JP5311537B2 - AMP deaminase derived from algae - Google Patents

Description

  The present invention relates to algae-derived AMP deaminase and use thereof, and more particularly, to use of the enzyme for developing a food having excellent taste.

  It is known that the taste of seaweed involves components such as free sugars and organic acids, as well as amino acids and nucleic acids, and glutamate and 5'-inosinic acid (inosinic acid) are particularly closely related (non-patent literature). 1 and 2). Furthermore, since many laver always contains a saturating amount of glutamic acid, it is clarified that the level of inosinic acid is the most important ingredient that determines the quality of taste. (Non-patent Document 3).

  While inosinic acid is a substance commonly found as an umami component in animal and fish meat, the presence of inosinic acid in plants is extremely rare. For example, even in seaweed, which is a marine plant, inosinic acid is not detected by the conventional 80% ethanol extraction method. However, inosinic acid is detected when the water is extracted as a dried seaweed or a baked seaweed and then extracted with water (Non-patent Document 3). In this case, the detected amount of inosinic acid is temperature-dependent, but since it was not confirmed when the water temperature was 75 ° C. or higher, it was considered that inosinic acid was generated enzymatically during the hot water extraction process (Non-patent Document 3). ).

  As described above, in algae such as seaweed, although the existence of an enzyme involved in biosynthesis of inosinic acid, which is an important element of taste, has been suggested, it has not yet been identified.

Prior art document information related to the invention of the present application is shown below.
Noda H, Horiguchi Y, Araki S, 1975, Studies on the flavor substances of 'Nori' the dried laver Porphyra spp.-II Free amino acids and 5'-nucleotides, Bull Japanese Soc Sci Fish 41: 1299-1303. Shigeru Araki, Takeshi Sakurai, Tomoka Izumi, Koushiko Takahashi, 1996, 5'-inosinic acid in dry seaweed and its enzymatic production, Nippon Shokuhin Kagaku KogakuKaishi 43: 956-961. Shigeru Araki, Tomoka Izumino, Takeshi Sakurai, Kosuke Takahashi, 1997, Taste evaluation of grilled seaweed with hot water extract, Nippon Shokuhin Kagaku KogakuKaishi 44: 430-437. Merkler, DJ., Wali, AS., Taylor, J., and Schramm, VL., 1989, AMP deaminase from Yeast.Role in AMP degradation, large scale purification, and properties of the native and proteolyzed enzyme.J. Biol. Chem 264: 21422-21430.

  This invention is made | formed in view of such a condition, The objective is to identify and isolate the enzyme and its gene which are concerned in the biosynthesis of inosinic acid in algae. A further object of the present invention is to evaluate and breed plants using the obtained enzyme and its gene, and to develop useful crops excellent in taste and the like.

As described above, inosinic acid in seaweed is not detected by using the ethanol extraction method, but is detected by hot water extraction from dried or baked seaweed. The biosynthesis of inosinic acid is carried out by the reaction of AMP → IMP + NH ₃ with AMP deaminase (AMPD). From these facts, the present inventors have inferred that when nori is chewed, inosinic acid is newly generated in the mouth by the action of AMPD, which affects the taste of nori.

  Assuming this reasoning, since the activity of AMPD and the abundance of the enzyme may be an indicator of the taste of nori, the present inventors first attempted to identify AMPD in nori. As a result of various investigations on the purification conditions and creative ideas, the inventors finally succeeded in obtaining a purified preparation of AMPD that retains its activity. Furthermore, the present inventors have succeeded in obtaining DNA encoding AMPD by determining the partial amino acid sequence of the acquired AMPD and performing the RACE method using a primer prepared based on the sequence information. Furthermore, the present inventors have obtained a recombinant protein of AMPD based on the obtained DNA, and have also obtained an antibody against AMPD using the recombinant protein.

  Analysis of the primary structure of AMPD derived from seaweed was found to have low homology with AMPD of plants, animals, and fungi that had been reported so far, and not to have high molecular systematic affinity (Fig. 3). ). So far, AMPD has been cloned in animals and fungi, and in plants, the gene has only been identified in Shiroizuna and rice by the Genome Project, and this enzyme has not been obtained in algae including laver. It wasn't. The reason for this is that AMPD research has been carried out mainly as part of nucleic acid metabolism research (Non-patent Document 4), and inosinic acid was not detected in normal conditions in laver. Even if there were almost no attempts to acquire a gene, and even if it was attempted, it was difficult to acquire it because of its low homology with known counterpart genes (see Table 3 below). It was mentioned that there was.

  Under these circumstances, the present inventors succeeded in obtaining the world's first AMPD and its gene in algae, and using them, the plant was evaluated and bred, and as a result, the taste was improved. The present inventors have found that it is possible to develop excellent useful agricultural products and the like and have completed the present invention.

  More specifically, the present invention provides the following <1> to <17>.

<1> The DNA according to any one of (a) to (d) below, which encodes algae-derived AMP deaminase.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2
(B) DNA comprising the coding region of the base sequence set forth in SEQ ID NO: 1
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 2
(D) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1.

  <2> The DNA according to <1>, wherein the AMP deaminase is thermostable.

  <3> A vector comprising the DNA according to <1> or <2>.

  <4> A transformed cell into which the DNA according to <1> or <2> or the vector according to <3> is introduced.

  <5> A protein encoded by the DNA according to <1> or <2>.

  <6> The method for producing a protein according to <5>, comprising a step of culturing the transformed cell according to <4> and recovering the protein according to <5> from the culture or the culture supernatant.

  <7> An antibody that binds to the protein according to <5>.

  <8> A plant cell into which the DNA according to <1> or <2> or the vector according to <3> is introduced.

  <9> A transformed plant comprising the plant cell according to <8>.

  <10> A transformed plant that is a descendant or clone of the transformed plant according to <9>.

  <11> A propagation material for a transformed plant according to <9> or <10>.

  <12> A food produced by processing the transformed plant according to <9> or <10> or the propagation material according to <11>.

  <13> A food to which the protein according to <5> is added.

  <14> A method for evaluating or sorting algae, comprising detecting the amount of the protein according to <5> in the algae.

  <15> The method according to <14>, wherein the amount of the protein according to <5> is used as an indicator of the taste of algae.

  <16> The protein detection reagent according to <5>, comprising the antibody according to <7> as an active ingredient.

  <17> The detection reagent according to <16>, which is used for evaluating the taste of algae.

  According to the present invention, AMPD in seaweed and a gene encoding it are provided. As a result, it has become possible to simply and objectively evaluate the taste of nori, which has been performed in sensory tests, by using AMPD quantification. In addition, it has become possible to efficiently select and screen nori strains with high taste by using such “quantification of taste”. In addition, by quantifying the quality of the products obtained in nori culture using AMPD as an index, it is possible to evaluate the quality degradation due to illness or discoloration, and promptly take measures against them. Became possible. Furthermore, the present enzyme and its gene can greatly contribute to the development of new foods with excellent taste by adding or introducing them into seaweed itself or other agricultural products. In particular, laver AMPD is excellent in long-term storage and heat resistance, and is an extremely stable enzyme, so it can be said that it is suitable for various food engineering applications.

  The present invention provides DNA encoding algae-derived AMPD. In the present invention, the term “algae” refers to an organism that performs photosynthesis that generates oxygen, excluding moss plants, fern plants, and seed plants (Ryohanabo, Biodiversity Series 3, Diversity and systems, edited by Mitsuo Chihara). The algae of the present invention are not particularly limited, but preferred algae from the viewpoint of industrial application include nori, kombu, wakame, hijiki, green nori, blue seaweed, mozuku, tengusa (agar), tosakanori, mucadenori, sea grapes Examples thereof include those used for food such as akamoku, and those used for health food such as chlorella and spirulina. In the present invention, DNA derived from laver is most preferable. In addition, the base sequence of cDNA encoding AMPD derived from Susabiori, which is one embodiment of the DNA of the present invention, is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the DNA is shown in SEQ ID NO: 2. Supadinori-derived AMPD does not deactivate even when it is dried or baked, and exhibits heat resistance. It is advantageous for AMPD to exhibit heat resistance in this way, for example, in processing and storage into foods. Therefore, the DNA of the present invention preferably encodes heat-resistant AMPD. Here, “heat resistance” means that the enzyme activity is not inactivated even when heat treatment is performed at 180 ° C. for 10 seconds or treatment at 60 ° C. for 5 minutes (Non-Patent Document 2, Fisheries Science 66: 101-116, 2000.).

  The DNA of the present invention includes genomic DNA, cDNA, and chemically synthesized DNA. Genomic DNA and cDNA can be prepared using methods known to those skilled in the art. For genomic DNA, for example, genomic DNA is extracted from algae, a genomic library (plasmids, phages, cosmids, BACs, PACs, etc. can be used as vectors) is developed, and the AMPD of the present invention is developed. It is possible to prepare by performing colony hybridization or plaque hybridization using a probe prepared based on a DNA encoding (eg, SEQ ID NO: 1). Moreover, it is also possible to prepare by preparing a primer specific for DNA encoding the AMPD of the present invention (for example, SEQ ID NO: 1) and performing PCR using this primer. In addition, for example, cDNA is synthesized based on mRNA extracted from algae, inserted into a vector such as λZAP to create a cDNA library, developed, and colony hybridization in the same manner as described above. Alternatively, it can be prepared by performing plaque hybridization or by performing PCR.

The present invention also includes a DNA that encodes a protein functionally equivalent to the AMPD derived from Susvinori described in SEQ ID NO: 2. Here, “functionally equivalent” means that the target protein has AMP deaminase activity. Such DNA includes, for example, mutants, derivatives, and the like that encode proteins consisting of amino acid sequences in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 2. Alleles, variants and homologs are included. As a method well known to those skilled in the art for preparing DNA encoding a protein having a modified amino acid sequence, for example, site-directed mutagenesis can be mentioned. In addition, the amino acid sequence of the encoded protein may be mutated in nature due to the mutation of the base sequence. Thus, even if the DNA encoding a protein having an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence encoding the natural AMPD, as long as it encodes a functionally equivalent protein Included in the DNA of the present invention. Moreover, even if the base sequence is mutated, it may not be accompanied by amino acid mutation in the protein (degenerate mutation), and such a degenerate mutant is also included in the DNA of the present invention. In such mutants, the number of amino acids to be mutated is usually within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (for example, within 5 amino acids). The amino acid residue to be mutated is preferably mutated to another amino acid in which the properties of the amino acid side chain are conserved from the viewpoint of maintaining its activity. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), an amino acid having an aliphatic side chain (G, A, V, L, I), an amino acid having a hydroxyl group-containing side chain (S, T, Y), and a sulfur atom-containing side chain Amino acids (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), amino acids with aromatic-containing side chains (F, Y, W) can be mentioned (the parentheses indicate single letter amino acids). Whether or not a protein encoded by a certain DNA has AMP deaminase activity is determined, for example, as described in the Examples, at 25 ° C. under a reaction solution containing the target protein (for example, the target protein, 50 10 mL of 3 mM 5'-AMP was added to 1 mL of a reaction solution containing mM Tris-HCl, 100 mM CaCl ₂ , and 1 mM 2-ME, and the time course of absorbance at 265 nm was measured using a spectrophotometer (Shimazdu UV2200). It can be evaluated by measuring with. Moreover, it is also possible to evaluate activity by quantifying the amount of AMPD produced | generated by HPLC (nonpatent literature 2).

  In order to prepare DNA encoding a protein functionally equivalent to the AMPD described in SEQ ID NO: 2, other methods well known to those skilled in the art include hybridization techniques and polymerase chain reaction (PCR) techniques. The method of using is mentioned. That is, for those skilled in the art, the base sequence of the AMPD gene (SEQ ID NO: 1) or a part thereof as a probe, and an oligonucleotide that specifically hybridizes to the AMPD gene (SEQ ID NO: 1) as a primer, It is usually possible to isolate DNA with high homology from seaweed and other algae. The DNA encoding the protein functionally equivalent to the AMPD described in SEQ ID NO: 2, which can be isolated by the hybridization technique or the PCR technique, is also included in the DNA of the present invention.

  In order to isolate such DNA, the hybridization reaction is preferably carried out under stringent conditions. In the present invention, stringent hybridization conditions refer to hybridization conditions of 6M urea, 0.4% SDS, 0.5xSSC or equivalent stringency. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, conditions of 6M urea, 0.4% SDS, 0.1 × SSC. The DNA thus isolated is considered to have high homology with the amino acid sequence (SEQ ID NO: 2) of AMPD derived from Susvinori at the amino acid level. High homology means a sequence of at least 50% or more, more preferably 70% or more, particularly preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) in the entire amino acid sequence. Refers to the identity of The identity of the amino acid sequence and base sequence was determined using the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990., Proc Natl Acad Sci USA 90: 5873, 1993.) by Carlin and Arthur. Can be determined. Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (J Mol Biol 215: 403, 1990.). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

  The DNA of the present invention can be used, for example, for the preparation of recombinant proteins, the development of useful crops excellent in plant evaluation, breeding, taste and the like.

  When preparing a recombinant protein, usually, a protein encoding the protein of the present invention is inserted into an appropriate expression vector, the vector is introduced into an appropriate cell, and transformed cells are cultured and expressed. Is purified. The recombinant protein can also be expressed as a fusion protein with another protein for the purpose of facilitating purification or the like. For example, a method for preparing a fusion protein with maltose binding protein using E. coli as a host (vector pMAL series released by New England BioLabs, USA), a method for preparing a fusion protein with glutathione-S-transferase (GST) (Amersham Pharmacia Biotech Vector pGEX series released by the company), a method of preparing by adding a histidine tag (pET series from Novagen), etc. can be used. The host cell is not particularly limited as long as it is a cell suitable for the expression of a recombinant protein. For example, yeast, various animal and plant cells, insect cells and the like can be used in addition to the above-mentioned E. coli. Various methods known to those skilled in the art can be used to introduce a vector into a host cell. For example, an introduction method using calcium ions can be used for introduction into E. coli. The recombinant protein expressed in the host cell can be purified and recovered from the host cell or its culture supernatant by methods known to those skilled in the art. When the recombinant protein is expressed as a fusion protein with the maltose-binding protein or the like, affinity purification can be easily performed. It is also possible to prepare a transformed plant into which the DNA of the present invention has been introduced by the method described later and prepare the protein of the present invention from the plant.

  If the obtained recombinant protein is used, an antibody that binds to the protein can be prepared. For example, a polyclonal antibody can be prepared by immunizing an immunized animal such as a rabbit with the purified protein of the present invention or a partial peptide thereof, collecting blood after a certain period of time, and removing the clot. is there. In addition, the monoclonal antibody is obtained by fusing an antibody-producing cell of an animal immunized with the protein or peptide and a bone tumor cell, and isolating a single clone cell (hybridoma) that produces the target antibody. Can be prepared. The antibody thus obtained can be used for purification and detection of the protein of the present invention. The present invention includes antibodies that bind to the proteins of the present invention. These antibodies can be used as a research reagent for detecting the protein of the present invention, and as described later, for example, evaluation of the taste of biological varieties and foods, In breeding, it can also be used for the purpose of detecting AMPD protein as an indicator of taste.

  When producing a transformed plant body in which algae-derived AMPD is expressed using the DNA of the present invention, for example, the DNA of the present invention is inserted into an appropriate vector and introduced into a plant cell. Then, the transformed plant cell thus obtained is regenerated. As a result, it is possible to improve the taste of a plant or a processed food product. The vector is not particularly limited as long as it contains a promoter sequence that can be transcribed in plant cells and a terminator sequence including a polyadenylation site necessary for the stabilization of the transcript.For example, plasmids `` pBI121 '', `` pBI221 '', Examples include “pBI101” (all manufactured by Clontech). The vector used for the transformation of plant cells is not particularly limited as long as the inserted gene can be expressed in the cells. For example, it is also possible to use a vector having a promoter for performing constitutive gene expression in a plant cell or a vector having a promoter that is inducibly activated by an external stimulus. Moreover, it is possible to use not only a promoter that is activated in the whole plant body but also a promoter that is specifically activated in a specific plant tissue, if necessary. As used herein, “plant cells” include various forms of plant cells, such as suspension culture cells, protoplasts, leaf sections, and callus. Depending on the type of plant, a plant cell form suitable for gene transfer may be selected and used for plant production.

  Examples of promoters for expressing the protein of the present invention include cauliflower mosaic virus 35S promoter, SV40 promoter, GDP promoter, rice actin promoter, maize ubiquitin promoter, and the like. In addition, promoters for inducible expression are known to be expressed by external factors such as infection and invasion of filamentous fungi, bacteria and viruses, low temperature, high temperature, drying, UV irradiation, and spraying of specific compounds. Promoters and the like. Specifically, for example, rice chitinase gene promoter expressed by infection or invasion of filamentous fungus, bacteria, virus, promoter of tobacco PR protein gene, rice lip19 gene promoter induced by low temperature, high temperature Induced rice “hsp80” and “hsp72” gene promoters, Arabidopsis thaliana “rab16” gene promoters induced by drying, parsley chalcone synthase gene promoters induced by UV irradiation, anaerobic conditions And the promoter of the corn alcohol dehydrogenase gene induced by. The rice chitinase gene promoter and tobacco PR protein gene promoter are also induced by specific compounds such as salicylic acid, and “rab16” is also induced by spraying the plant hormone abscisic acid.

  The plant cell into which the vector of the present invention is introduced is not particularly limited. In addition to cells such as chlorella and spirulina, cells such as rice, Arabidopsis, corn, potato, and tobacco can also be exemplified. The plant cells according to the present invention include cells in plant bodies in addition to cultured cells. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots. For introduction of a vector into a plant cell, various methods known to those skilled in the art such as polyethylene glycol method, electroporation method (electroporation), Agrobacterium-mediated method, particle gun method and the like can be used. Transformed plant cells can regenerate plant bodies by redifferentiation.

  As a technique for producing a transformed plant in laver, for example, a plasmid in which a target gene and a promoter (for example, CamV35S promoter, SV40 promoter, etc.) are incorporated into laver protoplasts is introduced by electroporation and transformed by homologous recombination. The technique is well known (J Appl Phycol 15: 1-7,2003., Aquacult Res, 38: 681-688,2007.) In brown algae, for example, particles are found on crushed macombe filamentous gametophyte cell fragments. A method of introducing a plasmid incorporating a target gene and a promoter (for example, SV40 promoter) by a cancer method is known (Plant Cell Rep. 21: 1211-1216, 2003.). In fungi, for example, a method using agarisk has been established (Mol. Gen. Genet. 250: 252-258, 1996.). As the promoter, for example, a GDP promoter is used, and a plasmid incorporating the target gene and the promoter can be introduced by electroporation. In Bacillus subtilis, for example, a transformation technique of B. subtilis has been established, and a commercially available vector such as pHY300PLK (Takara Bio) can be used. Other techniques for producing transformed plants include the method of Fujimura et al. (Plant Tissue Culture Lett. 2:74, 1995.) for rice, and Shillito et al. (Bio / Technology 7: 581, for maize). 1989.) and Gorden-Kamm et al. (Plant Cell 2: 603, 1990.), and for potatoes, Visser et al. (Theor. Appl. Genet 78: 594, 1989.). In the case of tobacco, the method of Nagata and Takebe (Planta 99: 12,1971.) Can be cited, and in the case of Arabidopsis, the method of Akama et al. (Plant Cell Reports 12: 7-11, 1992.) can be cited. For example, the method of Toi et al.

  Once a transformed plant into which the DNA of the present invention is introduced into the genome is obtained, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant body, its descendants or clones, and mass-produce the plant body based on them. Is possible. The present invention includes plant cells into which the DNA of the present invention has been introduced, plants containing the cells, progeny and clones of the plants, and propagation materials for the plants, their progeny, and clones.

  In the transformed plant produced in this way and the food processed from the same, inosinic acid is newly produced in the mouth when chewed by the action of AMPD of the present invention, thereby improving the taste. Can be done. Moreover, the taste of the food can also be improved by adding the purified preparation of AMPD of the present invention to the food. The present invention also provides a food prepared in this manner.

  The present invention also provides a method for evaluating and selecting algae targeting the AMPD of the present invention. The AMPD of the present invention can affect the taste by producing inosinic acid. Therefore, by detecting the amount and activity of the AMPD present in the algae, the quality evaluation of the algae and the selection of excellent varieties Can be done. Therefore, the method for evaluating and selecting algae according to the present invention is a method characterized by detecting the amount and activity of AMPD in algae. Protein mass can be detected by known methods such as ELISA, Western blotting, Biacore, etc., while protein activity can be detected by the above-described detection method of AMP deaminase activity. Can be implemented. As a result, individuals evaluated to have a high amount and activity of the protein of the present invention can be expected to produce more inosinic acid when chewed compared to normal individuals, thereby improving taste. Can be done. It is also possible to improve the breed by selecting and breeding such individuals. Thus, the amount and activity of the protein of the present invention can be used as an indicator of the taste of algae, but the present invention is not limited to this, and the amount and activity of the protein of the present invention have an effect. It also includes using it as an indicator of various other phenomena and phenotypes.

  In order to detect the protein of the present invention in such evaluation and selection, it is convenient to use an antibody that binds to the protein of the present invention. That is, the antibody of the present invention can be used as a reagent for detecting the protein of the present invention as an indicator of algae taste. When the antibody of the present invention is used as a detection reagent, various detectable labels may be provided on the antibody as necessary.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples.

  In addition, the sample Sabbinori (Porphyra yezoensis) used for an Example was provided from the Yamamoto Nori Institute (Yamamoto Nori store). Algae bodies used for purification were collected, transported in an ice-cooled state, and stored at −80 ° C. until used for experiments. Samples used for cloning were immediately frozen in liquid nitrogen after collection, then transferred to a -80 ° C freezer and stored until use. The basic experimental method in the present embodiment is as follows.

  All operations in RNA extraction were performed without RNase, and the equipment and reagents used were also treated with RNase. 3 g of algal cells are crushed under liquid nitrogen, and 45 mL of Apt buffer (100 mM Tris-HCl pH = 8.0, 1.5 M NaCl, 20 mM EDTA, 20 mM DTT, 2% CTAB, Apt et al 1995) is added. After shaking at rpm x 15 min, an equal amount of chloroform was added and stirred with a voltex mixer. After centrifugation (5000 g × 30 min, 4 ° C.), the upper layer was recovered, 1 / 3-dissolved 95% ethanol was added, and the mixture was centrifuged (5000 g × 30 min, 4 ° C.). An equal amount of chloroform was added to the obtained sample, vigorously stirred, and then separated into two phases by centrifugation, and the upper layer was recovered. This operation was repeated four times until no red color disappeared from the upper layer. To the obtained sample, 0.5 M of 9M LiCl and 0.01 M of 2-ME were added, stirred, and stored overnight at −20 ° C., followed by centrifugation (20000 g × 30 min) to obtain a precipitate. Thereafter, the sample was purified and purified using an RNA extraction kit (RNAgents Total RNA Isolation System, Promega) and an mRNA purification kit (PolyATtract mRNA Isolation Systems, Promega) according to the attached manual.

  The Escherichia coli was basically cultured in LB medium at 37 ° C. with amberite, and in some experiments, added with ampicillin (50 μg / mL). Agar culture was performed in a medium containing 1.5% agar. Ligation was performed using Takara Ligation Kit V2.1 (Takara Bio). Extraction and purification of the plasmid from Escherichia coli was performed according to a standard method using the QIAprep Spin Miniprep Kit (Qiagen) or the miniprep method. Agarose gel electrophoresis was performed according to a conventional method using mupid-2 (advance) in the electrophoresis tank, 1.0% agarose in the gel, and TAE in the electrophoresis buffer. The gel after electrophoresis was stained with 0.1 mg / L ethidium bromide aqueous solution, and the band was visualized with UV light. Nucleic acid was extracted from the agarose gel using Suprec-EZ (Takara Bio). Colony selection was performed according to a standard method using blue-white selection or colony direct PCR.

<Example 1> Purification of AMPD All operations were carried out at 4 ° C or on ice. Centrifugation was performed at 12000 g × 10 min. After adding 200 mL of buffer A (50 mM Tris-HCl pH 7.5, 1 mM 2-mercaptoethanol, 20 mM CaCl ₂ ) to 100 g of algae, crush it in an automatic mortar, remove the residue with nylon gauze, and centrifuge ( 12,000 g × 10 min) was performed to obtain a fine extract as a crude extract. The crude extract was salted out with 45% -80% saturated ammonium sulfate, and then the precipitate was dissolved in 150 mL of extraction buffer. This was dialyzed against buffer A, and the supernatant resulting from the treatment with 20% polyethylene glycol / 15% ammonium sulfate (Phycol Res 53: 164-168, 2005.) was recovered and dialyzed again against buffer A. After that, it was applied to a Blue Sepharose CL6B column (2 cm x 5 cm, GE Healthcare) equilibrated with buffer B (50 mM Tris-HCl pH 7.5, 1 mM 2-mercaptoethanol, 20 mM CaCl ₂ , 10% sorbitol) After washing with buffer B, elution was performed with buffer B containing 2 M NaCl. Active fractions were collected and dialyzed against buffer B, followed by cation exchange chromatography using MonoQ HR5 / 5 (GE Healthcare). The column was pre-equilibrated with buffer B, washed with the same buffer after sample addition, and gradient elution was performed with buffer B containing 250 mM NaCl. Active fractions were collected and added to a Superdex HR column (GE Healthcare) equilibrated with buffer B. After elution with buffer B, active fractions were collected to obtain purified samples (Table 1). .

The enzyme activity was determined by adding 10 μL of 3 mM 5′-AMP to 1 mL of a reaction solution containing 50 mM Tris-HCl, 100 mM CaCl ₂ and 1 mM 2-ME at 25 ° C., and measuring the absorbance at 265 nm. The time course was measured by measuring with a spectrophotometer (Shimazdu UV2200).

<Example 2> Isolation and structural analysis of cDNA encoding AMPD After the purified sample was subjected to SDS-PAGE (% T = 10%), the separated protein was visualized by zinc staining, and the band was excised. Extraction was performed using an electrophoresis buffer for SDS-PAGE (Jpn. J. Phycol. 52 (Supplement): 95-100, 2004). After concentration of the obtained sample by ultrafiltration (Millipore, Centricon YM-10), add 20 ng of lysyl endopeptidase to 70 μg of the sample, incubate at 37 ° C for 13 h, and SDS-PAGE (% T = 15%) The fragments were separated by the method described above and transferred to a PVDF membrane (J. Biol. Chem. 280: 18462-18468, 2004.). The transcribed protein fragment was visualized by CBB, the band was cut out, and its N-terminal amino acid sequence was analyzed by Edman degradation (model 477A, Applied Biosystem, Caloglossa continua (Ceramiales, Rhodophyta). Mar. Biotechnol. 3: 493 -500,2001.). The result is shown in FIG.

  This internal amino acid sequence was searched with a database (Porphyra yezoensis EST index, Kazusa DNA Laboratory), and the obtained partial nucleic acid sequence was used to obtain the full-length cDNA sequence by the RACE method. Specifically, the RACE method uses mRNA purified as 5'-RACE and 3'-RACE samples, primer R1 for 5'-RACE and primer left for 3'-RACE as specific primers. (Table 2, FIG. 2), each was performed using SMART RACE cDNA Amplification Kit (Clontech) and RNA LA PCR Kit (AMV) Ver 1.1 (Takara Bio).

  The gene fragment thus amplified was cloned by introducing it into a pGEM-T Easy vector using pGEM-T and pGEM-T Easy Vector Systems (Promega). Sequencing was performed using BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and DNA sequencer model 310 or model 3130 (Applied Biosystems).

  As a result, it was found that the Suprabinori AMPD gene encodes a protein consisting of 522 amino acids. The determined nucleotide sequence of cDNA encoding Susvinori AMPD is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 2. When the Suprabinori AMPD was compared with known AMPDs (human, yeast, Arabidopsis thaliana, rice), the similarity was low (Table 3).

  For this reason, in the blast search, a gene significantly similar in nucleotide sequence was not found. Although no similar gene was found in the protein sequence as a whole, a similar sequence was searched for Pseudoalteromonas adenosine deaminase only in the end of the sequence (Expect = 3e-24, Identities = 52/135 (38 %), Positives = 79/135 (58%)). In addition to adenosine deaminase (ADA (cd01320, pfam, eularyota, 4e-29)), this part includes adenosine / AMP deaminase (ADA_AMPD (cd00443, pfam, eukaryota, 2e-28)) and AMP deaminase (AMPD (AMP Similar to cd01319, pfam, eukaryota, 2e-12)). However, when molecular phylogenetic analysis between AMPDs, which has been clarified in plants, animals, and fungi, was performed using MEGA4 (Molecular Biology and Evolution 24: 1596-1599, 2007.), this protein was related to them. It was found that the properties were not high (FIG. 3). Furthermore, since the last amino acid (P) of [SA]-[LIVM]-[NGS]-[STA] -DDP, which is the motif sequence of AMPD, is changed to "E" in this protein, It was considered to be a type of AMPD.

<Example 3> Production of recombinant protein and antibody
A gene corresponding to the 25th amino acid to the last amino acid of AMPD is reverse-transcribed for 3'-RACE, and a Bam HI cleavage region is added to the 5'-end and an Xba I cleavage region is added to the 3'-end. After amplification using the primer left_Bam and primer right_Xba (Table 4), amplification using the pGEM-T Easy vector, and finally integration into the pCold II vector (Takara Bio), Expression of the replacement protein was performed.

  The expressed protein was purified by Ni-NTA Spin Kit (Qiagen). Purified protein is subjected to SDS-PAGE using a Rapidas mini slab electrophoresis tank (AE-6500, Ato) according to a conventional method, and the gel after electrophoresis is stained by CBB or zinc staining to visualize the protein band. (Jpn. J. Phycol. 52 (Supplement): 95-100, 2004.). As a result, a band was detected at the position of the molecular weight estimated from the amino acid sequence (FIG. 4).

  Antibody production was performed by SDS-PAGE of the protein serving as an antigen, followed by CBB staining, purification by cutting out the band, and injection into the mouse subcutaneously. For the initial immunization, the antigen was mixed with the adjuvant complete, and thereafter the mixture was mixed with the adjuvant incomplete and injected. Infusions were performed a total of 8 times at weekly intervals. The blood was collected, incubated at room temperature for 1 h, centrifuged (12000 g × 15 min), and the supernatant was collected as serum.

The reactivity between the antibody thus obtained and the recombinant protein was confirmed by Western blotting. Specifically, the protein was transferred to a PVDF membrane using SDS-PAGE followed by mini-trans blot cell (BioRad).
Immunostaining was performed by using an alkaline phosphatase-conjugated rabbit anti-mouse IgG antibody as a secondary antibody and coloring with an AP color development kit (BioRad). As a result, the obtained antibody was specifically bound to the recombinant protein (FIG. 4).

  Traditionally, the taste of seaweed has been performed by sensory tests, but by using the quantification of AMPD, it is possible to “quantify the taste (quantification)”, and (1) high taste. Can be used as a tool for easy selection and screening of nori strains. (2) In cultivating nori, quantifying the quality of the product obtained, quantifying deterioration in quality due to disease or discoloration The industrial value is very high, such as being able to make an evaluation and taking measures quickly. It also leads to the development of foods with excellent taste by introducing the gene of this enzyme into seaweed itself and other agricultural products. Furthermore, laver AMPD is an extremely stable enzyme that retains its activity even during long-term storage and baked laver. Therefore, it can be applied to various food engineering.

FIG. 1 is a drawing showing the results of electrophoresis showing limited degradation of nori AMP deaminase by lysyl endopeptidase, and the N-terminal amino acid sequence of the obtained peptide. Amino acids represented in lower case are less reliable. FIG. 2 shows the primers used for laver AMP deaminase cloning and sequencing. FIG. 3 shows the molecular phylogenetic tree of animals (human, AMPD1_Human P23109, AMPD2 HUMAN Q01432, AMPD3 HUMAN Q01432), plants (rice, ORYSJ Q84NP7, Shiroizuna, ARATH O80452), fungi (yeast, YEAST P15274) and Nori AMPD FIG. FIG. 4 is an electrophoresis photograph showing the reactivity of anti-AMP deaminase mouse serum. Serum was purified to 1/500 (A), 1/1 / P. YezoensisAMPD purified protein (after MonoQ, purifiedAMPD), P. yezoensis crude extract (crude), and their mixture (crude + puri), respectively. It diluted to 5000 (B) and 1/50000 (C), and performed immunostaining. In the figure, “CBB” indicates CBB staining, “PSM” indicates a prestained marker, and “LMW” indicates a molecular weight marker.

Claims (17)

The DNA according to any one of (a) to ( c ) below, which encodes algae-derived AMP deaminase.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2
(B) DNA consisting of positions 1 to 1569 of the base sequence set forth in SEQ ID NO: 1
(C) DNA encoding a protein comprising an amino acid sequence having 90% or more homology with the amino acid sequence of SEQ ID NO: 2
  The DNA according to claim 1, wherein the AMP deaminase is thermostable.   A vector comprising the DNA according to claim 1 or 2.   A transformed cell into which the DNA according to claim 1 or 2 or the vector according to claim 3 has been introduced.   A protein encoded by the DNA according to claim 1 or 2.   A method for producing the protein according to claim 5, comprising a step of culturing the transformed cell according to claim 4 and recovering the protein according to claim 5 from the culture or the culture supernatant.   An antibody that binds to the protein of claim 5.   A plant cell into which the DNA according to claim 1 or 2 or the vector according to claim 3 is introduced.   A transformed plant comprising the plant cell according to claim 8.   A transformed plant that is a descendant or clone of the transformed plant according to claim 9.   The propagation material of the transformed plant body of Claim 9 or 10.   A food produced by processing the transformed plant according to claim 9 or 10 or the propagation material according to claim 11.   A food to which the protein according to claim 5 is added.   A method for evaluating or sorting algae, comprising detecting the amount of the protein according to claim 5 in the algae.   The method according to claim 14, wherein the amount of the protein according to claim 5 is used as an indicator of the taste of algae.   The protein detection reagent according to claim 5, comprising the antibody according to claim 7 as an active ingredient.   The detection reagent of Claim 16 used for evaluation of the taste of algae.