).

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention provides a composition for promoting reproduction of mollusks comprising a gene encoding the separotosine represented by the amino acid sequence of SEQ ID NO: 1.

In the present invention, separotocin is a peptide belonging to the vasopressin/oxytocin superfamily. The present inventors analyzed the full-length genome and transcriptome information of the previously published California two-spotted octopus ( Octopus bimaculoides ) and octopus ( Octopus minor ). It was predicted that the separotosine active peptide portion of was predicted to have the same amino acid sequence as that of the octopus. Since the octopus-derived cephalotosine dealt with in the present invention has two amino acids different from vasopressin and three different from oxytocin, it is obvious that the effectiveness and effect of cephalotosin in mammals can also be known only through actual experiments.

In an embodiment of the present invention, the separotosine is preferably derived from at least one selected from the group consisting of octopus, octopus, white snail, siamese octopus, Japanese octopus, jukkumi, octopus, and cricket, more preferably Is from octopus.

Separotosine according to the present invention may be in a form in which the N-terminus or C-terminus is modified or protected by various organic groups in order to protect from protein cleavage enzymes in vivo and increase stability. That is, if the C-terminus of the separotosine is in a form that can be modified to increase stability, there is no particular limitation, but preferably it may be modified with a hydroxy group (-OH) or an amino group (-NH ₂ ). In addition, if the N-terminal of the separotosine is in a form that can be modified to increase stability, there is no particular limitation, but preferably an acetyl group, a fluorenyl methoxy carbonyl (Fmoc) group, or a formyl group. ) Group, palmitoyl (Palmitoyl) group, myristyl group, stearyl (Stearyl) group, and may be a group selected from the group consisting of polyethylene glycol (PEG).

The separotosine may consist of an amino acid sequence represented by SEQ ID NO: 1, and includes a functional equivalent of the peptide. The functional equivalent is at least 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably with the amino acid sequence represented by SEQ ID NO: 1 as a result of addition, substitution or deletion of amino acids. It has 95% or more sequence homology, and refers to a protein that exhibits substantially the same physiological activity as a protein consisting of the amino acid sequence represented by SEQ ID NO: 1. In addition, the protein having its native amino acid sequence as well as its amino acid sequence variants are also included in the scope of the present invention in the separotosine of the present invention. A separotosine variant refers to a protein having a different sequence from the natural amino acid sequence of separotocin and one or more amino acid residues by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchanges in proteins and peptides that do not totally alter the activity of the molecule are known in the art. The separotosine or a variant thereof may be extracted from nature or synthesized (Merrifleld, J. Amer. chem. Soc. 85:2149-2156, 1963) or prepared by a method of genetic recombination based on a DNA sequence (Sambrook). et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd ed., 1989).

In an embodiment of the present invention, the gene encoding the separotosine is preferably a separotosine isoform-1 comprising the nucleotide sequence of SEQ ID NO: 2 or a separotosine isoform-2 comprising the nucleotide sequence of SEQ ID NO: 3 And, more preferably, isoform-2.

In addition, variants of the base sequence are included within the scope of the present invention. Specifically, the gene has a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequences of SEQ ID NOs: 2 and 3, It means a sequence that shows substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NOs: 2 and 3. The "% of sequence homology" for a polynucleotide is identified by comparing two optimally aligned sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region is a reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include additions or deletions (ie, gaps) compared to (not including).

In an embodiment of the present invention, the concentration of the separotosine is preferably 1 to 1000 μg/kg, more preferably 10 to 500 μg/kg, more preferably 100 μg/kg.

In the present invention, the mollusk refers to terrestrial and aquatic mollusk invertebrates without a segment.

In an embodiment of the present invention, the molluscs may belong to the octopus family (Octopodidae) or the Loliginidae family, preferably belong to the octopus family, more preferably octopus ( Octopus minor ).

The octopus is characterized by having the most developed brain structure and high intelligence among invertebrates, but little sociality. For example, a mouse used as an experimental animal performs actions such as smelling when two individuals are reared in one farm. However, even if two individuals are placed in one tank, the movement of octopus is almost unchanged. Exceptionally, during the spawning period, the octopus exhibits a rapid increase in motor behavior and sociality, in order to seduce the opposite sex and then mate. In addition, after spawning, female octopuses increase their motility rapidly, because they constantly shake and care for the eggs in the shelter until they hatch. As above, the behavior of the octopus to care for the eggs is called brooding.

When separotocin according to the present invention was administered once to an octopus among mollusks, it was confirmed that the behavior of moving around with a head-lifted position increased significantly. In addition, it was confirmed that continuous administration of separotocin to octopus not only increased motor behavior, but also significantly increased access and contact (ie, sociality) to other octopuses. Therefore, separotocin can promote reproduction by remarkably increasing the motor behavior and sociality of octopus.

The route of administration and the method of administration of the composition according to the present invention may each be independent, and the method is not particularly limited, and any route of administration and method of administration may be applied as long as the pharmaceutical composition can reach the desired site. You can follow. In addition, the composition of the present invention can be administered in various dosage forms at the time of actual administration, and when formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In addition, it is preferable to use those disclosed in Remington's Pharmaceutical Science, recently Mack Publishing Company, Easton PA, as suitable formulations known in the art.

In an embodiment of the present invention, the composition for promoting reproduction may further include at least one component known to have an effect of inducing or promoting the reproduction of mollusks.

According to another aspect of the present invention, the present invention provides a method for promoting the reproduction of mollusks comprising administering the composition for promoting the reproduction of mollusks to the mollusks.

In an embodiment of the present invention, the administration of the composition may be single or multiple administration, preferably multiple administration, more preferably continuous administration for a certain time, but is not limited thereto.

In an embodiment of the present invention, the concentration of the separotosine is preferably 1 to 1000 μg/kg, more preferably 10 to 500 μg/kg, more preferably 100 μg/kg.

According to another aspect of the present invention, the present invention relates to a biomarker composition for confirming the incubation state of mollusks, including separotosine represented by the amino acid sequence of SEQ ID NO: 1 or a gene encoding the same.

In a specific embodiment of the present invention, the gene encoding the separotosine is a separotosine isoform-1 gene including the nucleotide sequence of SEQ ID NO: 2 or the separotosine isoform-2 gene including the nucleotide sequence of SEQ ID NO: 3 Preferably, it is the isoform-2 gene more preferably.

In the present invention, brooding refers to an act of warming or protecting an egg by using its own body until the egg hatches after the animal has spawned. In the present invention, incubation refers to an action of a mollusk constantly shaking and caring for an egg.

In the present invention, a biomarker is a substance capable of confirming the incubation state of a mollusk in a biological sample, and compared to a normal sample, a polypeptide or nucleic acid showing an increase or decrease in separotosine in a sample taken from a mollusk in an incubation ( Examples: mRNA, etc.), lipids, glycolipids, glycoproteins, or organic biomolecules such as sugars (monosaccharides, disaccharides, oligosaccharides, etc.).

In an embodiment of the present invention, the relationship between expression of separotocin and incubation behavior in octopus, which is a mollusk, was confirmed. As a result, it was confirmed that separotosine, in particular, isoform-2, was overexpressed in the incubating female octopus. Therefore, separotocin can be used as a biomarker for checking the egg state.

According to another aspect of the present invention, the present invention provides a composition for checking the incubation state of mollusks comprising an agent for measuring the expression of cepharotosine represented by the amino acid sequence of SEQ ID NO: 1 or a gene encoding the same.

In the present invention, the agent for measuring the expression of cepharotosine represented by the amino acid sequence of SEQ ID NO: 1 or a gene encoding the same is a marker that increases expression in a biological sample of a mollusk in incubation as described above, or It refers to a molecule that can be used for detection of a marker by specifically binding to the mRNA encoding it and confirming its expression level.

In an embodiment of the present invention, the agent for measuring the expression of separotosine is an oligopeptide, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, which specifically binds to the separotosine, It may be a peptide nucleic acid (PNA) or an aptamer.

In the present invention, an antibody is a term known in the art and refers to a specific protein molecule directed against an antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to separotosine, a marker of the present invention, and such an antibody clones each gene into an expression vector according to a conventional method and uses the marker gene. The encoded protein can be obtained and prepared from the obtained protein by a conventional method. This includes partial peptides that can be made from these proteins. The form of the antibody of the present invention is not particularly limited, and a polyclonal antibody, monoclonal antibody, or any one having antigen-binding properties is also included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention also includes special antibodies such as humanized antibodies.

Antibodies used for the detection of biomarkers of the present invention include functional fragments of antibody molecules as well as complete forms having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment that has at least an antigen-binding function, and includes Fab, F(ab'), F(ab') 2 and Fv.

In an embodiment of the present invention, the agent measuring the expression of a gene encoding separotosine may be an antisense oligonucleotide, a primer pair, or a probe that specifically binds to the gene encoding the separotosine.

In the present invention, the primer is a nucleic acid sequence having a short free 3'hydroxyl group and can form a complementary template and a base pair, and function as a starting point for template strand copying. It means a short nucleic acid sequence. Primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer and temperature. Specifically, by performing PCR amplification using sense and antisense primers of the separotosine polynucleotide, the incubation state can be diagnosed through whether a desired product is produced. The PCR conditions, the length of the sense and antisense primers can be modified based on those known in the art.

In the present invention, the probe refers to a nucleic acid fragment such as RNA or DNA corresponding to a short number of bases to a few hundred bases that can achieve specific binding with the mRNA, and is labeled so that the presence or absence of a specific mRNA can be confirmed. . The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA (DNA) probe, a double stranded DNA (DNA) probe, or an RNA probe. Selection of suitable probes and conditions for hybridization can be modified based on those known in the art.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, encapsulation, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, e.g., uncharged linkers (e.g., methyl phosphonate, phosphorester, phosphoro Amidates, carbamates, etc.) or to charged linkers (eg phosphorothioate, phosphorodithioate, etc.).

According to another aspect of the present invention, the present invention provides a kit for checking the incubation status, including the composition for checking the incubation status of the mollusk.

In an embodiment of the present invention, the kit may be an RT-PCR kit, a DNA chip kit, or a protein chip kit, but is not limited thereto.

The kit may include not only an agent measuring the expression of separotosine or a gene encoding it, but also tools and reagents generally used in the art used for immunological analysis.

Examples of the tool or reagent include, but are not limited to, a suitable carrier, a labeling substance capable of generating a detectable signal, chromophores, solubilizers, detergents, buffers, stabilizers, and the like. When the labeling material is an enzyme, a substrate capable of measuring enzyme activity and a reaction terminator may be included. The carrier includes a soluble carrier, an insoluble carrier, and an example of a soluble carrier includes a physiologically acceptable buffer solution known in the art, for example, PBS, and an example of an insoluble carrier includes polystyrene, polyethylene, polypropylene, polyester, poly It may be acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymers such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

Since the kit of the present invention comprises the above-described biomarker composition and diagnostic composition, duplicates are omitted in order to avoid excessive complexity of the present specification.

According to another aspect of the present invention, the present invention provides a method for confirming the incubation state of a mollusk comprising measuring the expression of a gene encoding the separotocin represented by the amino acid sequence of SEQ ID NO: 1 in a biological sample. do.

In an embodiment of the present invention, the gene encoding the separotosine is preferably a separotosine isoform-1 comprising the nucleotide sequence of SEQ ID NO: 2 or a separotosine isoform-2 comprising the nucleotide sequence of SEQ ID NO: 3 And, more preferably, isoform-2.

This step provides measurement information on the expression of separotosine or the gene encoding it, and for the purposes of the present invention, when it is determined that the expression is increased, mating is caused by increasing the motor behavior and sociality of the mollusk. Based on this, the present invention provides information on the reproduction of a subject (eg, mating status, mating time, spawning period, etc.).

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

Example 1. Octopus breeding

Mature octopus was purchased from the southwest coast of Korea in May 2019 for the purpose of distributing aquatic products, put one female and one male in an onion net, and fertilized for one week in a farm where natural seawater was supplied to induce mating. After that, only female octopus was left in the farm and spawning was induced. The female octopus was fed with clams before spawning, and after spawning, feeding was stopped and adapted.

Example 2. Prediction and verification of the presence of separotocin in octopus

From the results of the transcriptome analysis of octopus tissues, it was confirmed that two genes annotated as similar to snail's conopressin/neurophysin exist in octopus brain tissue. The base sequences of the two types of conopressin mRNA were different, but some identical parts existed. The conopressin was assumed to be separotosine, and as described later, the real substance was secured to perform a nucleotide sequence verification procedure.

Specifically, the brain of the female octopus prepared in Example 1 was completely separated from the cartilage, wrapped in foil, and then placed in 3 ml of TRIzol Reagent (Invitrogen, USA) and stored in a -70°C freezer until RNA extraction. The brain tissue of the isolated octopus was crushed using a Lysing kit with beads (Bertin, France) and Precellys Evolution homogenizer (Bertin, France). Total RNA was extracted from 1 ml of the lysate using chloroform and isopropanol according to the manufacturer's manual and stored in DEPC (diethyl pyrocarbonate) water. CDNA was synthesized from 1 μg of RNA treated with DNase (Takara, Japan). The cDNA synthesis was performed using SuperScriptTM IV First strand synthesis system (Invitrogen, USA).

RT-PCR was performed to produce amplification products of two types of conopressin genes (isoform-1 is 519 bp, isoform-2 is 560 bp) from the octopus brain cDNA template. The primer set used in the RT-PCR was designed to amplify a cDNA portion including two types of ORF (Open Reading Frame) sites of conopressin based on the result of transcript analysis. The designed primer set is as follows.

-Cloning_Forward: 5'- CAG TTG CGA AAA TGT CTC AG -3'

-Cloning_Reverse_isoform-1: 5'- GTC ATC AGA TAT TGT GCT CC -3'

-Cloning_Reverse_isoform-2: 5'- CAT TTT GCT GAT GAG GGT AG -3

The PCR product purified through electrophoresis on a 1% agarose gel was inserted into pGEM-T easy vector system I (Promega, USA), cloned, and the nucleotide sequence was decoded (Macrogen). Using MEGA X software, the nucleotide sequence decoding result and the transcriptome analysis result were compared, and the results are shown in FIG. 1.

As shown in Fig. 1, it was confirmed that the base sequences of isoform-1 and isoform-2 mostly matched the conopressin base sequence on the transcript analysis result.

Separotosine Isoform-1 has a single base level mutation, but the number of nucleotides in the ORF is the same as the nucleotide sequence information obtained through transcript analysis. In addition, it was confirmed that the nucleotide sequence of the region presumed to be an active peptide was also identical in isoform-1 except for one clone. In addition, it was confirmed that the full length nucleotide sequence of the separotosine isoform-1 is about 1 kb, and a part of the region including the ORF is represented by the nucleotide sequence of SEQ ID NO: 2.

Separotosine Isoform-2 was found in two single-base mutations in one clone that was successfully secured, but like isoform-1, the number of nucleotides in the ORF and the nucleotide sequence of the estimated active peptide matched the transcript information. In particular, it was confirmed that the active peptide presumed region is the same as that of Separotosine previously known in true octopus. In addition, it was confirmed that the full-length nucleotide sequence of Separotosine isoform-2 is about 1 kb, and a part of the region including the ORF is represented by the nucleotide sequence of SEQ ID NO: 3.

Separotosine isoform-1 and isoform-2 had 373 nucleotide sequences of the region including ORF starting from the 5'end and was exactly matched. Therefore, the active peptide made through this is also the same. However, 100 to 150 nucleotide sequences at the 3'end were completely different from each other, and there was no match in the middle. Therefore, there is no difference in the products produced by isoform-1 and isoform-2, but isoform-1 and isoform-2 are separate genes expressed, and it can be expected that their expression is regulated according to circumstances.

The above results indicate that there are a total of two types of octopus separotocin-expressing genes, and the estimated regions of the active peptides of each gene are the same.

As described above, the sequence and structure of the active peptides expected from the two types of separotocin-expressing genes are shown in FIG. 2. The active peptide is composed of 9 amino acids like other oxytocin/vasopressin superfamily, a disulfide bond is formed between amino acid 1 (cysteine) and amino acid 6 (cysteine), and amino acid 9 (glycine ) It is expected that amidation will occur in which -NH ₂ is added to the C-terminal site. Based on this prediction, the active peptide was synthesized (Anygen, Korea) and used in subsequent experiments.

Example 3. Confirmation of the relationship between expression of separotocin and incubation behavior

It was confirmed whether the expression of the separotocin gene and the incubation behavior were related. Specifically, 9 female octopuses having an incubation behavior prepared in Example 1, and 5 female octopuses whose incubation activity was terminated due to the dissolution of their eggs although they were laid were initially used in the experiment. In the case of pre-scattering samples, it was not possible to specify whether or not to spawn in the future and the timing of spawning. From the octopus brain cDNA of Example 2, sqRT-PCR was performed to produce an amplification product (426 bp for isoform-1, 326 bp for isoform-2) including some sequences of two types of separotocin genes. The primer sets used for the sqRT-PCR were designed to confirm the expression patterns of the two types of separotocin genes verified in Example 2. The designed primer set is as follows.

-Expression_Forward: 5'- GTT GCT ATT TCC GAA ACT GTC -3'

-Expression_Reverse_isoform-1: 5'- GTC ATC AGA TAT TGT GCT CC -3'

-Expression_Reverse_isoform-2: 5'- GCT GCG ATG ATT CAC TTT GTC -3'

The PCR product was electrophoresed on 1% agarose gel, and the brightness of the band in the agarose gel photograph was quantified with ImageJ software. In addition, the 18sRNA PCR results of each individual were corrected to a quantitative value, and the expression level of separotocin for each individual was analyzed. Data mean calculation and statistical comparison between groups using Student's t-test were performed using Excel software (Microsoft, USA). The results of confirming the relationship between expression of separotocin and incubation behavior in octopus are shown in FIG. 3.

As shown in Figure 3, isoform-1 was confirmed that there was no difference between the mother octopus group during incubation and the female octopus group after incubation, while isoform-2 was statistically significantly more expressed in the mother octopus during incubation. . This suggests that Separotosine isoform-2 is associated with the incubation behavior of octopus, and may contribute to inducing or maintaining the incubation behavior.

Example 4. Changes in movement and sociality of octopus according to administration of separotocin

4-1. Observation of movement of octopus according to single administration

In April 2019, female octopus caught for the purpose of distributing aquatic products on the southwest coast of Korea was purchased, and 10 individuals weighing more than 200g were used in the experiment. The prepared octopus was put in a white plastic container (35 x 48 x 24 (h) cm ³ ) for behavior observation and the weighed octopus was placed in a new environment for at least 40 minutes. At the same time, another octopus was trapped in a black mesh and fixed in the inner corner of a plastic container. In a plastic container, 6L of artificial seawater was put in advance so that the octopus could only move in two dimensions while submerged in water. When the behavior of the octopus was stable, the octopus was injected with the separotocin synthesized in Example 2 (100 μg/kg) and the behavior was observed.

As a result, when separotocin was administered once, the octopus showed the behavior of moving around with the head raised. The effect of separotocin was observed regardless of sex, appeared between 1-5 minutes after injection, lasted 10-20 minutes, peaked between 5-10 minutes after injection, but there were differences in the type and duration of behavior between individuals. .

4-2. Observation of changes in octopus movement and sociality according to continuous administration

Separotocin was injected regularly for a long period of time rather than a single injection, and behavioral changes were quantitatively analyzed. A schematic diagram of this experiment is shown in FIG. 4. Specifically, the separotocin powder synthesized in Example 2 was dissolved in tertiary distilled water at a concentration of 10 mg/ml, diluted 100 times in artificial seawater filtered through a 0.20 μm filter, and 0.1 mg/ml (ie, 100 μg/kg) Concentration injection solutions were prepared. A sufficient amount of injection solution was put in a 10 ml syringe and a KDS-210 syringe pump (KDS scientific) was fixed. The syringe and 26 gauge needle were connected with PE20 tubing (Becton Dickinson). When the octopus was stable and almost no movement, a needle connected to the syringe pump was inserted into the center between the octopus's head and body at a depth of 1cm. After measuring the weight of the octopus, separotocin was injected for 5 minutes at a concentration of 100 μg/kg. When separotocin was injected, the injection rate of the syringe pump was set to 50 μl/min, and the behavior change was observed by injecting at the same rate for 1 hour. The entire experimental process including environmental adaptation was observed after recording with an FDR-AXP55 camcorder (Sony) fixed on a tripod.

Based on the data recorded by the camcorder, the major behavioral changes observed during separotocin injection were divided into motor behavior and social behavior. A list was prepared in detail in the order of large and complex movements of body parts (FIG. 5), and the time spent for each action was quantified in seconds. Each action was given a weight value and used to quantify the effect of behavior regulation in later experiments. When different actions occur simultaneously, larger actions are quantified (e.g., when moving an arm with the head raised, only the amount of time the arm is moved). The results of quantifying the time spent by each individual to perform each motor and social behavior during 1 hour of administration of separotocin are shown in FIGS. 6 and 7, respectively.

As shown in Figs. 6 and 7, the control octopus (ASW) injected with artificial seawater barely moved, whereas the octopus injected with separotocin (CPT) increased movement, which is different from the movement of the arm (leg). It was confirmed that the approach and contact with octopus increased.

Overall, the present inventors have confirmed that two types of separotosine genes are expressed in octopus, and in particular, it has been confirmed that the expression of separotosine isoform-2 is increased in female octopus in incubation, and that movement and sociality of octopus are increased. This means that Separotosine isoform-2 can be used as a marker for confirming the incubation status of octopus and for promoting reproduction.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the art, it is obvious that these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> National Marine Biodiversity Institute of Korea <120> Compositions for promoting breeding of mollusks comprising cephalotocin <130> 1-4P <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 9 <212> PRT <213> Unknown <220> <223> Octopus minor <400> 1 Cys Tyr Phe Arg Asn Cys Pro Ile Gly 1 5 <210> 2 <211> 519 <212> DNA <213> Unknown <220> <223> Octopus minor <400> 2 cagttgcgaa aatgtctcag aattgtttta ccatcgtcca acttttgttt gtggtgttca 60 ccgtatgcgg tctgttcgtt gccacagcag atggttgcta tttccgaaac tgtcccattg 120 gtggcaaaag atctctaccc gtgacagagg aagacaatcg atcacaaaag tgtatgtcct 180 gcggacccaa aggtgaaggc cagtgtgttg gacccaacat ttgctgccac aaagacggtt 240 gtatcatagg cttacttgga aaagaatgca atgctgaaaa cgagagtacg acaccatgtt 300 ctgtgacagg tttggcatgc ggaatcgacg gacaaggacg atgtgtttcc gatggtgttt 360 gctgcgatga atctgcctgc tcttcgaaca ctcgctgtaa caccagtgga ggccgtagta 420 agagtctgaa agaattactt gcggttctaa acaaaatgta taggaacaaa atttaaagac 480 cctacccccc agatcccagg gagcacaata tctgatgac 519 <210> 3 <211> 560 <212> DNA <213> Unknown <220> <223> Octopus minor <400> 3 cagttgcgaa aatgtctcag aattgtttta ccatcgtcca acttttgttt gtggtgttca 60 ccgtatgcgg tctgttcgtt gccacagcag atggttgcta tttccgaaac tgtcccattg 120 gtggcaaaag atctctaccc gtgacagagg aagacaatcg atcacaaaag tgtatgtcct 180 gcggacccaa aggtgaaggc cagtgtgttg gacccaacat ttgctgccac aaagacggtt 240 gtatcatagg cttacttgga aaagaatgca atgctgaaaa cgagagtacg acaccatgtt 300 ctgtgacagg tttggcatgc ggaatcgacg gacaaggacg atgtgtttcc gatggtgttt 360 gctgcgatga atcctcctgt gttgcaaccg atagatgtga caaagtgaat catcgcagca 420 tcgcgatgca gaaattattg gcaatgcgag atggatttta ttacaagaaa taaatgaaat 480 atctcataaa aggaccattt tcaacacgag atttcatatg aaactggaaa aaaacatcca 540 ctaccctcat cagcaaaatg 560

Claims (14)

delete delete delete delete delete Biomarker composition for checking the incubation state of mollusks comprising the cepharotosine isoform-2 gene represented by the nucleotide sequence of SEQ ID NO: 3. The method of claim 6,
The cepharotosine isoform-2 gene is a biomarker composition for checking the incubation state of a mollusk that is overexpressed when the mollusk is incubating. delete A composition for checking the incubation state of mollusks comprising an agent measuring the expression of the cepharotosine isoform-2 gene represented by the nucleotide sequence of SEQ ID NO: 3. delete The method of claim 9,
The agent for measuring the expression of the gene is an antisense oligonucleotide, a primer pair, or a probe that specifically binds to the gene encoding the separotosine, a composition for checking the incubation state of mollusks. A kit for checking the incubation status of mollusks comprising the composition of claim 9 or 11. The method of claim 12,
The kit is an RT-PCR kit, a DNA chip kit, or a protein chip kit, a kit for checking the incubation status of mollusks. A method for confirming the incubation state of a mollusk comprising the step of measuring the expression of the separotosine isoform-2 gene represented by the nucleotide sequence of SEQ ID NO: 3 in a biological sample.

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