KR20180024184A - GnRH gene from Haliotis discus hannai and uses thereof - Google Patents

Abstract

The present invention relates to a gonadotropin releasing hormone (GnRH) gene from Nordotis discus hannai and to uses thereof, and in the present invention, it was confirmed that GnRH gene in the Nordotis discus hannai can be specifically detected using a specific primer set to determine the developmental status of abalone. Therefore, by using a maturation index of abalone obtained from the present invention, it is judged that it is possible to mass-produce healthy abalone spats by selecting healthy mothers which are suitable for seedling production, reaching the highest maturity level and securing eggs which is completely mature from the mothers, thereby being expected to be very useful for industry.

Description

GnRH gene derived from northern abalone and its use {GnRH gene from Haliotis discus hannai and uses thereof}

The present invention relates to a GnRH gene derived from northern abalone and its use.

In vertebrates, mature-related endocrine mechanisms are well studied, and the hormonal and receptor signaling regimes associated with breeding control are relatively detailed. Reproductive functions occur along the hypothalamus-pituitary-gonad (HPG) axis of the brain and various endocrine hormones ranging from maturation promotion, ovulation induction, and birth are suitable for agriculture, . Hormones and neuropeptides involved in maturation are typically melatonin, kisspeptin, neurokinin B, gonadotropin-inhibitory hormone (GnIH), gonadotropin Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and growth hormone (Inhibin). These hormones are also known as gonadotropin-releasing hormone (GnRH) To date, molecular biology studies related to reproductive physiological maturation and neuroendocrine development of the abalone have been reported in Haliotis asinina) they have been limited to the report on the expression of neuropeptide four kinds (APGWamide, myomodulin, FMRFamide, shistomomin ) that are considered higher the maturity and relevance.

On the other hand, the development of gender maturity indicators of abalone can be used to secure sexually healthy offspring for "healthy abalone production". As of 2014, the amount of abalone production in Korea is about 700 billion won, which is about 900-100 billion won. One of the reasons for the death of this massive spore is that the spoil of abalone is not healthy. In order to produce a healthy rollover, first fully mature eggs must be obtained from mature mothers. The main spawning season of abalone in nature is the early autumn (September-October), and it is spawned in early spring (April to April) in an artificial way at abalone farming site. Therefore, the egg - laying for egg laying is less maturing in early spring compared with early fall, and unhealthy egg - laying eggs are produced in immature eggs laid out from the mother of immature stage.

Korean Patent No. 0806208 discloses a method for determining the classification system of fishes belonging to the group of the sponges or the stigma, and related polynucleotide probes, DNA chips and kits. In Korean Patent No. 1211068, A polynucleotide probe, a DNA chip and a kit for discrimination of fish species have been disclosed. However, as in the present invention, the GnRH gene derived from northern abalone and its use have never been disclosed.

As a result of attempting to develop a maturity indicator of abalone, the present invention has found that the gene for gonadotropin releasing hormone (GnRH) among the maturation-related genes of northern abalone (Ganglion ganglion, ganglion ganglion and gill ganglion), gonad (ovary and testis), gill, hematocyte, mantle, shell, and visceral tissue Were collected by sampling the gonads at the gonadal development stage (Stage I, recovery period and early growth stage; stage II, late stage; stage III, mature stage; Stage IV, . In the case of northern abalone of female, it was found that the expression of GnRH transcripts in the metatarsal ganglia and gill ganglia at the mature and diffusive stages (Stage III and IV) And the expression of GnRH transcript was significantly increased in the gill ganglia of Stage IV in male Northern abalone. Thus, the inventors of the present invention confirmed that the developmental status of Jeonbuk could be distinguished by using the GnRH specific primer developed in the present invention, thereby completing the present invention.

In order to solve the above-mentioned problems, the present invention relates to a method for producing an amino acid sequence of Haliotis discus hannai- derived gonadotropin releasing hormone (GnRH) protein.

The present invention also provides a gene encoding the GnRH protein.

The invention also Northern abalone (Haliotis consisting of the amino acid sequence of SEQ ID NO: 2 discus The present invention provides a composition for discriminating the degree of maturation of northern abalone comprising a substance for measuring the expression level of a gene encoding hannai- derived gonadotropin releasing hormone (GnRH) protein.

The present invention also provides a method for determining the maturation development of Northern abalone comprising measuring the expression level of a gene encoding GnRH (gonadotropin releasing hormone) protein consisting of the amino acid sequence of SEQ ID NO: 2 from Haliotis discus hannai . ≪ / RTI >

In the present invention, it was confirmed that the degree of maturation of abalone could be discriminated by using a specific primer set capable of specifically detecting GnRH (gonadotropin releasing hormone) gene of northern abalone. Therefore, by using the maturation index of the abalone obtained from the present invention, it is judged that it is possible to mass-produce healthy abalone spots by selecting healthy moths which are suitable for seedling production and reaching the highest maturity and securing mature eggs completely from them. It is expected to be very useful for industry.

Figure 1 shows the HE staining (C) of the ganglion (A) and gonad (B) of the northern abalone and of the ganglion (CG, cranial nerve, PPG, BG, gill ganglion).
FIG. 2 shows the results of Hdh - GnRH (prepro- Hdh - GnRH ) The nucleotide sequence of the cDNA and the predicted amino acid sequence are shown. Prospective signal peptide sequences are indicated by a solid underline, and mature GnRH peptide regions are delineated into a dark box. The three basic amino acid cleavage sites of RKR are shown in italics, and the GAP sequence is indicated by a wavy line underlined. * Represents the termination codon. The polyadenylation site at the 3'-end is indicated by a bold solid underline.
Fig. 3 shows HE staining at the maturation stage of the northern abalone ovary. A, recovery / early growth; B, late growing stage, C, mature stage; D, scatterer / deflector, E, retrograde; F,
Fig. 4 shows HE staining at the maturation stage of northern abalone testis. A, recovery / early growth; B, late growing stage, C, mature stage; D, scatterer / deflector, E, retrograde; F,
FIG. 5 shows the results of various abalone tissues in the mature abalone ovary (A) and testis (B) development stages (Stage III and IV) GnRH gene expression by real-time PCR (RT-PCR). The RPL5 gene was used as a reference gene for the purpose of correcting the cDNA extracted tissue. Each figure is expressed as mean ± standard error and different letters on the bar mean statistically different at significance level 0.05. CG, cranial nerve; PPG, ovarian ganglia, O, ovary; T, testimony; G, gill; I, visceral tissue; H, hemocytes; AM; M, the mantle.
FIG. 6 shows the expression of Hdh- GnRH transcripts in female (A) and male (B) rolled ganglia at the gonadal maturation stage by qRT-PCR. (CG, cranial nerve; PPG, lateral ganglion; BG, gill ganglion). Stage I, recovery and early growth phase; Stage II, late growing season; Stage III, maturity; Stage IV: Spreader / deflector; Stage V, retrograde; Stage VI, Unsteady Period

In order to achieve the object of the present invention, the present invention provides a gonadotropin releasing hormone (GnRH) protein derived from Haliotis discus hannai comprising the amino acid sequence of SEQ ID NO: 2.

The present invention also includes fragments, derivatives and analogues of such proteins. As used herein, the terms "fragments", "derivatives" and "analogs" refer to polypeptides having substantially the same biological function or activity as the protein of the invention. The fragments, derivatives, and analogs of the present invention may be used in conjunction with (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted (the substituted amino acid residues are encoded Or (ii) a polypeptide having substituent (s) at one or more amino acid residues, or (iii) another compound (a compound capable of extending the half-life of the polypeptide, such as polyethylene glycol) (Iv) an additional amino acid sequence (e. G., A leader sequence, a secretory sequence, a sequence used to purify the polypeptide, a proteinogen sequence or a fusion protein) and a polypeptide derived from the mature polypeptide Or a polypeptide derived from said polypeptide. Such fragments, derivatives and analogs as defined herein are well known to those skilled in the art.

A polynucleotide encoding a protein having an amino acid sequence represented by SEQ ID NO: 2 comprises a coding sequence which encodes only a mature polypeptide; Sequences encoding mature polypeptides and various additional coding sequences; Mature polypeptides (and any additional coding sequences) and sequences coding for noncoding sequences.

The term "polynucleotide encoding a polypeptide" refers to a polynucleotide encoding a polypeptide, or a polynucleotide further comprising additional coding and / or noncoding sequences.

The invention also relates to variants of said polynucleotides encoding polypeptides comprising the same amino acid sequences as described herein, or fragments, analogs, and derivatives thereof. Polynucleotide variants can be naturally occurring allelic variants or non-naturally occurring variants. The nucleotide mutant includes a substitution mutant, a deletion mutant, and an insertion mutant. As is known in the art, an allelic variant is an alternative to a polynucleotide, which may comprise one or more substituted, deleted or inserted nucleotides and which does not result in a substantial functional change in the polypeptide encoded by the variant Do not.

The GnRH protein of the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous physiological activity" means activity as gonadotropin releasing hormone (GnRH) in northern abalone.

The present invention also provides a gene encoding the GnRH protein. Preferably, the gene of the present invention may comprise the nucleotide sequence of SEQ ID NO: 1. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

The present invention also relates to a polynucleotide which hybridizes with a sequence having at least 50%, preferably at least 70%, more preferably at least 80% identity with the nucleotide sequence of SEQ ID NO: 1 described above. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present invention, "stringent conditions" means (1) hybridization and washing under lower ionic strength and higher temperature such as 0.2 x SSC, 0.1% SDS, 60 ° C; Or (2) in the presence of a denaturant such as 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficoll and 42 ° C; Or (3) at least 80%, preferably at least 90%, more preferably at least 95%. In addition, the biological function and activity of the polypeptide encoded by the hybridizable polynucleotide is the same as the biological function and activity of the mature polypeptide represented by SEQ ID NO: 2.

The gonadotropin releasing hormone (GnRH) protein coding gene derived from Haliotis discus hannai of the present invention was isolated for the first time through the present invention, and no relation to the discrimination of maturation development of northern abalone was known at all.

The present inventors have established that the GnRH protein coding gene can be used as a marker for determining the maturation development of northern abalone through the following proofs.

Specifically, a gene for GnRH (gonadotropin releasing hormone) gene was newly isolated from the maturation-related genes of northern abalone, and a specific primer set capable of detecting the gene was prepared, and ganglion tissues (cranial nerves, (Ganglia and gill ganglia), gonads (ovaries and testes), gills, hematocytes, mantle cells, shellfishes and visceral tissues were divided into gonadal development stages (Stage I, (Stage III and IV) in the case of northern abalone of female, as a result of PCR on the total RNAs obtained by sampling each strain (Stage V, IV, Strain V, Stage V, , The expression of GnRH transcripts was high in ovarian ganglia and gill nerve ganglia, and in GnRH transcripts of male northern abalone, GnRH transcripts were remarkably expressed in gill ganglia of Stage IV Respectively.

Therefore, the developmental status of northern abalone could be determined by carrying out reverse transcription polymerase chain reaction (PCR) using a primer designed by the inventors of GnRH (gonadotropin releasing hormone) protein coding gene derived from Haliotis discus hannai .

In the present invention, the term "substance for measuring the expression level of GnRH (gonadotropin releasing hormone) protein coding gene derived from Haliotis discus hannai " refers to a substance which is a marker for determining the maturation development of northern abalone Refers to a molecule that can be used for the detection of a marker by confirming the expression level of the coding gene, and preferably refers to a marker-specific primer.

The expression level of the GnRH protein coding gene can be determined by confirming the expression level of the mRNA of the GnRH protein coding gene. In the present invention, "measurement of mRNA expression level" is a process of determining the presence and expression level of a GnRH protein coding gene in a biological sample in order to determine the degree of maturation of northern abalone by measuring the amount of mRNA. RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting (Northern blotting) blotting, and DNA chips, but are not limited thereto.

In the present invention, the term "primer" refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming a base pair with a complementary template and having a starting point for template strand copying ≪ / RTI > The primer can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures. In the present invention, PCR amplification using a sense and antisense primer of GnRH protein coding polynucleotide can be used to determine the degree of maturation of northern abalone through the production of a desired product. The PCR conditions, the lengths of the sense and antisense primers can be modified based on what is known in the art.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).

According to a specific embodiment of the present invention, the composition for determining the maturation development degree of northern abalone comprises respective primer pairs specific for the GnRH protein coding gene. Specifically, the GnRH protein coding gene is a primer represented by SEQ ID NOs: 8 and 9.

In another aspect, the present invention provides a method for determining the degree of maturation of northern abalone comprising measuring the expression level of a GnRH protein coding gene consisting of the amino acid sequence of SEQ ID NO: 2 from northern abalone.

The expression level of the GnRH protein coding gene of the present invention can be determined by confirming the expression level of the mRNA of the GnRH protein coding gene.

The process of separating mRNA from a biological sample can be carried out using known processes, and the level of mRNA can be measured by various methods.

Methods for measuring mRNA levels include, but are not limited to, reverse transcriptase polymerase, competitive reverse transcriptase polymerase, real-time reverse transcriptase polymerase, RNase protection assay, northern blotting, and DNA chip.

Through the above detection methods, mRNA expression of the GnRH protein coding gene in northern abalone samples can be examined to determine the degree of maturation of northern abalone.

The measurement of the mRNA expression level is preferably performed using a reverse transcriptase polymerase reaction method or a DNA chip using a primer specific to a gene used as a marker for determining the maturation development degree of northern abalone.

The method according to one embodiment of the present invention may be a method of determining the expression level of the gene encoding the GnRH protein and determining the highest level in the gill ganglia of female and male of the Northern abalone as a spawner season, The highest level in the females and the gill ganglia of the female are identified as mature and diffuse, and the highest in the gills of the northern abalone is determined as the ganglia.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Gonorrhea  And ganglion dissection and extraction

The gonad tissues of the mature male / female abalone were removed and a pair of cranial nerve (CG) and metatarsal ganglion (PPG) corresponding to ganglion tissues were extracted and stored at -80 ° C until RNA extraction. The paraffin sections of the tissues were prepared and then subjected to H / E staining to determine the suitability of the tissues.

mRNA  Extraction and cDNA synthesis

Total RNA was extracted using RNeasy Mini Kit (Qiagen, USA) for ganglion tissues, CG and PPG. First strand cDNA was synthesized from total RNA extracted using the SMART RACE (rapid amplification of cDNA ends) cDNA amplification kit (Clontech, USA) for the cloning of genes coding for maturation related hormones.

Mature Hormone Molecules Cloning

To elucidate the length of the total sequence of gonadotropin releasing hormone (GnRH) genes among the maturation related genes of gastropods belonging to abalone, H. laevigata (GenBank accession No. KP719129), which is homologous with Haliotis discus hannai (F) and reverse (R) primers were constructed based on the previously known partial sequence of GnRH mRNA and PCR was performed with LA Taq DNA polymerase (Takara Bio, Japan) using CG and PPG first strand cDNA as a template The partial nucleotide sequence of the GnRH gene of northern abalone was confirmed. Based on this sequence, 5'- / 3'-RACE PCR was performed using the first strand cDNA of CG and PPG as a template together with Universal Primer A Mix (UPM; Clontech). As a result, a cDNA encoding GnRH of 868 nucleotides (nt) (poly (A) -tail not included) was identified. The total length of the GnRH cDNA was found to contain 75 nt of 5'-untranslated region (UTR), 306 nt of coding sequence (corresponding to 101 amino acids) and 487 nt of 3'-UTR.

Utilization and search of mollusc DB

In order to identify a group with a sequence similar to that of GnRH of northern abalone, BLAST search was performed on the NCBI database and the ORF (open reading frame) of cDNA encoding GnRH of northern abalone using the CLUSTALW program and BLAST In order to investigate the phylogenetic tree through comparison with the GnRH-ORF of the highly similar biota. The prediction of post-translational processing of the GnRH neuropeptide was also performed using the NeuroPred, SignalIP program.

Acquisition of abalone samples per mature stage

In order to secure the abalone for maturation stage, H-E staining of the gonads was carried out on abalone collected monthly in the Koguryo area of Yangyang, Gangwon-do, and classified into 6 stages according to gonadal development stages. Stage I, recovery and early growth phase (January-March); Stage II, late growing (March to May); Stage III, maturity (June - August); Stage IV, Spreader / Spreader (September to October); Stage V, retrograde (October to November); Stage VI, unsteady period (November to February) (Najmudeen 2007, Molluscan Res 27 (3): 140-146).

In order to confirm the expression of GnRH mRNA in the tissues of the stages of maturation (Stage III and Stage IV), ganglion tissues (cranial nerves, osteophyte ganglia) and gonads, gills, hematocytes, mantle, And stored at -80 ° C until RNA extraction. The ganglion tissues of each stage were extracted to identify gingival developmental stages in the ganglion tissues (cranial nerves (CG), proximal ganglia (PPG), and gill ganglia (BG)), the main expression machinery of GnRH mRNA.

GnRH  Oligo primer  Production and primer  Identification of specificity

GnRH of northern abalone identified using Primer Express v3.0 Software (Applied Biosystems) GnRH oligonucleotide primers (Hdh-GnRH-F3 and R3, Table 1) specific for northern abalone were prepared. The primer selection criteria are as follows: primer length, 18-25 bp; Melting point (Tm) 63 캜; The Tm difference between the forward and reverse primers was 1 [deg.] C; Amplification length is 75-250 bp; GC content is 40-60%.

Reference gene selection

Primers (Hdh-RPL5-F and Hdh-RPL5-R, Table 1) were prepared by selecting the abalone RPL5 gene as a reference gene for the correction of the cDNA level extracted from each tissue.

Real-time polymerase chain reaction (real-time PCR ) analysis

Quantitative real-time PCR (qRT-PCR) was performed to identify mRNA expression patterns of maturation-related GnRH genes by tissue and maturation stages. Total RNA was extracted from cranial nerves, medial ganglia, gill ganglia, gonads, gills, hematopoietic cells, mantle membrane, excisional muscle and visceral tissue using RNeasy Kit (QIAGEN, Valencia, CA, USA). To remove genomic DNA, 5X gDNA Eraser Buffer was treated at 42 ° C for 2 minutes. First strand cDNA synthesis was carried out at 37 ° C for 15 minutes using PrimeScript RT reagent Kit (TaKaRa Bio, Shiga, Japan). Real-time PCR was performed using 7500 Real-Time PCR Instrument System (Applied Biosystem, Boston, USA). The reaction mixture was incubated at 20 ° C with 10 ng of each synthesized cDNA, 2x SYBR Premix Ex Taq and 50x Reference Dye II TaKaRa Bio. Japan) and a set of 10 uM GnRH oligopeptide primers. Two-step PCR was used for PCR, and the amplification was performed for 2 minutes at 50 ° C, 10 minutes at 95 ° C, 40 seconds at 95 ° C for 15 seconds, and 60 ° C for 1 minute.

Example  1. Abalone (Scientific name: Haliotis  discus hannai ) GnRH ( gonadotropin -releasing hormone) cDNA separation

1-1. Gonorrhea / Ganglion dissection and extraction

The nervous system of the northern abalone is composed of a number of ganglia with nerve cells, including the cranial nerves, the medial ganglia, and the ganglia. Tissue sections of extracted cranial nerve, metatarsal ganglia and gill ganglia were stained with hematoxyline-eosin and observed under an optical microscope (Fig. 1A and Fig. 1C).

1-2. Hdh - GnRH  cDNA Cloning

The 5 'and 3'-RACE PCR method was used to identify prepro - Hdh -GnRH cDNA in the metatarsal ganglia of mature northern abalone. The north upset prepro - Hdh - and named GnRH, Hdh - - a GnRH cDNA Hdh GnRH overall length of length 868 consisting of a base of the new clay lactide (nt), by using the ORF finder tool of the NCBI translated region (CDS, coding As a result of the prediction of the DNA sequence, a 75 nt 5 'untranslated region (UTR), a 306 nt translation region (corresponding to 101 amino acids: SEQ ID NO: 2) Region (except for the poly A terminal region) (Fig. 2). Using the SinalP 4.1 server and the NeuroPred program, the Hdh - GnRH Analysis of the amino acid sequence predicted from the genetic information revealed that GnRH protein precursors of northern abalone, similar to known GnRH precursors, had one signal peptide, one GnRH peptide (consisting of 12 amino acids) and one processing Region, and one GnRH-associated peptide (GAP) region.

Example  2. Real-time polymerase chain reaction (real-time PCR ) Abalone GnRH (gonadotropin-releasing hormone) mRNA  For expression studies GnRH  Specific Fry Merge production

2-1. Acquisition of abalone samples per mature stage

H-E staining of the gonads was performed and classified into six consecutive stages (Stage I to VI) according to gonadal developmental stage. In order to confirm the expression of GnRH mRNA in tissues belonging to the mature stage (Stage III and Stage IV), ganglion tissues (cranial nerves, gyro ganglia) and gonads, gills, hematocytes, mantle, And then stored at -80 ° C until RNA extraction. In addition, ganglion tissues (cranial nerve, medial ganglia, and gill ganglion) at each stage were extracted to identify the gestational developmental stage of gonadal development in the ganglia tissues, the main expression machinery of GnRH mRNA.

Abnormal gonadal development stages were classified into 6 consecutive stages. Tissue sections of ovary and testes were prepared and HE stained and examined by optical microscope. The gonadal developmental stage was based on the method of Najmudeen (Najmudeen 2007, Molluscan Res 27 (3): 140-146), the germ cell formation process, morphological changes in reproductive epithelium, undifferentiated hepatic tissue in gonadal lumen, (Fig. 3 and Fig. 4). These cells were classified into the convalescent / early growth phase, late growth phase, mature phase, diffusive / irregular phase, retrograde phase and irregular phase depending on the appearance frequency of yellow granular cells present in the reproductive epithelium.

2-2. Northern abalone GnRH Transpositional  Expression Patterns by Organization

The expression of Hdh -1 in the representative tissues (cranial nerves, gonads, gills, visceral cells, hematopoietic cells, osteoblasts , and endothelium ) in female and male abalones of the mature stages (Stage III and IV) The expression pattern of GnRH transcript was highly expressed in ganglion tissues (FIG. 5), particularly in the lateral ganglion (FIG. 6).

2-3. Northern abalone by breeding step GnRH Transpositional  Expression pattern

qRT-PCR method GnRH The changes of expression in ganglia of transcripts were examined. In female abalone, in particular, in maturation and egg layers (Stage III and IV), GnRH Expression of the transcript was high, and expression in the cranial nerve of the abdomen was not clear (Fig. 6). Interestingly, the expression of GnRH transcripts in the male abalone gillet of Stage IV was significantly increased. These results suggest that GnRH transcripts of abalone are highly expressed in the ganglion tissues during the developmental stage of gonad, especially at the maturation stage. GnRH abortion can be utilized as an indicator of maturation development of abalone in the future.

PCR And real-time PCR qRT - PCR ), primer Primer Sequence (5'- 3 ') (SEQ ID NO) Application Hdh-GnRH-F1 GCTGCTGCTCACGGTACATG (3) Partial PCR Hdh-GnRH-R1 CTGCATTCTGGCAGCGATGG (4) Hdh-GnRH-F2 GGAATCATCTCGTGTGGCCCATCGCTGC (5) RACE PCR Hdh-GnRH-R2 CTGGCAGCGATGGGCCACACGAGATG (6) UPM (F / R) CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT (7) Hdh-GnRH-F3 CATACGCACCAAACCACAGAA (8) Quantitative Real-time PCR (qPCR) Hdh-GnRH-R3 CGTGCCAGCCGTTGCT (9) Hdh-RPL5-F TCACCAACAAGGACATCATTTGTC (10) Hdh-RPL5-R CAGGAGGAGTCCAGTGCAGTATG (11) Note. F, forward; R, reverse; UPM, universal primer mix

Through the present invention, by examining the expression pattern of GnRH gene in the stages of maturation of abalone, it can be utilized as an indicator of maturation development of abalone. The development of gender maturation indicators of abalone can be used to secure a healthy mother abductor suitable for the production of high quality spouses because the mature state for "healthy abalone production" is complete.

<110> INDUSTRY FOUNDATION OF CHONNAM NATIONAL UNIVERSITY <120> GnRH gene from Haliotis discus hannai and uses thereof <130> PN16104 <160> 11 <170> Kopatentin 2.0 <210> 1 <211> 306 <212> DNA <213> Haliotis discus hannai <400> 1 atgtcagtac tatcaagaca aggggtgact gtgtcggtgc tgctgctgct gctcacagta 60 cacgctgttg gaggccagaa ctaccacttc agcaacggct ggcacgcagg caggaaacgt 120 ggcggggaca gcaccagctg cgtcttcagg aaagacatcc tcatgcttgt caacaaactg 180 atcatggagg aatcatctcg tgtggcccat cgctgccaga acggagtgcc gcacatggac 240 ttcacagagc ctgacacagt tgaagctgat tctgccaacc agctgacaga caagcgatgg 300 aagtga 306 <210> 2 <211> 101 <212> PRT <213> Haliotis discus hannai <400> 2 Met Ser Val Leu Ser Arg Gln Gly Val Thr Val Ser Val Leu Leu Leu   1 5 10 15 Leu Leu Thr Val His Ala Val Gly Gly Gln Asn Tyr His Phe Ser Asn              20 25 30 Gly Trp His Ala Gly Arg Lys Arg Gly Gly Asp Ser Thr Ser Cys Val          35 40 45 Phe Arg Lys Asp Ile Leu Met Leu Val Asn Lys Leu Ile Met Glu Glu      50 55 60 Ser Ser Arg Val Ala His Arg Cys Gln Asn Gly Val Pro His Met Asp  65 70 75 80 Phe Thr Glu Pro Asp Thr Val Glu Ala Asp Ser Ala Asn Gln Leu Thr                  85 90 95 Asp Lys Arg Trp Lys             100 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gctgctgctc acggtacatg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ctgcattctg gcagcgatgg 20 <210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggaatcatct cgtgtggccc atcgctgc 28 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ctggcagcga tgggccacac gagatg 26 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 catacgcacc aaaccacaga a 21 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cgtgccagcc gttgct 16 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tcaccaacaa ggacatcatt tgtc 24 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 caggaggagt ccagtgcagt atg 23

Claims (9)

2, which consists of the amino acid sequence of SEQ ID NO: 2 ( Haliotis gonadotropin releasing hormone (GnRH) protein derived from the discus hannai . A gene encoding the GnRH protein of claim 1. 3. The gene according to claim 2, wherein the gene consists of the nucleotide sequence shown in SEQ ID NO: 1. A Northern Abalone ( Haliotis) consisting of the amino acid sequence of SEQ ID NO: 2 A composition for discriminating the degree of maturation of Northern abalone comprising a substance for measuring the expression level of a gene encoding a gonadotropin releasing hormone (GnRH) protein derived from a discus hannai . The composition according to claim 1, wherein the substance for measuring the expression level of the gene is a primer set that specifically binds to the gene encoding the GnRH protein. 6. The composition of claim 5, wherein the primer set is a set of oligonucleotide primers of SEQ ID NOS: 8 and 9. Northern abalone ( Haliotis discus determining the level of expression of a gene encoding a gonadotropin releasing hormone (GnRH) protein consisting of the amino acid sequence of SEQ ID NO: 2 from hannai . The method according to claim 7, wherein the expression level of the gene is measured by a reverse transcriptase polymerase, a competitive reverse transcriptase polymerase, a real-time reverse transcriptase polymerase, an RNase protection assay, a Northern blotting or a DNA chip A method of determining the degree of maturation development of northern abalone characterized by. [Claim 7] The method according to claim 7, wherein the expression level of the gene coding for the GnRH protein is measured to determine that the expression level is highest in the ovarian ganglia and gill ganglia of the female of the Northern Abalone, In which the expression level is the highest, is identified as a reporter.

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