JPWO2020017504A1 - How to Screen for Preference Ingredients for Eels - Google Patents

Abstract

The present invention is a method for screening a palatability component for an eel, which is (a) a heterodimer composed of any one member of the taste receptor T1R2 family derived from eel and any one member of the T1R3 family. Provided is a method including a step of contacting a quanta with a candidate substance of a palatable component, and (b) a step of evaluating a candidate substance that increases the activity of a heterodimer as a palatable component.

Description

The present invention broadly relates to methods for screening palatable components for eels and the like.

The catch of Japanese eels is declining year by year, and there is concern about resource depletion. In 2014, the Japanese eel was finally designated as an endangered species by the International Union for Conservation of Nature (IUCN). Although efforts have been made toward complete aquaculture for many years, there are still many unclear points about the ecology of the larvae spending time in the sea, and the dietary habits were unknown. The current situation is that it is far from being put on the aquaculture base.

Currently, as the feed for eel larvae, a feed mainly composed of shark eggs, to which soybean peptides, krill decomposition products, and / or minerals are added is used. Research on the search for alternative substances is underway because it is not realistic to use shark eggs in the future from the viewpoint of resources. On the other hand, looking at the whole aquaculture feed, palatability is also considered to be one of the important factors. Preference includes various factors such as physical characteristics, shape, size, and taste.

Fish have generally been shown to respond to L-amino acids. It is known that the chemical substances that are perceived as taste substances differ depending on the fish species. For example, which of the L-amino acids has a strong response differs depending on the fish species, and it is considered that the difference in the amino acid response spectrum between the fish species of the taste organ reflects the difference in the eating habits between the species. There is. In recent years, studies on taste receptors in various animal species have suggested that there is a deep relationship between taste receptors and eating habits.

In vertebrates, it is known that sweet and umami substances are accepted by molecules of the T1R family, which are G protein-coupled receptors (GPCRs), and bitter substances are accepted by molecules of the T2R family. The T1R family consists of three molecular species, T1R1, T1R2 and T1R3. In mammals, the heterodimer of T1R1 + T1R3 is a umami substance such as L-amino acid, and the heterodimer of T1R2 + T1R3 is a sweet substance such as sugar and artificial sweetener. Accept. It is known that only T1R3 is expressed in some taste cells and accepts a high concentration of sugar. On the other hand, in fish, it has become clear that T1R2 + T1R3 also functions as a receptor for L-amino acids (Oike et al., J. Neurosci., 2007). Further, Japanese Patent Application Laid-Open No. 2006-204214 discloses sequences that are considered to correspond to T1R2 and T1R3 of medaka, and it is also described that these are related to the preference of amino acids.

Although there is a deep relationship between taste receptors and eating habits, the relationship between the two is complex. As mentioned above, in mammals, T1R2 + T1R3 functions as a sweetness receptor, but in some animals such as birds, although T1R2 is absent, it cannot be inferred that sweetness cannot be perceived, and in hummingbirds that accept flower honey (sweetness). It has been shown that T1R1 + T1R3 accept sweetness (Baldwin et al., Science, 2014).

Japanese Unexamined Patent Publication No. 2006-2021414

Oike et al. , J. Neurosci. , 2007 Baldwin et al. , Science, 2014

In view of the above circumstances, it is an object of the present invention to provide a novel method for screening a palatable component for an eel and a feed for an eel containing the screened palatable component.

We found that a heterodimer consisting of any one member of the taste receptor T1R2 family and any one member of the T1R3 family responds to stimuli of palatable components for eels. The present invention has been completed.

That is, the present application includes the following inventions.
[1] A method for screening palatability components for eels.
(A) A step of contacting a heterodimer composed of any one member of the taste receptor T1R2 family derived from eel and any one member of the T1R3 family with a candidate substance of a palatability component, and (b). ) A method comprising the step of evaluating a candidate substance that increases the activity of a heterodimer as a palatability component.
[2] The method according to [1], wherein the heterodimer is conjugated to the G protein α subunit.
[3] The G protein α subunit is Gα15 or Gα16 alone, or the C-terminal 5 residues, 25 residues or 44 residues of Gα15 or Gα16 correspond to members belonging to the family of Gi, Gq or Gs. The method according to [2], which is a chimeric protein substituted with a C-terminal residue.
[4] The method according to any one of [1] to [3], wherein the members of the T1R2 family are proteins selected from the group consisting of (a1) to (a6).
(A1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9;
(A2) A protein encoded by a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10;
(A3) A protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9, which responds to L-amino acids;
(A4) A protein encoded by a gene consisting of a base sequence having 80% or more identity with the base sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10, and which responds to L-amino acids;
(A5) A protein consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9 is deleted, substituted and / or added, and responds to L-amino acids. (A6) A protein encoded by a base sequence that hybridizes with the base sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 under stringent conditions and responds to L-amino acids. protein.
[5] Members of the T1R2 family are selected from the group consisting of AjT1R2a, AjT1R2b-1, AjT1R2b-2, AjT1R2b-3 and AjT1R2b-4.
The method according to [4].
[6] The method according to any one of [1] to [5], wherein the members of the T1R3 family are proteins selected from the group consisting of (b1) to (b6):
(B1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 11;
(B2) A protein encoded by a gene consisting of the nucleotide sequence shown in SEQ ID NO: 12;
(B3) A protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 11 and responsive to L-amino acids;
(B4) A protein encoded by a gene consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 12, which responds to L-amino acids;
(B5) A protein consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 11 are deleted, substituted and / or added, and which responds to L-amino acids; or (b6) sequence. A protein encoded by a base sequence that hybridizes with the base sequence of No. 12 under stringent conditions and that responds to L-amino acids.
[7] The method according to [6], wherein the member of the T1R3 family is AjT1R3.
[8] The method according to any one of [1] to [7], wherein the activity is measured using a calcium-sensitive photoprotein.
[9] The method according to [8], wherein the photoprotein is aequorin.
[10] A feed for eels, which comprises a palatability component screened using the method according to any one of [1] to [9].
[11] A feed for eels containing one or more of creatinine, N, N-dimethylglycine and sarcosine as a palatable ingredient.
[12] The feed according to [11], wherein the eel is a Japanese eel (A. japonica) or a European eel (A. anguilla), an Indonesian eel (A. bicolor bicor), or a New Guinea eel (A. bicolor pacifica).
[13] The feed according to any one of [10] to [12], which is for larvae, fry, immature fish or adult fish.

According to the present invention, since a substance that directly acts on the taste receptor of eel can be screened as a palatable component, a new component that has not been used in eel farming has been used in place of the conventional component such as shark eggs. Can be searched for.

FIG. 1 shows the alignment of the Japanese eel T1R. FIG. 2 shows the evaluation of G proteins in AjT1R2a + AjT1R3 and each AjT1R2b + AjT1R3. FIG. 3 shows an evaluation of the concentration dependence of AjT1R2b-1 + AjT1R3 on sarcosine, creatinine and N, N-dimethylglycine.

(Screening method for palatable ingredients)
In a first embodiment, the present invention is a method of screening for palatable ingredients for eels.
(A) A step of contacting a heterodimer composed of any one member of the taste receptor T1R2 family and any one member of the T1R3 family with a candidate substance of a palatability component, and (b) heterodi. Provided is a method comprising the step of evaluating a candidate substance that increases the activity of a meter as a palatability component.

As used herein, a "preference component for an eel" is a negative control that does not contain a ligand such as its candidate substance, eg, a substance that significantly acts on the taste receptor of an eel when compared to a buffer. Or, it means a taste substance preferred by eels or an ingredient that promotes the feeding of eels. L-amino acids such as alanine, arginine, glycine, proline, lysine, serine, histidine, and betaine are known as taste substances preferred by eels or components that promote the feeding of eels. Such known palatability components can also be used as an index for screening.

As used herein, "eel" is broadly referred to as a member of any one of the taste receptor T1R2 family and any one of the T1R3 family of Eel fish, especially Anguilla. It means an eel having a heterodimer consisting of. As long as the effects of the present invention are exhibited, other T1R families may be used instead of the T1R2 family. When classified by habitat, for example, the main eels are roughly classified into four types: Japanese eel (A. japonica), European eel (A. anguilla), American eel (A. rostora), and giant mottled eel (A. marmorata). NS. Other eels include New Guinea eel (A. bicolor pacifica), Indonesian eel (A. bicolor bicolor), Mozambique eel (A. mossamica), Australian eel (A. , Australian eel (A. reinhardtii), Celebes eel (A. celebesensis), Polynesia eel (A. megastoma), Pacific short eel (A. obscura), New Guinea alpine eel (A. intersior) A. nebulosa, New Zealand eel (A. diffenbachii), Luson eel (A. luzonensis), Bengal eel (A. bengalensis bengalensis), African eel (A. bengalensis eel) A. nigricans), Indonesian eel (A. malgumora) and the like can also be mentioned. There are two main types of edible eels, Japanese eels and European eels.

Eels include other eel fish, conger eels, moray eels, conger eels, etc. (Synaphobranchus kaupii), moray eel (Gymnothorax kidako), conger eel (Muraenesox cinereus), and conger eel (Muraenesox bagio) may also be included in the eel of the present invention.

Among the above eels, Japanese eels hatch in the open sea, pass through hatched larvae called pre-leptocephalus, become transparent floating larvae called leptocephalus or larvae, and become adult fish via fry (glass eels, crocodile). .. Eels that are expected to feed on the screened palatable components may be at any stage of growth as long as they express any one member of the T1R2 family and members of the T1R3 family, and are artificially hatched. It may be either fresh or natural.

Taste Receptor Protein A receptor for taste is a membrane protein called a 7-transmembrane protein, which is also called a G protein-coupled receptor (GPCR) because it binds to a G protein. Taste receptor proteins are roughly classified into two types according to their structure, those having a large extracellular region and those not having this region. The former is the T1R family (Taste Receptor type-1), and the latter is the T2R family (Taste Receptor type-1). It is called Taste Receptor type-2). The T1R family consists of three molecular species, T1R1, T1R2, and T1R3.

Regarding the eel T1R, the present inventors obtained the full-length sequence of five types of T1R2 and the full-length sequence of one type of T1R3, and used them as AjT1R2a, AjT1R2b-1, AjT1R2b-2, AjT1R2b-3, and AjT1R2b-4, respectively. , AjT1R3. These AjT1R2a, AjT1R2b-1, AjT1R2b-2, AjT1R2b-3, AjT1R2b-4, and AjT1R3 have the amino acid sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11 and SEQ ID NOs: 2, 4, 6, respectively. It has 8, 10 and 12 base sequences. The alignment of the obtained full-length amino acid sequence is shown in FIG.

他の魚類Ｔ１Ｒとの相同性The above six sequences had 44.2 to 52.0% homology for T1R2 and 51.5 to 53.7% for T1R3 when compared with the sequences of fish whose T1R2 and T1R3 sequences were known (2). Table 1).

Homology with other fish T1R

ニホンウナギＴ１Ｒ間における相同性The homology between Japanese eels T1R was 76.9 to 91.3% between T1R2 and 30.0 to 31.2% between T1R2 and T1R3 (Table 2).

Homology between Japanese eels T1R

As a result of creating a molecular phylogenetic tree based on the obtained full-length sequence, it was clarified that all of them were contained not in the mammalian T1R cluster but in the fish T1R cluster (results not shown).

In mammals, one member of each of the T1R1, T1R2, and T1R3 families has been found, and T1R1 + T1R3 receives umami substances and T1R2 + T1R3 receives sweet substances. On the other hand, fish such as medaka and tiger puffer, which have been reported on taste receptors, have one member in the T1R1 and T1R3 families and multiple members in the T1R2 family. Are known. In eels, as in other fish, it is possible that T1R2 is diversified due to gene duplication. Since the draft genome of the genome database targeted for this search contains incomplete sequences and does not cover all genome sequences, receptor candidates other than the sequences found this time are candidates. It is quite possible that there is a gene sequence that serves as.

In the present invention, the taste receptors that are contacted with the candidate substance of the palatability component include any one member of the T1R2 family such as AjT1R2a, AjT1R2b-1, AjT1R2b-2, AjT1R2b-3 and AjT1R2b-4, and AjT1R3 and the like. It is a heterodimer consisting of any one member of the T1R3 family.

The combination of AjT1R2a and AjT1R3 responds to L-alanine, L-serine, L-arginine, glycine, L-proline. The combination of AjT1R2b-1 and AjT1R3 responds to L-proline, L-alanine. The combination of AjT1R2b-1 and AjT1R3 also responds to sarcosine, creatinine, N, N-dimethylglycine. The combination of AjT1R2b-2 and AjT1R3 responds to L-histidine, L-serine, L-phenylalanine, L-alanine, L-asparagine. The combination of AjT1R2b-4 and AjT1R3 responds to L-alanine, glycine, L-serine, L-proline.

Members of the T1R2 family may be selected from the group consisting of the following (a1)-(a6):
(A1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9;
(A2) A protein encoded by a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10;
(A3) A protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9, which responds to L-amino acids;
(A4) A protein encoded by a gene consisting of a base sequence having 80% or more identity with the base sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10, and which responds to L-amino acids;
(A5) A protein consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9 is deleted, substituted and / or added, and responds to L-amino acids. (A6) A protein encoded by a base sequence that hybridizes with the base sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 under stringent conditions and responds to L-amino acids. protein.

Members of the T1R3 family may be selected from the group consisting of the following (b1)-(b6):
(B1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 11;
(B2) A protein encoded by a gene consisting of the nucleotide sequence shown in SEQ ID NO: 12;
(B3) A protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 11 and responsive to L-amino acids;
(B4) A protein encoded by a gene consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 12, which responds to L-amino acids;
(B5) A protein consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 11 are deleted, substituted and / or added, and which responds to L-amino acids; or (b6) sequence. A protein encoded by a base sequence that hybridizes with the base sequence of No. 12 under stringent conditions and that responds to L-amino acids.

The amino acid sequence is wild, such as SEQ ID NO: 1, 3, 5, 7, 9 or 11, as long as it responds to the stimulus of a known preference component such as L-amino acid or a preference component identified by screening. In the amino acid sequence of the type protein, if one to a plurality, for example, 100 amino acids in the amino acid sequence are regarded as one unit, 1, 2, 3, 4, 5, 6, 7, 8, per unit. Even those consisting of an amino acid sequence having 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, preferably several amino acids deleted, substituted, added, etc. good.

Here, the range of "1 to several" in "deletion, substitution, addition of one to several amino acids" of the amino acid sequence is not particularly limited, but one unit is preferably 1, 2, 3, 4 per unit. It means about 5, 6, 7, 8, 9 or 10, more preferably about 1, 2, 3, 4 or 5. In addition, "amino acid deletion" means that an amino acid residue in the sequence is missing or eliminated, and "amino acid substitution" means that an amino acid residue in the sequence is replaced with another amino acid residue. However, "addition of amino acid" means that a new amino acid residue is added to the sequence so as to be inserted.

Specific embodiments of "amino acid deletion, substitution, addition" include wild-type, as long as the response to the stimulus of known palatable components such as L-amino acids and the palatable components identified by screening is maintained. An embodiment in which the amino acid in is replaced with another chemically similar amino acid can be mentioned. For example, a case where one hydrophobic amino acid is replaced with another hydrophobic amino acid, a case where a certain polar amino acid is replaced with another polar amino acid having the same charge, and the like can be mentioned. Such chemically similar amino acids are known in the art for each amino acid.

Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Examples of positively charged basic amino acids include arginine, histidine, and lysine. Examples of negatively charged acidic amino acids include aspartic acid and glutamic acid.

Further, in the above amino acid sequence, as a specific embodiment of "deletion, substitution, addition of amino acid", it is constant with the amino acid sequence possessed by the wild type protein such as SEQ ID NO: 1, 3, 5, 7, 9 or 11. Examples thereof include amino acid sequences having the above sequence identity, for example, 75% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more, most preferably 90% or more, the amino acid sequence of wild-type protein. Amino acid sequences having 95% or more identity can be mentioned.

The base sequence used may be one that produces a desired sequence via splicing after transcription of the base sequence when introduced into a host organism, or one that produces a desired sequence after transcription without going through splicing. good.

The base sequence used does not have to be exactly the same as the wild-type sequence, and is wild-type as long as it maintains a response to the stimulus of a known palatability component such as L-amino acid or a palatability component identified by screening. It may be a base sequence that hybridizes with a base sequence complementary to the base sequence of the gene under stringent conditions.

As used herein, the term "base sequence that hybridizes under stringent conditions" refers to DNA that corresponds to a part of the base sequence of a wild-type gene such as SEQ ID NO: 2, 4, 6, 8, 10 or 12. It is used as a probe and means a base sequence of DNA obtained by using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like.

As used herein, the "stringent condition" is a condition in which a specific hybrid signal is clearly distinguished from a non-specific hybrid signal, and the hybridization system to be used, the type and sequence of the probe are used. And depends on the length. Such conditions can be determined by varying the hybridization temperature, washing temperature and salt concentration.

For example, when a non-specific hybrid signal is strongly detected, the specificity can be increased by raising the temperature of hybridization and washing and, if necessary, lowering the salt concentration of washing. If no specific hybrid signal is also detected, the hybrid can be stabilized by lowering the hybridization and wash temperatures and, if necessary, increasing the wash salt concentration.

In some embodiments, specific examples of stringent conditions include: For example, a DNA probe is used as a probe, and hybridization is performed with 5 × SSC, 1.0% (w / v) nucleic acid hybridization blocking reagent (manufactured by Beringa Mannheim), 0.1% (w / v) N. -Lauroyl sarcosine, 0.02% (w / v) SDS, overnight (about 8 to 16 hours). Washing was performed twice with 0.1 × 0.5 × SSC, 0.1% (w / v) SDS, preferably 0.1 × SSC, 0.1% (w / v) SDS for 15 minutes. conduct. The temperature at which hybridization and washing are performed is 65 ° C. or higher, preferably 68 ° C. or higher.

As the DNA having a base sequence that hybridizes under stringent conditions, for example, a DNA having a base sequence of a wild-type gene derived from a colony or plaque or a filter in which a fragment of the DNA is immobilized is used to use the above-mentioned string. After hybridization at 40-75 ° C. in the presence of DNA obtained by hybridization under gentle conditions and 0.5-2.0 M NaCl, preferably 0.7-1.0 M. After hybridization was performed at 65 ° C. in the presence of NaCl, a filter was used under 65 ° C. conditions using a 0.1 to 1 × SSC solution (1 × SSC solution was 150 mM sodium chloride, 15 mM sodium citrate). DNA and the like that can be identified by washing the above can be mentioned. Methods for probe preparation and hybridization are described in Molular Cloning: A laboratory Manual, 2nd-Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. , 1989, Current Protocols in Molecular Biology, Supplement 1-38, John Willey & Sons, 1987-1997 and the like.

If you are a person skilled in the art, in addition to the conditions such as the salt concentration and temperature of the buffer, other conditions such as the probe concentration, the probe length, and the reaction time are taken into consideration, and the base sequence of the wild-type gene is taken into consideration. Conditions for obtaining a DNA having a base sequence complementary to the above and a base sequence that hybridizes under stringent conditions can be appropriately set.

As a DNA containing a base sequence complementary to the base sequence of a wild-type gene and a base sequence that hybridizes under stringent conditions, a DNA having a certain degree of sequence identity with the base sequence of the wild-type gene used as a probe. For example, a DNA having a sequence identity of 75% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more, still more preferably 95% or more with the base sequence of a wild-type gene. Can be mentioned.

As a base sequence that hybridizes with a base sequence complementary to the base sequence of the wild type gene under stringent conditions, for example, if the number of bases in the base sequence is 500 as one unit, the base sequence of the wild type gene , 1 to plural, for example, 1 to 125, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 20, preferably 1 to several, per unit. For example, it contains a base sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base deletions, substitutions, additions, and the like.

Among the stringent conditions, a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed, that is, a high stringent condition is preferable. Examples of the high stringent condition include a condition in which nucleic acids having high identity hybridize with each other and nucleic acids having lower identity do not hybridize with each other.

Here, "base deletion" means that a base in the sequence is missing or lost, and "base substitution" means that the base in the sequence is replaced with another base. "Addition of base" means that a new base has been added to be inserted.

The protein encoded by the base sequence complementary to the base sequence of the wild type gene and the base sequence that hybridizes under stringent conditions is one to more than one in the amino acid sequence of the protein encoded by the base sequence of the wild type gene. It is likely that the protein has an amino acid sequence having deletions, substitutions, additions, etc. of several amino acids, preferably several amino acids, but is considered to have the same effect as a protein encoded by the base sequence of a wild-type gene.

In addition, the gene encoding the protein is a base sequence encoding an amino acid sequence that is the same as or similar to the amino acid sequence possessed by the enzyme encoded by the wild-type gene by utilizing the fact that there are several types of codons corresponding to one amino acid. It may contain a base sequence different from that of the wild-type gene. The base sequence may be modified to have the optimum codon depending on the codon usage frequency of the host used.

The method for determining the sequence identity of the base sequence and the amino acid sequence is not particularly limited, but for example, a commonly known method is used to target the amino acid sequence of the wild-type gene or the enzyme encoded by the wild-type gene. It is obtained by aligning a base sequence or an amino acid sequence and using a program for calculating the matching rate of both sequences.

As a program for calculating the matching rate between two amino acid sequences and base sequences, for example, Karlin and Altschul's algorithm (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993), and a BLAST program using this algorithm has been developed by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Furthermore, Gapped BLAST, which is a program for determining sequence identity more sensitively than BLAST, is also known (Nucleic Acids Res. 25: 3389-3402, 1997). Therefore, a person skilled in the art can, for example, use the above program to search the database for a sequence showing high sequence identity for a given sequence. These are available, for example, on the National Center for Biotechnology Information website on the Internet (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Each of the above methods can be usually used to search a database for sequences showing sequence identity, but as a means for determining the sequence identity of individual sequences, Genetyx network version version 12.0. Homology analysis of 1 (manufactured by Genetics) can also be used. This method is based on the Lipman-Pearson method (Science 227: 1435-1441, 1985). When analyzing the sequence identity of the base sequence, the region encoding the protein (CDS or ORF) is used if possible.

Since the heterodimer consisting of the G proteins T1R2 and T1R3 is coupled to the heterotrimeric G protein specifically expressed in the taste receptor cells, the heterodimer is used in the cells during screening. It may be co-expressed with G protein. As such G proteins, the Gq family, gustducin, transducin and the like are known.

G proteins consist of Gα (α subunit) and Gβγ subunit. When screening for palatability components, it is sufficient that at least the Gα subunit is expressed. In addition, the Gβγ subunit may be expressed together with the Gα subunit. It is known that the Gα subunit includes a group of Gi, Gq or Gs, G12 / 13. Among them, chimeric proteins in which Gα15 or Gα16 alone or their C-terminal 5 residues, 25 residues or 44 residues are replaced with corresponding C-terminal residues of members belonging to the family of Gi, Gq or Gs. Can be preferably used. Such substitutions of C-terminal residues are known to those of skill in the art (see, eg, Li et al., PNAS April 2, 2002. 99 (7) 4692-469, etc.). By substituting the C-terminal residue in this way, it is considered that coupling with the receptor becomes easier.

The "corresponding C-terminal residue" means, for example, 5 amino acids constituting the C-terminal sequence of the Gα subunit when the 5 amino acids in the C-terminal sequence of Gα15 are replaced with another Gα subunit. Regarding the notation of the chimeric protein, for example, the expression rG15-rGi3-5 may describe rat Gα15 (hereinafter, Gα15 is referred to as “G15”, rat Gα15 is “rG15”, mouse Gα15 is “mG15”, and so on. It means that the C-terminal sequence 5 amino acids of (human Gα16 may be referred to as “hG16” or the like) are replaced with the C-terminal sequence 5 amino acids of rat Gi3.

In selecting a G protein, for known literature, for example, rG15-rGi2-5, two tastes of ligands of ligand specialty in T1R1 / T1R3 (the umami taste receptor) Takahiro Okada, Takahiro Okada Imai H, Shimaru Y, Misaka T. et al. J Biol Chem. 2013 Dec 27; 288 (52): 36863-77. Doi et al., And for mG15, Sensory biology. Evolution of sweet taste perception in hummingbirds by transformation of the ancestor umami receptor. Baldwin MW, Toda Y, Nakagita T, O'Connell MJ, Klasing KC, Misaka T, Edwards SV, Liberles SD. Science. 2014 August 22; 345 (6199): 929-33. doi: 10.1126 / science. 1255097 and the like can be referred to.

The ACSESSION numbers (NCBI Reference Sequence) of major G proteins are shown below.
Mouse G15: NM_010304
Rat G15: NM_053542
Human G16: NM_002068
Human Ggust: NM_001102386
Rat Ggust: NM_173139
Rat Gi2: NM_031035
Rat Gi3: NM_013106
Rat Gs: NM_0191132

The sequence information of the chimeric protein used in the examples is described below.
Amino acid sequence:
rG15-rGi3-5 (SEQ ID NO: 13);
rG15-rGi2-5 (SEQ ID NO: 15);
hG16-rGi3-5 (SEQ ID NO: 17);
mG15 (SEQ ID NO: 19);
rG15 (SEQ ID NO: 21);
rG15-rGgust-5 (SEQ ID NO: 23);
hG16 (SEQ ID NO: 25);
hG16-rGgust-25 (SEQ ID NO: 27);
hG16-rGi2-44 (SEQ ID NO: 29);
hG16-rGs-25 (SEQ ID NO: 31);
hG16-rGs-44 (SEQ ID NO: 33);
rG15-rGgust-25 (SEQ ID NO: 35)

Base sequence:
rG15-rGi3-5 (SEQ ID NO: 14);
rG15-rGi2-5 (SEQ ID NO: 16);
hG16-rGi3-5 (SEQ ID NO: 18);
mG15 (SEQ ID NO: 20);
rG15 (SEQ ID NO: 22);
rG15-rGgust-5 (SEQ ID NO: 24);
hG16 (SEQ ID NO: 26);
hG16-rGgust-25 (SEQ ID NO: 28);
hG16-rGi2-44 (SEQ ID NO: 30);
hG16-rGs-25 (SEQ ID NO: 32);
hG16-rGs-44 (SEQ ID NO: 34);
rG15-rGgust-25 (SEQ ID NO: 36)

The protein used in the screening can be prepared by operably linking each base sequence alone or in any combination in any combination and expressing it in the desired cell. Each sequence can be inserted into the same or different vectors.

The nucleotide sequence encoding the heterodimer is expressed in a suitable host, such as a mammal such as a human, an animal cell such as an amphibian such as a frog, an insect cell or a yeast. Preferably, the host is a cell such as an insect or a mammal. More preferably, the mammalian cell is a human cell. Examples of mammalian cells include Human Embryonic Kidney (HEK), such as HEK293T, Madein-Darby Canine Kidney (MDCK), Chinese Hamster Ovary Cell (CHO), and the like.

Introduction of a nucleotide sequence encoding a heterodimer into a host cell is carried out by a known method, such as Sambrook, J. Mol. , Fritsch, E.I. F. , And Maniatis, T.M. , "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, (1989) and the like.

The screening method of the present invention may include any step used for screening in addition to the following steps.
(A) A step of contacting a heterodimer composed of any one member of the taste receptor T1R2 family and any one member of the T1R3 family with a candidate substance of a palatability component, and (b) heterodi. A step of evaluating a candidate substance that increases the activity of a meter as a palatable component

Increased activity of the heterodimer, for example, increased calcium ion concentration in cells can be measured using a calcium ion measuring reagent or the like. As such a reagent, for example, a calcium-sensitive photoprotein, specifically a protein such as aequorin that emits light depending on the concentration of calcium ions is preferable. Instead of the reagent, calcium imaging can also be used in which receptor activation is observed based on an image, the number of responding cells is counted from the obtained image, and the degree of response is determined. Devices for measuring such activation include the CCD camera CoolSNAP HQ2 (Nippon Roper), the inverted microscope IX-81 (OLYMPUS), and software for imaging includes MetaMorph and MetaFlour (Molecular Devices).

Alternatively, a well-based assay may be used to obtain the cellular response on the multi-well plate numerically. FlexStation3 (Molecular Devices), FLIPR (Molecular Devices) and the like can be used for such a well-based assay.

GPCRs such as T1R2 and T1R3 undergo structural changes (activation) when bound to a ligand, a palatable component, attract G proteins such as gastoducin, and in addition to elevated intracellular calcium, various downstream. (For example, fluctuation of cyclic AMP concentration, GTP binding to low molecular weight G protein Rho, etc.). By detecting these intracellular events, the activity of the heterodimer can also be measured.

In the step of evaluating the palatability component, a sample containing no ligand such as a candidate substance, for example, a buffer can be used as a control. Candidate substances with similar or higher activity in comparison with such controls can be evaluated as palatable components. Instead of negative controls such as buffers, use known palatable ingredients such as L-amino acids such as L-alanine, L-arginine, glycine, L-proline, L-lysine, L-serine, L-histidine. You may.

(Feed for eels)
In a second embodiment, the present invention provides a feed for eels, which comprises a palatability component screened using the method described above.

The feed may be for larvae, fry, or adult fish. Since eel larvae become active in the secretion of digestive enzymes around 7 days after hatching at a water temperature of 23 ° C., the feed of the present invention is considered to be useful at such a stage.

Preference components screened by the present invention include sarcosine, N, N-dimethylglycine or creatinine, or analogs thereof.
As the palatability component, one or more of sarcosine, N, N-dimethylglycine or creatinine may be contained.

Sarcosine is one of the components of snow crab leg meat extract (Kawai, 2011), and it has been reported that puffer fish responds (Kiyohara et al., 1985). It has also been reported that creatinine is mainly contained in the muscles of higher animals above fish and is contained as a guanidine compound contained in Scombridae fish. Although there is a report that creatinine has an effect of imparting a strong meat-like taste in humans and improving the soup stock flavor, there is no report of examining the taste response in fish, and the present inventors have newly found it. It is likely to be a taste component.

The amount of the palatable component to be blended can be appropriately determined by those skilled in the art in consideration of the type of eel to be fed, its growth state, the number of feedings, and the like. For example, when N, N-dimethylglycine is given to adult Japanese eel fish once a day, the blending amount in the feed is preferably 3% by mass or more, for example.

The feed for eels is mainly fish-derived ingredients based on the conventionally used raw feed such as sardines, dried fish meal such as sardines, mackerel, herring or horse mackerel, and shark egg powder. You may use a mixed feed or the like. In addition to the fish-derived ingredients used in conventional feeds, soybean peptides, krill decomposition products, starch, calcium phosphate, minerals such as salt, yeast, and herbal extracts, as long as the effects of the palatable ingredients added to the feed are not impaired. Etc. may be added to the feed.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

(Construction of evaluation system using taste receptor expressing cells)
Each AjT1R2 was co-expressed with AjT1R3 and the presence or absence of a response to amino acids was examined using a luminescence detection system by a well-based assay. Aequorin was used as the photoprotein.

As the chimeric G protein, known rG15-rGi3-5, rG15-rGi2-5, and hG16-rGi3-5 were used. According to the following procedure, how each receptor responds to 15 kinds of amino acids in combination with each chimeric G protein was investigated.

1) HEK293T cells were seeded on a 12-well plate and cultured overnight at _{37 ° C. and 5% CO 2.}
2) For the cells of 1), use each plasmid (pEAK10 vector (Edge Biosystems, Gaithersburg, MD) expressing each AjT1R2, AjT1R3, chimeric G protein, apoaequorin). Was transfected with Lipovectorine 2000 (Invitrogen, Carlsbad, CA).
3) After 6 to 7 hours, the medium was changed and the cells were cultured overnight at _{37 ° C. and 5% CO 2.}
4) The next day, the cells were reseeded on a 96-well plate for measurement (Corning, CellBIND (registered trademark) Surface) and cultured overnight at _{37 ° C. and 5% CO 2.}
5) On the day of the assay, cells were washed with assay buffer and then allowed to stand at 27 ° C. for 4 hours in assay buffer (containing 0.1% BSA) containing 10 μM coelenterazine (under shading).
6) The change in luminescence intensity when the ligand was added was detected by FlexStation 3 (Molecular Devices).

(Results and discussion)
As a preliminary study, the concentration dependence of AjT1R2a + AjT1R3 on L-alanine was evaluated. As a result, the EC ₅₀ value was calculated to be around 1 mM in each case, but there was a difference in response intensity. Table 3 shows some results among the G proteins that showed a somewhat high response.

For AjT1R2a + AjT1R3 and each AjT1R2b + AjT1R3, the response results to a specific amino acid among 15 kinds of amino acids are shown in FIG. In FIG. 2, the change in luminescence intensity during ligand administration was detected by FlexStation 3 (mean value, n = 2). The ligand concentration is 50 mM. The vertical axis represents the emission intensity.

As shown in FIG. 2, the degree of response was different depending on the type of chimeric G protein to be co-expressed, but the response profile was similar for almost all G proteins.

Considering the results not shown in FIG. 2, among the 15 kinds of amino acids used, AjT1R2a + AjT1R3 is L-alanine, L-serine, L-arginine, glycine, L-proline, AjT1R2b-1 + AjT1R3 is L-proline. L-alanine, AjT1R2b-2 + AjT1R3 responded to L-alanine, L-serine, L-phenylalanine, L-alanine, L-asparagin, AjT1R2b-4 + AjT1R3 responded to L-alanine, glycine, L-serine, L-proline. rice field. AjT1R2b-3 + AjT1R3 tended to respond to L-serine, L-threonine, L-alanine, and L-arginine, but the overall response was low.

From the evaluation results of the concentration dependence on L-alanine in AjT1R2a + AjT1R3 and each AjT1R2b + AjT1R3, it was found that any chimeric G protein can be used with a difference in response intensity. Regarding the chimeric G protein, hG16-rGi3-5, which has a low baseline and shows a somewhat high response, was used in the subsequent experiments, which is common to all receptor combinations.

(Evaluation of response to sarcosine, N, N-dimethylglycine, creatinine)
The assay method was the same as for confirming the response to amino acids described above.

AjT1R2b-1 + AjT1R3 responded to sarcosine, N, N-dimethylglycine, and creatinine in a concentration-dependent manner. FIG. 3 shows the concentration response curves of AjT1R2b-1 + AjT1R3 to sarcosine, N, N-dimethylglycine, and creatinine (mean ± SEM, n = 3). No response was observed to any of the ligands in the cultured cells into which only hG16-rGi3-5 was introduced, demonstrating that these ligands are accepted via AjT1R2b-1 + AjT1R3 (results not shown). ).

(Feeding test)
Each test plot was set up by accommodating 10 immature Japanese eels in a 200L aquarium. Feeding was once a day, and feeding was performed daily.
A mixed feed for growing eels (for eels, sold by Hayashikane Sangyo Co., Ltd.) excluding betaine, glycine, alanine, and krill meal, which are known palatable components, was used as a palatable component-removed feed. A feed obtained by adding 3% of N, N-dimethylglycine to a palatable component-removed feed (hereinafter referred to as "N, N-dimethylglycine-added feed") was fed to immature Japanese eel fish as described above, and they were fed. The palatability of N, N-dimethylglycine was evaluated by measuring the amount of feed, feed efficiency, etc. As a comparison group, a feed group from which palatable components were removed and a mixed feed group for eel breeding were included.
The test period was 31 days. The water temperature during the test period was 30-34 ° C.

The results are shown in Table 4 (Group A: Preference component-free feed group, Group B: N, N-dimethylglycine-added feed group, Group C: eel breeding compound feed group). The amount of food intake was evaluated based on the amount of residual food and was shown by dry weight.
From this result, it was suggested that N, N-dimethylglycine induces the feeding of Japanese eels and has a growth promoting effect.

According to the screening method of the present invention, it becomes possible to search for new components such as N, N-dimethylglycine, which have not been used in conventional eel farming.

Claims (13)

A method of screening for palatable ingredients for eels
(A) A step of contacting a heterodimer composed of any one member of the T1R2 family of taste receptors derived from eel and any one member of the T1R3 family with a candidate substance of a palatability component, and (b). ) A method comprising the step of evaluating a candidate substance that increases the activity of a heterodimer as a palatability component. The method of claim 1, wherein the heterodimer is conjugated to the G protein α subunit. The G protein α subunit is Gα15 or Gα16 alone, or the C-terminal 5 residues, 25 or 44 residues of Gα15 or Gα16 are the corresponding C-terminus of a member belonging to the Gi, Gq or Gs family. The method of claim 2, wherein the chimeric protein is substituted with a residue. The method according to any one of claims 1 to 3, wherein the members of the T1R2 family are proteins selected from the group consisting of (a1) to (a6):
(A1) A protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9;
(A2) A protein encoded by a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10;
(A3) A protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9, which responds to L-amino acids;
(A4) A protein encoded by a gene consisting of a base sequence having 80% or more identity with the base sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10, and which responds to L-amino acids;
(A5) A protein consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9 is deleted, substituted and / or added, and responds to L-amino acids. (A6) A protein encoded by a base sequence that hybridizes with the base sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10 under stringent conditions and responds to L-amino acids. protein. The method of claim 4, wherein the members of the T1R2 family are selected from the group consisting of AjT1R2a, AjT1R2b-1, AjT1R2b-2, AjT1R2b-3 and AjT1R2b-4. The method according to any one of claims 1 to 5, wherein the members of the T1R3 family are proteins selected from the group consisting of (b1) to (b6):
(B1) A protein consisting of the amino acid sequence shown in SEQ ID NO: 11;
(B2) A protein encoded by a gene consisting of the nucleotide sequence shown in SEQ ID NO: 12;
(B3) A protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 11 and responsive to L-amino acids;
(B4) A protein encoded by a gene consisting of a base sequence having 80% or more identity with the base sequence shown in SEQ ID NO: 12, which responds to L-amino acids;
(B5) A protein consisting of an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 11 are deleted, substituted and / or added, and which responds to L-amino acids; or (b6) sequence. A protein encoded by a base sequence that hybridizes with the base sequence of No. 12 under stringent conditions and that responds to L-amino acids. The method of claim 6, wherein the member of the T1R3 family is AjT1R3. The method according to any one of claims 1 to 7, wherein the activity is measured using a calcium-sensitive photoprotein. The method of claim 8, wherein the photoprotein is aequorin. A feed for eels comprising a palatability component screened using the method according to any one of claims 1-9. A diet for eels containing one or more of creatinine, N, N-dimethylglycine and sarcosine as palatable ingredients. The feed according to claim 11, wherein the eel is a Japanese eel (A. japonica), a European eel (A. anguilla), an Indonesian eel (A. bicolor bicolor), or a New Guinea eel (A. bicolor pacifica). The feed according to any one of claims 10 to 12, which is for larvae, fry, immature fish or adult fish.

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