High-temperature-tolerant strain of pyropia yezoensis for land-based cultivation

Abstract

The present disclosure provides a high-temperature-tolerant strain of Pyropia yezoensis. The novel high-temperature-tolerant strain of Pyropia yezoensis according to the present disclosure has tolerance for high temperatures and exhibits a high growth rate under land-based cultivation conditions using an artificial light source, and is thus highly useful for land-based cultivation and dealing with increases in seawater temperature.

Description

Radial-patterned seaweed with high temperature tolerance, suitable for land-based aquaculture

[0001] The present disclosure claims the benefit of priority based on Korean Patent Application No. 10-2024-0130235 filed September 25, 2024, and all contents disclosed in the document of said Korean patent application are incorporated as part of the present disclosure.

[0002] Throughout this disclosure, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this disclosure in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0003] The present disclosure relates to a radial seaweed (Pyropia yezoensis) having high temperature resistance and a food containing the same.

[0004] Gim belongs to the Kingdom Plantae, Division Rhodophyta, Class Rhodophyceae, Order Bangiales, and Family Bangiacea, with about 140 species known worldwide. Among these, the types of gim mainly cultivated in South Korea include Pyropia yezoensis, Pyropia dentata, and Pyropia seriata.

[0005] The optimal growth temperature for the radial laver, which accounts for 70-80% of farmed laver in Korea, is around 10℃, and if the water temperature rises to around 20℃, the growth of the thallus is inhibited.

[0006] Meanwhile, according to a research report by the National Institute of Fisheries Science (Report on the Impact of Climate Change on the Fisheries Sector and Research, 2023), the global average surface water temperature rose by 0.52°C (0.0094°C / yr) over the past 55 years (1968–2022), whereas the annual average surface water temperature in domestic waters rose by approximately 1.36°C (0.025°C / yr) during the same period. This indicates that the annual average surface water temperature rise in domestic coastal waters is about 2.5 times higher than the global average surface water temperature rise. Due to this continuous rise in domestic seawater temperatures, the annual production of laver is decreasing, and research on land-based laver cultivation to replace marine aquaculture is currently being actively conducted.

[0007] A new variety of *Ibbadi* (a type of sea snail) for use in marine aquaculture has been proposed in the past (Registered Patent 10-1983724). It is a new variety of *Ibbadi* with high temperature tolerance, characterized by a higher thallus growth rate at 25°C compared to the optimal growth temperature of existing *Ibbadi* at around 15°C, and is a different species from *Pteris vernalis*. *Ibbadi* is an early-maturing variety that grows during the early stages of marine aquaculture; however, unlike *Pteris vernalis*, it does not release monospores (neutral spores), so cultivation ends after 1 to 2 harvests, making continuous production impossible. Due to these characteristics, there are limitations to its use in indoor land-based aquaculture.

[0008] With active research underway on land-based seaweed cultivation utilizing artificial light sources that enables quality, production timing, and environmental control, there is a need to develop seaweed varieties suitable for land-based farming that can cope with rising seawater temperatures.

[0009] [Prior Art Literature]

[0010] [Patent Literature]

[0011] (Patent Document 01) Republic of Korea Registered Patent No. 10-1983724

[0012] One objective of the present disclosure is to provide a radial seaweed (Pyropia yezoensis) having high temperature resistance.

[0013] Another objective of the present disclosure is to provide a food product comprising the radial seaweed (Pyropia yezoensis) of the present disclosure.

[0014] The present disclosure is summarized as follows:

[0015] [Claim 1]

[0016] Pyropia yezoensis, having high temperature resistance and comprising the nucleotide sequence of SEQ ID NO. 18 or a nucleotide sequence having 80% or more homology thereto.

[0017] [Claim 2]

[0018] The radial seaweed of claim 1, comprising 18S rRNA comprising the nucleotide sequence of SEQ ID NO. 1 or a nucleotide sequence having 80% or more homology therewith.

[0019] [Claim 3]

[0020] Radial seaweed according to claim 1 or 2, comprising the nucleotide sequence of SEQ ID NO. 19 or a nucleotide sequence having 80% or more homology thereto.

[0021] [Claim 4]

[0022] Radial pattern seaweed according to any one of claims 1 to 3, wherein the high temperature is 12.5℃ to 30℃.

[0023] [Claim 5]

[0024] In any one of paragraphs 1 to 4, radial seaweed having the following seaweed thallus characteristics:

[0025] (a) Tilia shape: lanceolate

[0026] (b) Mature leaf thallus length: 30.3±3.9cm

[0027] (c) Mature leaf thallus width: 1.3±0.2cm

[0028] (d) Mature leaf thallus thickness: 22±1.9μm

[0029] (e) Monospore formation: present

[0030] (f) Serrations at the thallus margin: None

[0031] (g) Mature leaf thallus color: brownish-purple

[0032] (h) Reproductive type of the thallus: Hermaphroditic.

[0033] [Claim 6]

[0034] Pyropia yezoensis, which is obtained by treating Pyropia yezoensis with ethyl methanesulfonate in any one of claims 1 to 5.

[0035] [Claim 7]

[0036] A radial seaweed according to any one of claims 1 to 6, obtained from neutral spores released by treating the thallus of the radial seaweed with ethyl methanesulfonate.

[0037] [Claim 8]

[0038] Radial seaweed according to claim 6 or 7, wherein the concentration of the ethyl methanesulfonate is 0.1% (w / v) to 3% (w / v).

[0039] [Claim 9]

[0040] Radial-patterned seaweed according to any one of claims 1 to 8, wherein the expression of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), or both is increased.

[0041] [Claim 10]

[0042] Radial-patterned seaweed according to any one of claims 1 to 9, wherein the expression of one or more selected from the group consisting of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase), and RPL19 (ribosomal protein L19) is increased at high temperature.

[0043] [Claim 11]

[0044] Radial patterned seaweed according to any one of claims 1 to 10, wherein the high temperature is 12.5℃ to 30℃.

[0045] [Claim 12]

[0046] Pyropia yezoensis, which is deposited under accession number KCTC 15932BP in any one of paragraphs 1 through 11.

[0047] [Claim 13]

[0048] A food containing the radial laver (Pyropia yezoensis) of any one of paragraphs 1 to 12.

[0049] [Claim 14]

[0050] In paragraph 13, the above-mentioned Pyropia yezoensis is a food deposited under accession number KCTC 15932BP.

[0051]

[0052] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this disclosure may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this disclosure fall within the scope of this disclosure. Furthermore, the scope of this disclosure is not to be limited by the specific descriptions provided below. Additionally, a person skilled in the art can recognize or identify numerous equivalents to the specific aspects of this disclosure described herein using only ordinary experiments. Moreover, such equivalents are intended to be included in this disclosure.

[0053] Furthermore, numerous papers and patent documents are referenced and cited throughout this disclosure. The disclosures of the cited papers and patent documents are incorporated by reference into this disclosure in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0054]

[0055] One aspect of the present disclosure provides a radial seaweed (Pyropia yezoensis) having high temperature resistance.

[0056] The inventors have made diligent research efforts to develop a laver variety suitable for use in land-based aquaculture while being able to cope with rising seawater temperatures. As a result, it was found that the radial laver of the present disclosure, in which mutations were induced by treatment with ethyl methanesulfonate, has resistance to high temperatures and a high growth rate under land-based aquaculture conditions using artificial light sources.

[0057] In the present disclosure, 'high temperature tolerance' means that normal growth of *Pyropia yezoensis* continues even under high temperature conditions or high temperature shock conditions exceeding the temperature range (about 10°C to 15°C) at which optimal growth generally occurs, normal growth continues, the thallus maintains a normal shape, and / or growth is possible through generational advancement.

[0058] In one embodiment, the high temperature may be 12.5°C to 30°C, and more specifically, 12.5°C to 30°C, 12.5°C to 26°C, 12.5°C to 25°C, 12.5°C to 24°C, 12.5°C to 20°C, 12.5°C to 17.5°C, 12.5°C to 15°C, 14°C to 30°C, 14°C to 26°C, 14°C to 25°C, 14°C to 24°C, 14°C to 20°C, 14°C to 17.5°C, 14°C to 15°C, 15°C to 30°C, 15°C to 26°C, 15°C to 25°C, 15°C to 24°C, 15°C to 20°C, 15°C to 17.5°C, It may be 17.5°C to 30°C, 17.5°C to 26°C, 17.5°C to 25°C, 17.5°C to 24°C, 17.5°C to 20°C, 20°C to 30°C, 20°C to 26°C, 20°C to 25°C, 20°C to 24°C, 24°C to 30°C, or 24°C to 26°C, but is not limited thereto.

[0059] In one embodiment, the radial seaweed of the present disclosure (or the genome thereof) may comprise 18S rRNA comprising a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology with the nucleotide sequence of SEQ ID NO. 1.

[0060] In another embodiment, the radial seaweed of the present disclosure may essentially include in its genome a nucleotide sequence of SEQ ID NO. 1 or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity with the same.

[0061] In another embodiment, the radial seaweed of the present disclosure may include 18S rRNA that essentially comprises the nucleotide sequence of SEQ ID NO. 1 or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity with the same.

[0062] The inventors prepared 18S rRNA gene primers for the radial-patterned seaweed of the present disclosure, performed a polymerase chain reaction (PCR), and subsequently performed sequencing to confirm that the radial-patterned seaweed of the present disclosure belongs to the radial-patterned seaweed and that the radial-patterned seaweed of the present disclosure contains 18S rRNA containing the nucleotide sequence of SEQ ID NO. 1.

[0063]

[0064] In one embodiment, the radial seaweed of the present disclosure (or the genome thereof) may comprise the nucleotide sequence of SEQ ID NO. 18 or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity with the same.

[0065] In another embodiment, the radial seaweed of the present disclosure may essentially include in its genome the nucleotide sequence of SEQ ID NO. 18 or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity with the same.

[0066] The nucleotide sequence of SEQ ID NO. 18 is a partial cDNA sequence of the glutamate dehydrogenase gene identified in the radial-patterned seaweed of the present disclosure, and contains specific variations when compared to the wild species. Specifically, when compared with the glutamate dehydrogenase gene sequence (partial cDNA sequence) of the corresponding wild species, T (thymine) at positions 147 and 587 of SEQ ID NO. 18 is C (cytosine) in the wild species, and A (adenine) at position 633 of SEQ ID NO. 18 is G (guanine) in the wild species. The variation at position 633 of SEQ ID NO. 18 causes an amino acid substitution from Ala (alanine) to Thr (threonine).

[0067] The glutamate dehydrogenase (GDH) mentioned above is an enzyme involved in intracellular nitrogen metabolism and amino acid metabolism, and is an enzyme that catalyzes the reaction of interconverting glutamate between α-ketoglutarate and ammonia.

[0068] In one embodiment, the wild strain may refer to the parental strain before treatment with ethyl methanesulfonate, but is not limited thereto.

[0069] The nucleotide sequence of the partial cDNA of the wild-type glutamate dehydrogenase gene corresponding to the nucleotide sequence of SEQ ID NO. 18 above is shown as SEQ ID NO. 20.

[0070] In one embodiment, the radial seaweed containing the nucleotide sequence of SEQ ID NO. 18 of the present disclosure may have increased high temperature tolerance compared to the radial seaweed not containing the nucleotide sequence of SEQ ID NO. 18. The radial seaweed not containing the nucleotide sequence of SEQ ID NO. 18 may be wild-type radial seaweed or general radial seaweed, such as radial seaweed not treated with ethyl methanesulfonate, parental strain before treatment with ethyl methanesulfonate, or Cheongpung No. 1 radial seaweed, but is not limited thereto.

[0071] In one embodiment, the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 18 of the present disclosure may have increased high temperature resistance compared to the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 20.

[0072]

[0073] In one embodiment, the radial seaweed of the present disclosure (or the genome thereof) may comprise the nucleotide sequence of SEQ ID NO. 19 or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity with the same.

[0074] In another embodiment, the radial seaweed of the present disclosure may essentially include in its genome the nucleotide sequence of SEQ ID NO. 19 or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity with the same.

[0075] The nucleotide sequence of SEQ ID NO. 19 above is a partial cDNA sequence of the heat shock protein 70 gene identified in the radial seaweed of the present disclosure, and includes a specific variant when compared to the Pyropia yezoensis heat shock protein 70 (HSP70) mRNA sequence (NCBI Accession No. KF574043.1) registered in the NCBI database or the wild species.

[0076] Specifically, NCBI Accession No. When comparing the sequence of KF574043.1 with the corresponding portion of the nucleotide sequence of SEQ ID NO. 19, the nucleotide variants occurring in the nucleotide sequence of SEQ ID NO. 19 are as follows (indicated in the order of the corresponding nucleotide in NCBI Accession No. KF574043.1, the reference variant position in SEQ ID NO. 19, and the nucleotide identified in SEQ ID NO. 19): T266C, G285A, T291C, C380T, C400T, C474T, G480A, T492C, T540C, G549C, C552T, G555C, T615C, G639A, T642C, R (meaning A or G as an IUPAC base code)678A, S (meaning G or C as an IUPAC base code)735G, Y (meaning C or T as an IUPAC base code Meaning) 781C, C810T, C963T, C1008T, A1444G, C1701T, G1983A. Of the above 24 mutations, 21 mutations did not result in a change in the amino acid sequence. Meanwhile, C (cytosine), the nucleotide at the 266th position of the nucleotide sequence of SEQ ID NO. 19, is T (thymine) in the sequence of NCBI Accession No. KF574043.1; in the radial-patterned seaweed of the present disclosure, a T→C mutation occurred, resulting in an amino acid substitution from Thr (Threonine) to Ile (Isoleucine), and C (cytosine), the nucleotide at the 781st position, is the above NCBI Accession No. In the sequence of KF574043.1, Y (meaning C or T as an IUPAC base code) occurred, and in the radial-patterned seaweed of the present disclosure, a Y→C mutation occurred, resulting in an amino acid substitution from an unclear amino acid to Gln (glutamine), and G (guanine), the nucleotide at the 1,444th position, is the above NCBI Accession No. KF574043.In the sequence of 1, it was confirmed that A→G mutation occurred in the radial seaweed of the present disclosure, and as a result, an amino acid sequence change occurred from Lys (lysine)→Glu (glutamic acid).

[0077] When the nucleotide sequence of SEQ ID NO. 19 above is compared with the corresponding wild-species heat shock protein 70 gene sequence (partial cDNA sequence), there were variations at the following nucleotide positions (indicated in the order of the corresponding nucleotide in the wild-species sequence (SEQ ID NO. 21), the variation position of SEQ ID NO. 19, and the nucleotide identified in SEQ ID NO. 19): C474T, G480A, C810T, C963T, C1008T, A1444G, C1701T, G1983A. Although 7 of the above 8 mutations did not affect the change in amino acid sequence, it was confirmed that G (guanine), the nucleotide at the 1,444th position of the nucleotide sequence of SEQ ID NO. 19, was changed to A (adenine) in the wild species, and that an A→G mutation occurred in the radial seaweed of the present disclosure, resulting in a change in the amino acid sequence from Lys (lysine) to Glu (glutamic acid).

[0078] The heat shock protein 70 (HSP70) mentioned above is a molecule that plays an important role in the cellular stress response, and its expression increases when exposed to environmental stimuli such as high temperature, salt, or oxidative stress. HSP70 acts as a molecular chaperone to help the proper folding of denatured proteins, prevent abnormal protein aggregation, and promote the repair or degradation of damaged proteins.

[0079] The above wild strain may refer to the parental strain before treatment with ethyl methanesulfonate, but is not limited thereto.

[0080] The nucleotide sequence of the partial cDNA of the wild species heat shock protein 70 gene corresponding to the nucleotide sequence of SEQ ID NO. 19 above is shown as SEQ ID NO. 21.

[0081] In one embodiment, the radial seaweed containing the nucleotide sequence of SEQ ID NO. 19 of the present disclosure may have increased high temperature tolerance compared to the radial seaweed not containing the nucleotide sequence of SEQ ID NO. 19. The radial seaweed not containing the nucleotide sequence of SEQ ID NO. 19 may be wild-type radial seaweed or general radial seaweed, such as radial seaweed not treated with ethyl methanesulfonate, parental strain before treatment with ethyl methanesulfonate, or Cheongpung No. 1 radial seaweed, but is not limited thereto.

[0082] In one embodiment, the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 19 of the present disclosure may have increased high temperature resistance compared to the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 21.

[0083]

[0084] In the present disclosure, the phrase “a polynucleotide or polypeptide comprises a specific nucleotide sequence (nucleic acid sequence, base sequence) or amino acid sequence” may mean that the polynucleotide or polypeptide is composed of or essentially comprises the specific nucleotide sequence (nucleic acid sequence, base sequence) or amino acid sequence.

[0085] In this disclosure, the terms 'homology' or 'identity' refer to the degree of similarity between two given amino acid sequences or nucleotide sequences and may be expressed as a percentage. The terms homology and identity may often be used interchangeably.

[0086] Sequence homology or identity of conserved polynucleotides or polypeptides is determined by standard arrangement algorithms, and a default gap penalty established by the program used may be utilized. Practically, homologous or identical sequences can generally be hybridized with the entire sequence or a part thereof under moderate or high stringent conditions. It is evident that hybridization also includes hybridization with polynucleotides containing common codons or codons that account for codon degeneracy.

[0087] Whether any two polynucleotide or polypeptide sequences have homology or identity can be determined using a known computer algorithm, such as the “FASTA” program, using default parameters as in, for example, Pearson et al (1988) [Proc.Natl. Acad. Sci. USA 85]: 2444. Alternatively, it can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later) (GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Homology or identity can be determined, for example, using BLAST from the National Biotechnology Information Database Center or ClustalW.

[0088] The homology or identity of polynucleotides or polypeptides can be determined by comparing sequence information using a GAP computer program, such as that described in, for example, Smith and Waterman, Adv. Appl. Math (1981) 2:482, or Needleman et al. (1970), J Mol Biol. 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of the two sequences divided by the number of similarly arranged symbols (i.e., nucleotides or amino acids). The default parameters for a GAP program are (1) a binary comparison matrix (containing values ​​of 1 for identity and 0 for non-identity) and, as disclosed by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979), or Gribskov et al. (1986) Nucl. Acids Res. 14: A weighted comparison matrix of 6745 (or an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for terminal gaps.

[0089] In one example, a polynucleotide comprising a specific nucleotide sequence provided in the present disclosure may be interpreted to comprise a polynucleotide fragment comprising not only the specific nucleotide sequence or a substantially equivalent nucleotide sequence, but also a nucleotide sequence complementary to the specific nucleotide sequence. Specifically, a polynucleotide having such complementarity can be identified under the conditions described below: such conditions are specifically described in the known literature. For example, conditions in which genes with high complementarity of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more hybridize with each other, and genes with lower complementarity do not hybridize with each other, or the washing conditions of conventional Southern hybridization, which are 60°C, 1x SSC (saline-sodium citrate buffer), and 0.1% (w / v) SDS (Sodium Dodecyl Sulfate); Conditions for washing once, specifically two to three times, at a salt concentration and temperature equivalent to 60°C, 0.1x SSC, and 0.1% SDS; or 68°C, 0.1x SSC, and 0.1% SDS may be listed, but are not limited thereto. Hybridization requires that two nucleotides have complementary sequences, but some base mismatches may be permitted depending on the strictness of hybridization. The term "complementary" may be used to describe the relationship between nucleotide bases that can hybridize with each other. For example, in the case of DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine.The strictness of hybridization between polynucleotides depends on the length and degree of complementarity of the polynucleotides, which is well known in the relevant technical field (see Sambrook et al., supra, 9.50-9.51, 11.7-11.8).

[0090]

[0091] In one embodiment, the radial laver of the present disclosure may be the one deposited under accession number KCTC 15932BP. The inventors confirmed the high temperature tolerance and high growth rate of the radial laver of the present disclosure under land-based cultivation conditions, and deposited it with the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Biotechnology and Bioengineering on June 11, 2024, under accession number KCTC 15932BP.

[0092]

[0093] In one embodiment, the radial patterned laver of the present disclosure is characterized by having the following laver thallus characteristics:

[0094] (a) Tilia shape: lanceolate

[0095] (b) Mature leaf thallus length: 30.3±3.9cm

[0096] (c) Mature leaf thallus width: 1.3±0.2cm

[0097] (d) Mature leaf thallus thickness: 22±1.9μm

[0098] (e) Monospore formation: present

[0099] (f) Serrations at the thallus margin: None

[0100] (g) Mature leaf thallus color: brownish-purple

[0101] (h) Reproductive type of the thallus: Hermaphroditic.

[0102] In one embodiment, the results of comparing the traits of the radial seaweed of the present disclosure and a control variety (wild type) are as follows:

[0103]

[0104] In one embodiment, the radial seaweed of the present disclosure can be obtained by treating radial seaweed (Pyropia yezoensis) with ethyl methanesulfonate.

[0105] In one embodiment, the control strain (wild type) may refer to the parental strain before treatment with ethyl methanesulfonate, but is not limited thereto.

[0106] In a specific embodiment, the radial seaweed of the present disclosure can be obtained from neutral spores released by treating the thallus of radial seaweed with ethyl methanesulfonate.

[0107] In a specific embodiment, the concentration of the ethyl methanesulfonate is 0.1% (w / v) to 3% (w / v), more specifically 0.1% (w / v) to 3% (w / v), 0.1% (w / v) to 2% (w / v), 0.1% (w / v) to 1% (w / v), 0.1% (w / v) to 0.75% (w / v), 0.1% (w / v) to 0.5% (w / v), 0.2% (w / v) to 3% (w / v), 0.2% (w / v) to 2% (w / v), 0.2% (w / v) to 1% (w / v), 0.2% (w / v) to 0.75% (w / v), and 0.2% (w / v) to 0.5% (w / v). It may be 0.3%(w / v) to 3%(w / v), 0.3%(w / v) to 2%(w / v), 0.3%(w / v) to 1%(w / v), 0.3%(w / v) to 0.75%(w / v), 0.3%(w / v) to 0.5%(w / v), 0.4%(w / v) to 3%(w / v), 0.4%(w / v) to 2%(w / v), 0.4%(w / v) to 1%(w / v), 0.4%(w / v) to 0.75%(w / v), or 0.4%(w / v) to 0.5%(w / v), more specifically 0.5%(w / v), but is not limited thereto.

[0108] In a specific embodiment, the ethyl methanesulfonate may be treated for 30 minutes to 6 hours, and more specifically, for 30 minutes to 6 hours, 30 minutes to 4 hours, 30 minutes to 3 hours, 30 minutes to 2 hours 30 minutes, 1 hour to 6 hours, 1 hour to 4 hours, 1 hour to 3 hours, 1 hour to 2 hours 30 minutes, 1 hour 30 minutes to 6 hours, 1 hour 30 minutes to 4 hours, 1 hour 30 minutes to 3 hours, 1 hour 30 minutes to 2 hours 30 minutes, 2 hours to 6 hours, 2 hours to 4 hours, 2 hours to 3 hours, or 2 hours to 2 hours 30 minutes, but is not limited thereto.

[0109] In one embodiment, the high temperature may be 12.5°C to 30°C, and more specifically, 12.5°C to 30°C, 12.5°C to 26°C, 12.5°C to 25°C, 12.5°C to 24°C, 12.5°C to 20°C, 12.5°C to 17.5°C, 12.5°C to 15°C, 14°C to 30°C, 14°C to 26°C, 14°C to 25°C, 14°C to 24°C, 14°C to 20°C, 14°C to 17.5°C, 14°C to 15°C, 15°C to 30°C, 15°C to 26°C, 15°C to 25°C, 15°C to 24°C, 15°C to 20°C, 15°C to 17.5°C, It may be 17.5°C to 30°C, 17.5°C to 26°C, 17.5°C to 25°C, 17.5°C to 24°C, 17.5°C to 20°C, 20°C to 30°C, 20°C to 26°C, 20°C to 25°C, 20°C to 24°C, 24°C to 30°C, or 24°C to 26°C, but is not limited thereto.

[0110] As demonstrated in the examples to be described below, the radial seaweed of the present disclosure exhibits a high growth rate even at temperatures higher than the optimal growth temperature of 10°C, ranging from 12.5°C to 30°C, and grows normally even after receiving a high-temperature shock, thus having resistance to high temperatures.

[0111] In a specific embodiment, the radial seaweed of the present disclosure may have high temperature resistance compared to radial seaweed that has not been induced with mutations using ethane methylsulfonate.

[0112] In a specific embodiment, the radial seaweed that has not induced mutation with the ethanemethylsulfonate may be the parental strain before treatment with ethyl methanesulfonate or Cheongpung No. 1 radial seaweed, but is not limited thereto.

[0113] In one embodiment, the radial seaweed of the present disclosure may have increased expression of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), or both.

[0114] In a specific embodiment, the radial patterned seaweed of the present disclosure may have increased expression of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), or both, compared to radial patterned seaweed that has not been mutated with ethane methylsulfonate. The radial patterned seaweed that has not been mutated with ethane methylsulfonate may be a parental strain or Cheongpung No. 1 radial patterned seaweed prior to treatment with ethyl methanesulfonate, but is not limited thereto.

[0115] In a specific embodiment, the radial seaweed containing the nucleotide sequence of SEQ ID NO. 18 of the present disclosure may have increased expression of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), or both compared to the radial seaweed not containing the nucleotide sequence of SEQ ID NO. 18. The radial seaweed not containing the nucleotide sequence of SEQ ID NO. 18 may be wild-type radial seaweed or general radial seaweed, such as radial seaweed not treated with ethyl methanesulfonate, parental strain before treatment with ethyl methanesulfonate, or Cheongpung No. 1 radial seaweed, but is not limited thereto.

[0116] In a specific embodiment, the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 18 of the present disclosure may have increased expression of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), or both, compared to the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 20.

[0117] In a specific embodiment, the radial seaweed containing the nucleotide sequence of SEQ ID NO. 19 of the present disclosure may have increased expression of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), or both compared to the radial seaweed not containing the nucleotide sequence of SEQ ID NO. 19. The radial seaweed not containing the nucleotide sequence of SEQ ID NO. 19 may be wild-type radial seaweed or general radial seaweed, such as radial seaweed not treated with ethyl methanesulfonate, parental strain before treatment with ethyl methanesulfonate, or Cheongpung No. 1 radial seaweed, but is not limited thereto.

[0118] In a specific embodiment, the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 19 of the present disclosure may have increased expression of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), or both, compared to the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 21.

[0119] HSP 70 (heat shock Protein 70) is a heat shock protein produced when cells are under stress, and it plays an important role in maintaining the proper folding and stability of proteins within the cell.

[0120] D6D (delta-6-desaturase) is an enzyme that plays an important role in fatty acid metabolism and is also known as FADS2 (fatty acid desaturase 2). D6D is particularly involved in the biosynthesis of polyunsaturated fatty acids (PUFA) and primarily catalyzes the conversion of linoleic acid (omega-6 fatty acid) into gamma-linolenic acid (GLA).

[0121] As demonstrated in the examples to be described below, the radial seaweed of the present disclosure has increased gene expression of HSP 70 (heat shock Protein 70) and D6D (delta-6-desaturase) compared to radial seaweed (wild type) that was not induced with ethane methylsulfonate.

[0122]

[0123] In one embodiment, the radial seaweed may have increased expression of one or more selected from the group consisting of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase) and RPL19 (ribosomal protein L19) at high temperature.

[0124] In a specific embodiment, the radial laver of the present disclosure may have increased expression of one or more selected from the group consisting of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase), and RPL19 (ribosomal protein L19) compared to radial laver that has not been mutated with ethane methylsulfonate. The radial laver that has not been mutated with ethane methylsulfonate may be a parental strain or Cheongpung No. 1 radial laver before treatment with ethyl methanesulfonate, but is not limited thereto.

[0125] In a specific embodiment, the radial seaweed containing the nucleotide sequence of SEQ ID NO. 18 of the present disclosure may have increased expression of one or more selected from the group consisting of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase), and RPL19 (ribosomal protein L19) compared to the radial seaweed not containing the nucleotide sequence of SEQ ID NO. 18. The radial seaweed not containing the nucleotide sequence of SEQ ID NO. 18 may be wild-type radial seaweed or general radial seaweed, such as radial seaweed not treated with ethyl methanesulfonate, parental strain before treatment with ethyl methanesulfonate, or Cheongpung No. 1 radial seaweed, but is not limited thereto.

[0126] In a specific embodiment, the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 18 of the present disclosure may have increased expression of one or more selected from the group consisting of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase), and RPL19 (ribosomal protein L19) compared to the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 20.

[0127] In a specific embodiment, the radial seaweed containing the nucleotide sequence of SEQ ID NO. 19 of the present disclosure may have increased expression of one or more selected from the group consisting of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase), and RPL19 (ribosomal protein L19) compared to the radial seaweed not containing the nucleotide sequence of SEQ ID NO. 19. The radial seaweed not containing the nucleotide sequence of SEQ ID NO. 19 may be wild-type radial seaweed or general radial seaweed, such as radial seaweed not treated with ethyl methanesulfonate, parental strain before treatment with ethyl methanesulfonate, or Cheongpung No. 1 radial seaweed, but is not limited thereto.

[0128] In a specific embodiment, the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 19 of the present disclosure may have increased expression of one or more selected from the group consisting of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase), and RPL19 (ribosomal protein L19) compared to the radial seaweed comprising the nucleotide sequence of SEQ ID NO. 21.

[0129] D5D (putative delta-5 desaturase) is an enzyme that plays an important role in fatty acid metabolism and is also known as FADS1 (fatty acid desaturase 1). D5D is particularly involved in the biosynthesis of polyunsaturated fatty acids and catalyzes the conversion of eicosatrienoic acid (ETA) to eicosapentaenoic acid (EPA) in the omega-3 fatty acid pathway and the conversion of dihomo-gamma-linolenic acid (DGLA) to arachidonic acid in the omega-6 fatty acid pathway.

[0130] RPL19 (ribosomal protein L19) is one of the components of the 60S ribosomal subunit and plays a role in increasing protein synthesis and ribosomal stability.

[0131] In a specific embodiment, the high temperature may be 12.5°C to 30°C, and more specifically, 12.5°C to 30°C, 12.5°C to 26°C, 12.5°C to 25°C, 12.5°C to 24°C, 12.5°C to 20°C, 12.5°C to 17.5°C, 12.5°C to 15°C, 14°C to 30°C, 14°C to 26°C, 14°C to 25°C, 14°C to 24°C, 14°C to 20°C, 14°C to 17.5°C, 14°C to 15°C, 15°C to 30°C, 15°C to 26°C, 15°C to 25°C, 15°C to 24°C, 15°C to 20°C, 15°C to 17.5°C, It may be 17.5°C to 30°C, 17.5°C to 26°C, 17.5°C to 25°C, 17.5°C to 24°C, 17.5°C to 20°C, 20°C to 30°C, 20°C to 26°C, 20°C to 25°C, 20°C to 24°C, 24°C to 30°C, or 24°C to 26°C, but is not limited thereto.

[0132] As demonstrated in the examples to be described below, the radial seaweed of the present disclosure shows increased gene expression of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase), and RPL19 (ribosomal protein L19) at high temperatures. In particular, unlike the case of radial seaweed (wild type) that was not induced with ethane methylsulfonate, where the expression of D5D (putative delta-5 desaturase) and RPL19 (ribosomal protein L19) decreases at high temperatures, the radial seaweed of the present disclosure shows a tendency for increased expression of D5D (putative delta-5 desaturase) and RPL19 (ribosomal protein L19) at high temperatures.

[0133] More specifically, the radial-patterned seaweed of the present disclosure, when cultured at high temperature for 24 hours, shows an increase in the expression of HSP 70 (heat shock Protein 70) of about 25% or more, about 27% or more, about 29% or more, or about 29.5% or more (the upper limit is not particularly limited and may be, for example, about 60% or less, about 50% or less, about 40% or less, about 35% or less, or about 30% or less), an increase in the expression of D6D (delta-6-desaturase) of about 40% or more, about 45% or more, or about 50% or more (the upper limit is not particularly limited and may be, for example, about 70% or less, about 60% or less, about 55% or less, or about 52% or less), and an increase in the expression of D5D (putative delta-5 desaturase) of about 35% or more, about 40% or more, or about 42% or more (the upper limit is not particularly limited and may be, for example, about (It may be 60% or less, about 50% or less, or about 45% or less) and / or the expression of RPL19 (ribosomal protein L19) may increase by about 20% or more, about 25% or more, about 30% or more, or about 34% or more (the upper limit is not specifically limited, e.g., about 50% or less, about 40% or less, or about 35% or less).

[0134] In this specification, "about" is a range including all of ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all numerical values ​​in a range equivalent to or similar to the numerical value following the term "about," but is not limited thereto.

[0135] In one embodiment, the expression of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase) and RPL19 (ribosomal protein L19) may be measured at the mRNA level or the protein level, but is not limited thereto. When the expression of the above HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase) and RPL19 (ribosomal protein L19) is measured at the mRNA level, the measurement may be performed by conventional nucleic acid analysis methods, such as reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), real-time quantitative polymerase chain reaction (real-time qPCR), Northern blotting, RNA sequencing (RNA-seq), RNA microarray, digital droplet PCR (ddPCR), in situ hybridization (ISH), fluorescent in situ hybridization (FISH), but is not limited thereto.When the expression of the above HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase), and RPL19 (ribosomal protein L19) is measured at the protein level, the measurement may be performed by conventional protein measurement methods, such as immunochromatography, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting, protein microarray, and flow cytometry, but is not limited thereto.

[0136]

[0137] Another aspect of the present disclosure provides a method for producing Pyropia yezoensis having high temperature resistance, comprising the step of treating Pyropia yezoensis with ethyl methanesulfonate.

[0138] In one embodiment, the step of treating the radial seaweed with ethyl methanesulfonate may be a step of treating the thallus of the radial seaweed with ethyl methanesulfonate.

[0139] In a specific embodiment, the concentration of the ethyl methanesulfonate is 0.1% to 3%, more specifically 0.1% (w / v) to 3% (w / v), 0.1% (w / v) to 2% (w / v), 0.1% (w / v) to 1% (w / v), 0.1% (w / v) to 0.75% (w / v), 0.1% (w / v) to 0.5% (w / v), 0.2% (w / v) to 3% (w / v), 0.2% (w / v) to 2% (w / v), 0.2% (w / v) to 1% (w / v), 0.2% (w / v) to 0.75% (w / v), 0.2% (w / v) to 0.5% (w / v), 0.3% (w / v) to It may be 3%(w / v), 0.3%(w / v) to 2%(w / v), 0.3%(w / v) to 1%(w / v), 0.3%(w / v) to 0.75%(w / v), 0.3%(w / v) to 0.5%(w / v), 0.4%(w / v) to 3%(w / v), 0.4%(w / v) to 2%(w / v), 0.4%(w / v) to 1%(w / v), 0.4%(w / v) to 0.75%(w / v), or 0.4%(w / v) to 0.5%(w / v), more specifically 0.5%(w / v), but is not limited thereto.

[0140] In a specific embodiment, the step of treating the ethyl methanesulfonate may be performed for 30 minutes to 6 hours, and more specifically, for 30 minutes to 6 hours, 30 minutes to 4 hours, 30 minutes to 3 hours, 30 minutes to 2 hours 30 minutes, 1 hour to 6 hours, 1 hour to 4 hours, 1 hour to 3 hours, 1 hour to 2 hours 30 minutes, 1 hour 30 minutes to 6 hours, 1 hour 30 minutes to 4 hours, 1 hour 30 minutes to 3 hours, 1 hour 30 minutes to 2 hours 30 minutes, 2 hours to 6 hours, 2 hours to 4 hours, 2 hours to 3 hours, or 2 hours to 2 hours 30 minutes, but is not limited thereto.

[0141] In one embodiment, the manufacturing method may additionally include, after the step of treating the thallus of the radial seaweed with ethyl methanesulfonate, a step of inducing the release of neutral spores of the radial seaweed treated with ethyl methanesulfonate at a high temperature.

[0142] In one embodiment, the manufacturing method may additionally include a step of culturing the released neutral spores to grow into thallus after the step of inducing the release of neutral spores of the radial seaweed treated with ethyl methanesulfonate at high temperature.

[0143] In a specific embodiment, the high temperature may be 12.5°C to 30°C, and more specifically, 12.5°C to 30°C, 12.5°C to 26°C, 12.5°C to 25°C, 12.5°C to 24°C, 12.5°C to 20°C, 12.5°C to 17.5°C, 12.5°C to 15°C, 14°C to 30°C, 14°C to 26°C, 14°C to 25°C, 14°C to 24°C, 14°C to 20°C, 14°C to 17.5°C, 14°C to 15°C, 15°C to 30°C, 15°C to 26°C, 15°C to 25°C, 15°C to 24°C, 15°C to 20°C, 15°C to 17.5°C, It may be 17.5°C to 30°C, 17.5°C to 26°C, 17.5°C to 25°C, 17.5°C to 24°C, 17.5°C to 20°C, 20°C to 30°C, 20°C to 26°C, 20°C to 25°C, 20°C to 24°C, 24°C to 30°C, or 24°C to 26°C, but is not limited thereto.

[0144] In one embodiment, the manufacturing method may additionally include a step of selecting a thallus with a high growth rate after the step of culturing the released neutral spores to grow into a thallus.

[0145] In a specific embodiment, the thallus with a high growth rate may be a thallus that has grown to 5 cm or more, 10 cm or more, 15 cm or more, 17.5 cm or more, 20 cm or more, or 25 cm or more (the upper limit is not specifically limited and may be, for example, 50 cm or less, 40 cm or less, or 30 cm or less), but is not limited thereto.

[0146] In one embodiment, the manufacturing method may additionally include a step of inducing neutral spores after the step of selecting a thallus with a high growth rate.

[0147] After inducing the above neutral spores, the second generation can be obtained to verify high temperature resistance.

[0148] In one embodiment of the present aspect, the radial pattern seaweed having high temperature resistance may include the nucleotide sequence of SEQ ID NO. 18.

[0149] In one embodiment of the present aspect, the radial pattern seaweed having high temperature resistance may include 18S rRNA containing the nucleotide sequence of SEQ ID NO. 1.

[0150] In one embodiment of the present aspect, the radial pattern seaweed having high temperature resistance may include the nucleotide sequence of SEQ ID NO. 19.

[0151] In one embodiment of the present aspect, the radial pattern seaweed having high temperature resistance may be the one deposited under accession number KCTC 15932BP.

[0152] In this embodiment, the radial pattern seaweed having high temperature resistance is as described in the previous embodiment.

[0153]

[0154] Another aspect of the present disclosure provides a method for increasing the high temperature resistance of Pyropia yezoensis, comprising the step of treating Pyropia yezoensis with ethyl methanesulfonate.

[0155] The step of treating the ethyl methanesulfonate and the radial seaweed are as described in other embodiments.

[0156]

[0157] Another aspect of the present disclosure provides a food containing the radial seaweed (Pyropia yezoensis) having high temperature resistance according to the above-described present disclosure. In one embodiment, the food may be a processed seaweed food. More specifically, the processed seaweed food may include, but is not limited to, dried seaweed, seasoned seaweed, roasted seaweed, seaweed chips, seasoned seaweed flakes, and seaweed powder.

[0158]

[0159] Another aspect of the present disclosure provides a novel polynucleotide comprising the nucleotide sequence of SEQ ID NO. 18 described above or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity therewith.

[0160] Another aspect of the present disclosure provides a novel polypeptide comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 18 described above or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity therewith. The polypeptide may function as a part of glutamate dehydrogenase.

[0161] Another aspect of the present disclosure provides a novel polynucleotide comprising the nucleotide sequence of SEQ ID NO. 19 or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity therewith.

[0162] Another aspect of the present disclosure provides a novel polypeptide comprising an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO. 19 described above or a nucleotide sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 98.75% or more, 99% or more, 99.2% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more homology or identity therewith. The polypeptide may function as a part of heat shock protein 70.

[0163] Another aspect of the present disclosure provides an expression vector comprising the nucleotide sequence of SEQ ID NO. 18 or SEQ ID NO. 19, and / or a host cell comprising the same.

[0164] A polynucleotide comprising the nucleotide sequence of SEQ ID NO. 18 or SEQ ID NO. 19 or a polypeptide encoded by the same may be used for increasing the high temperature resistance of radial seaweed (composition for increasing high temperature resistance and / or method for increasing high temperature resistance).

[0165] A polynucleotide comprising the nucleotide sequence of SEQ ID NO. 18 or SEQ ID NO. 19 or a polypeptide encoded by the same may be used for one or more expression-increasing uses selected from the group consisting of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase) and RPL19 (ribosomal protein L19) in radial seaweed (composition and / or method for expression-increasing uses selected from the group consisting of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase) and RPL19 (ribosomal protein L19).

[0166] A polynucleotide comprising the nucleotide sequence of SEQ ID NO. 18 or SEQ ID NO. 19 or a polypeptide encoded by the same may be used for the purpose of selecting and / or detecting seaweed having high temperature tolerance and / or increased high temperature tolerance (a composition for selecting and / or detecting seaweed having high temperature tolerance and / or increased high temperature tolerance, and / or a method for selecting and / or detecting seaweed having high temperature tolerance and / or increased high temperature tolerance). The seaweed may be *P. tenera*, *P. yezoensis*, *P. suborbiculata*, *P. pseudolinearis*, *P. dentata*, or *P. seriata*, and more specifically may be *P. yezoensis*, but is not limited thereto.

[0167] The nucleotide sequence of SEQ ID NO. 18, the nucleotide sequence of SEQ ID NO. 19, glutamate dehydrogenase, heat shock protein 70, high temperature resistance, HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase), and RPL19 (ribosomal protein L19), etc., are as described in other embodiments above.

[0168] The present disclosure provides a radial seaweed (Pyropia yezoensis) having high temperature tolerance. The radial seaweed (Pyropia yezoensis) of the present disclosure has high temperature tolerance and shows a high growth rate under land-based aquaculture conditions using artificial light sources, making it very useful for land-based aquaculture while responding to rising seawater temperatures.

[0169] Figure 1 is a figure showing the thallus length measured during the process of selecting high-temperature resistant candidates.

[0170] Figures 2a and 2b show photographs of thalluses having selected high-temperature tolerance traits. Figure 2a shows the results of culture at 20°C for 4 weeks, and Figure 2b shows the results of culture after 2 generations. In Figures 2a and 2b, 1 represents the wild species, and 2 represents the thalluses of the candidate group having selected high-temperature tolerance traits.

[0171] Figure 3 shows the results of gene expression pattern analysis through RT-PCR.

[0172] Figure 4 shows the results of comparing the increase in average leaf length (length) of the new high-temperature tolerant variety of radial-patterned seaweed, wild species, and ordinary radial-patterned seaweed according to temperature.

[0173] Figure 5 shows images of the thallus of a new high-temperature resistant variety of radial-patterned seaweed, a wild variety, and a regular radial-patterned seaweed at different temperatures.

[0174] Figure 6 shows images of the thallus of a new high-temperature resistant variety of radial patterned seaweed and a general radial patterned seaweed at different temperatures.

[0175] Figure 7 shows the results of comparing the increase in average leaf length (length) of the new high-temperature resistant variety of radial-patterned seaweed and the general radial-patterned seaweed according to temperature.

[0176]

[0177] The present application will be described in more detail below through examples. These examples are intended solely to illustrate the present application more specifically, and it will be obvious to those skilled in the art that the scope of the present application is not limited by these examples according to the gist of the application.

[0178]

[0179] Examples

[0180]

[0181] (Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.)

[0182]

[0183] Example 1: Selection of a new high-temperature tolerant variety of *Pyropia yezoensis*

[0184] We obtained wild-collected thallus of *Pyropia yezoensis* from the National Institute of Fisheries Science and subsequently mass-cultured them (Effect of Shell-type, Light and Temperature on the Shell Infiltration of Free-living Conchocelis of Three Pyropia Species, Korean J Fish Aquat Sci 54(1),23-30,202) to obtain thallus through sexual and asexual reproduction.

[0185] High-temperature resistant varieties were induced by treating the obtained thallus with ethyl methanesulfonate (EMS) (sigma).

[0186] Specifically, wild laver thallus measuring 1 cm or more was treated with seawater in which EMS was dissolved at 0.5% (w / v) for 2–3 hours. The thallus was washed with seawater in which sodium thiosulfate was dissolved and recovered for 8–24 hours under room temperature and dark conditions. Subsequently, neutral spore release was induced in each thallus in a 2L flask containing fresh seawater at a high temperature of 20°C. The released neutral spores were then grown into thallus at 20°C. The thallus of most groups dissociated and released; however, only those thallus that grew normally to over 20 cm without releasing were selected for isolation culture (Fig. 1). After culturing at 20°C for 4 weeks and measuring growth, it was found that the thallus grew to a maximum length of 30 cm.

[0187] To determine whether high-temperature tolerance traits are maintained through generational progression, the release of neutral spores, a form of asexual reproduction, was induced in the isolated candidate group to obtain a second generation, and the obtained neutral spores were cultured into mature leaves. As a result, it was confirmed that the second generation was also capable of growing to approximately 25 cm at 20°C (Figs. 2a and 2b).

[0188]

[0189] Example 2: Identification of the new high-temperature tolerant variety *Porphyra radiata* and comparison of traits with the wild species

[0190] Example 2-1: Identification of the new high-temperature tolerant variety of *Porphyra radiata*

[0191] It was verified whether the candidate group selected through DNA analysis matched *Pyropia (Porphyra) yezoensis*. After extracting DNA from the final high-temperature tolerant candidate group (high-temperature tolerant new variety *Pyropia yezoensis*) of Example 1 described above, 18S rRNA gene primers capable of identifying the species of *Pyropia* were designed, and polymerase chain reaction (PCR) was performed, followed by sequencing. The sequences of the primers used are shown in Table 1 below, and the 18S rRNA gene sequences are shown in Table 2 below.

[0192] Primer Sequence SEQ ID NO:Py-SSU-KF2 forward primerTAAGCCATGCATGTCTAAG2Py-SSU-KR2 reverse primerACGACTTCTCCTTCCTCTAAATG3Py-SSU-KF3 forward primerACCATGGTGTCGACGGGTGAC4Py-SSU-KR3 reverse primerCCTTCCGACCCAGGACKATC5G01 forward primerCACCTGGTTGATCCTGCCAG6G14 reverse primerCCTTGGCAGACGGCTTTCGCAG7G04 forward primerCAGAGGTGAAATTCTTGGAT8G07 reverse primerGCTTGATCCTTCTGCAGGTTCACCTAC9

[0193]

[0194]

[0195] As a result, the 18S rRNA sequence (Sequence No. 1) of the selected high-temperature tolerant new variety of *Porphyra yezoensis* was 99.96% identical to the 18S rRNA sequence of *Porphyra yezoensis* KPH (Genetic polymorphism within Porphyra yezoensis (Bangiales,Rhodophyta) and related species from Japan and Korea detected by cleaved amplified polymorphic sequence analysis, Eur. J. Phycol. 42, 29-40 (2007)), confirming that it was *Porphyra yezoensis*. Accordingly, the acquired high-temperature tolerant new variety of *Porphyra yezoensis* was deposited with the National Center for Biological Resources at the Korea Research Institute of Biotechnology and Bioengineering on June 11, 2024, and was assigned accession number KCTC 15932BP.

[0196]

[0197] Example 2-2: Comparison of Traits Between High-Temperature Tolerant Novel Radiata-Patterned Porphyra and Wild Species

[0198] The traits of the new high-temperature tolerant variety of Porphyra radiata and the wild species were compared and are shown in Table 3 below. The above traits were investigated according to the characteristics investigation guidelines for Porphyra spp. (Laver) in the characteristics investigation guidelines for aquatic plants for new variety evaluation by the National Institute of Fisheries Science of the Ministry of Oceans and Fisheries (https: / www.nifs.go.kr / apvc / 06_manage / 02_04.ap). As shown in Table 3, the new variety of Porphyra radiata of the present invention has a thallus length approximately 2.5 times longer than the control variety (Porphyra radiata collected from the wild in Example 1 above), indicating vigorous growth, and it was confirmed that the thallus shape is formed in a lanceolate form, distinguishing it from the linear thallus of the control variety.

[0199] Characteristics Invented New Variety: Radial Pattern Kim Dae-jo (Wild type) Thalliform Shape: Lanceolate / Linear Mature Leaf: Tallus Length 30.3±3.9cm 12.1±2.7cm Mature Leaf: Tallus Width 1.3±0.2cm 1.5±0.1cm Mature Leaf: Tallus Thickness 22±1.9μm 24±1.7μm Single Spore Formation: Present / Present Toothed Margins: Absent / Absent Mature Leaf: Tallus Color: Purple / Purple Reproductive Type: Hermaphroditic

[0200]

[0201] Example 3: Obtaining a novel partial cDNA sequence

[0202] RNA sequencing was performed to confirm genetic differences between the wild type (wild type, the wild-collected *Pterocarya radiata* from the National Institute of Fisheries Science in Example 1 above) and the new high-temperature tolerant *Pterocarya radiata* variety. To this end, total RNA was extracted from 100 mg of each conchocelis using the RNeasy Plant Mini Kit (Qiagen) according to the protocol provided in the kit. The quality of the extracted RNA was verified using a 2200 TapeStation (Agilent Technology), and sequencing was performed using Illumina NovaSeq X with 1 µg of RNA from each sample. The differences between the wild type and the new variety were confirmed by analyzing the obtained sequence variants.

[0203] As a result, a partial cDNA sequence of the glutamate dehydrogenase gene was obtained from a new high-temperature tolerant variety of radial-patterned laver. The sequence is shown in SEQ ID NO. 18 below.

[0204]

[0205] When the above sequence was compared with the partial cDNA sequence of the glutamate dehydrogenase gene of the wild species of Example 1 (Sequence No. 20), it was confirmed that the nucleotide T at the 147th position of the nucleotide sequence of Sequence No. 18 was C in the wild species, and the nucleotide T at the 587th position was C in the wild species, indicating that a C→T mutation occurred in each case. In addition, it was confirmed that the nucleotide A at the 633rd position was G in the wild species, indicating that a G→A mutation occurred. (The mutation locations are indicated by underlining and bolding in Table 4.) It was confirmed that the mutations at the 147th position T and the 587th position did not affect the amino acid sequence change (maintaining Leu and Ile, respectively), while the mutation at the 633rd position caused an amino acid sequence change from Ala to Thr.

[0206] In addition, a partial cDNA sequence of the heat shock protein 70 gene was obtained from a new high-temperature tolerant variety of radial-patterned laver. The sequence is shown in SEQ ID NO. 19 below.

[0207]

[0208]

[0209] As a result of comparing the above sequence with the Pyropia yezoensis heat shock protein 70 (HSP70) mRNA sequence registered in the NCBI database (NCBI Accession No. KF574043.1), approximately 99% of the corresponding portions showed identity (1,983 bp match out of 2,007 bp), and the above NCBI Accession No. When compared to the sequence KF574043.1, the nucleotide mutations occurring in the high-temperature tolerant new variety of *Porphyra radiata* disclosed in this disclosure are as follows (indicated in the order of the corresponding nucleotide of NCBI Accession No. KF574043.1, the reference mutation position of SEQ ID NO. 19, and the nucleotide identified in SEQ ID NO. 19): T266C, G285A, T291C, C380T, C400T, C474T, G480A, T492C, T540C, G549C, C552T, G555C, T615C, G639A, T642C, R (meaning A or G as an IUPAC base code) 678A, S (meaning G or C as an IUPAC base code) 735G, Y (meaning C or T as an IUPAC base code) 781C, C810T, C963T, C1008T, A1444G, C1701T, G1983A. Of these, 21 mutations did not affect the amino acid sequence change. Meanwhile, it was confirmed that the above T266C mutation caused a change in the amino acid sequence from Thr to Ile, the above Y781C mutation caused a change in the amino acid sequence from an unclear amino acid to Gln, and the above A1,444G mutation caused a change in the amino acid sequence from Lys to Glu.

[0210] In addition, when the nucleotide sequence of SEQ ID NO. 19 was compared with the partial cDNA sequence of the wild species of Example 1 (SEQ ID NO. 21), the following nucleotide position variations were found (indicated in the order of the corresponding nucleotide in the wild species sequence (SEQ ID NO. 21), the variation position relative to SEQ ID NO. 19, and the nucleotide identified in SEQ ID NO. 19): C474T, G480A, C810T, C963T, C1008T, A1444G, C1701T, G1983A. Among the above eight variations, it was confirmed that the only change in amino acid sequence was the amino acid substitution from Lys to Glu caused by G at the 1,444th position (A→G variation relative to the wild species).

[0211]

[0212] Example 4: Analysis of gene expression via RT-PCR

[0213] To compare the gene expression of the wild species (the wild-collected sea bream from the National Institute of Fisheries Science in Example 1 above) and the new high-temperature tolerant variety of sea bream at high temperatures and to find candidate group-specific genes, gene expression was analyzed via RT-PCR. Tearpieces of 3 cm to 5 cm were used, and gene expression was confirmed after 0, 1 h, 4 h, 6 h, 24 h, and 48 h under conditions of 20℃.

[0214] Primers were designed for the four obtained genes (Table 6), and differences in gene expression between the wild species and the new high-temperature tolerant variety *Porphyra radiata* were verified via reverse transcription polymerase chain reaction (RT-PCR). PCR was performed using the primers designed for each gene, and the resulting products were loaded onto the same gel for electrophoresis. The gel images obtained after electrophoresis were analyzed to measure the band intensity of each band; the measured values ​​were normalized by the intensity of the actin band, which was used as an internal control, to calculate the final gene expression values. The primers used are shown in Table 6 below.

[0215] As a result, the gene expression of HSP70 (heat shock protein 70), D5D (putative delta-5 fatty acid desaturase), D6D (delta-6 fatty acid desaturase), and RPL19 (ribosomal protein L19) in the new heat-tolerant variety of radial-patterned seaweed showed a significant difference from that of the wild variety (WT) (Figure 3 and Table 7). Specifically, when the new heat-tolerant variety of radial-patterned seaweed was cultured at high temperature for 24 hours, the expression of the HSP70 gene and the D6D gene increased by approximately 29.6% and approximately 50.6%, respectively, showing a greater increase in expression at high temperatures compared to the wild variety (WT). In contrast to the decrease in expression in the wild variety (WT) at high temperatures, the expression of the D5D gene and the RPL19 gene increased at high temperatures (approximately 43.0% and approximately 34.7%, respectively).

[0216] Primer used in RT-PCRPrimerPrimer sequenceSEQ ID NO:D5D (putative delta-5 desaturase) forward primerTCCACGGCAACGTCTATGAC10D5D (putative delta-5 desaturase) reverse primerCTTGCTCGTGAACGGATGGTA11D6D (delta-6-desaturase) forward primerTTAAACAAGCTCGGCGTCAC12D6D (delta-6-desaturase) reverse primerGCGTGGTCTTTACCGTAGGC13HSP70 (heat shock protein 70) forward primerCTGGAGGGGCTATCTGTACAACG14HSP70 (heat shock protein 70) reverse primerGTTGACCACCTCCTGTACCTCA15RPL19 (ribosomal protein L19) forward primerCGGGAACAATTATGTGAAGGA16RPL19 (ribosomal protein L19) reverse primerTGTTTTAATCGTCCGGCTTT17Actin forward primerCAAGCAGAAGGGCATCAT22Actin reverse primerCCGAGTAGAAAGCGTGGT23

[0217] -Putative delta-5 fatty acid desaturase Ribosomal protein L19HSP70Delta-6 fatty acid desaturase 0h24h0h24h0h24h0h24hWT1.210.761.040.710.550.680.610.61High temperature tolerant variety 1.001.430.981.320.710.920.791.19

[0218]

[0219] Example 5: Land-based cultivation test of a new high-temperature tolerant variety of radial-patterned laver

[0220] A land-based culture test (Algae 2023, 38(2): 141-150, https: / doi.org / 10.4490 / algae.2023.38.5.25) was performed using a new high-temperature tolerant variety of radial-patterned laver and a common radial-patterned laver (Cheongpung No. 1, name registration number 08-0001-33; thalamus obtained from Kongju National University, a marine red algae resource management institution). Tear thallus of 3-5 cm in length were introduced into tanks at the same weight and cultured in the same manner for 3 weeks. At each weekly measurement, the thallus was harvested using a net, its weight was recorded, and the culture medium was added. The daily relative growth rates of the two varieties were compared using the Specific Growth Rate (SGR) formula below based on the recorded weights.

[0221]

[0222] W e =T e The weight on the measurement date corresponding to, W i =T i The weight of the initial day corresponding to

[0223] As a result of calculating the daily relative growth rate, the new high-temperature tolerant variety of radial patterned seaweed showed 8.18% / day, while the general radial patterned seaweed showed 6.91% / day, confirming that the new high-temperature tolerant variety of radial patterned seaweed has 18.4% higher productivity than the general radial patterned seaweed variety used in marine aquaculture. Thus, the new high-temperature tolerant variety of radial patterned seaweed disclosed in this disclosure showed a high growth rate in land-based aquaculture as well, confirming that it is suitable for use in land-based aquaculture.

[0224]

[0225] Example 6: Confirmation of cultivation of the new high-temperature tolerant variety of radial-patterned laver at different temperatures

[0226] 6-1. Confirmation of culture by temperature

[0227] High-temperature tolerant new varieties of radial-patterned laver were cultured aerated at 10°C, 15°C, and 20°C for 14 days. Wild type (radial-patterned laver collected from the wild at the National Institute of Fisheries Science in Example 1 above) and general radial-patterned laver (Cheongpung No. 1 in Example 5 above) were used as controls. The thallus length and cell condition of each thallus were measured periodically (days 0, 3, 7, and 14) (Figs. 4 and 5).

[0228] As a result, when cultured at 10℃ for 14 days, the new high-temperature tolerant variety of radial-patterned seaweed grew to an average leaf length of 30.3±3.9cm, the wild species to an average leaf length of 12.1±2.7cm, and the ordinary radial-patterned seaweed to an average leaf length of 9.5±3.2cm. When cultured at 15℃ for 14 days, the new high-temperature tolerant variety of radial-patterned seaweed grew to an average leaf length of 31.9±4.4cm, the wild species to an average leaf length of 21.1±1.5cm, and the ordinary radial-patterned seaweed to an average leaf length of 7.0±1.2cm. When cultured at 20℃ for 14 days, the new high-temperature tolerant variety of radial-patterned seaweed grew to an average leaf length of 31.3±4.0cm, the wild species to an average leaf length of 11.1±4.0cm, and the ordinary radial-patterned seaweed to an average leaf length of 6.9±1.2cm.

[0229] As such, it was confirmed that the high-temperature resistant new variety of radial patterned seaweed of the present disclosure exhibits a higher growth rate than wild species and general radial patterned seaweed not only at 10°C, which is the general optimal growth temperature for radial patterned seaweed, but also at temperature conditions of 15°C and 20°C, which are higher than the optimal growth temperature.

[0230]

[0231] 6-2. Confirmation of culture at different temperatures after short-term high-temperature shock

[0232] A test was conducted to confirm whether the new high-temperature tolerant variety of radial-patterned seaweed would continue to grow without dying even after a short-term high-temperature shock (24–26°C, 3 hours). The new high-temperature tolerant variety of radial-patterned seaweed and the general radial-patterned seaweed (Cheongpung No. 1 of Example 5 above) were cultured at 10°C, 15°C, and 20°C. On the third day of culture, the new high-temperature tolerant variety of radial-patterned seaweed and the general radial-patterned seaweed being cultured at 10°C were subjected to a high-temperature shock (24–26°C, 3 hours), and then moved back to 10°C and cultured for 21 days.

[0233] As a result, it was confirmed that the new high-temperature tolerant variety of radial-patterned seaweed subjected to high-temperature shock grew normally for 21 days at 10°C, whereas the thallus of the ordinary radial-patterned seaweed subjected to high-temperature shock gradually disintegrated starting from 7 days at 10°C (Fig. 6). Overall, when cultured for 21 days at 10°C, the new high-temperature tolerant variety of radial-patterned seaweed subjected to high-temperature shock grew to an average thallus length of 40.5±4.6 cm, while the ordinary radial-patterned seaweed subjected to high-temperature shock showed significantly poor growth with an average thallus length of 3.6±1.6 cm. At 15°C, the new high-temperature tolerant variety of radial-patterned seaweed grew to an average thallus length of 20.6±5.6 cm, while all thallus of the ordinary radial-patterned seaweed disintegrated, making measurement impossible. At 20℃, the new high-temperature tolerant variety of radial-patterned seaweed grew to an average leaf length of 19.1±8.0cm, while all thallus of the ordinary radial-patterned seaweed dissociated and could not be measured (Figs. 6 and 7).

[0234]

[0235] [Consignment Number]

[0236] Depository Name: Korea Research Institute of Biotechnology and Bioengineering Biological Resource Center (KCTC)

[0237] Trustee Number: KCTC15932BP

[0238] Date of Trust: 20240611

[0239]

Claims

Pyropia yezoensis, having high temperature resistance and comprising the nucleotide sequence of SEQ ID NO. 18 or a nucleotide sequence having 80% or more homology thereto. The radial seaweed of claim 1, comprising 18S rRNA comprising the nucleotide sequence of SEQ ID NO. 1 or a nucleotide sequence having 80% or more homology therewith. In claim 1, radial seaweed comprising the nucleotide sequence of SEQ ID NO. 19 or a nucleotide sequence having 80% or more homology thereto. Radial patterned seaweed according to claim 1, wherein the high temperature is 12.5℃ to 30℃. In paragraph 1, radial seaweed having the following seaweed thallus characteristics: (a) Tilia shape: lanceolate (b) Mature leaf thallus length: 30.3±3.9cm (c) Mature leaf thallus width: 1.3±0.2cm (d) Mature leaf thallus thickness: 22±1.9μm (e) Monospore formation: present (f) Serrations at the thallus margin: None (g) Mature leaf thallus color: brownish-purple (h) Reproductive type of the thallus: Hermaphroditic. The radial seaweed of claim 1, which is obtained by treating radial seaweed (Pyropia yezoensis) with ethyl methanesulfonate. In paragraph 6, the radial laver is obtained from neutral spores released by treating the thallus of the radial laver with ethyl methanesulfonate. In claim 7, the radial patterned seaweed, wherein the concentration of the ethyl methanesulfonate is 0.1% (w / v) to 3% (w / v). In claim 1, the radial seaweed having increased expression of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), or both. The radial seaweed of claim 1, wherein the expression of one or more selected from the group consisting of HSP 70 (heat shock Protein 70), D6D (delta-6-desaturase), D5D (putative delta-5 desaturase), and RPL19 (ribosomal protein L19) is increased at high temperatures. In item 10, the above high temperature is 12.5℃ to 30℃, radial pattern seaweed. Pyropia yezoensis, which is deposited under accession number KCTC 15932BP in any one of paragraphs 1 through 11. A food containing the radial laver (Pyropia yezoensis) of claim 1. In paragraph 13, the above-mentioned Pyropia yezoensis is a food deposited under accession number KCTC 15932BP.

Images