TW202014516A - Method for screening palatable component for eels - Google Patents
Method for screening palatable component for eels Download PDFInfo
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- TW202014516A TW202014516A TW108125278A TW108125278A TW202014516A TW 202014516 A TW202014516 A TW 202014516A TW 108125278 A TW108125278 A TW 108125278A TW 108125278 A TW108125278 A TW 108125278A TW 202014516 A TW202014516 A TW 202014516A
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Abstract
Description
本發明係關於一種廣泛篩選用於鰻魚之嗜好性成份之方法等。The present invention relates to a method for extensively screening preference components for eel.
日本鰻魚有捕魚量逐年減少之傾向，擔心資源枯竭，2014年終於被國際自然保護聯盟(IUCN)指定為瀕危物種。雖長年進行以完全養殖為目標之努力，但由於關於在海中度過幼體時期之其生態不明之處較多，食性不明等理由，成功完全養殖之現在亦有與達到商業化基準相距甚遠之現狀。Japanese eels have a tendency to decrease their fishing volume year by year. They are worried about the depletion of resources. In 2014, they were finally designated as an endangered species by the International Union for Conservation of Nature (IUCN). Although efforts have been made for the purpose of complete farming for many years, due to reasons such as the unclear ecology of the larval period in the sea and the unclear diet, there is still a long way to go to achieve the commercialization standard. .
現在，作為鰻魚之仔魚之餌食，使用以鯊魚之卵為主體，加入大豆肽、磷蝦分解物、及/或礦物等而成之餌食。由於將來使用鯊魚之卵即便就資源方面考慮亦並不現實等理由，進行關於其代替物質之探索之研究。另一方面，若著眼於養殖飼料整體，則嗜好性亦被認為係重要要素之一。嗜好性包括物性、形狀、尺寸、味道等多種因素。Currently, as a bait for eel larvae, a shark egg is used as a main body, and a soy peptide, a krill decomposition product, and/or minerals are added. Due to the fact that the use of shark eggs in the future is unrealistic even in terms of resources, research has been conducted on the exploration of alternative substances. On the other hand, if you focus on the whole cultured feed, the hobby is also considered to be one of the important elements. Preferences include physical properties, shape, size, taste and other factors.
明確魚類通常對L-胺基酸作出應答。已知作為呈味物質而感知之化學物質視魚種類各異。例如，認為對L-胺基酸中之哪一個之應答較強視魚種類各異，味覺器之魚種類間之胺基酸應答光譜之差異反映種間之食性之差異。近年來，藉由以各種動物種類為對象之味覺受體之研究，提示味覺受體與食性有較深之關係。It is clear that fish usually respond to L-amino acids. It is known that the chemical substances perceived as taste substances vary depending on the type of fish. For example, it is believed that the response to which of the L-amino acids is stronger depends on the fish species, and the difference in the amino acid response spectrum between the fish species of the taste organs reflects the difference in diet between the species. In recent years, studies on taste receptors targeting various animal species have suggested that taste receptors have a deep relationship with eating habits.
已知於脊椎動物中，甜味、鮮味物質由作為G蛋白質共軛型受體(GPCR)之T1R家族之分子接受，苦味物質由T2R家族之分子接受。已知T1R家族包含T1R1、T1R2及T1R3這3個分子種類，於哺乳類中，由T1R1＋T1R3之異質二聚物接受L-胺基酸等鮮味物質，由T1R2＋T1R3之異質二聚物接受糖及人工甜味料等甜味物質。於一部分之味覺細胞中僅表現T1R3，其接受高濃度之糖。另一方面，明確於魚類中T1R2＋T1R3亦作為L-胺基酸之受體發揮功能(Oike et al., J. Neurosci., 2007)。又，日本專利特開2006-204214號公報中揭示認為相當於稻田魚之T1R2及T1R3之序列，亦記載該等與胺基酸之嗜好相關。It is known that in vertebrates, sweet and umami substances are accepted by molecules of the T1R family as G protein conjugated receptors (GPCRs), and bitter substances are accepted by molecules of the T2R family. It is known that the T1R family contains three molecular species of T1R1, T1R2 and T1R3. In mammals, the heterodimer of T1R1+T1R3 receives umami and other umami substances, and the heterodimer of T1R2+T1R3 receives sugar and artificial sweetness. Flavoring and other sweet substances. Only a part of taste cells express T1R3, which accepts high concentration of sugar. On the other hand, it is clear that T1R2+T1R3 in fish also function as L-amino acid receptors (Oike et al., J. Neurosci., 2007). In addition, Japanese Patent Laid-Open No. 2006-204214 discloses sequences that are equivalent to T1R2 and T1R3 of rice field fish, and also describes that these are related to amino acid preferences.
味覺受體與食性有較深之關係，但兩者之關係複雜。如上所述，於哺乳類中，T1R2＋T1R3作為甜味受體發揮功能，但於鳥類等一部分動物中沒有T1R2，但無法推測其無法察覺甜味，明確接受花蜜(甜味)之蜂鳥藉由T1R1＋T1R3接受甜味(Baldwin et al., Science, 2014)。 [先前技術文獻] [專利文獻]Taste receptors have a deep relationship with eating habits, but the relationship between the two is complicated. As mentioned above, in mammals, T1R2+T1R3 functions as a sweet taste receptor, but there is no T1R2 in some animals such as birds, but it is impossible to speculate that it cannot detect sweetness. Hummingbirds that clearly receive nectar (sweetness) receive sweetness through T1R1+T1R3 Taste (Baldwin et al., Science, 2014). [Prior Technical Literature] [Patent Literature]
[專利文獻1]日本專利特開2006-204214號公報 [非專利文獻][Patent Document 1] Japanese Patent Laid-Open No. 2006-204214 [Non-patent literature]
[非專利文獻1]Oike et al., J. Neurosci., 2007 [非專利文獻2]Baldwin et al., Science, 2014[Non-Patent Literature 1] Oike et al., J. Neurosci., 2007 [Non-Patent Document 2] Baldwin et al., Science, 2014
[發明所欲解決之問題][Problems to be solved by the invention]
鑒於上述情況，本發明之目的在於提供一種用以篩選用於鰻魚之嗜好性成份之新穎方法、以及含有篩選之該嗜好性成份之用於鰻魚之飼料。 [解決問題之技術手段]In view of the above circumstances, an object of the present invention is to provide a novel method for screening preference components for eel and a feed for eel containing the screening preference components. [Technical means to solve the problem]
本發明人等發現包含味覺受體T1R2家族之任一成員及T1R3家族之任一成員之異質二聚物對用於鰻魚之嗜好性成份之刺激作出應答，從而完成了本發明。The present inventors have found that a heterodimer comprising any member of the T1R2 family of taste receptors and any member of the T1R3 family responds to the stimulation of a preference component used for eel, thereby completing the present invention.
即，本申請案包含以下之發明。 [1]一種篩選用於鰻魚之嗜好性成份之方法，其包括： (a)使來自鰻魚之包含味覺受體T1R2家族之任一成員及T1R3家族之任一成員之異質二聚物與嗜好性成份之候選物質接觸之步驟；及 (b)將使異質二聚物之活性增大之候選物質評價為嗜好性成份之步驟。 [2]如[1]所記載之方法，其中異質二聚物與G蛋白質α次單元共軛。 [3]如[2]所記載之方法，其中G蛋白質α次單元為單獨之Gα15或Gα16、或者為Gα15或Gα16之C末端5殘基、25殘基或44殘基由屬於Gi、Gq或Gs之家族之成員的對應之C末端殘基置換之嵌合蛋白質。 [4]如[1]至[3]中任一項所記載之方法，其中T1R2家族之成員為選自由(a1)～(a6)所組成之群中之蛋白質： (a1)包含序列編號1、3、5、7或9中記載之胺基酸序列之蛋白質； (a2)由包含序列編號2、4、6、8或10中記載之鹼基序列之基因編碼之蛋白質； (a3)包含與序列編號1、3、5、7或9中記載之胺基酸序列具有80%以上之同一性之胺基酸序列，且對L-胺基酸作出應答之蛋白質； (a4)由包含與序列編號2、4、6、8或10中記載之鹼基序列具有80%以上之同一性之鹼基序列之基因編碼，且對L-胺基酸作出應答之蛋白質； (a5)包含序列編號1、3、5、7或9中記載之胺基酸序列之1個或複數個胺基酸缺失、置換及/或附加所得之胺基酸序列，且對L-胺基酸作出應答之蛋白質；或者 (a6)由與序列編號2、4、6、8或10中記載之鹼基序列於嚴格之條件下雜交之鹼基序列編碼，且對L-胺基酸作出應答之蛋白質。 [5]如[4]所記載之方法，其中T1R2家族之成員選自由AjT1R2a、AjT1R2b-1、AjT1R2b-2、AjT1R2b-3及AjT1R2b-4所組成之群。 [6]如[1]至[5]中任一項所記載之方法，其中T1R3家族之成員為選自由(b1)～(b6)所組成之群中之蛋白質： (b1)包含序列編號11中記載之胺基酸序列之蛋白質； (b2)由包含序列編號12中記載之鹼基序列之基因編碼之蛋白質； (b3)包含與序列編號11中記載之胺基酸序列具有80%以上之同一性之胺基酸序列，且對L-胺基酸作出應答之蛋白質； (b4)由包含與序列編號12中記載之鹼基序列具有80%以上之同一性之鹼基序列之基因編碼，且對L-胺基酸作出應答之蛋白質； (b5)包含序列編號11中記載之胺基酸序列之1個或複數個胺基酸缺失、置換及/或附加所得之胺基酸序列，且對L-胺基酸作出應答之蛋白質；或者 (b6)由與序列編號12中記載之鹼基序列於嚴格之條件下雜交之鹼基序列編碼，且對L-胺基酸作出應答之蛋白質。 [7]如[6]所記載之方法，其中T1R3家族之成員為AjT1R3。 [8]如[1]至[7]中任一項所記載之方法，其中活性使用鈣敏感性之發光蛋白質進行測定。 [9]如[8]所記載之方法，其中發光蛋白質為水母發光蛋白。 [10]一種用於鰻魚之飼料，其包含使用如[1]至[9]中任一項所記載之方法篩選之嗜好性成份。 [11]一種用於鰻魚之飼料，其包含肌酸酐、N,N-二甲基甘胺酸及肌胺酸中之1種以上作為嗜好性成份。 [12]如[11]所記載之飼料，其中鰻魚為日本鰻魚(A. japonica)或歐洲鰻魚(A. anguilla)、印度尼西亞鰻魚(A. bicolor bicolor)或新幾內亞鰻魚(A. bicolor pacifica)。 [13]如[10]至[12]中任一項所記載之飼料，其為仔魚用、稚魚用、未成魚用或成魚用。 [發明之效果]That is, this application includes the following inventions. [1] A method for screening preference components for eels, including: (a) The step of contacting the heterodimer containing any member of the taste receptor T1R2 family and any member of the T1R3 family from the eel with the candidate substance of the preference component; and (b) A step of evaluating candidate substances that increase the activity of the heterodimer as a preference component. [2] The method according to [1], wherein the heterodimer is conjugated to the G protein alpha subunit. [3] The method as described in [2], wherein the G protein alpha subunit is Gα15 or Gα16 alone, or the C-terminal 5-residue, 25-residue or 44-residue of Gα15 or Gα16 belongs to Gi, Gq or A chimeric protein in which the corresponding C-terminal residues of members of the Gs family are replaced. [4] The method as described in 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 containing the amino acid sequence described in SEQ ID No. 1, 3, 5, 7 or 9; (a2) A protein encoded by a gene containing the base sequence described in SEQ ID No. 2, 4, 6, 8 or 10; (a3) A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence described in SEQ ID No. 1, 3, 5, 7 or 9 and responding to L-amino acid; (a4) A protein encoded by a gene containing a base sequence having 80% or more identity with the base sequence described in SEQ ID No. 2, 4, 6, 8 or 10, and responding to L-amino acid; (a5) The amino acid sequence obtained by deleting, replacing and/or appending one or more amino acids of the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7 or 9 Proteins whose base acids respond; or (a6) A protein encoded by a base sequence that hybridizes to the base sequence described in SEQ ID NO. 2, 4, 6, 8 or 10 under stringent conditions and responds to L-amino acid. [5] The method as described in [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. [6] The method as described in 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 containing the amino acid sequence described in SEQ ID NO: 11; (b2) A protein encoded by a gene containing the base sequence described in SEQ ID NO: 12; (b3) A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence described in SEQ ID NO: 11 and responding to L-amino acid; (b4) A protein encoded by a gene containing a base sequence having 80% or more identity with the base sequence described in SEQ ID NO: 12, and responding to L-amino acids; (b5) A protein comprising one or more amino acid deletions, replacements and/or additions of the amino acid sequence described in SEQ ID NO: 11 and a protein that responds to L-amino acid; or (b6) A protein encoded by a base sequence that hybridizes with the base sequence described in SEQ ID NO. 12 under stringent conditions and responds to L-amino acid. [7] The method as described in [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 luminescent protein. [9] The method according to [8], wherein the luminescent protein is aequorin. [10] A feed for eel, which contains a preference component selected using the method described in any one of [1] to [9]. [11] A feed for eel, which contains at least one of creatinine, N,N-dimethylglycine and creatine as a preference component. [12] The feed as described in [11], wherein the eel is Japanese eel (A. japonica) or European eel (A. anguilla), Indonesian eel (A. bicolor bicolor) or New Guinea eel (A. bicolor pacifica). [13] The feed as described in any one of [10] to [12], which is for larval fish, juvenile fish, non-adult fish or adult fish. [Effect of invention]
根據本發明，可篩選直接作用於鰻魚之味覺受體之物質作為嗜好性成份，因此可探索迄今為止於鰻魚之養殖中未使用之新穎成份代替鯊魚之卵等先前之成份。According to the present invention, substances directly acting on the taste receptors of eels can be screened as hobby components, so that novel components that have not been used in eel farming so far can be used to replace previous components such as shark eggs.
(嗜好性成份之篩選方法) 於第一實施形態中，本發明提供一種篩選用於鰻魚之嗜好性成份之方法，其包括： (a)使包含味覺受體T1R2家族之任一成員及T1R3家族之任一成員之異質二聚物與嗜好性成份之候選物質接觸之步驟；及 (b)將使異質二聚物之活性增大之候選物質評價為嗜好性成份之步驟。(Screening method for preference components) In the first embodiment, the present invention provides a method for screening preference components for eel, which includes: (a) The step of contacting a heterodimer comprising any member of the taste receptor T1R2 family and any member of the T1R3 family with a candidate substance of a preference component; and (b) A step of evaluating candidate substances that increase the activity of the heterodimer as a preference component.
於本說明書中使用之情形時，「用於鰻魚之嗜好性成份」意指不含其候選物質等配位基之陰性對照、例如於與緩衝液之比較中顯著作用於鰻魚之味覺受體之物質、或者鰻魚喜歡之呈味物質或促進鰻魚之飲食之成份。再者，作為鰻魚喜歡之呈味物質或促進鰻魚之飲食之成份，已知有L-胺基酸，例如丙胺酸、精胺酸、甘胺酸、脯胺酸、離胺酸、絲胺酸、組胺酸及甜菜鹼等。亦可使用此種既知之嗜好性成份作為篩選之指標。In the case of use in this specification, "preference component for eel" means a negative control that does not contain ligands such as its candidate substance, for example, it is significantly used in the taste receptor of eel in comparison with buffer Substances, or taste substances preferred by eels or ingredients that promote eel diets. Furthermore, L-amino acids, such as alanine, arginine, glycine, proline, lysine, serine, are known as flavoring substances that eels like or ingredients that promote eel diets , Histidine and betaine. This known preference component can also be used as a screening indicator.
於本說明書中使用之情形時，「鰻魚」意指廣泛鰻魚目魚類、尤其是鰻魚屬之魚類(Anguilla)中，具有包含味覺受體T1R2家族之任一成員及T1R3家族之任一成員之異質二聚物者。只要發揮本發明之效果，則亦可使用其他T1R家族代替T1R2家族。若按照棲息地進行分類，則例如主要之鰻魚大致分為日本鰻魚(A. japonica)、歐洲鰻魚(A. anguilla)、美洲鰻魚(A. rostorata)、花鰻魚(A. marmorata)這4種。作為其他鰻魚，亦可列舉：新幾內亞鰻魚(A. bicolor pacifica)、印度尼西亞鰻魚(A. bicolor bicolor)、非洲鰻(A. mossambica)、澳洲鰻魚(A. australis australis)、新澳鰻魚(A. australis schmidtii)、雷恩東澳鰻(A. reinhardtii)、西里伯斯鰻(A. celebesensis)、大口鰻魚(A. megastoma)、灰鰻(A. obscura)、內唇鰻(A. interioris)、雲紋鰻魚(A. nebulosa)、紐西蘭大鰻(A. diffenbachii)、呂宋鰻(A. luzonensis)、孟加拉鰻魚(A. bengalensis bengalensis)、東印度洋鰻魚(A.bengalensis labiata)、短頭鰻魚(A. breviceps)、烏耳鰻魚(A. nigricans)、印尼長鰭鰻魚(A. malgumora)等。作為食用，主要為日本鰻魚及歐洲鰻魚這兩種。As used in this manual, "eel" means a wide range of eel fishes, especially fish of the genus Eel (Anguilla), with heterogeneity including any member of the T1R2 family of taste receptors and any member of the T1R3 family Dimers. As long as the effect of the present invention is exerted, other T1R families may be used instead of the T1R2 family. When classified according to habitat, for example, the main eels are roughly classified into four types: Japanese eel (A. japonica), European eel (A. anguilla), American eel (A. rostorata), and flower eel (A. marmorata). As other eels, New Guinea eels (A. bicolor pacifica), Indonesian eels (A. bicolor bicolor), African eels (A. mossambica), Australian eels (A. australis australis), and New Australia eels (A. bicolor pacifica) australis schmidtii), Rennes East Australian eel (A. reinhardtii), West ribe eel (A. celebesensis), large mouth eel (A. megastoma), gray eel (A. obscura), inner lip eel (A. interioris), Moray eel (A. nebulosa), New Zealand big eel (A. diffenbachii), Luzon eel (A. luzonensis), Bengal eel (A. bengalensis bengalensis), East Indian eel (A. bengalensis labiata), short head Eel (A. breviceps), ear eel (A. nigricans), Indonesian albacore eel (A. malgumora), etc. As food, they are mainly Japanese eel and European eel.
鰻魚包含除上述以外之鰻魚目魚類、康吉鰻、蠕紋裸胸鱔、海鰻等，例如本發明之鰻魚亦可包含星康吉鰻(Conger. myriaster)、日本康吉鰻(C. japonica)、米克氏康吉鰻(Ariosoma meeki)、尼氏頜吻鰻(Gnathophis nystromi nystoromi)、柯氏合鰓鰻(Synaphobranchus kaupii)、蠕紋裸胸鱔(Gymnothorax kidako)、海鰻(Muraenesox cinereus)及百吉海鰻(Muraenesox bagio)。The eel includes eel-order fish other than the above, conger eel, porpoise eel, moray, etc. For example, the eel of the present invention may also include conger. myriaster, Japanese conger eel (C. japonica), rice Ariosoma meeki, Gnathophis nystromi nystoromi, Synaphobranchus kaupii, Gymnothorax kidako, Morayesox cinereus, and bagel moray (Muraenesox bagio).
上述鰻魚中，日本鰻魚於遠洋孵化，經過前柳葉型鰻之孵化仔魚，成為稱為狹首型幼魚或仔魚之透明浮游幼體，經由稚魚(幼鰻、未成年鰻)成為成魚。預想飲食篩選之嗜好性成份之鰻魚只要表現T1R2家族之任一成員及T1R3家族之成員，則可為任一生長階段者，又，亦可為人工孵化者、天然者之任一者。Among the above-mentioned eels, Japanese eels incubate in the ocean, and the hatchling larvae after the pre-willow-leaf eels become transparent planktonic larvae known as narrow-headed larvae or larvae, and become adult fishes through juvenile fish (eel eels, juvenile eels). The eel, which is expected to be a favorite component of diet screening, can be of any growth stage as long as it represents any member of the T1R2 family and a member of the T1R3 family, and can also be any of artificial incubators and natural ones.
味覺受體蛋白質 對味覺之受體係稱為7次跨膜型蛋白質之膜蛋白質，由於與G蛋白質連結，故而亦稱為G蛋白質共軛受體(GPCR)。味覺受體蛋白質根據其結構大致分為具有較大之細胞外區域者及不具有該區域者這兩種，前者稱為T1R家族(Taste Receptor type-1)，後者稱為T2R家族(Taste Receptor type-2)。T1R家族包含T1R1、T1R2及T1R3這3個分子種類。 Taste receptor protein The receptor system for taste is called the membrane protein of the 7th transmembrane type protein, and it is also called G protein conjugated receptor (GPCR) because it is linked to G protein. Taste receptor proteins are roughly divided into those with a large extracellular region and those without a region according to their structure. The former is called the T1R family (Taste Receptor type-1), and the latter is called the T2R family (Taste Receptor type -2). The T1R family includes three molecular species: T1R1, T1R2, and T1R3.
關於鰻魚之T1R，本發明人等獲取5種T1R2之全長序列及1種T1R3之全長序列，分別命名為AjT1R2a、AjT1R2b-1、AjT1R2b-2、AjT1R2b-3、AjT1R2b-4、AjT1R3。該等AjT1R2a、AjT1R2b-1、AjT1R2b-2、AjT1R2b-3、AjT1R2b-4、AjT1R3分別具有序列編號1、3、5、7、9、11之胺基酸序列及序列編號2、4、6、8、10、12之鹼基序列。將獲得之全長胺基酸序列之對準示於圖1。Regarding the T1R of eel, the inventors obtained five full-length sequences of T1R2 and one full-length sequence of T1R3, and named them AjT1R2a, AjT1R2b-1, AjT1R2b-2, AjT1R2b-3, AjT1R2b-4, AjT1R3. The AjT1R2a, AjT1R2b-1, AjT1R2b-2, AjT1R2b-3, AjT1R2b-4, AjT1R3 have the amino acid sequences of
關於上述6種序列，若T1R2及T1R3之序列與既知之魚類之各序列相比較，則T1R2具有44.2～52.0%之同源性，T1R3具有51.5～53.7%之同源性(表1)。
[表1]
又，關於日本鰻魚T1R間之同源性，T1R2彼此為76.9～91.3%，T1R2及T1R3為30.0～31.2%(表2)。
[表2]
根據獲得之全長序列製成分子系統樹，結果明確皆不是哺乳類之T1R簇，而包含於魚類T1R之簇(結果未示出)。Based on the obtained full-length sequence, a molecular phylogenetic tree was prepared, and the results clearly showed that they were not mammalian T1R clusters, but included in fish T1R clusters (results not shown).
於哺乳類中，T1R1、T1R2、T1R3家族分別發現1個成員，由T1R1＋T1R3接受鮮味物質，由T1R2＋T1R3接受甜味物質。另一方面，於迄今為止關於味覺受體有報告之稻田魚及虎河豚等魚類中，T1R1、T1R3家族存在1個成員，T1R2家族存在複數個成員，已知除T1R1＋T1R3以外，T1R2＋T1R3亦接受鮮味物質之例。考慮於鰻魚中，亦與其他魚類同樣地藉由發生基因重複而T1R2多樣化之可能性。再者，此次探索時，作為對象之基因組數據庫之基因組草圖包含不完全序列，而且並非覆蓋所有之基因組序列，因此認為除此次發現之序列以外，亦充分具有存在成為受體候選之基因序列之可能性。In mammals, one member of the T1R1, T1R2, and T1R3 families were found, T1R1 + T1R3 receiving umami substances, and T1R2 + T1R3 receiving sweet substances. On the other hand, in the fish such as rice field fish and tiger puffer fish which have been reported on taste receptors so far, there is one member in the T1R1 and T1R3 families, and there are multiple members in the T1R2 family. Examples of substances. Considering the possibility of eel divergence in T1R2 by gene duplication as in other fish. In addition, during this exploration, the genomic sketch of the target genomic database contains incomplete sequences and does not cover all genomic sequences. Therefore, in addition to the sequences discovered this time, it is considered that there are sufficient gene sequences that exist as receptor candidates. Possibility.
於本發明中，與嗜好性成份之候選物質接觸之味覺受體係包含AjT1R2a、AjT1R2b-1、AjT1R2b-2、AjT1R2b-3、AjT1R2b-4等T1R2家族之任一成員及AjT1R3等T1R3家族之任一成員之異質二聚物。In the present invention, the taste-receptive system in contact with the candidate substance of the preference component includes any member of the T1R2 family such as AjT1R2a, AjT1R2b-1, AjT1R2b-2, AjT1R2b-3, AjT1R2b-4 and any one of the T1R3 family such as AjT1R3 Heterogeneous dimer of members.
AjT1R2a與AjT1R3之組合對L-丙胺酸、L-絲胺酸、L-精胺酸、甘胺酸、L-脯胺酸作出應答。AjT1R2b-1與AjT1R3之組合對L-脯胺酸、L-丙胺酸作出應答。AjT1R2b-1與AjT1R3之組合對肌胺酸、肌酸酐、N,N-二甲基甘胺酸亦作出應答。AjT1R2b-2與AjT1R3之組合對L-組胺酸、L-絲胺酸、L-苯丙胺酸、L-丙胺酸、L-天冬醯胺作出應答。AjT1R2b-4與AjT1R3之組合對L-丙胺酸、甘胺酸、L-絲胺酸、L-脯胺酸作出應答。The combination of AjT1R2a and AjT1R3 responds to L-alanine, L-serine, L-arginine, glycine, and L-proline. The combination of AjT1R2b-1 and AjT1R3 responds to L-proline and L-alanine. The combination of AjT1R2b-1 and AjT1R3 also responded to creatine, creatinine, and N,N-dimethylglycine. The combination of AjT1R2b-2 and AjT1R3 responds to L-histidine, L-serine, L-phenylalanine, L-alanine, and L-aspartamide. The combination of AjT1R2b-4 and AjT1R3 responds to L-alanine, glycine, L-serine, and L-proline.
T1R2家族之成員可選自由以下之(a1)～(a6)所組成之群： (a1)包含序列編號1、3、5、7或9中記載之胺基酸序列之蛋白質； (a2)由包含序列編號2、4、6、8或10中記載之鹼基序列之基因編碼之蛋白質； (a3)包含與序列編號1、3、5、7或9中記載之胺基酸序列具有80%以上之同一性之胺基酸序列，且對L-胺基酸作出應答之蛋白質； (a4)由包含與序列編號2、4、6、8或10中記載之鹼基序列具有80%以上之同一性之鹼基序列之基因編碼，且對L-胺基酸作出應答之蛋白質； (a5)包含序列編號1、3、5、7或9中記載之胺基酸序列之1個或複數個胺基酸缺失、置換及/或附加所得之胺基酸序列，且對L-胺基酸作出應答之蛋白質；或者 (a6)由與序列編號2、4、6、8或10中記載之鹼基序列於嚴格之條件下雜交之鹼基序列編碼，且對L-胺基酸作出應答之蛋白質。Members of the T1R2 family can choose from the following groups (a1) to (a6): (a1) A protein containing the amino acid sequence described in SEQ ID No. 1, 3, 5, 7 or 9; (a2) A protein encoded by a gene containing the base sequence described in SEQ ID No. 2, 4, 6, 8 or 10; (a3) A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence described in SEQ ID No. 1, 3, 5, 7 or 9 and responding to L-amino acid; (a4) A protein encoded by a gene containing a base sequence having 80% or more identity with the base sequence described in SEQ ID No. 2, 4, 6, 8 or 10, and responding to L-amino acid; (a5) The amino acid sequence obtained by deleting, replacing and/or appending one or more amino acids of the amino acid sequence described in SEQ ID NOs: 1, 3, 5, 7 or 9 Proteins whose base acids respond; or (a6) A protein encoded by a base sequence that hybridizes to the base sequence described in SEQ ID NO. 2, 4, 6, 8 or 10 under stringent conditions and responds to L-amino acid.
T1R3家族之成員可選自由以下之(b1)～(b6)所組成之群： (b1)包含序列編號11中記載之胺基酸序列之蛋白質； (b2)由包含序列編號12中記載之鹼基序列之基因編碼之蛋白質； (b3)包含與序列編號11中記載之胺基酸序列具有80%以上之同一性之胺基酸序列，且對L-胺基酸作出應答之蛋白質； (b4)由包含與序列編號12中記載之鹼基序列具有80%以上之同一性之鹼基序列之基因編碼，且對L-胺基酸作出應答之蛋白質； (b5)包含序列編號11中記載之胺基酸序列之1個或複數個胺基酸缺失、置換及/或附加所得之胺基酸序列，且對L-胺基酸作出應答之蛋白質；或者 (b6)由與序列編號12中記載之鹼基序列於嚴格之條件下雜交之鹼基序列編碼，且對L-胺基酸作出應答之蛋白質。Members of the T1R3 family can choose from the following groups (b1) to (b6): (b1) A protein containing the amino acid sequence described in SEQ ID NO: 11; (b2) A protein encoded by a gene containing the base sequence described in SEQ ID NO: 12; (b3) A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence described in SEQ ID NO: 11 and responding to L-amino acid; (b4) A protein encoded by a gene containing a base sequence having 80% or more identity with the base sequence described in SEQ ID NO: 12, and responding to L-amino acids; (b5) A protein comprising one or more amino acid deletions, replacements and/or additions of the amino acid sequence described in SEQ ID NO: 11 and a protein that responds to L-amino acid; or (b6) A protein encoded by a base sequence that hybridizes with the base sequence described in SEQ ID NO. 12 under stringent conditions and responds to L-amino acid.
胺基酸序列只要為對L-胺基酸等既知之嗜好性成份或藉由篩選鑑定之嗜好性成份之刺激作出應答者，則亦可為包含於如序列編號1、3、5、7、9或11之野生型蛋白質所具有之胺基酸序列中，具有1至複數個，例如若將胺基酸序列中之胺基酸數100個設為一單元，則該每一單元具有1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20個、較佳為數個胺基酸之缺失、置換、附加等之胺基酸序列者。The amino acid sequence may also be included in
此處，胺基酸序列之「1至數個胺基酸之缺失、置換、附加」中之「1至數個」之範圍並無特別限定，意指上述每一單元較佳為1、2、3、4、5、6、7、8、9或10個左右、更佳為1、2、3、4或5個左右。又，「胺基酸之缺失」意指序列中之胺基酸殘基之缺失或消失，「胺基酸之置換」意指序列中之胺基酸殘基被置換成其他胺基酸殘基，「胺基酸之附加」意指以於序列中插入新胺基酸殘基之方式附加。Here, the range of "1 to several" in the "deletion, substitution, addition of 1 to several amino acids" of the amino acid sequence is not particularly limited, meaning that each unit above is preferably 1, 2 , 3, 4, 5, 6, 7, 8, 9 or 10, more preferably about 1, 2, 3, 4 or 5. Also, "deletion of amino acids" means the deletion or disappearance of amino acid residues in the sequence, and "substitution of amino acids" means that the amino acid residues in the sequence are replaced with other amino acid residues , "Addition of amino acids" means the addition by inserting new amino acid residues in the sequence.
作為「胺基酸之缺失、置換、附加」之具體態樣，可列舉如下態樣：以維持對L-胺基酸等既知之嗜好性成份以及藉由篩選鑑定之嗜好性成份之刺激之應答為限度，野生型中之胺基酸被其他化學上類似之胺基酸置換。例如，於將某疏水性胺基酸置換為其他疏水性胺基酸之情形時，可列舉將某極性胺基酸置換為具有相同電荷之其他極性胺基酸之情形等。此種化學上類似之胺基酸之每種胺基酸於該技術領域已知。Specific examples of the "deletion, replacement, and addition of amino acids" include the following: maintaining the response to the stimuli known for L-amino acids and other known preference components and the preference components identified by screening To limit, the amino acids in the wild type are replaced with other chemically similar amino acids. For example, when a certain 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 is mentioned. Each amino acid of such chemically similar amino acids is known in the art.
若列舉具體例，則作為非極性(疏水性)胺基酸，可列舉：丙胺酸、纈胺酸、異白胺酸、白胺酸、脯胺酸、色胺酸、苯丙胺酸、甲硫胺酸等。作為極性(中性)胺基酸，可列舉：甘胺酸、絲胺酸、蘇胺酸、酪胺酸、麩醯胺、天冬醯胺、半胱胺酸等。作為具有正電荷之鹼性胺基酸，可列舉：精胺酸、組胺酸、離胺酸等。又，作為具有負電荷之酸性胺基酸，可列舉：天冬胺酸、麩胺酸等。If specific examples are given, examples of the nonpolar (hydrophobic) amino acids include alanine, valine, isoleucine, leucine, proline, tryptophan, amphetamine, and methionine. Acid and so on. Examples of polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, aspartamide, and cysteine. Examples of the positively charged basic amino acids include spermine, histidine, and lysine. In addition, examples of the acidic amino acid having a negative charge include aspartic acid and glutamic acid.
又，於上述胺基酸序列中，作為「胺基酸之缺失、置換、附加」之具體態樣，可列舉與如序列編號1、3、5、7、9或11之野生型蛋白質所具有之胺基酸序列具有一定以上之序列同一性之胺基酸序列，例如可列舉與野生型蛋白質所具有之胺基酸序列具有75%以上、較佳為80%以上、更佳為85%以上、更佳為90%以上、最佳為95%以上之同一性之胺基酸序列。In addition, in the above-mentioned amino acid sequence, specific examples of "amino acid deletion, substitution, and addition" include wild type proteins such as
使用之鹼基序列於導入至宿主生物時，可為於鹼基序列之轉錄後經由剪接而生成所需之序列者，亦可為於轉錄後不經由剪接而生成所需之序列者。The base sequence used when introduced into the host organism may be one that generates the desired sequence by splicing after transcription of the base sequence, or may generate the desired sequence without splicing after transcription.
使用之鹼基序列亦可不與野生型序列完全相同，只要維持對L-胺基酸等既知之嗜好性成份或者藉由篩選鑑定之嗜好性成份之刺激之應答，亦可為與對野生型基因之鹼基序列互補之鹼基序列於嚴格之條件下雜交之鹼基序列。The base sequence used may not be exactly the same as the wild-type sequence, as long as it maintains a response to the known preference components such as L-amino acids or the preference components identified by screening, it can also be the same as the wild-type gene. The base sequence complementary to the base sequence that hybridizes under strict conditions.
本說明書中之「於嚴格之條件下雜交之鹼基序列」意指藉由使用相當於如序列編號2、4、6、8、10或12之野生型基因之鹼基序列之一部分之DNA作為探針，使用菌落雜交法、噬菌斑雜交法、南方墨點雜交法等所得之DNA之鹼基序列。The "base sequence that hybridizes under strict conditions" in this specification means that by using DNA equivalent to a part of the base sequence of a wild-type gene such as
本說明書中之「嚴格之條件」係指將特異性雜交之訊號與非特異性雜交之訊號明確識別之條件，視使用之雜交之系以及探針之種類、序列及長度而不同。此種條件可藉由改變雜交之溫度，改變洗淨之溫度及鹽濃度而決定。The "strict conditions" in this specification refer to the conditions for clearly identifying the signal of specific hybridization and the signal of non-specific hybridization, depending on the hybridization system used and the type, sequence and length of the probe. Such conditions can be determined by changing the temperature of hybridization, changing the washing temperature and the salt concentration.
例如，於強烈地檢測出非特異性雜交之訊號之情形時，可藉由提高雜交及洗淨之溫度，並且視需要降低洗淨之鹽濃度而提高特異性。又，於亦未檢測出特異性雜交之訊號之情形時，可藉由降低雜交及洗淨之溫度，並且視需要提高洗淨之鹽濃度，而使雜交穩定化。For example, when the signal of non-specific hybridization is strongly detected, the specificity can be improved by increasing the temperature of hybridization and washing, and reducing the concentration of the washed salt as necessary. In addition, when the signal of specific hybridization is not detected, the hybridization can be stabilized by lowering the temperature of hybridization and washing, and increasing the salt concentration of washing if necessary.
於一部分之態樣中，作為嚴格條件之具體例包含以下情形。例如，使用DNA探針作為探針，雜交使用5×SSC(Saline Sodium Citrate，檸檬酸鈉)、1.0%(w/v)核酸雜交用黏連試劑(Boehringer Mannheim公司製造)、0.1%(w/v)N-月桂醯肌胺酸、0.02%(w/v)SDS(Sodium Dodecyl Sulfate，十二烷基硫酸鈉)，進行一晩(8～16小時左右)。洗淨使用0.1～0.5×SSC、0.1%(w/v)SDS、較佳為0.1×SSC、0.1%(w/v)SDS，耗時15分鐘，實施2次。進行雜交及洗淨之溫度為65℃以上，較佳為68℃以上。In some aspects, specific examples of strict conditions include the following. For example, using a DNA probe as a probe, hybridization using 5×SSC (Saline Sodium Citrate, sodium citrate), 1.0% (w/v) nucleic acid hybridization adhesion reagent (made by Boehringer Mannheim), 0.1% (w/ v) N-lauryl sarcosinate, 0.02% (w/v) SDS (Sodium Dodecyl Sulfate, sodium dodecyl sulfate), for one night (about 8 to 16 hours). For washing, 0.1 to 0.5×SSC, 0.1% (w/v) SDS, preferably 0.1×SSC, 0.1% (w/v) SDS, which takes 15 minutes and is performed twice. The temperature at which hybridization and washing are performed is 65°C or higher, preferably 68°C or higher.
作為具有於嚴格之條件下雜交之鹼基序列之DNA，例如可列舉如下DNA等：藉由使用使具有來自菌落或噬菌斑之野生型基因之鹼基序列之DNA或該DNA之片段固定化之過濾器，於上述嚴格之條件下雜交所得；以及可藉由於0.5～2.0 M之NaCl存在下，於40～75℃實施雜交後，較佳為於0.7～1.0 M之NaCl存在下，於65℃下實施雜交後，使用0.1～1×SSC溶液(1×SSC溶液為150 mM之氯化鈉、15 mM之檸檬酸鈉)，於65℃條件下將過濾器洗淨而鑑定。探針之製備及雜交之方法可依據Moleular Cloning： A laboratory Manual, 2nd - Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989、Current Protocols in Molecular Biology, Supplement 1 - 38, John Wiley & Sons, 1987 - 1997等中記載之方法而實施。Examples of DNA having a base sequence that hybridizes under stringent conditions include, for example, the following DNA: immobilizing DNA having a base sequence of a wild-type gene derived from a colony or plaque or a fragment of the DNA The filter is obtained by hybridization under the above strict conditions; and can be performed by hybridization at 40-75°C in the presence of 0.5-2.0 M NaCl, preferably in the presence of 0.7-1.0 M NaCl at 65 After performing hybridization at ℃, 0.1 to 1×SSC solution (1×SSC solution is 150 mM sodium chloride and 15 mM sodium citrate) was used, and the filter was washed at 65°C for identification. Probe preparation and hybridization methods can be based on Molecular Cloning: A laboratory Manual, 2nd-Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., 1989, Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons, 1987-1997 and other methods described in the implementation.
再者，業者可除此種緩衝液之鹽濃度及溫度等條件以外，亦考慮其他探針濃度、探針長度、反應時間等諸條件，適當設定用以獲得具有與對野生型基因之鹼基序列互補之鹼基序列於嚴格之條件下雜交之鹼基序列之DNA之條件。Furthermore, in addition to the salt concentration and temperature of the buffer, the manufacturer can also consider other conditions such as probe concentration, probe length, reaction time, etc., and set them appropriately to obtain bases with wild-type genes. The condition of the DNA of the base sequence to which the sequence complementary base sequence hybridizes under strict conditions.
作為包含與對野生型基因之鹼基序列互補之鹼基序列於嚴格之條件下雜交之鹼基序列之DNA，可列舉與作為探針使用之野生型基因之鹼基序列具有一定以上之序列同一性之DNA，例如可列舉與野生型基因之鹼基序列具有75%以上、較佳為80%以上、更佳為85%以上、更佳為90%以上、進而較佳為95%以上之序列同一性之DNA。Examples of the DNA containing a base sequence complementary to the base sequence complementary to the base sequence of the wild-type gene under stringent conditions include the same sequence as the base sequence of the wild-type gene used as a probe with a certain degree or more DNA, for example, a sequence having a base sequence of 75% or more, preferably 80% or more, more preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more of the base sequence of the wild-type gene DNA of identity.
作為與對野生型基因之鹼基序列互補之鹼基序列於嚴格之條件下雜交之鹼基序列，例如包含如下鹼基序列，即若將鹼基序列中之鹼基數500個設為一單元，則於野生型基因之鹼基序列中，該每一單元具有1至複數個、例如1至125個、1至100個、1至75個、1至50個、1至30個、1至20個、較佳為1至數個、例如1、2、3、4、5、6、7、8、9或10個鹼基之缺失、置換、附加等之鹼基序列。The base sequence that is complementary to the base sequence complementary to the base sequence of the wild-type gene under strict conditions includes, for example, the following base sequence, that is, if the number of bases in the base sequence is 500 as a unit , In the base sequence of the wild-type gene, each unit has 1 to plural, such as 1 to 125, 1 to 100, 1 to 75, 1 to 50, 1 to 30, 1 to 20, preferably 1 to several, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base deletions, substitutions, additions, etc. base sequences.
於嚴格之條件中亦然，較佳為形成所謂之特異性雜交，不形成非特異性雜交之條件，即，高嚴格之條件。作為高嚴格條件，可列舉同一性高之核酸彼此雜交，但同一性較其為低之核酸彼此不雜交之條件。The same applies to stringent conditions, and it is preferable to form so-called specific hybridization, and to not form non-specific hybridization conditions, that is, highly stringent conditions. Examples of highly stringent conditions include conditions in which nucleic acids with high identity are hybridized with each other, but nucleic acids with lower identity are not hybridized with each other.
此處，「鹼基之缺失」意指序列中之鹼基欠缺或消失，「鹼基之置換」意指序列中之鹼基被置換成其他鹼基，「鹼基之附加」意指以插入新鹼基之方式附加。Here, "deletion of bases" means the lack or disappearance of bases in the sequence, "substitution of bases" means that the bases in the sequence are replaced with other bases, and "addition of bases" means to insert The new base is added.
認為由與對野生型基因之鹼基序列互補之鹼基序列於嚴格之條件下雜交之鹼基序列編碼之蛋白質，具有為以下蛋白質 (即具備於由野生型基因之鹼基序列編碼之蛋白質所具有之胺基酸序列中具有1至複數個、較佳為數個胺基酸之缺失、置換、附加等之胺基酸序列之蛋白質) 之或然性，但與由野生型基因之鹼基序列編碼之蛋白質具有相同效果。It is considered that a protein encoded by a base sequence that is complementary to a base sequence complementary to the base sequence of a wild-type gene under strict conditions has the following protein (that is, a protein encoded by a base sequence encoded by a wild-type gene Possibility of amino acid sequence having 1 to plural, preferably several amino acid deletions, substitutions, additions, etc. of amino acid sequence), but not with the base sequence of wild type gene The encoded protein has the same effect.
又，編碼蛋白質之基因亦可含有利用1個胺基酸所對應之密碼子具有數種，而編碼與野生型基因所編碼之酵素具有之胺基酸序列相同或近似之胺基酸序列，且不同於野生型基因之鹼基序列。鹼基序列亦可改變成根據使用之宿主之密碼子使用頻度而具有最適之密碼子。In addition, the protein-encoding gene may contain several amino acid codons corresponding to one amino acid, and the amino acid sequence that encodes the amino acid sequence that is the same as or similar to that of the enzyme encoded by the wild-type gene, and The base sequence is different from the wild type gene. The base sequence can also be changed to have the most suitable codon according to the frequency of codon usage of the host used.
求出鹼基序列及胺基酸序列之序列同一性之方法並無特別限定，但例如藉由如下方式求出：利用通常廣為人知之方法，使用用以將野生型基因或由野生型基因編碼之酵素之胺基酸序列與成為對象之鹼基序列或胺基酸序列對準，而算出兩者序列之一致率之程式。The method for determining the sequence identity of the base sequence and the amino acid sequence is not particularly limited, but, for example, it can be obtained by using a method that is commonly known and used to encode or encode a wild-type gene. The amino acid sequence of the enzyme is aligned with the target base sequence or amino acid sequence, and the formula for calculating the coincidence rate of the two sequences.
作為用以算出2個胺基酸序列或鹼基序列之一致率之程式，例如已知Karlin及Altschul之演算法(Proc. Natl. Acad. Sci. USA 87: 2264 - 2268、1990；Proc. Natl. Acad. Sci. USA 90: 5873 - 5877、1993)，使用該演算法之BLAST(Basic Local Alignment Search Tool，基本局部對準搜索工具)程式由Altschul等開發(J. Mol. Biol. 215: 403 - 410、1990)。進而，亦已知由BLAST感度良好地決定序列同一性之程式即Gapped BLAST(Nucleic Acids Res. 25: 3389 - 3402、1997)。因此，業者例如可利用上述程式，自資料庫中檢索對於提供之序列顯示較高之序列同一性之序列。該等可例如可於美國國家生物技術信息中心(National Center for Biotechnology Information)之網際網路上之web網站(http://blast.ncbi.nlm.nih.gov/Blast.cgi)上利用。As a program for calculating the agreement rate of two amino acid sequences or base sequences, for example, the algorithm of Karlin and Altschul is known (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990; Proc. Natl Acad. Sci. USA 90: 5873-5877, 1993), the BLAST (Basic Local Alignment Search Tool) program using this algorithm was developed by Altschul et al. (J. Mol. Biol. 215: 403 -410, 1990). Furthermore, Gapped BLAST (Nucleic Acids Res. 25: 3389-3402, 1997), which is a program for determining sequence identity with good BLAST sensitivity, is also known. Therefore, for example, the operator can use the above program to retrieve a sequence from the database that shows a higher sequence identity for the provided sequence. These can be used, for example, on a web site (http://blast.ncbi.nlm.nih.gov/Blast.cgi) on the Internet of the National Center for Biotechnology Information.
上述各方法通常可用以自資料庫中檢索顯示序列同一性之序列，但作為決定個別序列之序列同一性之方法，亦可使用Genetyx網路版version 12. 0. 1(GENETYX公司製造)之同源性分析。該方法基於Lipman-Pearson法(Science 227: 1435 - 1441，1985)。分析鹼基序列之序列同一性時，可使用編碼蛋白質之區域(CDS(Coding Sequence，編碼序列)或ORF(Open Reading Frame，開放閱讀框架))。The above methods can usually be used to retrieve sequences showing sequence identity from the database, but as a method for determining the sequence identity of individual sequences, Genetyx Internet version 12.0.1 (manufactured by GENETYX) can also be used. Source analysis. This method is based on the Lipman-Pearson method (Science 227: 1435-1441, 1985). When analyzing the sequence identity of a base sequence, a region encoding a protein (CDS (Coding Sequence, Coding Sequence) or ORF (Open Reading Frame)) can be used.
G 蛋白質 包含T1R2及T1R3之異質二聚物與於味覺受體細胞特異地表現之異型三聚物之G蛋白質共軛，因此於篩選時可於細胞內共同表現異質二聚物及G蛋白質。作為此種G蛋白質，已知Gq家族、味蛋白(Gustducin)、轉導蛋白(Transducin)等。 G protein The heterodimer including T1R2 and T1R3 is conjugated with the G protein of the heterotrimer specifically expressed in taste receptor cells, so that the heterodimer and G protein can be expressed together in the cell during screening. As such G proteins, the Gq family, Gustducin, Transducin, etc. are known.
G蛋白質包含Gα(α次單元)及Gβγ次單元。篩選嗜好性成份時，該等之中至少表現Gα次單元即可。又，亦可與Gα次單元一起表現Gβγ次單元。已知Gα次單元包含Gi、Gq或Gs、G12/13之基團。其中，可較佳地使用單獨之Gα15或Gα16、或者其等之C末端5殘基、25殘基或44殘基由屬於Gi、Gq或Gs之家族之成員的對應之C末端殘基置換之嵌合蛋白質。此種C末端殘基之置換為業者所公知(例如參照Li et al., PNAS April 2, 2002. 99 (7) 4692 - 469等)。認為如此藉由置換C末端殘基而容易與受體偶合。The G protein includes Gα (α subunit) and Gβγ subunit. When screening preference components, at least Gα subunits should be represented among these. In addition, the Gβγ subunit may be expressed together with the Gα subunit. It is known that the Gα subunit contains groups of Gi, Gq or Gs, and G12/13. Among them, it may be preferable to use Gα15 or Gα16 alone, or the C-terminal 5 residues, 25 residues or 44 residues of which are replaced by corresponding C-terminal residues belonging to members of the Gi, Gq or Gs family Chimeric protein. Such substitution of C-terminal residues is well known to the industry (for example, refer to Li et al., PNAS April 2, 2002. 99 (7) 4692-469, etc.). It is considered that by replacing the C-terminal residue, it is easy to couple with the receptor.
「對應之C末端殘基」意指例如於Gα15之C末端序列5胺基酸被置換成其他Gα次單元之情形時，構成該Gα次單元之C末端序列之5胺基酸。又，關於嵌合蛋白質之記法，例如rG15-rGi3-5之表述意指大鼠Gα15(以下，有時將Gα15記為「G15」，有時將大鼠Gα15記為「rG15」、將小鼠Gα15記為「mG15」、將人Gα16記為「hG16」等)之C末端序列5胺基酸被大鼠Gi3之C末端序列5胺基酸置換。"Corresponding C-terminal residue" means, for example, when the 5 amino acid of the C-terminal sequence of Gα15 is replaced with another Gα subunit, the 5 amino acid constituting the C-terminal sequence of the Gα subunit. In addition, regarding the notation of chimeric protein, for example, the expression rG15-rGi3-5 means rat Gα15 (hereinafter, sometimes Gα15 is referred to as “G15”, and sometimes rat Gα15 is referred to as “rG15”, and mouse Gα15 is denoted as “mG15”, and human Gα16 is denoted as “hG16” etc.) the C-terminal sequence 5 amino acid is replaced with the C-terminal sequence 5 amino acid of rat Gi3.
於G蛋白質之選擇時，可參照公知文獻，例如關於rG15-rGi2-5，可參照Two distinct determinants of ligand specificity in T1R1/T1R3 (the umami taste receptor) Toda Y, Nakagita T, Hayakawa T, Okada S, Narukawa M, Imai H, Ishimaru Y, Misaka T. J Biol Chem. 2013 Dec 27; 288 (52): 36863-77. doi等，又，關於mG15，可參照Sensory biology. Evolution of sweet taste perception in hummingbirds by transformation of the ancestral umami receptor. Baldwin MW, Toda Y, Nakagita T, O'Connell MJ, Klasing KC, Misaka T, Edwards SV, Liberles SD. Science. 2014 Aug 22; 345 (6199): 929 - 33. doi: 10. 1126/science. 1255097等。For the selection of G protein, you can refer to well-known literature, such as rG15-rGi2-5, you can refer to Two distinct determinants of ligand specificity in T1R1/T1R3 (the umami taste receptor) Toda Y, Nakagita T, Hayakawa T, Okada S, Narukawa M, Imai H, Ishimaru Y, Misaka T. J Biol Chem. 2013 Dec 27; 288 (52): 36863-77. doi, etc. Also, regarding mG15, please refer to Sensory biology. Evolution of sweet taste perception in hummingbirds by transformation of the ancestral umami receptor. Baldwin MW, Toda Y, Nakagita T, O'Connell MJ, Klasing KC, Misaka T, Edwards SV, Liberles SD. Science. 2014 Aug 22; 345 (6199): 929-33. doi: 10. 1126/science. 1255097 and so on.
將主要G蛋白質之登錄號(NCBI參考序列)示於以下。 小鼠G15：NM_010304 大鼠G15：NM_053542 人G16：NM_002068 人Ggust：NM_001102386 大鼠Ggust：NM_173139 大鼠Gi2：NM_031035 大鼠Gi3：NM_013106 大鼠Gs：NM_019132The accession numbers of major G proteins (NCBI reference sequence) are shown below. Mouse G15: NM_010304 Rat G15: NM_053542 G16: NM_002068 Ggust: NM_001102386 Rat Ggust: NM_173139 Rat Gi2: NM_031035 Rat Gi3: NM_013106 Rat Gs: NM_019132
實施例中使用之嵌合蛋白質之序列資訊記載於以下。 胺基酸序列： rG15-rGi3-5(序列編號13)； rG15-rGi2-5(序列編號15)； hG16-rGi3-5(序列編號17)； mG15(序列編號19)； rG15(序列編號21)； rG15-rGgust-5(序列編號23)； hG16(序列編號25)； hG16-rGgust-25(序列編號27)； hG16-rGi2-44(序列編號29)； hG16-rGs-25(序列編號31)； hG16-rGs-44(序列編號33)； rG15-rGgust-25(序列編號35)The sequence information of the chimeric protein used in the examples is described below. Amino acid sequence: rG15-rGi3-5 (serial number 13); rG15-rGi2-5 (sequence number 15); hG16-rGi3-5 (sequence number 17); mG15 (serial number 19); rG15 (serial number 21); rG15-rGgust-5 (serial number 23); hG16 (serial number 25); hG16-rGgust-25 (serial number 27); hG16-rGi2-44 (serial number 29); hG16-rGs-25 (serial number 31); hG16-rGs-44 (sequence number 33); rG15-rGgust-25 (Serial number 35)
鹼基序列： rG15-rGi3-5(序列編號14)； rG15-rGi2-5(序列編號16)； hG16-rGi3-5(序列編號18)； mG15(序列編號20)； rG15(序列編號22)； rG15-rGgust-5(序列編號24)； hG16(序列編號26)； hG16-rGgust-25(序列編號28)； hG16-rGi2-44(序列編號30)； hG16-rGs-25(序列編號32)； hG16-rGs-44(序列編號34)； rG15-rGgust-25(序列編號36)Base sequence: rG15-rGi3-5 (sequence number 14); rG15-rGi2-5 (sequence number 16); hG16-rGi3-5 (serial number 18); mG15 (serial number 20); rG15 (serial number 22); rG15-rGgust-5 (serial number 24); hG16 (serial number 26); hG16-rGgust-25 (serial number 28); hG16-rGi2-44 (serial number 30); hG16-rGs-25 (serial number 32); hG16-rGs-44 (sequence number 34); rG15-rGgust-25 (Serial number 36)
篩選中使用之蛋白質可藉由如下方式製備：將各鹼基序列單獨或以共同任意組合以可作用之方式連結，於所需之細胞內表現。各序列可插入至相同或不同之載體內。The proteins used in the screening can be prepared by linking each base sequence individually or in any combination together in a functional manner and performing in the desired cells. Each sequence can be inserted into the same or different vectors.
編碼異質二聚物之鹼基序列可於適當之宿主、例如人等哺乳動物、青蛙等兩栖類動物細胞、昆蟲細胞或酵母中表現。較佳為宿主為昆蟲或哺乳動物等之細胞。更佳為哺乳動物細胞為人細胞。哺乳動物細胞之例有人胚胎腎(HEK，Human Embryonic Kidney)、例如HEK293T、馬-達二氏犬腎(MDCK，Madin-Darby Canine Kidney)、中國倉鼠卵巢細胞(CHO)等。The base sequence encoding the heterodimer can be expressed in a suitable host, such as mammals such as humans, amphibians cells such as frogs, insect cells, or yeast. Preferably, the host is a cell such as an insect or mammal. More preferably, the mammalian cells are human cells. Examples of mammalian cells include human embryonic kidney (HEK, Human Embryonic Kidney), such as HEK293T, Madin-Darby Canine Kidney (MDCK), Chinese hamster ovary cells (CHO), and the like.
對宿主細胞導入編碼異質二聚物之鹼基序列可使用公知之方法、例如Sambrook, J., Fritsch, E. F., and Maniatis, T., “Molecular Cloning A Laboratory Manual, Second Edition”, Cold Spring Harbor Laboratory Press, (1989)等中記載之技術進行。For introducing a base sequence encoding a heterodimer into a host cell, a well-known method such as Sambrook, J., Fritsch, EF, and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory can be used Press, (1989) and other techniques described in.
本發明之篩選方法除以下步驟以外，可包含用於篩選之任意步驟。 (a)使包含味覺受體T1R2家族之任一成員及T1R3家族之任一成員之異質二聚物與嗜好性成份之候選物質接觸之步驟；及 (b)將使異質二聚物之活性增大之候選物質評價為嗜好性成份之步驟The screening method of the present invention may include any steps for screening in addition to the following steps. (a) The step of contacting a heterodimer comprising any member of the taste receptor T1R2 family and any member of the T1R3 family with a candidate substance of a preference component; and (b) Steps for evaluating candidate substances that increase the activity of heterodimers as hobby components
異質二聚物之活性增大、例如細胞中之鈣離子濃度之增大可使用鈣離子測定試劑等測定。作為此種試劑，例如較佳為鈣敏感性之發光蛋白質、具體而言根據鈣離子之濃度發光之水母發光蛋白等蛋白質。亦可使用基於圖像觀察受體之活化，自獲得之圖像計數應答細胞數，判斷應答程度之鈣成像代替試劑。作為此種測定活化之機器，有CCD(Charge-Coupled Device，電荷耦合裝置)攝影機CoolSNAP HQ2(NIPPON ROPER)、倒置顯微鏡IX-81(OLYMPUS)，而且，作為用於成像之軟體，有MetaMorph, MetaFlour(分子裝置)等。The increase in the activity of the heterodimer, for example, the increase in the concentration of calcium ions in the cells, can be measured using a calcium ion measurement reagent. As such a reagent, for example, a calcium-sensitive luminescent protein, specifically, a protein such as aequorin that emits light according to the concentration of calcium ions. It is also possible to use calcium imaging instead of reagents to observe the activation of the receptor based on the image, count the number of responding cells from the obtained image, and judge the degree of response. As such a device for measuring activation, there are a CCD (Charge-Coupled Device, charge coupled device) camera CoolSNAP HQ2 (NIPPON ROPER), an inverted microscope IX-81 (OLYMPUS), and as software for imaging, MetaMorph, MetaFlour (Molecular device) etc.
除此以外，亦可使用將孔板上之細胞應答作為數值獲取之孔分析。此種孔分析可使用FlexStation3(分子裝置)、FLIPR(分子裝置)等。In addition to this, well analysis using the cell response on the well plate as a value can also be used. For such pore analysis, FlexStation3 (molecular device), FLIPR (molecular device), etc. can be used.
如T1R2及T1R3之GPCR若與配位基、即嗜好性成份結合，則引起結構變化(活化)，叫出如味蛋白之G蛋白質，除細胞內鈣之上升以外，亦誘導各種下流訊號(例如，GTP結合於環狀AMP(Adenosine Monophosphate，單磷酸腺苷)濃度之變動低分子量G蛋白質Rho等)。藉由檢測該等細胞內事件，亦可測定異質二聚物之活性。If the GPCRs of T1R2 and T1R3 are combined with ligands, that is, hobby components, they will cause structural changes (activation), called G proteins like taste protein, in addition to the increase of intracellular calcium, it also induces various downstream signals (such as , GTP binds to cyclic AMP (Adenosine Monophosphate, adenosine monophosphate) concentration changes low molecular weight G protein Rho, etc.). By detecting these intracellular events, the activity of the heterodimer can also be determined.
於評價為嗜好性成份之步驟中，作為對照組，可使用不含有候選物質等配位基之樣品、例如緩衝液。可將於與此種對照組之比較中活性為相同程度或其以上之候選物質評價為嗜好性成份。亦可使用L-胺基酸、例如L-丙胺酸、L-精胺酸、甘胺酸、L-脯胺酸、L-離胺酸、L-絲胺酸、L-組胺酸等既知之嗜好性成份代替緩衝液等陰性對照。In the step of evaluating as a preference component, as a control group, a sample that does not contain a ligand such as a candidate substance, such as a buffer solution, can be used. Candidate substances with the same or higher activity in comparison with such a control group can be evaluated as addictive components. L-amino acids such as L-alanine, L-arginine, glycine, L-proline, L-ionine, L-serine, and L-histidine can also be used Preference components replace the negative controls such as buffer.
(用於鰻魚之飼料) 於第二實施形態中，本發明提供一種包含使用上述方法篩選之嗜好性成份之用於鰻魚之飼料。(For eel feed) In the second embodiment, the present invention provides a feed for eel containing the preference components screened by the above method.
飼料可為仔魚用、稚魚用、成魚用之任一者。認為鰻魚之仔魚於在水溫23℃下孵化後第7天左右消化酵素之分泌變得活躍，因此若為此種階段，則本發明之飼料有用。The feed can be any of larval fish, juvenile fish and adult fish. The larvae of eel are believed to be active in the secretion of digestive enzymes on the seventh day after hatching at a water temperature of 23°C. Therefore, if it is at this stage, the feed of the present invention is useful.
作為藉由本發明篩選之嗜好性成份，有肌胺酸、N,N-二甲基甘胺酸或肌酸酐、或者其等之類似物等。
作為嗜好性成份，亦可含有肌胺酸、N,N-二甲基甘胺酸或肌酸酐中之一種或兩種以上。
[化1]
有肌胺酸為雪蟹腿肉汁之成份之一種(河合，2011)，河豚作出應答之報告(Kiyohara et al., 1985)。又，有肌酸酐主要包含於魚類以上之高等動物之肌肉中，作為青花魚科魚類所包含之胍基化合物而包含之報告。關於肌酸酐，有對於人有賦予較強之肉質類之呈味，提高湯汁風味之效果之報告，但沒有研究魚類之味覺應答之報告，本發明人等新發現之呈味成份之可能性較高。There is a report that sarcosine is one of the ingredients of the snow crab leg gravy (Hehe, 2011), and the pufferfish responded (Kiyohara et al., 1985). In addition, there are reports that creatinine is mainly contained in muscles of higher animals above fish, and is included as a guanidinium compound contained in mackerel fish. Regarding creatinine, there are reports on humans that impart a stronger flavour to enhance the flavor of soups, but there is no report on the taste response of fish, and the possibility of newly discovered flavour ingredients by the inventors Higher.
關於嗜好性成份之調配量，業者可考慮投餌之鰻魚之種類或其生長狀態、投餌次數等而適當決定。例如於對日本鰻魚之成魚以一天一次之方式投予N,N-二甲基甘胺酸之情形時，飼料中之其調配量例如較佳為3質量%以上。Regarding the blending amount of the hobby ingredients, the industry can make appropriate decisions in consideration of the type of eel to be fed, its growth status, and the number of times of feeding. For example, when N,N-dimethylglycine is administered to the adult fish of Japanese eel once a day, the blending amount in the feed is preferably 3% by mass or more, for example.
作為鰻魚之餌食，亦可使用先前使用之以鯡魚等生餌食、或使鯡魚、青花魚、鯡魚或者竹莢魚等青背魚類乾燥所得之魚粉、鯊魚卵粉末等為基礎，以來自魚之成份為主之調配飼料等。以不損害添加至飼料之嗜好性成份之效果為限度，除先前之餌食所使用之來自魚之成份以外，亦可將大豆肽、磷蝦分解物、澱粉、磷酸鈣、食鹽等礦物、酵母、藥草萃取物等成份調配至飼料。As a bait for eels, fish meal and shark egg powder obtained from raw bait such as herring or dried herring, mackerel, herring or horse mackerel, etc. can also be used based on fish-derived ingredients Mainly for the distribution of feed, etc. To the extent that the effect of the addictive ingredients added to the feed is not impaired, in addition to the fish-derived ingredients used in the previous bait, minerals such as soybean peptides, krill decomposition products, starch, calcium phosphate, table salt, yeast, Herbal extracts and other ingredients are formulated into feed.
以下列舉實施例進一步對本發明具體地進行說明，但本發明並不限定於該等。 [實施例]The following examples further illustrate the present invention in detail, but the present invention is not limited to these. [Example]
(使用味覺受體表現細胞之評價系統之構築) 使各AjT1R2與AjT1R3共同表現，使用利用孔分析之發光檢測系統，調查是否對胺基酸作出應答。使用水母發光蛋白作為發光蛋白質。(Construction of an evaluation system for expressing cells using taste receptors) Let each AjT1R2 and AjT1R3 perform together, and use a luminescence detection system using pore analysis to investigate whether they respond to amino acids. As the luminescent protein, aequorin is used.
嵌合G蛋白質使用公知之rG15-rGi3-5、rG15-rGi2-5、hG16-rGi3-5。按照以下之順序，調查各受體與各嵌合G蛋白質之組合對15種胺基酸如何作出應答。As the chimeric G protein, well-known rG15-rGi3-5, rG15-rGi2-5, and hG16-rGi3-5 are used. In the following order, investigate how the combination of each receptor and each chimeric G protein responds to 15 amino acids.
1)將HEK293T細胞播種至12孔培養盤，於37℃、5%CO2 下培養一晩。 2)使用Lipofectamine 2000(Invitrogen, Carlsbad, CA)對1)之細胞轉染表現各AjT1R2、AjT1R3、嵌合G蛋白質、脫輔基水母發光蛋白之各質體(使用pEAK10載體(Edge Biosystems, Gaithersburg, MD)；於AscI與NotI之限制酵素部位插入各序列)。 3)於6～7小時後交換培養基，於37℃、5%CO2 下培養一晩。 4)第二天，向測定用96孔培養盤(Corning，CellBIND(註冊商標)Surface)重新播種細胞，於37℃、5%CO2 下培養一晩。 5)分析當天，於藉由檢測緩衝液洗淨細胞後，於包含10μM coelenterazine之檢測緩衝液(包含0.1%之BSA(Bovine Serum Albumin，牛血清白蛋白))中，於27℃下靜置4小時(遮光下)。 6)藉由FlexStation3(分子裝置)檢測配位基添加時之發光強度之變化。1) Seed HEK293T cells to a 12-well culture plate and culture overnight at 37°C and 5% CO 2 . 2) Use Lipofectamine 2000 (Invitrogen, Carlsbad, CA) to transfect the cells of 1) to express each plastid of each AjT1R2, AjT1R3, chimeric G protein, and apoprotein (using pEAK10 vector (Edge Biosystems, Gaithersburg, MD); insert each sequence at the restriction enzyme site of AscI and NotI). 3) After 6 to 7 hours, the medium was exchanged and cultured overnight at 37°C and 5% CO 2 . 4) The next day, the cells were reseeded into a 96-well culture plate for measurement (Corning, CellBIND (registered trademark) Surface), and cultured overnight at 37°C and 5% CO 2 . 5) On the day of analysis, after washing the cells with the detection buffer, let it stand at 27°C in detection buffer containing 10 μM coelenterazine (containing 0.1% BSA (Bovine Serum Albumin, bovine serum albumin)) 4 Hours (under shading). 6) Use FlexStation3 (molecular device) to detect the change in luminous intensity when ligands are added.
(結果與探討)
作為事前研究，評價了AjT1R2a＋AjT1R3對L-丙胺酸之濃度依賴性。其結果，EC50
值皆算出為1 mM左右，但應答強度可見差異。於顯示某種程度高應答之G蛋白質之中，將若干結果示於表3。
[表3]
關於AjT1R2a＋AjT1R3及各AjT1R2b＋AjT1R3，將對15種類胺基酸之中特定之胺基酸之應答結果示於圖2。於圖2中，藉由FlexStation3檢測出配位基投予時之發光強度之變化(平均值，n＝2)。配位基濃度皆為50 mM。縱軸表示發光強度。Regarding AjT1R2a+AjT1R3 and each AjT1R2b+AjT1R3, the results of responses to specific amino acids among 15 types of amino acids are shown in FIG. 2. In FIG. 2, the change in luminous intensity when the ligand is administered is detected by FlexStation3 (average value, n=2). The ligand concentration is 50 mM. The vertical axis represents the luminous intensity.
如圖2所示，根據共同表現之嵌合G蛋白質之種類應答之程度可見差異，但大致任一G蛋白質皆應答分佈相似。As shown in FIG. 2, there are differences in the degree of response according to the types of chimeric G proteins that are commonly expressed, but roughly the response distribution of any G protein is similar.
若亦根據圖2未顯示之結果探討，則使用之15種胺基酸之中，觀察到AjT1R2a＋AjT1R3對L-丙胺酸、L-絲胺酸、L-精胺酸、甘胺酸、L-脯胺酸作出應答，AjT1R2b-1＋AjT1R3對L-脯胺酸、L-丙胺酸作出應答，AjT1R2b-2＋AjT1R3對L-組胺酸、L-絲胺酸、L-苯丙胺酸、L-丙胺酸、L-天冬醯胺作出應答，AjT1R2b-4＋AjT1R3對L-丙胺酸、甘胺酸、L-絲胺酸、L-脯胺酸作出應答。觀察到AjT1R2b-3＋AjT1R3對L-絲胺酸、L-蘇胺酸、L-丙胺酸、L-精胺酸作出應答之傾向，但整體應答較低。If also based on the results not shown in Figure 2, among the 15 amino acids used, AjT1R2a+AjT1R3 was observed for L-alanine, L-serine, L-arginine, glycine, L-pro Aminic acid responds, AjT1R2b-1+AjT1R3 responds to L-proline and L-alanine, AjT1R2b-2+AjT1R3 responds to L-histidine, L-serine, L-phenylalanine, L-alanine, L- Asparagine responded, and AjT1R2b-4+AjT1R3 responded to L-alanine, glycine, L-serine, and L-proline. The tendency of AjT1R2b-3+AjT1R3 to respond to L-serine, L-threonine, L-alanine, and L-arginine was observed, but the overall response was lower.
根據AjT1R2a＋AjT1R3及各AjT1R2b＋AjT1R3對L-丙胺酸之濃度依賴性之評價結果，可知應答強度存在差異，任一嵌合G蛋白質皆可使用。又，關於嵌合G蛋白質，於以後之試驗中使用對所有之受體之組合共通，基準線低，顯示某種程度高應答之hG16-rGi3-5。According to the evaluation results of the concentration dependence of AjT1R2a+AjT1R3 and each AjT1R2b+AjT1R3 on L-alanine, it is known that there is a difference in response intensity, and any chimeric G protein can be used. In addition, regarding the chimeric G protein, hG16-rGi3-5, which is common to all receptor combinations in the subsequent experiments, has a low baseline and shows a certain degree of high response.
(對肌胺酸、N,N-二甲基甘胺酸、肌酸酐之應答評價) 分析之方法與上述對胺基酸之應答之確認同樣地進行。(Evaluation of response to creatine, N,N-dimethylglycine and creatinine) The method of analysis is the same as the confirmation of the above-mentioned response to the amino acid.
AjT1R2b-1＋AjT1R3分別對肌胺酸、N,N-二甲基甘胺酸、肌酸酐進行了濃度依賴性應答。圖3表示AjT1R2b-1＋AjT1R3對肌胺酸、N,N-二甲基甘胺酸及肌酸酐之濃度應答曲線(mean±SEM，n＝3)。於僅導入hG16-rGi3-5之培養細胞中對任一配位基均未觀察到應答，因此明確了該等配位基經由AjT1R2b-1＋AjT1R3而被接受(結果未示出)。AjT1R2b-1+AjT1R3 responded to creatine, N,N-dimethylglycine and creatinine in a concentration-dependent manner. Figure 3 shows the concentration response curve of AjT1R2b-1 + AjT1R3 to sarcosine, N,N-dimethylglycine and creatinine (mean±SEM, n=3). No response to any ligand was observed in cultured cells into which hG16-rGi3-5 was introduced, so it was clear that these ligands were accepted via AjT1R2b-1+AjT1R3 (results not shown).
(投餌試驗) 於200 L容水槽設置各收容10尾日本鰻魚未成魚之各試驗區。投餌設為1天1次，每天進行投餌。 將自鰻魚培育用調配飼料(成鰻用，林兼產業股份有限公司銷售)除去作為公知之嗜好性成份之甜菜鹼、甘胺酸、丙胺酸、磷蝦粉所得者作為嗜好性成份去除飼料。將向嗜好性成份去除飼料添加3%之N,N-二甲基甘胺酸所得之飼料(以下稱為「N,N-二甲基甘胺酸添加飼料」)如上所述向日本鰻魚未成魚投餌，測定其等之攝餌量、飼料效率等，藉此評價N,N-二甲基甘胺酸之嗜好性。作為比較群，設置了嗜好性成份去除飼料群、鰻魚培育用調配飼料群。 試驗期間設為31天。試驗期間中之水溫為30～34℃。(Bait test) A test area containing 10 unfinished Japanese eel fish was installed in a 200 L water tank. The bait is set once a day, and the bait is carried out every day. The feed obtained by removing the formulated feed for eel cultivation (for eel production, sold by Linhe Industry Co., Ltd.), which is a well-known preference component, is betaine, glycine, alanine, and krill meal as a preference component. The feed obtained by adding 3% of N,N-dimethylglycine to the feed for the removal of hobby ingredients (hereinafter referred to as "N,N-dimethylglycine to add feed") as described above is not successful for Japanese eels Fish are fed, and their feed intake, feed efficiency, etc. are measured to evaluate the preference of N,N-dimethylglycine. As a comparison group, a preference group removal feed group and a mixed feed group for eel cultivation are provided. The test period was set to 31 days. The water temperature during the test period was 30 to 34°C.
將結果示於表4(A群：嗜好性成份去除飼料群；B群：N,N-二甲基甘胺酸添加飼料群；C群：鰻魚培育用調配飼料群)。攝餌量以剩餘餌食量為基礎進行評價，以乾燥重量表示。
根據該結果，提示N,N-二甲基甘胺酸引誘日本鰻魚攝餌，具有成長促進效果。
[表4]
根據本發明之篩選方法，可探索N,N-二甲基甘胺酸等於先前鰻魚之養殖中未使用之新穎成份。According to the screening method of the present invention, it can be explored that N,N-dimethylglycine is equal to a novel ingredient not previously used in the cultivation of eel.
圖1係表示日本鰻魚T1R之對準。 圖2係表示AjT1R2a＋AjT1R3及各AjT1R2b＋AjT1R3中之G蛋白質之評價。 圖3係表示AjT1R2b-1＋AjT1R3對肌胺酸、肌酸酐及N,N-二甲基甘胺酸之濃度依賴性之評價。Figure 1 shows the alignment of the Japanese eel T1R. Figure 2 shows the evaluation of the G protein in AjT1R2a+AjT1R3 and each AjT1R2b+AjT1R3. Figure 3 shows the evaluation of the concentration dependence of AjT1R2b-1 + AjT1R3 on creatine, creatinine and N,N-dimethylglycine.
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