Peptides having octopus octopressin activity and use thereof

Abstract

The present invention relates to an Octopus minor-specific octopressin peptide comprising the amino acid sequence of SEQ ID NO: 3, and the peptide, by promoting the brooding behaviors of mother Octopus minor to protect her eggs, may increase the hatched larva rate when applied to partial cultivation of octopuses, and thus may be used for the growth of octopus resources.

Claims

1. A method of promoting brooding behaviors of Octopus minor, the method comprising administering an effective amount of a composition comprising a peptide consisting of an amino acid sequence of SEQ ID NO: 3 as an active ingredient to Octopus minor as needed.

2. The method of claim 1, wherein the brooding behavior lasts for about 5 minutes to about 30 minutes.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows images of brooding behavior of a mother Octopus minor;

(2) FIG. 2 is a schematic diagram that shows a structure of an octopressin gene in an Octopus minor genome (intron size in 1/100 reduction);

(3) FIG. 3 shows the result of amplifying a partial octopressin gene in a brain of Octopus minor;

(4) FIG. 4 shows the result of amplifying the whole octopressin gene (including encoding nucleotide sequences) in a brain of Octopus minor;

(5) FIG. 5 shows the result of amplifying a part of a cloned vector corresponding to the octopressin gene;

(6) FIG. 6A are images of behavior change of Octopus minor after injection of a synthetic peptide that satisfies octopressin amino acid sequence and modification; and

(7) FIG. 6B are images of the Octopus minor injected with octopressin showing behavior similar to the brooding behavior.

MODE OF DISCLOSURE

(8) Hereinafter, the present disclosure will be described in more detail with reference to examples. The examples are for only descriptive purposes, and it will be understood by those skilled in the art that the scope of the present disclosure is not construed as being limited to the examples.

EXAMPLE

Example 1. Analysis of Brooding Behavior of Mother Octopus minor and Detection of Regulatory Gene

(9) 1-1. Analysis of Brooding Behavior of Mother Octopus minor

(10) In order to observe brooding behaviors of Octopus minor, mother Octopus minors were obtained through cooperation with the western branch Resource Creation Department of the Jeonnam Institute of Ocean and Fisheries Science. After purchasing mature individuals (180 to 330 g in weight) collected in April to June 2017 from the sea around Sinan-gun, Jeollanam-do, one female and one male were put into an onion net and then in the natural sea farm in the Resource Creation Department for 1 week to induce copulation, and then only female Octopus minors were remained to induce spawning. The clams were fed as food before spawning (once per week), the feeding was stopped after spawning, and the Octopus minors were maintained for 1 month or more. Then, the Octopus minors were moved to an artificial seawater breeding facility (18° C.) in the resource center for maintenance and observation.

(11) FIG. 1 shows images of brooding behaviors of a mother Octopus minor. As shown in FIG. 1, the mother Octopus minor, after laying eggs, exhibited unique behaviors including anchoring the eggs on the wall using a green cement secreted from her and frequently shaking the eggs using her arms (legs) and suckers. It was observed that the mother Octopus minor continued these behaviors for about 3 months until the fry hatch, during which the female Octopus minor kept the side of the eggs without eating food. The Octopus minor in nature is known as performing the same spawning brooding behaviors in the deep cave beneath the mud flat, and these behaviors of the mother Octopus minor has an effect of cleaning surfaces of the eggs and circulating the surrounding seawater.

(12) 1-2. Detection of Regulatory Gene

(13) A full-length genome analysis was performed to identify genes related to the brooding behavior of Octopus minor. In particular, after extracting the DNA of the octopus, the full-length genome analysis was performed through next-generation base sequencing techniques (NGS) and bioinformatics methods to obtain a large number of genetic data. Subsequently, the presence of a gene annotated with a gene similar to oxytocin or octopressin of octopus was confirmed by mining the data. From the results, oxytocin-like gene information of about 147 kb in length was obtained, which was named octopressin of Octopus minor (Table 1).

(14) TABLE-US-00001 TABLE 1 Tran- scriptome/ Scaffold Shape Start End protein ID oct000694F gene 156,242 303,030 Omin007228 oct000694F mRNA 156,242 303,030 Omin007228 oct000694F exon 156,242 156,317 Omin007228 oct000694F CDS 156,242 156,317 Omin007228 oct000694F exon 203,999 204,083 Omin007228 oct000694F CDS 203,999 204,083 Omin007228 oct000694F exon 231,093 231,231 Omin007228 oct000694F CDS 231,093 231,231 Omin007228 oct000694F exon 302,349 303,030 Omin007228 oct000694F CDS 302,349 302,483 Omin007228 oct000694F five_prime_UTR 302,484 303,030 Omin007228

(15) FIG. 2 is a diagram analyzing a structure of an octopressin gene in an Octopus minor. As shown in FIG. 2, a structure similar to that of an oxytocin gene was confirmed from the full-length nucleotide sequence information of the genome of the octopressin gene.

(16) In addition, from the result of transcriptome analysis, it was confirmed that octopressin mRNA of Octopus minor was not expressed at the beginning of development, but the expression level gradually increased as the development process progressed. In particular, it was confirmed that octopressin was expressed more in the brain and intestine in adults, less in the spermatophore, stomach, liver, and mouth, and not in the arms (legs), gills, eyes, heart, kidneys, skin, ovaries, respiratory tract, and poison glands.

(17) In this regard, it was possible to predict that the octopressin of Octopus minor may be similar to oxytocin of human. That is, when an expression level of octopressin in a mother Octopus minor is significantly increased, it is expected that the octopressin induces brooding behaviors and suppresses the appetite during the period of incubation, and thus this may be applied to the Octopus minor partial cultivation and increase a hatched larva rate.

Example 2. Analysis of Regulatory Gene

(18) 2-1. Analysis of Coding Nucleic Acid Sequence of Octopressin Gene

(19) The coding sequences of octopressin gene predicted by the genome analysis on the Octopus minor in Example 1 were analyzed. In particular, the coding nucleic acid sequence (CDS) sites obtained from the genomic analysis result were entered into the ORF finder program (www.ncbi.nlm.nih.gov) of NCBI to obtain a list of possible open reading frames (ORFs). An amino acid sequence of the longest ORF among these was analyzed using the protein blast (blastX) program of NCBI, and thus it was confirmed that the sequence had a part similar to that of other oxytocin-like genes of other species, indicating that it was an octopressin precursor protein.

(20) 2-2. Analysis of Octopressin Active Amino Acid Sequence

(21) The amino acid sequence of the active peptide analyzed in Example 2-1 was analyzed, and the amino acid sequence is as follows;

(22) CFWTNCPVG(SEQ ID NO:3).

(23) Particularly, the amino acid sequence was compared with an amino acid sequence of human oxytocin precursor protein, and the similar sites were found and predicted. As a result, the amino acid sequence predicted as an active peptide had a signal peptide in the front part of the amino acid sequence, and a cleavage site and a neurophysin-like site at the end, showing a structure similar to that of the human oxytocin precursor and octopressin of Octopus vulgaris. The amino acid sequence of the octopressin active peptide of Octopus minor was composed of 9 amino acids, and the two cysteine positions were the same, respectively, and it was confirmed that the amino acid sequence was similar to other oxytocin-based peptides in terms of being terminated with glycine. That is, when the octopressin of Octopus minor undergoes amidation, in which a disulfide bond is formed between amino acid 1 (cysteine) and amino acid 6 (cysteine), and NH.sub.2 is added at the C-terminal site of amino acid 9 (glycine), the octopressin may act as an active peptide. Also, it was confirmed that the amino acid sequence of the octopressin active peptide of Octopus minor was a novel octopressin different from the octopressin amino acid sequence of Octopus vulgaris (Table 2).

(24) TABLE-US-00002 TABLE 2 SEQ ID NO: Amino acid sequence 4 Homo sapiens Oxytocin C Y I Q N C P L G 5 Homo sapiens Vasopressin C Y P Q N C P R G 6 Octopus vulgaris Octopressin C F W T S C P I G 7 Octopus vulgaris Cephalotosine C Y F R N C P I G 13 Octopus minor Octopressin C F W T N C P V G 8 Sepia officinalis Sepiatocin C F W T T C P I G

Example 3. Synthesis of cDNA of Octopus minor-Specific Octopressin Gene and Confirmation of Expression of Octopressin

(25) 3-1. Synthesis of cDNA of Octopus minor-Specific Octopressin Gene

(26) In the spring of 2017, eight female Octopus minors (average weight of 183.5 g) caught for the purpose of distribution of seafood on the southwest coast of Korea were purchased and placed in the breeding tank for more than 3 months. Then, the brain of the Octopus minor was separated, wrapped in foil, and then rapidly frozen at −70° C. The frozen brain was crushed into a powder using a hammer, put in 3 ml of Isoplus (Takara) solution and dissolved, and RNA was extracted from 1 ml of the mixed solution. Thereafter, cDNA was synthesized from 1 μg of RNA using SuperScript™ IV First strand synthesis system (Invitrogen), and primers were designed to amplify the octopressin gene from the synthesized cDNA (Table 3).

(27) TABLE-US-00003 TABLE 3  SEQ ID NO: Gene Nucleic acid sequence  9 Om-S011E-F1 Forward 5′-GTT TCT GGA CAA ACT GCC-3′ 10 Om-S011E-R1 Reverse 5′-GCT GCG ATG ATT CAC TTT GTC-3′ 11 Om-S011C-F1 Forward 5′-GGA AAT ATT CCC GTG AAA CC-3′ 12 Om-S011C-R1 Reverse 5′-CAT TTT GCT GAT GAG GGT AG-3′

(28) 3-2. Confirmation of Expression of Octopressin

(29) In order to confirm whether the octopressin gene is expressed in the brain of the Octopus minor, RT-PCR was performed on a part of the octopressin gene using the cDNA primers (SEQ ID NOS: 9 and 10) designed in Example 3-1 (5 minutes at 95° C..fwdarw.(30 seconds at 94° C..fwdarw.30 seconds at 67° C..fwdarw.30 seconds at 72° C.)×35 cycles.fwdarw.10 minutes at 72° C..fwdarw.∞ at 10° C.). Next, the amplified PCR product was electrophoresed on a 1.2% agarose gel to confirm the DNA band.

(30) As a result, as shown in FIG. 3, the same band of 317 bp was confirmed in all of the eight Octopus minors. Thereafter, in all Octopus minors, the coding sequence containing the full-length ORF of the octopressin gene was amplified for cloning.

(31) 3-3. Amplification and Confirmation of Coding Sequence Part of Octopressin Gene

(32) In order to obtain the full-length ORF coding sequence of octopressin, the cDNA primers (SEQ ID NOS: 11 and 12) synthesized in Example 3-1 and K-2016 AccuPower PCR premix (Bioneer) were mixed, and RT-PCR was performed on the full-length coding sequence of the octopressin gene (10 minutes at 94° C..fwdarw.(30 seconds at 94° C..fwdarw.30 seconds at 60° C..fwdarw.45 seconds at 72° C.)×30 cycles.fwdarw.10 minutes at 72° C..fwdarw.∞ at 10° C.). Next, the amplified PCR product was electrophoresed on a 1.2% agarose gel to confirm the DNA band.

(33) As a result, as shown in FIG. 4, the same band of 687 bp was confirmed in all of the Octopus minors. This gene was used in cloning as the octopressin full-length ORF coding sequence was predicted to be included in the gene.

Example 4. Preparation of Recombinant Gene

(34) 4-1. Preparation of Octopus minor-Derived Recombinant Octopressin Gene

(35) In the agarose gel where the PCR product of Example 3-1 was confirmed, the band was cut with a knife while confirming the position of the band with UV. The cut gel was purified using a QIAquick Gel extraction kit (QIAGEN) to purify the amplified DNA contained in the band. The purified PCR product DNA was inserted into the vector using pGEM-T easy vector system I (Promega), transformed into E. coli DH5a competent cells, and cultured in an LB/amp plate. Since the transformed E. coli with the ligation vector grows into white colonies, 5 colonies per Octopus minor were selected and further cultured in LB/amp medium. Then, colony PCR was performed form the same colony (10 minutes at 95° C..fwdarw.(30 seconds at 94° C..fwdarw.30 seconds at 55° C..fwdarw.50 seconds at 72° C.)×30 cycles.fwdarw.5 minutes at 72° C..fwdarw.∞ at 10° C.) to confirm whether the octopressin ORF coding sequence was properly inserted into the cultured colony. The PCR was performed by adding commercially available sp6 primers and T7 primers. Next, the amplified PCR product was electrophoresed on a 1 agarose gel to confirm the DNA band.

(36) As a result, as shown in FIG. 5, the recombinant plasmid DNA having an octopressin coding sequence inserted in the vector showed the band of 837 bp, and the recombinant plasmid DNA having self-ligated vector showed the band of 150 bp.

(37) 4-2. Confirmation of Coding Nucleic Acid Sequence of Recombinant Octopressin Gene

(38) Among the E. coli cultured in Example 4-1, 2 colonies exhibiting the band of 837 bp, that are colonies to which the octopressin coding sequence was inserted, were selected per one Octopus minor, and plasmid DNA was extracted therefrom using a QIAprep spin miniprep kit (QIAGEN). Then, the concentration of the extracted DNA was measured, and the nucleic acid sequence was analyzed to confirm inclusion of the octopressin full-length coding sequence, and the nucleic acid sequences predicted in Example 2 were verified. Here, the same primers as those used in Example 2-2 were used, and the nucleic acid sequences were analyzed by Macrogen. Next, the results of the analysis were aligned using the MEGA6 program.

(39) As a result, the nucleic acid sequences of the cloned parts mostly matched the octopressin coding nucleic acid sequences of Example 2-1. Some mutated sequences were also found, but the number of nucleotide sequences in the ORF site was the same in all 8 Octopus minors. Also, the nucleic acid sequences of the cloned parts mostly matched. Some mutated sequences were also found, but the number of nucleotide sequences in the ORF site was the same in all 8 Octopus minors. In addition, it was confirmed that the octopressin active peptide (9 amino acids) and the part corresponding to the signal peptide have the same nucleic acid sequence in all the 8 Octopus minors, and were exactly the same as the sequences predicted through genome analysis in Example 2-2. Although the number of the nucleic acid sequences was the same for the part corresponding to neuropicin and glycopeptides, differences between Octopus minor individuals were found in the nucleic acid sequence, and, diversity of amino acid sequences was confirmed. From these results, it may be confirmed that a recombinant plasmid DNA containing the octopressin full-length coding sequence was successfully prepared from 8 Octopus minors.

Example 5. Analysis of Bioactivity of Octopressin Peptide

(40) Peptides satisfying the octopressin amino acid sequence and modification predicted in Example 4 were ordered and purchased from a peptide synthesizing company and used. A dried peptide powder was dissolved in tertiary distilled water to a concentration of 10 mg/ml, diluted 100 times of artificial seawater filtered through a 0.20 μm filter to prepare an injection solution. A disposable syringe and a 26 gauge needle were used to poke at a 1 cm deep, and the injection solution was administered in the center between the body of the head of the adult Octopus minor. The injection volume was 100 microgram/kg by weight of the Octopus minor. As a control group, Octopus minor without injection (before injection), Octopus minor injected with filtered artificial seawater only, and Octopus minor injected with octopus-derived octopressin were used.

(41) FIG. 6A shows images of behavior change in the Octopus minor after injection of the peptide. As shown in FIG. 6A, the Octopus minors exhibited repetitive movements of bending and stretching several arms (legs) were observed, and social behaviors including approaching other Octopus minors, and touching or wiping other Octopus minors by stretching the arms (legs) were induced. In addition, these behavior changes began to appear 1 to 5 minutes after injection and lasted for 10 to 20 minutes, and the effect peaked between 5 to 10 minutes after injection and lasted for 10 to 20 minutes. It was confirmed that the octopressin-activated peptide had bioactivity in 1 to 3 Octopus minors per day, a total of 12 Octopus minors, regardless of sex. Here, the used Octopus minor were discarded without reuse. On the other hand, no specific behavior changes were observed in the Octopus minor without injection at all, the Octopus minor injected with only filtered artificial seawater, and the Octopus minor injected with Octopus minor-derived octopressin.

(42) FIG. 6B shows images of the Octopus minor injected with octopressin exhibiting behaviors similar to the brooding behaviors. As shown in FIG. 6B, since the Octopus minor-derived octopressin may induce the brooding-like behaviors and social behaviors in Octopus minors, when the more Octopus minor eggs are hatched by promoting the copulation, production of Octopus minors may increase, and thus the peptide may be used in the growth of Octopus minor resources.

(43) It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art may readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are illustrative in all aspects and should not be construed as limiting the scope of the present invention.