JP5137052B2 - Trehalose transporter gene and method for introducing trehalose into cells using the gene - Google Patents

Description

  The present invention relates to a trehalose transporter gene and a method for introducing trehalose into a cell using the gene.

  Trehalose is a non-reducing sugar in which two glucoses are α1,1 linked. It is widely used in insects, crustaceans, fungi, bacteria, etc., and is used as an energy source and carbon source for these organisms. Since trehalose is a non-reducing sugar unlike other sugars, it does not have any toxicity like protein denaturation due to the Maillard reaction found in reducing sugars. In addition, trehalose functions as an osteoclast differentiation induction inhibitor to prevent osteoporosis (Patent Document 1, Non-Patent Document 1), and polyglutamine / polyalanine disease represented by Huntington's disease and ophthalopharyngeal muscular dystrophy. It prevents the accumulation of abnormal proteins in nerve cells and muscle cells (Non-Patent Documents 2 and 12), and has a function of protecting the structure of biological components such as proteins and cell membranes (Non-Patent Documents 3 and 4). Thus, trehalose has a special physiological activity not found in other sugars.

  Applied research utilizing the physiological activity of trehalose is underway. For example, the US Advanced Research Projects Agency (DARPA-the institution that oversees theoretical research by the Department of Defense) has developed a dry preservation method for blood for transfusion using the high biopolymer protection function of trehalose. In the case of platelets, it has been successfully dried and stored for about 2 years by the cell introduction method of trehalose by endocytosis and freeze-drying treatment (Non-patent Document 5). However, platelets are the only successful example, and other general nucleated cells do not work. In addition, development for practical use of therapeutic agents for osteoporosis and Huntington's disease using the physiological activity of trehalose has not progressed. This is because the cell membrane does not permeate saccharides containing trehalose under normal conditions. Therefore, at present, finding a method for introducing trehalose into cells easily and without adverse effects is the key to establishing a method of using trehalose targeting nucleated cells and living organisms (non- Patent Document 11).

  As a system for introducing trehalose into sexual animal cells, a method of introducing a gene for trehalose synthase (Non-patent Document 6), a pore or a channel for non-selectively permeating small molecules such as genetically modified hemolysin and P2X7, etc. The method of introducing using the method (Non-Patent Documents 7 and 13) has been reported. In both cases, trehalose is accumulated in the cells, but the former consumes intracellular carbon sources and is under the control of a constitutive expression promoter, so that trehalose remains permanently. Moreover, since the latter method has extremely low substrate selectivity for pores and channels, there is a problem that substances other than trehalose easily flow into and out of the cell. Recently, a method of introducing trehalose into animal cells using pinocytosis (drinking action) inherent in animal cells has been studied (Non-patent Document 8). However, in this method, even if it is taken up into cells, trehalose is once retained in the endosome and then diffuses into the cytoplasm after 24 hours or more, so it cannot be said to be a rapid introduction system. Moreover, since pinocytosis ability strongly depends on the cell type, it seems to be a system that is difficult to generalize. Therefore, in order to allow the trehalose to permeate selectively into cells and to control the trehalose concentration in the “cytoplasm” quickly and easily, some cell membrane-localized trehalose-specific transport carrier must be used. Need to use.

As carriers having trehalose transport activity, MalEGFK ₂ (Gene Bank no. P68187) (Non-patent document 9) and MAL11 / Agt1 (Gene Bank no. P53048) (Non-patent document 10) are already used from single-cell organisms such as bacteria and yeast. An active transporter having a trehalose transport activity has been isolated. However, there is no report on a method for introducing trehalose into animal cells using these transporters. It is not active unless it is a heteropentamer composed of different gene products (MalEGFK ₂ ) (Non-patent document 9), or the optimum pH is biased toward the acidic side (MAL11 / Agt1) (Non-patent document 10). This is because it can be easily inferred that even if these transporter genes are expressed in animal cells, they do not easily function. In addition, as the driving force for transport, hydrolysis of ATP (MalEGFK ₂ ) (Non-patent Document 9) and hydrogen ion concentration gradient (MAL11 / Agt1) (Non-patent Document 10) are used. Inevitable points are also disadvantages. Therefore, a novel trehalose transporter that is derived from a single gene product, shows activity near neutrality, does not consume ATP, is independent of substances other than trehalose, and does not depend on the type of cells to be expressed, In other words, if a facilitated transport type trehalose transporter is used, all of these problems are considered to be solved.

Prior art document information related to the invention of the present application is shown below.
JP 2000-38343, `` Anti-osteoporosis agent '', Yoshiyo Tomoyo, Nishizaki Taiji, Arai Naruyuki, Kurimoto Masashi. Nishizaki, Y. et al. (2000) Nutrition Research 20, 653. Tanaka, M. et al. (2004) Nat Med 10, 148-54. Crowe, JH, Crowe, LM, Carpenter, JF and Aurell Wistrom, C. (1987) Biochem J 242, 1-10. Crowe, JH, Carpenter, JF and Crowe, LM (1998) Annu Rev Physiol 60, 73-103. Wolkers, WF, Walker, NJ, Tablin, F. and Crowe, JH (2001) Cryobiology 42, 79-87. Guo, N., Puhlev, I., Brown, DR, Mansbridge, J. and Levine, F. (2000) Nat Biotechnol 18, 168-71. Eroglu, A., Russo, MJ, Bieganski, R., Fowler, A., Cheley, S., Bayley, H. and Toner, M. (2000) Nat Biotechnol 18, 163-7. Oliver, AE, Jamil, K., Crowe, JH and Tablin, F. (2004) Cell Preservation Technology 2, 35-49. Boos, W. and Shuman, H. (1998) Microbiol Mol Biol Rev 62, 204-29. Stambuk, BU, De Araujo, PS, Panek, AD and Serrano, R. (1996) Eur J Biochem 237, 876-81. Brumfiel, G. Nature, (2004) 428, 14-15 Davies JE, Sarkar S, Rubinsztein DC. (2006) Hum Mol Genet. 15 (23-31) Elliott GD, Liu XH, Cusick JL, Menze M, Vincent J, Witt T, Hand S, Toner M. (2006) Cryobiology 52, 114-127

  The present invention has been made in view of such a situation, and the problem to be solved by the present invention is to isolate and identify a novel trehalose transporter gene, and to use trehalose in a cell using the gene. Is to provide a way to introduce.

  The inventors of the present application have attempted to isolate a facilitated transport-type trehalose transporter that has not yet been isolated, using Nemur chironomid that accumulates trehalose in large quantities as it dries. Specifically, the present inventors first identified a plurality of EST clones that were annotated on the sugar transporter from the uniquely generated Japanese chironomid EST database. These clones were found to form one cluster. Therefore, the inventors of the present application tried to isolate a gene using the RACE (rapid amplification of cDNA ends) method based on the base sequence information of the cluster. As a result, we succeeded in isolating about 2.3 kb of full-length cDNA from Nemulus chironomid. And this new gene was named PvTRET1 (meaning Trehalose transporter of P. vanderplanki).

  The isolated cDNA contained one 1512 bp ORF encoding a protein of 504 amino acid residues. As a result of Pfam search, it was confirmed that the protein translated from the ORF has a conserved domain of the sugar transporter. Moreover, when the three-dimensional structure of PvTRET1 protein was estimated by the SOSUI algorithm, it was shown to be a 12-transmembrane membrane protein typical of sugar transporters. Furthermore, it was also confirmed that the expression of the PvTRET1 gene in the chironomid chironomid showed drought-induced expression fluctuations as well as trehalose accumulation changes. From these results, it was considered that this PvTRET1 is closely related to trehalose transport.

  Therefore, the inventors of the present invention examined whether or not the isolated PvTRET1 protein really has a trehalose transport ability in an expression system using Xenopus oocytes. As a result, it was confirmed that the translation product of cDNA isolated from the chironomid was a trehalose transport activity. In addition, the present inventors have found that PvTRET1 exhibits specific activity against trehalose among various disaccharides. When the trehalose transport kinetics of PvTRET1 was examined, Km was 114.5 ± 27.9 mM and Vmax was 7.84 ± 0.77 nmol / 15 min / oocyte. This indicates that PvTRET1 is a highly functional transporter that does not saturate even at very high concentrations of substrate. In addition, it was considered that the reaction was independent of pH because it showed activity regardless of whether the reaction conditions were neutral or acidic. Furthermore, since this transport activity did not change even in the presence of an ionophore or an uncoupler that inhibits active transport, PvTRET1 was also confirmed to be a facilitated transport type transporter. In general, the transport direction of the facilitated transport transporter is determined depending on the substrate concentration difference between the inside and outside of the membrane. In fact, in cells expressing PvTRET1, it was possible to remove the trehalose in the cells once incorporated by simply changing the trehalose medium to a trehalose-free medium. In addition, hamster, mouse, and human cells exhibited functions similar to those expressed in Xenopus oocytes, suggesting that the activity of PvTRET1 does not depend on the type of cells that are expressed.

  By the way, since trehalose is the blood sugar of insects, it was considered that insects other than Nemurisumika also have trehalose transporters. Therefore, the inventors of the present application tried to search for an ortholog of the PvTRET gene using a gene database that has already been published. As a result, we succeeded in finding orthologs of the PvTRET1 gene in Drosophila melanogaster, Gambier spruce, honeybees and silkworms. These clones showed high homology with PvTRET1, but were not annotated and all of their activities were unidentified. Therefore, genes were isolated from these insects and named DmTRET1, AgTRET1, AmTRET1, and BmTRET1, respectively.

  Next, the present inventors examined whether or not these gene products have trehalose transport activity. As a result, it was confirmed that Xenopus laevis oocytes in which the insect facilitating transport type trehalose transporter (TRET1) ortholog was expressed permeate trehalose. Therefore, it was suggested that the TRET1 gene is not a specific gene of Chironomidae but a gene that exists universally in insects.

  For the purpose of examining the physiological function of TRET1, in situ hybridization was performed using larvae 12 hours after drying using the PvTRET1 gene as a probe. As a result, it was found that the PvTRET1 gene was specifically expressed in the fat pad. Since the fat body is an organ that synthesizes trehalose, which is the blood sugar of insects, it was suggested that TRET1 releases trehalose from the fat body to hemolymph.

That is, the present invention relates to a novel trehalose transporter gene, and specifically provides the following inventions.
[1] DNA according to any one of (a) to (d) below;
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10,
(B) DNA comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9;
(C) 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, 4, 6, 8, or 10; DNA encoding a protein having a function equivalent to that of a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9, comprising SEQ ID NO: 2, 4, 6, 8, or A DNA encoding a protein having a function equivalent to that of the protein consisting of the amino acid sequence according to 10.
[2] DNA according to (a) or (b) below;
(A) comprising the amino acid sequence set forth in SEQ ID NO: 21, wherein one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10 are substituted, deleted, added, and / or A DNA encoding a protein consisting of an inserted amino acid sequence, the protein having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10;
(B) It is encoded by DNA comprising the amino acid sequence set forth in SEQ ID NO: 21 and hybridizing under stringent conditions with DNA consisting of the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9. A DNA encoding a protein having a function equivalent to that of a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.
[3] DNA according to (a) or (b) below;
(A) including the amino acid sequence of the 12th transmembrane region, wherein one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10 A DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, or 10;
(B) a protein encoded by DNA comprising the amino acid sequence of the twelve transmembrane region and hybridized under stringent conditions with DNA consisting of the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9 A DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.
[4] A vector comprising the DNA according to any one of [1] to [3].
[5] A transformed cell that holds the DNA according to any one of [1] to [3] or the vector according to [4] in an expressible manner.
[6] The transformed cell according to [5], wherein the cell is derived from any one of Nemurus, Gambiae, Drosophila melanogaster, honeybee, and silkworm.
[7] A transgenic non-human organism into which the DNA according to any one of [1] to [3] or the vector according to [4] has been introduced.
[8] The transgenic non-human organism according to [7], wherein the non-human organism is selected from insects or vertebrates.
[9] The transgenic non-human organism according to [8], wherein the insect is selected from Nemurium, Gambier, Drosophila, Honeybee or Silkworm.
[10] An oligonucleotide comprising at least 15 consecutive bases complementary to the DNA according to any one of [1] to [3] or a complementary sequence thereof.
[11] An oligonucleotide comprising the base sequence according to any one of SEQ ID NOs: 11 to 20, and 34.
[12] A primer set for amplifying all or part of the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9.
[13] At least one primer set shown in any of (a) to (e) below;
(A) DNA containing the base sequence set forth in SEQ ID NO: 11 and DNA containing the base sequence set forth in SEQ ID NO: 12
(B) DNA containing the base sequence set forth in SEQ ID NO: 13 and DNA containing the base sequence set forth in SEQ ID NO: 14
(C) DNA containing the base sequence set forth in SEQ ID NO: 15 and DNA containing the base sequence set forth in SEQ ID NO: 16
(D) DNA containing the base sequence set forth in SEQ ID NO: 17 and DNA containing the base sequence set forth in SEQ ID NO: 18
(E) DNA containing the base sequence set forth in SEQ ID NO: 19 and DNA containing the base sequence set forth in SEQ ID NO: 20.
[14] A protein encoded by the DNA of any one of [1] to [3].
[15] The transformed cell according to [5] or [6] is cultured, and the expression product of the DNA according to any one of [1] to [3] is collected. A method for producing the described protein.
[16] An antibody that binds to the protein of [14].
[17] The antibody according to [16], which recognizes an epitope of a protein having the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.
[18] The antibody according to [17], wherein the epitope of the protein having the amino acid sequence set forth in SEQ ID NO: 2 is an epitope consisting of the amino acid sequence set forth in SEQ ID NO: 32.
[19] The antibody according to [17], wherein the epitope of the protein having the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10 is an epitope consisting of the amino acid sequence set forth in SEQ ID NO: 33.
[20] The antibody according to [18] or [19], wherein the antibody is a monoclonal antibody.
[21] A drug for introducing trehalose into a cell, comprising any one of the DNA of [1] to [3], the vector of [4], or the protein of [14].
[22] A drug that increases drought tolerance of cells, comprising any one of the DNA according to [1] to [3], the vector according to [4], or the protein according to [14].
[23] A method for increasing trehalose permeability of a cell, comprising a step of expressing the DNA according to any one of [1] to [3] in an arbitrary cell.
[24] A method for introducing trehalose into a cell, comprising a step of expressing the DNA according to any one of [1] to [3] in an arbitrary cell.
[25] A screening method for a test compound comprising the following steps (a) to (c):
(A) contacting the test compound with the transcription product of the DNA according to any one of [1] to [3],
(B) a step of detecting the binding between the transcription product of the DNA according to any one of [1] to [3] and a test compound;
(C) A step of selecting a test compound that binds to the transcription product of the DNA according to any one of [1] to [3].
[26] A screening method for a test compound comprising the following steps (a) to (c):
(A) contacting a test compound with a cell collected from an invertebrate,
(B) a step of measuring the expression level of the transcription product of the DNA according to any one of [1] to [3],
(C) A step of selecting a test compound in which the expression level of the transcript is increased or decreased as compared with the case where the test compound is not contacted.
[27] A screening method for a test compound comprising the following steps (a) to (d);
(A) a step of providing a cell or cell extract having a DNA to which a reporter gene is functionally linked downstream of the promoter region of the DNA according to any one of [1] to [3];
(B) contacting the test compound with the cells or the cell extract;
(C) measuring the expression level of the reporter gene in the cell or cell extract,
(D) A step of selecting a test compound in which the expression level of the reporter gene is increased or decreased as compared with the case where the test compound is not contacted.
[28] A kit for use in the screening method according to any one of [25] to [27].
[29] A method for expressing an arbitrary gene in a fat body, comprising a step of expressing a DNA in which the promoter of the DNA according to any one of [1] to [3] and an arbitrary gene are functionally linked.
[30] A DNA encoding an antisense RNA complementary to the transcription product of the DNA according to any one of [1] to [3].
[31] The DNA of [30], comprising the base sequence of SEQ ID NO: 35.

  The present invention provides a trehalose transporter gene and a method for introducing trehalose into a cell using the gene. PvTRET1 and other insect TRET1 genes have activity as facilitated transport type trehalose transporters, and by introducing these genes into animal cells, it is possible to easily incorporate extracellular trehalose into cells. It became.

  As shown in Table 1, insect TRET1 has many advantages compared to transporters having trehalose transport activity of unicellular organisms. For example, in the case of MalEGFK2 and MAL11 / Agt1, both transport directions are unidirectional from the outside to the inside of the cell, so that it is difficult to arbitrarily control the intracellular trehalose concentration without reaching the transport equilibrium, Energy is required for transport, and ATP must always be supplied to cells. On the other hand, since the transport direction of TRET1 is arbitrary, the transport stops when the transported trehalose concentration reaches equilibrium inside and outside the membrane, so the intracellular trehalose can be controlled by adjusting the trehalose concentration in the extracellular fluid. . TRET1 does not require energy for trehalose transport. Furthermore, MalEFGK2 is composed of four different gene products, so the gene transfer method is complicated, and MAL11 / Agt1 does not show effective activity at neutral pH, so it does not work even if the gene is introduced into animals. However, TRTET1 is composed of only a single gene product, is very easy to express, and works neutrally.

  In addition, as shown in Table 2, there are many advantages when comparing the method of introducing trehalose into existing animal cells and the method of introducing trehalose by the expression of TRET1. For example, when modified hemolysin or P2X7 is used, molecules smaller than the pore size permeate the membrane non-selectively, so that the flow of molecules other than trehalose cannot be stopped. On the other hand, TRET1 has a high substrate selectivity with respect to trehalose, so that unwanted molecular inflow and outflow does not occur. In addition, in the method of expressing trehalose synthase derived from E. coli in cells, gene expression is performed under the control of a constitutive expression promoter, so that a carbon source typified by intracellular glucose is consumed and trehalose is required. Even if it disappears, it cannot be removed. On the other hand, TRET1 can introduce trehalose from outside the cell without using energy, so it consumes no intracellular carbon source and easily and selectively removes unnecessary trehalose. Is possible. When pinocytosis (non-selective phagocytosis) is used, the ability of pinocytosis depends largely on the cell type, and the substance to be incorporated is not limited to trehalose. Therefore, any amount of trehalose can be applied to any cell. It is not suitable as a system to be introduced. On the other hand, TRET1 can be expressed in any cell as long as gene transfer is possible, and once expressed, trehalose transport ability can be added to the cell.

  From the above points, TRET1 is very advantageous as a system that can introduce trehalose easily and without causing unnecessary damage to cells compared to existing transporters and trehalose introduction systems with trehalose transport activity. It is.

  The establishment of a method for introducing trehalose into animal cells using TRET1 has made it possible to develop dry preservation techniques for animal cells and tissues. In addition, when trehalose derivatives are used in anti-osteoporosis drugs, anti-huntington drugs, etc., it may be possible to contribute to the development of a new drug delivery system (DDS). In addition, when synthesizing trehalose derivatives (so-called drug design), it may be used as an index to determine whether the derivatives can have the same activity as trehalose using the substrate selectivity of trehalose transporter. It has become possible.

Furthermore, the trehalose transporter is expected to become a target molecule for the development of new insecticides because it controls the release of trehalose, which is the blood sugar of insects, from the fat body.
As described above, the present invention is important for imparting and utilizing the excellent biological activity of trehalose effectively to animal cells to the maximum extent.

The present invention provides a novel trehalose transporter gene TRET1 isolated from insects. Specifically, the TRET1 gene of the present invention includes
(1) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10,
(2) DNA comprising the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9;
(3) 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 set forth in SEQ ID NO: 2, 4, 6, 8, or 10; DNA encoding a protein having a function equivalent to that of a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.
(4) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9, comprising SEQ ID NO: 2, 4, 6, 8, or DNA encoding a protein having a function equivalent to that of the protein consisting of the amino acid sequence described in 10,
Is included.

  The TRET1 gene of the present invention is a TRET1 gene isolated from Nemurian medaka, Gambier anopheles, Drosophila melanogaster, honeybee, and silkworm. SEQ ID NO: 1 shows the base sequence of TRET1 isolated from Japanese chironomid, and SEQ ID NO: 2 shows the amino acid sequence of the protein generated from the base sequence. In addition, the base sequences of TRET1 isolated from Gambier spp., Drosophila melanogaster, honeybees, and silkworms are shown in SEQ ID NOs: 3, 5, 7, and 9, respectively, and the amino acid sequences of proteins generated from these base sequences. Are shown in SEQ ID NOs: 4, 6, 8, and 10, respectively.

The TRET1 protein produced from the TRET1 gene has an amino acid sequence shown in SEQ ID NO: 21 as compared with a human glucose transporter. Therefore, the TRET1 gene of the present invention includes
(A) comprising the amino acid sequence set forth in SEQ ID NO: 21, wherein one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10 are substituted, deleted, added, and / or A DNA encoding a protein consisting of an inserted amino acid sequence, the protein having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10;
Is included.

The amino acid sequence set forth in SEQ ID NO: 21 corresponds to the first transmembrane region in TRET1. In SEQ ID NO: 21, the 2nd, 6th, 10th, 13th, 14th, 18th and 20th amino acids are any hydrophobic amino acids, the 7th amino acid is serine or alanine, the 11th amino acid is glycine or alanine, 15 The amino acid is valine or isoleucine, the 17th amino acid is phenylalanine or tyrosine, the 26th amino acid is leucine or valine, the 27th amino acid is leucine or valine, and the 28th amino acid is serine or threonine. The amino acid sequences of the region corresponding to SEQ ID NO: 21 in Nemurus, Gambier, Drosophila, Honeybee, and silkworm are shown in SEQ ID NOs: 22, 23, 24, 25, and 26, respectively. Therefore, the TRET1 gene of the present invention includes
A protein comprising the amino acid sequence set forth in SEQ ID NO: 22 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 2; DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2;
A protein comprising the amino acid sequence set forth in SEQ ID NO: 23, wherein the amino acid sequence set forth in SEQ ID NO: 4 has an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted; DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 4;
A protein comprising the amino acid sequence of SEQ ID NO: 24, wherein the amino acid sequence of SEQ ID NO: 6 has an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted; DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
A protein comprising the amino acid sequence of SEQ ID NO: 25, wherein the amino acid sequence of SEQ ID NO: 8 comprises an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted; DNA encoding a protein having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 8;
A protein comprising an amino acid sequence represented by SEQ ID NO: 26, wherein the amino acid sequence represented by SEQ ID NO: 10 has an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted; DNA encoding a protein having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 10;
Is included.

Here, the base sequence encoding the amino acid sequence set forth in SEQ ID NO: 22 corresponds to the base sequence from position 247 to position 333 of the base sequence of SEQ ID NO: 1.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 23 corresponds to the base sequence of positions 133 to 219 of the base sequence of SEQ ID NO: 3.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 24 corresponds to the 127th to 213th base sequences of the base sequence of SEQ ID NO: 5;
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 25 corresponds to the 124th to 210th base sequences of the base sequence of SEQ ID NO: 7.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 26 corresponds to the 151st to 237th base sequences of the base sequence of SEQ ID NO: 9.

Furthermore, the TRET1 gene of the present invention includes
(B) It is encoded by DNA comprising the amino acid sequence set forth in SEQ ID NO: 21 and hybridizing under stringent conditions with DNA consisting of the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9. A DNA encoding a protein having a function equivalent to that of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10;
Is included. The description of the amino acid sequence of SEQ ID NO: 21 is as described above.

Here, as described above, the amino acid sequences of the region corresponding to SEQ ID NO: 21 in Nemurus, Gambiae, Drosophila, Apis mellifera, and silkworm are SEQ ID NOs: 22, 23, 24, 25, and 26, respectively. Therefore, the TRET1 gene of the present invention includes
A protein encoded by a DNA comprising the amino acid sequence set forth in SEQ ID NO: 22 and hybridizing under stringent conditions with the DNA consisting of the base sequence set forth in SEQ ID NO: 1, wherein the protein is set forth in SEQ ID NO: 2. DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence,
A protein encoded by a DNA comprising the amino acid sequence set forth in SEQ ID NO: 23 and hybridizing under stringent conditions with the DNA consisting of the base sequence set forth in SEQ ID NO: 3 and described in SEQ ID NO: 4. DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence,
A protein encoded by a DNA comprising the amino acid sequence set forth in SEQ ID NO: 24 and hybridizing under stringent conditions with the DNA consisting of the base sequence set forth in SEQ ID NO: 5; DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence,
A protein encoded by a DNA comprising the amino acid sequence set forth in SEQ ID NO: 25 and hybridizing under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 7; DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence,
A protein encoded by a DNA comprising the amino acid sequence set forth in SEQ ID NO: 26 and hybridizing under stringent conditions with a DNA consisting of the base sequence set forth in SEQ ID NO: 9 and described in SEQ ID NO: 10 DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence,
Is included. Here, the base sequences encoding the amino acid sequences described in SEQ ID NOs: 22 to 26 are as described above.

Furthermore, the TRET1 protein produced from the TRET1 gene of the present invention is characterized by having a 12-transmembrane region called a sugar transporter region. Therefore, the TRET1 gene of the present invention includes
(I) comprising the amino acid sequence of the 12th transmembrane region, wherein one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10 A DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, or 10;
Is included.

Here, the amino acid sequences of the twelve transmembrane regions in Nemurus, Gambier, Drosophila, Honeybee, and Silkworm correspond to SEQ ID NOs: 27, 28, 29, 30 and 31, respectively (see FIG. 10). Therefore, the TRET1 gene of the present invention includes
A protein comprising any amino acid sequence set forth in SEQ ID NO: 27 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 2. A DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2;
A protein comprising any amino acid sequence set forth in SEQ ID NO: 28 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 4. A DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 4;
A protein comprising any amino acid sequence set forth in SEQ ID NO: 29 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 6. A DNA encoding a protein having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
A protein comprising any one of the amino acid sequences set forth in SEQ ID NO: 30 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 8. A DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 8;
A protein comprising any amino acid sequence set forth in SEQ ID NO: 31 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 10. A DNA encoding a protein having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 10;
Is included.

Here, the base sequence encoding the amino acid sequence set forth in SEQ ID NO: 27 corresponds to the base sequence from position 250 to position 1572 of the base sequence of SEQ ID NO: 1.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 28 corresponds to the base sequence from position 136 to position 1458 of the base sequence of SEQ ID NO: 3.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 29 corresponds to the base sequence from position 130 to position 1452 of the base sequence of SEQ ID NO: 5.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 30 corresponds to the 127th to 1446th base sequences of the base sequence of SEQ ID NO: 7,
The base sequence encoding the amino acid sequence described in SEQ ID NO: 31 corresponds to the 154th to 1461th base sequences of the base sequence of SEQ ID NO: 9.

Furthermore, the TRET1 gene of the present invention includes
(Ii) a protein encoded by DNA comprising the amino acid sequence of the 12-transmembrane region and hybridized under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9 A DNA encoding a protein having a function equivalent to that of a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.
Is included.

Here, as mentioned above, the amino acid sequences of the 12-transmembrane region in Nemur Chironomidae, Gambier anopheles, Drosophila melanogaster, Apis mellifera and Silkworm correspond to SEQ ID NOs: 27, 28, 29, 30 and 31, respectively. From the TRET1 gene of the present invention,
A protein encoded by a DNA comprising any one of the amino acid sequences set forth in SEQ ID NO: 27 and hybridizing under stringent conditions with the DNA consisting of the base sequence set forth in SEQ ID NO: 1, comprising SEQ ID NO: 2 DNA encoding a protein having the same function as the protein consisting of the amino acid sequence described in 1.
A protein encoded by a DNA comprising any one of the amino acid sequences set forth in SEQ ID NO: 28 and hybridizing under stringent conditions with the DNA consisting of the base sequence set forth in SEQ ID NO: 3; SEQ ID NO: 4 DNA encoding a protein having the same function as the protein consisting of the amino acid sequence described in 1.
A protein encoded by a DNA comprising any one of the amino acid sequences set forth in SEQ ID NO: 29 and hybridizing under stringent conditions with the DNA comprising the base sequence set forth in SEQ ID NO: 5; SEQ ID NO: 6 DNA encoding a protein having the same function as the protein consisting of the amino acid sequence described in 1.
A protein encoded by a DNA comprising any one of the amino acid sequences set forth in SEQ ID NO: 30 and hybridizing under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 7; SEQ ID NO: 8 DNA encoding a protein having the same function as the protein consisting of the amino acid sequence described in 1.
A protein encoded by a DNA comprising any one of the amino acid sequences set forth in SEQ ID NO: 31 and hybridizing under stringent conditions with the DNA comprising the base sequence set forth in SEQ ID NO: 9; SEQ ID NO: 10 DNA encoding a protein having the same function as the protein consisting of the amino acid sequence described in 1.
Is included. Here, the base sequence of the DNA encoding the amino acid sequence described in SEQ ID NOs: 27 to 31 is as described above.
By using the TRET1 gene of the present invention, for example, preparation of a recombinant protein, production of a transgenic animal into which a trehalose transporter gene has been introduced, and the like are possible.

The “TRET1 gene” of the present invention is not particularly limited as long as it can encode a “TRET1 protein”. The “TRET1 gene” includes genomic DNA, chemically synthesized DNA, etc. in addition to cDNA. .
Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means. Genomic DNA is extracted from, for example, insects (eg, Nemurus, Gambier, Drosophila, Apis, or Silkworm), and genomic libraries (vectors include plasmids, phages, cosmids, BACs, PACs, etc.) Can be used to expand colony hybridization using a probe prepared based on the TRET1 gene (for example, the DNA described in SEQ ID NO: 1, 3, 5, 7, or 9). It can be prepared by performing plaque hybridization. It is also possible to prepare a primer specific to the TRET1 gene and perform PCR using this primer. In addition, for example, cDNA is synthesized based on mRNA extracted from insects (for example, Nemurus, Gambier, Drosophila, Apis mellifera, or silkworm), and inserted into a vector such as λZAP. It is possible to prepare a library by developing a library and performing colony hybridization or plaque hybridization in the same manner as described above, or by performing PCR.

  Methods well known to those skilled in the art for isolating the TRET1 gene include hybridization techniques (Southern, EM, Journal of Molecular Biology, Vol. 98, 503, 1975) and polymerase chain reaction (PCR) techniques (Saiki). , RK, et al. Science, vol. 230, 1350-1354, 1985, Saiki, RK et al. Science, vol. 239, 487-491, 1988). That is, for those skilled in the art, the base sequence of the TRET1 gene (for example, the sequence described in SEQ ID NO: 1, 3, 5, 7, or 9) or a part thereof is used as a probe and specifically hybridizes to the TRET1 gene. It is usually possible to isolate the TRET1 gene from insects using the oligonucleotide to be used as a primer.

  Furthermore, since the TRET1 gene is considered to be widely present in insects, the TRET1 gene includes homologous genes present in various insects as well as Nemur chironomid, Gambier spp., Drosophila melanogaster, honey bees, and silkworms. As used herein, the term “homologous gene” refers to a gene encoding a protein having a physiological function similar to that of the TRET1 gene product (eg, trehalose transport activity) in various insects, such as Nemurium, Gambier, Drosophila, Drosophila, and Bombyx mori. Point to.

  In order to isolate a homologous gene, a hybridization reaction is usually carried out under stringent conditions. Examples of stringent hybridization conditions include 6M urea, 0.4% SDS, 0.5 × SSC, or equivalent stringency hybridization conditions. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, conditions of 6M urea, 0.4% SDS, and 0.1 × SSC. The sequence of the isolated DNA can be determined by a known method. The homology of the isolated DNA has at least 50% or more homology over the entire amino acid sequence (see Table 8 in Example 8). Further, it has a sequence identity of 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more). Sequence homology can be determined using BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). Yes. When analyzing a base sequence by BLASTN, parameters are set to score = 100 and wordlength = 12, for example. Further, when the amino acid sequence is analyzed by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

  In the present invention, the types of insects (for example, Nemurian, Gambier medaka, Drosophila melanogaster, Apis mellifera, and silkworm) are not limited to cultivars / lines. For example, in the case of Nemur chironomid, it may be a Nigerian line, a Malawi line, or a hybrid or line of these. In the case of Drosophila melanogaster, it may be an Oregon R strain, a Canton S strain, or a hybrid of these. In the case of honey bees, it may be an Italian line, a carniolan line, or a hybrid of these lines. In the case of silkworms, it may be a large line or a C108 line, or a hybrid variety / line of these. In addition, in the case of Gambier anopheles, it may be a PEST line, a Yaounde line, or a hybrid line of these.

The present invention also provides a vector containing the TRET1 gene or a transformed cell containing the vector.
As the vector, for example, when Escherichia coli is used as a host, the vector is amplified in Escherichia coli in order to amplify it in large quantities in Escherichia coli (for example, JM109, DH5α, HB101, XL1Blue) and the like. There is no particular limitation as long as it has an ori ”and a transformed gene of E. coli transformed (for example, any drug (drug resistance gene that can be distinguished by ampicillin, tetracycline, kanamycin, chloramphenicol)). Examples include M13 vectors, pUC vectors, pBR322, pBluescript, pCR-Script, etc. For the purpose of cDNA subcloning and excision, in addition to the above vectors, for example, pGEM-T, pDIRECT PT7, etc. Especially when using a vector for the purpose of producing a protein produced from the TRET1 gene, For example, for the purpose of expression in E. coli, the expression vector is amplified in E. coli, and the host is defined as JM109, DH5α, HB101, XL1. In the case of E. coli such as -Blue, a promoter that can be expressed efficiently in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341, 544-546; FASEB J. (1992) 6, 2422-2427 ), The araB promoter (Better et al., Science (1988) 240, 1041-1043), or the T7 promoter, etc. In addition to the above vectors, pGEX-5X-1 (Amersham), “QIAexpress system” (Qiagen), pEGFP, or pET.

  The vector may contain a signal sequence for polypeptide secretion. As a signal sequence for polypeptide secretion, the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4379) may be used when the polypeptide is produced in the periplasm of E. coli. Introduction of a vector into a host cell can be performed using, for example, a calcium chloride method or an electroporation method.

  In addition to E. coli, examples of vectors for producing a protein generated from the TRET1 gene include mammalian-derived expression vectors (for example, pcDNA3 (manufactured by Invitrogen)) and pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322), pEF, pCDM8), insect cell-derived expression vectors (for example, “Bac-to-BAC baculovairus expression system” (manufactured by Gibco BRL), pBacPAK8), plant-derived expression vectors (for example, pMH1, pMH2), animal virus-derived expression vectors (eg, pHSV, pMV, pAdexLcw), retrovirus-derived expression vectors (eg, pZIPneo), yeast-derived expression vectors (eg, “Pichia Expression Kit” (Invitrogen) And pNV11, SP-Q01), Bacillus subtilis-derived expression vectors (for example, pPL608, pKTH50), and the like.

  When it is intended for expression in animal cells such as CHO cells, COS cells, NIH3T3 cells, etc., a promoter required for expression in the cells, such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108), It is essential to have MMLV-LTR promoter, EF1α promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322), CMV promoter, etc., and genes for selecting transformation into cells (for example, More preferably, it has a drug resistance gene that can be discriminated by a drug (neomycin, G418, etc.). Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

Those skilled in the art can introduce the DNA of the present invention into cells by a known method such as electroporation (electroporation).
In the present invention, the transformed cell containing the above-mentioned vector includes a transformed cell derived from Nemurus rika, Gambier mosquito, Drosophila melanogaster, honey bee, or silkworm, or a transformed cell derived from mouse, rat or human. Preferably there is. It is also possible to introduce a vector containing the TRET1 gene into a transformed cell derived from these insects or buzzard animals by the method described above.

  The present invention also provides a transgenic non-human organism comprising a transformed cell having a TRET1 gene or a vector comprising a TRET1 gene. Preferred non-human organisms in the transgenic non-human organisms of the present invention include, but are not limited to, insects and vertebrates. Insects include, but are not limited to, murmured chironomid, Gambier spp., Drosophila melanogaster, honey bees, silkworms, etc., and vertebrates include but are not limited to mice, rats , Zebrafish, or Xenopus laevis.

  For example, the production of a transgenic silkworm having a TRET1 gene is not limited to this, but can be performed as follows. That is, a vector having a TRET1 gene is introduced into a silkworm egg. For example, DNA can be introduced into silkworm eggs by injecting transposon into a silkworm early egg as a vector (Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E. , Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prudhomme, J.-C. and Couble, P , 2000, Nature Biotechnology 18, 81-84). For example, the above DNA is inserted between the transposon inverted terminal repeats (Handler AM, McCombs SD, Fraser MJ, Saul SH. (1998) Proc. Natl. Acad. Sci. USA 95 (13): 7520-5). A vector (helper vector) having a DNA encoding a transposon transferase is introduced into a silkworm egg together with the prepared vector. Helper vectors include pHA3PIG (Tamura, T., Thibert, C., Royer, C., Kanda, T., Abraham, E., Kamba, M., Komoto, N., Thomas, J.-L., Mauchamp, B., Chavancy, G., Shirk, P., Fraser, M., Prudhomme, J.-C. and Couble, P., 2000, Nature Biotechnology 18, 81-84). It is not limited.

The transposon in the present invention is preferably piggyBac, but is not limited to this, and mariner, minos, etc. can also be used (Shimizu, K., Kamba, M., Sonobe, H ., Kanda, T., Klinakis, AG, Savakis, C. and Tamura, T. (2000) Insect Mol. Biol., 9, 277-281; Wang W, Swevers L, Iatrou K. (2000) Insect Mol Biol 9 (2): 145-55).
In the present invention, transgenic silkworms can also be produced by using baculovirus vectors (Yamao, M., N. Katayama, H. Nakazawa, M. Yamakawa, Y. Hayashi et al., 1999, Genes Dev 13: 511-516).

  For example, the production of a transgenic mouse having a TRET1 gene is not limited to this, but can be performed as follows. In other words, the TRET1 gene is introduced into a mouse fertilized egg. Direct microinjection of TRET1 gene into the pronuclei of mouse fertilized eggs (Gordon, JH, Scangos, GA, Plotkin, DJ, Barbosa, JA, Ruddle, FH (1980) Proc. Natl. Acad. Sci. USA 77 7380 -7384). However, the method for producing a transgenic mouse in the present invention is not limited to this, and includes any method known to those skilled in the art.

  Production of transgenic non-human organisms can also be carried out by methods known to those skilled in the art for insects other than silkworms and vertebrates other than mice. The transgenic non-human organisms of the present invention include transgenic non-human organisms produced by any method known to those skilled in the art.

  The present invention also provides a primer set for amplifying all or part of the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9. In the primer of the present invention, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp, and more preferably 21, 24, 25, 26, 28, 29, 30 nucleotides, for example. The primer is not particularly limited as long as it can amplify at least part of the DNA of the present invention or its complementary strand. When used as a primer, the 3 ′ region can be complementary, and a restriction enzyme recognition sequence, a tag, or the like can be added to the 5 ′ side.

Specifically, as the primer set of the present invention,
(A) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 11 and DNA comprising the nucleotide sequence set forth in SEQ ID NO: 12 (primer for detecting a marker comprising all or part of the nucleotide sequence set forth in SEQ ID NO: 1 set),
(B) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 13 and DNA comprising the nucleotide sequence set forth in SEQ ID NO: 14 (primer for detecting a marker comprising all or part of the nucleotide sequence set forth in SEQ ID NO: 3 set),
(C) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 15 and DNA comprising the nucleotide sequence set forth in SEQ ID NO: 16 (primer for detecting a marker comprising all or part of the nucleotide sequence set forth in SEQ ID NO: 5 set),
(D) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 17 and DNA comprising the nucleotide sequence set forth in SEQ ID NO: 18 (primer for detecting a marker comprising all or part of the nucleotide sequence set forth in SEQ ID NO: 7 set),
(E) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 19 and DNA comprising the nucleotide sequence set forth in SEQ ID NO: 20 (primer for detecting a marker comprising all or part of the nucleotide sequence set forth in SEQ ID NO: 9 set),
Can be illustrated. By comparing the information characterized by the DNA sequence amplified by these primer sets with the base sequence of the TRET1 gene of the present invention, it is determined whether the cell from which the amplified DNA sequence is derived has the TRET1 gene. Is possible.

In addition to the primers described above, those skilled in the art can create a primer set having the same function using the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9. It is. Such primers are also included in the primer of the present invention.
The PCR primer of the present invention can be prepared by those skilled in the art using, for example, an automatic oligonucleotide synthesizer. Those skilled in the art can also carry out the method of the present invention by a well-known polymorphism detection method, for example, the PCR-SSCP method described later using the above PCR primers.

The present invention provides an isolated TRET1 protein encoded by the DNA described in (1) or (2) below.
(1) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10
(2) DNA comprising the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9

  The protein can be prepared, for example, as a recombinant. For example, if the chironomid TRET1 protein (PvTRET1), based on mRNA extracted from cells expressing PvTRET1 protein (eg, fat bodies, cultured cells derived from fat bodies, and cells and tissues capable of producing trehalose) A cDNA library is obtained (Short, JM et al., Nucleic Acid Research, 16, 7583, 1988). DNA encoding PvTRET1 protein is isolated by screening clones that hybridize from this library using a probe set based on the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9. be able to. The PvTRET1 protein encoded from the DNA can be obtained by using a protein expression system well known to those skilled in the art. PvTRET1 protein can be recovered and purified from cultures of cells expressing PvTRET1 protein. TRET1 protein derived from Gambier anopheles, Drosophila melanogaster, honeybee, and silkworm can be obtained by the same method.

  The present invention also provides a protein functionally equivalent to the above TRET1 protein. The biological species from which such a protein is derived is not particularly limited, and examples thereof include Tosonama grasshopper, American cockroach, tobacco tin mega, and cecropia moth that are used for insect research.

Examples of proteins functionally equivalent to the TRET1 protein include, for example,
(3) 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, 4, 6, 8, or 10; DNA encoding a protein having a function equivalent to that of a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.
(4) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9, comprising SEQ ID NO: 2, 4, 6, 8, or DNA encoding a protein having a function equivalent to that of the protein consisting of the amino acid sequence described in 10,
Isolated protein encoded by.

Here, a protein functionally equivalent to the TRET1 protein has an amino acid sequence represented by SEQ ID NO: 21 as compared with a human glucose transporter. Therefore, proteins functionally equivalent to the TRET1 protein of the present invention include
(A) comprising the amino acid sequence set forth in SEQ ID NO: 21, wherein one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10 are substituted, deleted, added, and / or A DNA encoding a protein consisting of an inserted amino acid sequence, the protein having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10;
The isolated protein encoded by is included. The description of the amino acid sequence of SEQ ID NO: 21 is as described above.

Here, the amino acid sequences of the region corresponding to SEQ ID NO: 21 in Nemuri chironika, Gambier spp., Drosophila melanogaster, honeybee and silkworm correspond to SEQ ID NOS: 22, 23, 24, 25 and 26, respectively. Therefore, proteins functionally equivalent to the TRET1 protein of the present invention include
A protein comprising the amino acid sequence set forth in SEQ ID NO: 22 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 2; DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2;
A protein comprising the amino acid sequence set forth in SEQ ID NO: 23, wherein the amino acid sequence set forth in SEQ ID NO: 4 has an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted; DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 4;
A protein comprising the amino acid sequence of SEQ ID NO: 24, wherein the amino acid sequence of SEQ ID NO: 6 has an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted; DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
A protein comprising the amino acid sequence of SEQ ID NO: 25, wherein the amino acid sequence of SEQ ID NO: 8 comprises an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted; DNA encoding a protein having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 8;
A protein comprising an amino acid sequence represented by SEQ ID NO: 26, wherein the amino acid sequence represented by SEQ ID NO: 10 has an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted; DNA encoding a protein having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 10;
The isolated protein encoded by is included.

Here, the base sequence encoding the amino acid sequence set forth in SEQ ID NO: 22 corresponds to the base sequence from position 247 to position 333 of the base sequence of SEQ ID NO: 1.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 23 corresponds to the base sequence of positions 133 to 219 of the base sequence of SEQ ID NO: 3.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 24 corresponds to the 127th to 213th base sequences of the base sequence of SEQ ID NO: 5;
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 25 corresponds to the 124th to 210th base sequences of the base sequence of SEQ ID NO: 7.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 26 corresponds to the 151st to 237th base sequences of the base sequence of SEQ ID NO: 9.

Furthermore, as a protein functionally equivalent to the TRET1 protein of the present invention,
(B) It is encoded by DNA comprising the amino acid sequence set forth in SEQ ID NO: 21 and hybridizing under stringent conditions with DNA consisting of the base sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9. A DNA encoding a protein having a function equivalent to that of a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10;
The isolated protein encoded by is included. The amino acid sequence set forth in SEQ ID NO: 21 is as described above.

Here, as described above, the amino acid sequences of the region corresponding to SEQ ID NO: 21 in Nemurus, Gambiae, Drosophila, Apis mellifera, and silkworm are SEQ ID NOs: 22, 23, 24, 25, and 26, respectively. Therefore, proteins functionally equivalent to the TRET1 protein of the present invention include:
A protein encoded by a DNA comprising the amino acid sequence set forth in SEQ ID NO: 22 and hybridizing under stringent conditions with the DNA consisting of the base sequence set forth in SEQ ID NO: 1, wherein the protein is set forth in SEQ ID NO: 2. DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence,
A protein encoded by a DNA comprising the amino acid sequence set forth in SEQ ID NO: 23 and hybridizing under stringent conditions with the DNA consisting of the base sequence set forth in SEQ ID NO: 3 and described in SEQ ID NO: 4. DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence,
A protein encoded by a DNA comprising the amino acid sequence set forth in SEQ ID NO: 24 and hybridizing under stringent conditions with the DNA consisting of the base sequence set forth in SEQ ID NO: 5; DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence,
A protein encoded by a DNA comprising the amino acid sequence set forth in SEQ ID NO: 25 and hybridizing under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 7; DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence,
A protein encoded by a DNA comprising the amino acid sequence set forth in SEQ ID NO: 26 and hybridizing under stringent conditions with a DNA consisting of the base sequence set forth in SEQ ID NO: 9 and described in SEQ ID NO: 10 DNA encoding a protein having a function equivalent to a protein consisting of an amino acid sequence,
The isolated protein encoded by is included. Here, the base sequence of the DNA encoding the amino acid sequence described in SEQ ID NOs: 22 to 26 is as described above.

Furthermore, the TRET1 protein produced from the TRET1 gene of the present invention is characterized by having a 12-transmembrane region called a sugar transporter region. Therefore, TRET1 protein produced from TRET1 of the present invention includes:
(I) comprising the amino acid sequence of the 12th transmembrane region, wherein one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10 A DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence described in SEQ ID NO: 2, 4, 6, 8, or 10;
The isolated protein encoded by is included.

Here, the amino acid sequences of the twelve transmembrane regions in Nemurus, Gambier, Drosophila, Honeybee, and Silkworm correspond to SEQ ID NOs: 27, 28, 29, 30 and 31, respectively (see FIG. 10). Therefore, the TRET1 protein produced from the TRET1 gene of the present invention includes:
A protein comprising any amino acid sequence set forth in SEQ ID NO: 27 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 2. A DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2;
A protein comprising any amino acid sequence set forth in SEQ ID NO: 28 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 4. A DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 4;
A protein comprising any amino acid sequence set forth in SEQ ID NO: 29 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 6. A DNA encoding a protein having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
A protein comprising any one of the amino acid sequences set forth in SEQ ID NO: 30 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence set forth in SEQ ID NO: 8. A DNA encoding a protein having a function equivalent to the protein consisting of the amino acid sequence set forth in SEQ ID NO: 8;
A protein comprising any amino acid sequence set forth in SEQ ID NO: 31 and comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 10. A DNA encoding a protein having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 10;
Is included.

Here, the base sequence encoding the amino acid sequence set forth in SEQ ID NO: 27 corresponds to the base sequence from position 250 to position 1572 of the base sequence of SEQ ID NO: 1.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 28 corresponds to the base sequence from position 136 to position 1458 of the base sequence of SEQ ID NO: 3.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 29 corresponds to the base sequence from position 130 to position 1452 of the base sequence of SEQ ID NO: 5.
The base sequence encoding the amino acid sequence set forth in SEQ ID NO: 30 corresponds to the 127th to 1446th base sequences of the base sequence of SEQ ID NO: 7,
The base sequence encoding the amino acid sequence described in SEQ ID NO: 31 corresponds to the 154th to 1461th base sequences of the base sequence of SEQ ID NO: 9.

Furthermore, the TRET1 protein produced from the TRET1 gene of the present invention includes:
(Ii) a protein encoded by DNA comprising the amino acid sequence of the 12-transmembrane region and hybridized under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7, or 9 A DNA encoding a protein having a function equivalent to that of a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10.
Is included.

Here, as mentioned above, the amino acid sequences of the 12-transmembrane region in Nemur Chironomidae, Gambier anopheles, Drosophila melanogaster, Apis mellifera and Silkworm correspond to SEQ ID NOs: 27, 28, 29, 30 and 31, respectively. From the TRET1 protein produced from the TRET1 gene of the present invention,
A protein encoded by a DNA comprising any one of the amino acid sequences set forth in SEQ ID NO: 27 and hybridizing under stringent conditions with the DNA consisting of the base sequence set forth in SEQ ID NO: 1, comprising SEQ ID NO: 2 DNA encoding a protein having the same function as the protein consisting of the amino acid sequence described in 1.
A protein encoded by a DNA comprising any one of the amino acid sequences set forth in SEQ ID NO: 28 and hybridizing under stringent conditions with the DNA consisting of the base sequence set forth in SEQ ID NO: 3; SEQ ID NO: 4 DNA encoding a protein having the same function as the protein consisting of the amino acid sequence described in 1.
A protein encoded by a DNA comprising any one of the amino acid sequences set forth in SEQ ID NO: 29 and hybridizing under stringent conditions with the DNA comprising the base sequence set forth in SEQ ID NO: 5; SEQ ID NO: 6 DNA encoding a protein having the same function as the protein consisting of the amino acid sequence described in 1.
A protein encoded by a DNA comprising any one of the amino acid sequences set forth in SEQ ID NO: 30 and hybridizing under stringent conditions with the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 7; SEQ ID NO: 8 DNA encoding a protein having the same function as the protein consisting of the amino acid sequence described in 1.
A protein encoded by a DNA comprising any one of the amino acid sequences set forth in SEQ ID NO: 31 and hybridizing under stringent conditions with the DNA comprising the base sequence set forth in SEQ ID NO: 9; SEQ ID NO: 10 DNA encoding a protein having the same function as the protein consisting of the amino acid sequence described in 1.
The isolated protein encoded by is included. Here, the base sequence of the DNA encoding the amino acid sequence described in SEQ ID NOs: 22 to 26 is as described above. Examples of the protein “having an equivalent function” to the TRET1 protein include a protein having trehalose transport activity.

  Examples of the protein having a function equivalent to that of the TRET1 protein include immunologically equivalent proteins. In the present invention, the “protein that is immunologically equivalent to the TRET1 protein” is not particularly limited as long as it reacts with an antibody that specifically recognizes the TRET1 protein. For example, a TRET1 protein epitope peptide, a TRET1 protein domain containing the epitope, or a protein containing the domain can be cited as a protein immunologically equivalent to the TRET1 protein.

  A TRET1 protein fragment can be obtained by digestion with a protease. In addition, by randomly cutting the DNA encoding the TRET1 protein shown in SEQ ID NO: 1, 3, 5, 7 or 9 and inserting it into a phage vector to create a phage library displaying domain peptides Can also be obtained. Immunologically active domains can be determined by immunoscreening these libraries with an antibody that recognizes the TRET1 protein. By determining the base sequence of the inserted fragment of the cloned phage, the amino acid sequence of the active domain can also be clarified.

  In addition, a protein functionally equivalent to the TRET1 protein according to the present invention is also defined based on the biochemical activity of the TRET1 protein. The biochemical activity of TRET1 protein mainly refers to trehalose transport activity that does not depend on substances other than trehalose such as ATP and protons, and in part, glucose transport activity and α-glucoside transport activity excluding sucrose and maltose. Point to.

  Proteins having functions equivalent to these TRET1 proteins can be used as fusion proteins with other proteins. For example, a protein having an additional amino acid sequence such as a FLAG tag, an HA tag, or a histidine tag and having at least one property as a protein having a function equivalent to the TRET1 protein has a function equivalent to the above. Included in proteins that have. Even if the protein to be added has an activity different from that of the TRET1 protein, the fusion protein is a protein having an equivalent function in the present invention when at least one function of the TRET1 protein is maintained. include.

  A protein having a function equivalent to that of the TRET1 protein can be isolated by a person skilled in the art by a known method (Experimental Medicine Separate Volume / Genetic Engineering Handbook, pp246-251, Yodosha, 1991). For example, if a desired library is screened using the nucleotide sequence (or fragment thereof) shown in SEQ ID NO: 1, 3, 5, 7 or 9 as a probe, DNA having a highly homologous nucleotide sequence is cloned. It is possible. As such a library, a library obtained by randomly mutating the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7 or 9 can be presented. In addition, fat shells and fat body-derived cultured cells such as Nemuri chironika, fat body and fat body-derived cultured cells such as Gambier spp. A cDNA library such as a fat body and a cultured cell derived from a fat body is used for a honey bee, and a cultured cell derived from a fat body and a fat body is used for a silkworm.

  As a method for randomly mutating a given base sequence, for example, substitution of base pairs by nitrite treatment of DNA is known (Hirose, S. et al., Proc. Natl. Acad. Sci. USA., 79: 7258-7260, 1982). In this method, base pair substitution can be randomly introduced into a specific segment by treating the segment into which a mutation is to be introduced with nitrous acid. Alternatively, there is a gapped duplex method or the like as a technique for bringing a target mutation to an arbitrary place (Kramer W. and Fritz HJ., Methods in Enzymol., 154: 350-367, 1987). A circular double-stranded vector in which a gene to be introduced with a mutation is cloned is made into a single strand, and a synthetic oligonucleotide having a mutation at the target site is hybridized. Complementary single-stranded DNA derived from a vector that has been linearized by cutting with a restriction enzyme is annealed to the circular single-stranded vector, and the gap between the synthetic nucleotide is filled with DNA polymerase and further ligated. To make a complete double-stranded circular vector.

The number of amino acids to be modified is typically within 50 amino acids, preferably within 30 amino acids, and more preferably within 5 amino acids (eg, 1 amino acid).
When an amino acid is artificially substituted, it is considered that the activity of the original protein is easily maintained if the amino acid is substituted with an amino acid having similar properties. The protein of the present invention includes a protein to which a conservative substitution is added in the above amino acid substitution and has a function equivalent to that of the TRET1 protein (SEQ ID NO: 2, 4, 6, 8 or 10). Conservative substitution is considered to be important, for example, when substituting amino acids of domains important for protein activity. Such amino acid conservative substitutions are well known to those skilled in the art.

Examples of amino acid groups corresponding to conservative substitutions include basic amino acids (eg, lysine, arginine, histidine), acidic amino acids (eg, aspartic acid, glutamic acid), uncharged polar amino acids (eg, glycine, asparagine, glutamine, serine, Threonine, tyrosine, cysteine), nonpolar amino acids (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched amino acids (eg threonine, valine, isoleucine), and aromatic amino acids (eg tyrosine, phenylalanine) , Tryptophan, histidine) and the like.
Moreover, it is also conceivable to increase the protein activity or the like by non-conservative substitution (for example, including a constitutively activated protein) or decrease (for example, by including a dominant negative).

  It consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10, and SEQ ID NO: 2, 4, 6, A protein functionally equivalent to a protein comprising the amino acid sequence described in 8 or 10 includes a naturally occurring protein. In general, eukaryotic genes have a polymorphism, as is known for interferon genes and the like. One or more amino acids may be substituted, deleted, inserted, and / or added due to a change in the base sequence caused by this polymorphism. Such a naturally occurring protein, and in the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8 or 10, one or more amino acids are substituted, deleted, inserted and / or added. A protein having the same amino acid sequence and having a function equivalent to that of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10 is included in the present invention.

  Alternatively, the amino acid sequence may not change even if the base sequence changes due to the polymorphism. Such a nucleotide sequence variation is called a silent variation. A protein encoded by a DNA comprising a base sequence having a silent mutation is also included in the present invention. In addition, the polymorphism mentioned here means that a certain gene has a different base sequence between individuals in a population. Polymorphism is independent of the rate at which different genes are found.

  In addition, examples of a method for obtaining a protein having a function equivalent to that of the TRET1 protein include a method using hybridization. Specifically, DNA encoding the TRET1 protein according to the present invention as shown in SEQ ID NO: 1, 3, 5, 7 or 9 or a fragment thereof is used as a probe, and DNA capable of hybridizing with this is isolated. When hybridization is performed under stringent conditions, DNA with high homology is selected as the base sequence, and as a result, the isolated protein may contain a protein having a function equivalent to that of the TRET1 protein. Rise.

  In addition, stringent conditions can show the above-mentioned conditions, for example. Stringency depends on conditions such as salt concentration, formamide concentration, or temperature, but those skilled in the art will readily set these conditions to obtain the required stringency.

  By utilizing hybridization, it is possible to isolate DNA encoding a TRET1 protein homolog in an insect species other than, for example, Chironomidae, Gambier spp., Drosophila melanogaster, honeybees, or silkworms. A homologue of TRET1 protein encoded by DNA obtained from insect species other than these, for example, Tonama grasshopper, American cockroach, tobacco tin mega, and cecropia moth, which are used for insect research, is a functionally equivalent protein in the present invention. Configure.

  Proteins obtained by introducing mutations into the TRET1 protein (SEQ ID NO: 2, 4, 6, 8 or 10), and proteins encoded by DNA isolated using the above-described hybridization techniques, Usually, it has high homology in amino acid sequence with the TRET1 protein (SEQ ID NO: 2, 4, 6, 8 or 10). High homology refers to sequence identity of at least 30% or more, preferably 50% or more, more preferably 80% or more (eg, 95% or more). As described above, the identity of the base sequence and amino acid sequence can be performed using a homology search site using the Internet. In addition, a search using BLAST can be performed in the National Center for Biotechnology Information (NCBI) (for example, the BLAST page of the NCBI website; http://www.ncbi.nlm.nih.gov/BLAST Altschul, SF et al., J. Mol. Biol., 1990, 215 (3): 403-10; Altschul, SF & Gish, W., Meth.Enzymol., 1996, 266: 460-480; Altschul , SF et al., Nucleic Acids Res., 1997, 25: 3389-3402).

  For example, the calculation of amino acid sequence identity in Advanced BLAST 2.1 uses blastp as the program, Expect value is 10, Filter is all OFF, BLOSUM62 is used for Matrix, Gap existence cost, Per residue gap cost, and Lambda ratio Can be set to 11, 1, 0.85 (default values), respectively, to obtain identity values (%) (Karlin, S. and SF Altschul (1990) Proc. Natl. Acad Sci. USA 87: 2264-68; Karlin, S. and SF Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7).

  The protein according to the present invention or a protein having an equivalent function is a protein to which various modifications such as physiological modification of sugar chains, labels such as fluorescence and radioactive substances, or fusion with other proteins are added. be able to. In the gene recombinants described later, there is a possibility that the modification by the sugar chain varies depending on the host to be expressed. However, even if there is a difference in the modification of the sugar chain, any TRET1 protein according to the present invention or a protein having an equivalent function can be used as long as it exhibits the same properties as the TRET1 protein disclosed in the present specification. It is.

  The TRET1 protein can be obtained not only as a biomaterial but also as a gene recombinant by incorporating a gene encoding it into an appropriate expression system. In order to obtain the TRET1 protein by a genetic engineering technique, the DNA encoding the TRET1 protein described above may be incorporated into an appropriate expression system for expression. Examples of host / vector systems applicable to the present invention include expression vector pGEX and E. coli. Since pGEX can express foreign genes as fusion proteins with glutathione S-transferase (GST) (Gene, 67: 31-40, 1988), pGEX incorporating the gene encoding TRET1 protein can be treated with BL21 by heat shock. And isopropylthio-β-D-galactoside (IPTG) is added after an appropriate culture time to induce the expression of GST-fused TRET1 protein. The gene encoding the TRET1 protein can be obtained by, for example, amplification by PCR or the like using a fat library and a cDNA library such as a fat cell-derived cultured cell as a template. Since GST according to the present invention is adsorbed to glutathione sepharose 4B, the expression product can be easily separated and purified by affinity chromatography.

  In addition to the above, the following can be applied as a host / vector system for obtaining a TRET1 protein recombinant. First, when a bacterium is used as a host, a fusion protein expression vector using a histidine tag, an HA tag, a FLAG tag or the like is commercially available. In yeast, it is known that yeasts of the genus Pichia are effective for the expression of proteins with sugar chains. In terms of glycosylation, an expression system using a baculovirus vector hosted by insect cells is also useful (Bio / Technology, 6: 47-55, 1988). In addition, mammalian cells are used to transfect vectors using promoters such as CMV, RSV, or SV40, and these host / vector systems are all used as TRET1 protein expression systems. can do. In addition, the gene can be introduced using a viral vector such as a retrovirus vector, an adenovirus vector, or an adeno-associated virus vector.

  The obtained protein of the present invention can be isolated from inside or outside the host cell (such as a medium) and purified as a substantially pure and homogeneous protein. The separation and purification of the protein may be performed using the separation and purification methods used in normal protein purification, and is not limited at all. For example, chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. are appropriately selected, When combined, proteins can be separated and purified.

  Examples of chromatography include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, etc. (Marshak et al., Strategies for Protein Purification and Characterization: A Laboratory Course Manual. Ed Daniel R. Cold Spring Harbor Laboratory Press, 1996). These chromatography can be performed using liquid phase chromatography, for example, liquid phase chromatography such as HPLC and FPLC.

  In addition, the protein of the present invention is preferably a protein to which a modification such as a physiological higher order structure, sugar chain addition, disulfide bond, lipid addition, methylation and the like is added. Moreover, the protein of the present invention is preferably a substantially purified protein. Here, “substantially purified” means that the degree of purification of the protein of the present invention (ratio of the protein of the present invention in the whole protein component) is 50% or more, 60% or more, 70% or more, 80% or more, It means 90% or more, 95% or more, 100% or close to 100%. The upper limit close to 100% depends on the purification technique and analysis technique of those skilled in the art, and is, for example, 99.999%, 99.99%, 99.9%, 99%, etc.

  Moreover, as long as it has said purity, what was refine | purified by what kind of purification method is contained in the substantially purified protein. For example, the above chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. Although substantially purified protein can be exemplified by selection or combination, it is not limited thereto.

  The present invention also provides a partial peptide (partial fragment) of the protein of the present invention described above. The length of the partial peptide is not particularly limited as long as it is functionally equivalent to the protein of the present invention. Partial peptides can be used as drugs that increase drought tolerance of cells, osteoclast differentiation inhibitors for the prevention of osteoporosis, and polyglutamine / polyalanine symptom-improving drugs represented by Huntington's disease. is there.

  The present invention provides an antibody that recognizes TRET1 protein and the like. The antibody of the present invention can be obtained by a known method using the TRET1 protein of the present invention or a fragment thereof as an immunogen (Harlow, E. & Lane, D .; Antibodies; A Laboratry manual. Cold Spring Harbor, New York , 1988, Kohler, G. & Milstein, C., Nature 256: 495-7, 1975).

  For immunization, an immunized animal is immunized with a TRET1 protein of the present invention or a fragment thereof together with an appropriate adjuvant. The TRET1 protein fragment can be combined with a carrier protein to serve as an immunogen. As the carrier protein for obtaining the immunogen, mussel hemocyanin (KLH), bovine serum albumin (BSA) or the like can be used.

  Rabbits, mice, rats, goats or sheep are generally used as immunized animals. Freund's complete adjuvant (FCA) or the like is generally used as an adjuvant (Adv. Tubercl. Res., 1: 130-148, 1956). Immunization is added at appropriate intervals, and when an increase in antibody titer is confirmed, blood is collected to obtain antiserum. Furthermore, if the antibody fraction is purified, a purified antibody (polyclonal antibody) can be obtained.

  Alternatively, monoclonal antibodies can also be obtained by collecting antibody-producing cells and cloning them by a cell fusion method or the like. Monoclonal antibodies are important tools for achieving high sensitivity and specificity in immunoassays.

  As antibody-producing cells, those derived from immunized animals can also be used. Furthermore, it is possible to construct a chimeric antibody or a humanized antibody based on the antibody gene of the monoclonal antibody-producing cells derived from the immunized animal thus obtained.

  The antibody of the present invention can be used as a reagent for immunological detection such as TRET1 protein. Furthermore, the antibody of the present invention can also be used for transformant determination when a transgenic mammal is produced. Methods for immunologically detecting proteins and the like present in tissues and blood using antibodies are known.

  The antibody of the present invention can be used for separation or detection of TRET1 protein or the like, or cells expressing TRET1 protein or the like. Methods for isolating and purifying proteins and the like using antibodies are well known to those skilled in the art. Moreover, the TRET1 protein and the like of the present invention can be used as a marker for fat bodies, fat body-derived cells and cells having trehalose-producing ability. That is, by using the expression of TRET1 protein or the like as an index, fat bodies, cells derived from fat bodies, and cells capable of producing trehalose can be detected and separated. The antibody is appropriately labeled with fluorescence or the like. For example, cells that express TRET1 protein or the like can be isolated by cell sorting or the like using an antibody against TRET1 protein or the like.

The present invention relates to a drug for introducing trehalose into a cell, which contains the gene of the present invention, a vector containing the gene, or a protein produced from the gene. The present invention also relates to a drug that increases the drought tolerance of cells, comprising the gene of the present invention, a vector containing the gene, a protein produced from the gene or a protein containing a partial sequence thereof. “Increasing drought tolerance of cells” is as described above. Furthermore, the present invention is represented by an agent that enhances drought tolerance of cells or tissues containing the protein of the present invention or a protein containing a partial sequence thereof, an osteoclast differentiation inhibitor for the prevention of osteoporosis, and Huntington's disease Provide symptom-improving drugs for polyglutamine and polyalanine diseases. As long as the protein in these drugs or the protein containing the partial sequence thereof is isolated, there is no particular limitation on the state thereof. Either a protein or a protein containing a crude state or a partial sequence thereof can be used. Examples of cells with enhanced drought tolerance include Sf-9 cells and Schneider cells for insects, and CHO-K1 cells, NIH / 3T3 cells, and HuH-7 cells for mammalian cells. Examples include, but are not limited to, fat bodies and muscles for insects, and a group of cells constituting the blood, liver, and muscle for mammals.
These drugs are osteoclast differentiation inhibitors for the prevention of osteoporosis in humans and non-human animals (eg laboratory animals, livestock animals, pets, etc.), and improve the symptoms of polyglutamine and polyalanine diseases represented by Huntington's disease It can also be used as a medicine.

The protein of the present invention or a protein containing a partial sequence thereof is, for example, a pharmacologically acceptable carrier or medium such as sterile water or physiological saline, a stabilizer, an excipient, an antiseptic, a surfactant, a chelating agent. (EDTA etc.), binders, etc. may be formulated and administered as appropriate. It is also conceivable that the gene of the present invention linked to an arbitrary tissue-specific promoter is incorporated into a vector such as adenovirus and administered to humans or animals other than humans.
Examples of the drug form (dosage form) of the present invention include, but are not limited to, injection dosage forms, lyophilized dosage forms, and solution dosage forms.

  Administration to a patient can be either oral or parenteral administration, but preferably is parenteral administration, for example, injection administration is possible. Examples of injection administration can be systemic or local administration by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like.

  The dose varies depending on the weight and age of the patient, the administration method, symptoms, etc., but those skilled in the art can appropriately select an appropriate dose. The general dose varies depending on the effective blood concentration of the drug and the metabolic time, but the daily maintenance dose is about 0.1 mg / kg to about 1.0 g / kg, preferably about 0.1 mg / kg to about 10 mg / kg. More preferably, it is considered to be about 0.1 mg / kg to about 1.0 mg / kg. Administration can be divided into 1 to several times.

  The present invention also relates to a method for increasing trehalose permeability of a cell, comprising the step of expressing the gene of the present invention in any cell. The present invention further relates to a method for introducing trehalose into a cell, comprising the step of expressing the gene of the present invention in any cell. The cell used in the present invention is not particularly limited, and any cell can be used. In addition, the method for expressing the gene of the present invention in a cell is not particularly limited, and various methods known to those skilled in the art can be used.

  The present invention also provides an oligonucleotide having a chain length of at least 15 nucleotides complementary to DNA comprising the TRET1 gene or its complementary strand. The chain length of the oligonucleotide in the present invention is not particularly limited as long as it is 15 nucleotides or more, but is preferably 20 nucleotides or more, and more preferably, for example, 21, 24, 25, 26, 28, 29 , 30 nucleotides.

  The “complementary strand” refers to the other strand with respect to one strand of a double-stranded nucleic acid consisting of A: T (U in the case of RNA) and G: C base pairs. Further, “complementary” is not limited to a completely complementary sequence with at least 15, more preferably 20 or more consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably It suffices to have 90% or more preferably homology on the base sequence of 95% or more. An algorithm for determining homology may be one known to those skilled in the art. Examples of the oligonucleotide of the present invention include DNA containing the base sequence described in any of SEQ ID NOs: 11 to 20 and 35.

  The oligonucleotide of the present invention can be used as a probe for detecting DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, 7 or 9. Moreover, the oligonucleotide of the present invention can be used in the form of a substrate of a DNA array.

When the oligonucleotide of the present invention is used as a probe, it is preferably used after appropriately labeling. As a labeling method, a method of labeling by phosphorylating the 5 ′ end of the oligonucleotide with ³² P using T4 polynucleotide kinase, and a random hexamer oligonucleotide or the like using a DNA polymerase such as Klenow enzyme are used. Examples of the primer include a method of incorporating a substrate base labeled with an isotope such as ³² P, a fluorescent dye, or biotin (random prime method or the like).

  The oligonucleotide of the present invention can be prepared, for example, by a commercially available oligonucleotide synthesizer. The probe can also be prepared as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like.

The present invention also provides a screening method for a test compound.
As a first aspect of the screening method of the present invention, a screening method for a test compound including the following steps (a) to (c) can be mentioned.
(A) contacting the test compound with the TRET1 gene transcript (b) detecting the binding between the TRET1 gene transcript and the test compound (c) selecting a test compound that binds to the TRET1 gene transcript Process

  In the first embodiment, first, a test compound is brought into contact with the transcription product of the TRET1 gene. The “transcription product of TRET1 gene” in the screening method of the present invention includes a transcription product of TRET1 gene and a translation product translated from the transcription product.

  There is no restriction | limiting in particular as "test compound" in the method of this invention, For example, a single compound, such as a natural compound, an organic compound, an inorganic compound, protein, a peptide, and an expression product of a compound library and a gene library And cell extracts, cell culture supernatants, fermented microorganism products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single cell extracts, animal cell extracts, and the like. The test compound can be appropriately labeled and used as necessary. Examples of the label include a radiolabel and a fluorescent label.

  In the present invention, “contact” is performed as follows. For example, if the TRET1 gene transcript is in a purified state, it can be carried out by adding a test compound to the purified preparation. Moreover, if it is the state expressed in the cell or the state expressed in the cell extract, it can be carried out by adding a test compound to the cell culture solution or the cell extract, respectively. The cells in the present invention are not particularly limited as long as TRET1 is expressed, and cells derived from any organism can be used. Examples of the cells used in the present invention include, but are not limited to, cells derived from insects including Nemur Chironomidae, Gambier anopheles, Drosophila melanogaster, honey bees, and silkworms. When the test compound is a protein, for example, a vector containing DNA encoding the protein is introduced into a cell in which the TRET1 gene is expressed, or the vector is introduced into a cell extract in which the TRET1 gene is expressed. It is also possible to carry out by adding. Also, for example, a two-hybrid method using yeast or animal cells can be used.

  In the first embodiment, the binding between the TRET1 gene transcript and the test compound is then detected. The means for detecting or measuring the binding between proteins can be performed, for example, by using a label attached to the protein. Examples of the type of label include a fluorescent label and a radiolabel. Further, it can also be measured by a known method such as an enzyme two-hybrid method or a measurement method using BIACORE. In this method, a test compound bound to the TRET1 gene transcription product is then selected. If the binding to the selected test compound changes the intracellular localization of TRET1 or the activity of TRET1, it can be used for the regulation of TRET1 activity using the test compound. Therefore, it is considered that the test compound is useful for providing a more precise trehalose introduction system. In addition, the selected test compound may be used as a test compound for the following screening.

In addition, as a second aspect of the screening method of the present invention, there is provided a screening method for a test compound comprising the following steps (a) to (c).
(A) a step of contacting a test compound with a cell collected from an invertebrate (b) a step of measuring the expression level of a transcription product of the TRET1 gene (c) as compared with a case where the test compound is not contacted And selecting a test compound having an increased or decreased expression level of the transcript.

  In the second embodiment, first, a test compound is brought into contact with cells collected from an invertebrate that uses trehalose as a blood sugar. Here, examples of the “invertebrates” include crustaceans, nemur chironomids, Gambier anopheles, Drosophila melanogaster, honey bees, and insects including silkworms. In addition, “a cell collected from an invertebrate animal” refers to any cell derived from an invertebrate animal that clearly has the TRET1 gene. The description of “test compound” and “contact” is as described above.

  In the second embodiment, the expression level of the TRET1 gene transcript is then measured. The expression level of the TRET1 gene transcript can be measured by methods known to those skilled in the art. For example, mRNA encoding a TRET1 gene transcript can be extracted according to a standard method, and the transcription level of the gene can be measured by performing Northern hybridization or RT-PCR using this mRNA as a template. . Furthermore, it is also possible to measure the expression level of the TRET1 gene transcript using DNA array technology.

  In addition, the fraction containing the TRET1 gene transcript can be collected according to a conventional method, and the expression level of the TRET1 gene transcript can be detected by electrophoresis such as SDS-PAGE to measure the translation level of the gene. . It is also possible to measure the translation level of the gene by performing Western blotting using an antibody against the TRET1 gene transcript and detecting the expression of the TRET1 gene transcript.

  The antibody used for detecting the transcription product of the TRET1 gene is not particularly limited as long as it is a detectable antibody. For example, both a monoclonal antibody and a polyclonal antibody can be used. The antibody can be prepared by methods known to those skilled in the art as described above.

  In the second embodiment, the test compound is then selected as a compound for controlling insect growth when the expression level of the TRET1 gene transcript changes compared to when the test compound is not contacted. . Specifically, when the test compound increases the expression of the TRET1 gene, the test compound is useful for promoting the growth of natural enemy insects such as mistletoe and industrial insects such as silkworms. When the expression is decreased, it is considered useful for delaying the growth of pests, that is, as an agrochemical.

A third aspect of the screening method of the present invention is a screening method for a test compound, which includes the following steps (a) to (d).
(A) a step of providing a cell or a cell extract having DNA to which a reporter gene is functionally linked downstream of the promoter region of the TRET1 gene (b) a step of bringing a test compound into contact with the cell or the cell extract ( c) a step of measuring the expression level of the reporter gene in the cell or the cell extract (d) a subject whose expression level of the reporter gene is increased or decreased as compared with the case where the test compound is not contacted Process of selecting test compound

In the third embodiment, first, a cell or a cell extract having DNA to which a reporter gene is operably linked downstream of the promoter region of the TRET1 gene is provided.
In the third embodiment, “functionally linked” means that the promoter region of the TRET1 gene and the reporter gene are such that expression of the reporter gene is induced by binding of a transcription factor to the promoter region of the TRET1 gene. Is connected. Therefore, even when the reporter gene is bound to another gene and forms a fusion protein with another gene product, expression of the fusion protein is caused by binding of a transcription factor to the promoter region of the TRET1 gene. If it is derived, it is included in the meaning of the above “functionally coupled”.

  The reporter gene is not particularly limited as long as its expression can be detected. For example, a CAT gene, a lacZ gene, a luciferase gene, a β-glucuronidase gene (GUS) and a GFP that are commonly used by those skilled in the art. A gene etc. can be mentioned.

  In the third embodiment, the test compound is then brought into contact with the cells or the cell extract. Next, the expression level of the reporter gene in the cell or the cell extract is measured. The description of “test compound” and “contact” is as described above.

  The expression level of the reporter gene can be measured by methods known to those skilled in the art depending on the type of reporter gene used. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting acetylation of chloramphenicol by the gene product. When the reporter gene is a lacZ gene, by detecting the color development of the dye compound catalyzed by the gene expression product, and when the reporter gene is a luciferase gene, the fluorescent compound catalyzed by the gene expression product By detecting fluorescence, and in the case of a β-glucuronidase gene (GUS), the luminescence of Glucuron (ICN) or 5-bromo-4-chloro-3-indolyl- By detecting the color development of β-glucuronide (X-Gluc), and in the case of a GFP gene, the expression level of the reporter gene can be measured by detecting fluorescence due to the GFP protein.

  In the third embodiment, finally, when the expression level of the reporter gene changes compared to when the test compound is not contacted, the test compound is selected as a compound for controlling the growth of insects. Specifically, when the test compound increases the expression of the TRET1 gene, the test compound is useful for promoting the growth of natural enemy insects such as mistletoe and industrial insects such as silkworms. When the expression is decreased, it is considered useful for delaying the growth of pests, that is, as an agrochemical.

  The present invention also relates to a kit for use in the screening method described above. Such a kit may include those used in the detection step and the measurement step of the screening method described above. For example, TRET1 gene, antisense RNA of TRET1, primer, antibody, staining solution and the like can be mentioned. In addition, distilled water, salt, buffer solution, protein stabilizer, preservative and the like may be contained.

  The present invention also provides a method for expressing any gene in the fat body, which comprises the step of expressing DNA in which the promoter of the gene of the present invention and any gene are functionally linked. The TRET1 gene of the present invention is expressed in a fat body-specific manner in Nemurian chironomid, Gambier anopheles, Drosophila melanogaster, honey bee, and silkworm. Therefore, by using the promoter of the gene of the present invention, it becomes possible to express any gene in a fat body-specific manner. Here, “functionally coupled” is as described above.

  In the method of the present invention, a vector constructed to express an arbitrary gene downstream of the promoter of the gene of the present invention is introduced into an insect. The gene that is functionally linked to the promoter of the gene of the present invention is not particularly limited, and examples thereof include GFP and beta-gal. Since the fat body is one of tissues in which gene expression is active even in insects, the TRET1 promoter of the present invention can be used for useful gene expression using insects.

  The present invention also provides a DNA encoding an antisense RNA complementary to the TRET1 gene transcript of the present invention. The DNA encoding an antisense RNA complementary to the TRET1 gene transcript of the present invention can be used for detection of the TRET1 gene and the like. The definition of “complementary” is as described above.

  The sequence of the antisense DNA of the present invention is preferably completely complementary to the TRET1 transcript, but may not be completely complementary as long as it can effectively inhibit gene expression. The transcribed RNA preferably has a complementarity of 90% or more (eg, 95%, 96%, 97%, 98%, 99% or more) to the transcript of the target gene. The length of the antisense DNA is at least 15 bases or more, preferably 100 bases or more, and more preferably 500 bases or more. Usually, the length of the antisense DNA used is shorter than 5 kb, preferably shorter than 2.5 kb. Examples of DNA encoding an antisense RNA complementary to the transcription product of TRET1 gene include DNA containing the base sequence set forth in SEQ ID NO: 34.

  In the present invention, there are no particular limitations on non-human organisms that suppress the expression of the TRET1 gene, but representatives include insects such as Gambier spp., Drosophila melanogaster, honey bees, silkworms, Tonama grasshoppers, American cockroaches, tobacco suzumega, and cecropia moths Invertebrates and the like are preferred.

  Antisense DNA is, for example, a phosphorothioate method (Stein, Nucleic Acids Res., 16: 3209-3221, 1988) based on the sequence information of DNA set forth in SEQ ID NO: 1, 3, 5, 7, or 9. ) And the like. The prepared DNA can be transformed into a desired plant by a known method.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Materials and methods
1. Facilitated transport-type trehalose transporter (PvTRET1, BmTRET1, AgTRET1, AmTRET1, DmTRET1) Isolation of gene and construction of expression vector Annotation by BLAST search using Nemuri chironomid EST database results in a sugar transporter motif An EST clone could be identified. Align the identified EST clones, design specific primers based on the obtained cluster sequence information, and determine the full-length cDNA sequence by RT-PCR and SMART-RACE (Clontech). Named PvTRET1. To construct an Xenopus oocyte expression vector, the ORF portion was amplified from PvTRET1 cDNA by PCR, and the pXbG-ev1 vector (Preston, GM, Carroll, TP, Guggino, WB and Agre, P. (1992) Science 256, 385-7.) Or between the Bgl II and Eco RI sites of the pIRES2AcGFP1 vector (Clontech). The obtained plasmids were named pPvTRET1-XbG-ev1 and pPvTRET1-IRES2-AcGFP1 in order.

  In addition, in order to identify the orthologue of the PvTRET1 gene, silkworms (KAIKOBLAST: http://kaikoblast.dna.affrc.go.jp/), Gambier mosquito (AnoBase: http://www.anobase.org/index. html), honeybee (BeeBase: http: // racerx00.tamu.edu/bee_resources.html) and Drosophila flyfly (Fly Base: http://shigen.lab.nig.ac.jp:7081/) Searched with tBlastn search for. Primers were prepared from the obtained clones, cDNA clones were obtained by RT-PCR and RACE, and were named BmTRET1, AgTRET1, AmTRET1, and DmTRET1, respectively. Similarly to PvTRET1, vectors such as pBmTRET1-XbG-ev1, pAgTRET1-XbG-ev1, pAmTRET1-XbG-ev1, pDmTRET1-XbG-ev1 were constructed for Xenopus oocyte expression.

2. RNA isolation and Northern blot analysis Total RNA was extracted using ISOGEN (Nippon Gene) from 0, 1, 3, 6, 24, and 48 hours of Chironomidae larvae. The obtained RNA was subjected to electrophoresis using a guanidine-denatured agarose gel and transferred to a nylon membrane using a negative pressure blotter. The nylon membrane was subjected to a hybridization reaction using a ³² P radiolabeled probe from the full-length PvTRET1 cDNA obtained this time using a Strip-EZ kit (Ambion). After washing to leave only specific bands, image analysis was performed using LAS-2500 (Fujifilm).

3. Trehalose transport assay using Xenopus oocytes
Xenopus containing the T7 polymerase binding region by PCR using vector-specific primers with pPvTRET1-XbG-ev1, pBmTRET1-XbG-ev1, pAgTRET1-XbG-ev1, pAmTRET1-XbG-ev1, pDmTRET1-XbG-ev1 PvTRET1, BmTRET1, AgTRET1, AmTRET1, and DmTRET1 DNA fragments having b-globin untranslated regions bound to the 5 ′ and 3 ′ ends were prepared. Using this DNA fragment as a template, cap RNA (cRNA) was synthesized using mMESSAGE mMACHINE (Ambion). The synthesized 1 ng / nl cRNA (40 nl) was microinjected into Xenopus oocytes (stage V-VI) treated with collagenase. At the same time, a small amount of 40 nl of sterilized ultrapure water was also injected as a negative control. The trehalose transport activity was assay buffer adjusted to be isosmotic pressure 72 to 96 hours after cRNA injection: 0.5 × MBS containing 105 mM trehalose (44.0 mM NaCl, 0.5 mM KCl, 1.2 mM NaHCO ₃ , 7.5 Quantification of the incorporated trehalose after leaving the oocytes in mM Tris-HCl (pH 7.6), 0.15 mM Ca (NO ₃ ) ₂ , 0.21 mM CaCl ₂ , 0.41 mM MgSO ₄ ) for 3 hours at 15 ° C Went. When the acidic trehalose transport activity was examined, the above-mentioned assay was performed by replacing the Tris buffer in MBS with an acetic acid-sodium acetate buffer (pH 4.2) having the same concentration. When an ionophore was used, the assay was performed by adding an arbitrary amount of nigericin, valinomycin and CCCP (Carbonyl cyanide 3-chlorophenylhydrazone) to the assay buffer. When performing a trehalose release assay, the oocytes were transferred to 1 × MBS after being left in 0.5 × MBS containing 105 mM trehalose for 3 hours, and the incorporated trehalose was quantified after being collected at an appropriate time.

4). Trehalose transport assay using animal cells CHO-K1, NIH / 3T3-3-4, and HuH-7 were used as animal cells. For culture, F-12 ham medium containing 10% FBS (for CHO-K1) or DMEM medium containing 10% FBA (for NIH / 3T3-3-4 and HuH-7), 5% carbon dioxide, 95 Performed in an environment of 37% RH and% humidity. Each of these cells was seeded in a 6-well culture dish of 1 to 5 × 10 ⁵ cells, and after overnight culture, pPvTRET1-IRES2-AcGFP1 or pIRES2AcGFP1 (negative control) was introduced into the cells using FuGene6 (Roche). Trehalose transport activity was determined by exchanging the medium with a medium containing 100 mM trehalose two days after gene introduction and culturing for 3 hours, and then quantifying the incorporated trehalose. Further, using part of the collected cells, AcGFP1-positive cells were counted with a flow cytometer (EPICS ALTRA, Beckman) to determine gene transfer efficiency.

5. Trehalose quantification Trehalose-incorporated oocytes or animal cells were rinsed several times with 1 x MBS or Dulbecco's PBS (Invitorogen), respectively, and ground in 80% ethanol. A trehalose fraction was obtained by dissolving in ml of ultrapure water. Trehalose was quantified by HPLC analysis. HPLC is fractionated with a HPLC system (LC-10A, Shimadzu) connected to a sugar analysis column (Aminex HPX-87C, BioRad) heated to 80 ° C using ultrapure water as the mobile phase, and a differential refractive index detector. Peaks were detected at (RID-6A, Shimadzu), and trehalose was quantified from the area values.

6). in situ hybridization
In situ hybridization was performed at Geno Staff (Tokyo). Specifically, larvae individuals 24 hours after the drying treatment were formalin-fixed to prepare paraffin sections (6 μM). A digoxigenin-labeled riboprobe encoding the antisense strand and sense strand was prepared using the DIG RNA labeling kit (Roche) by subcloning the region corresponding to positions 1298 to 1594 of PvTRET1 cDNA and hybridized to the paraffin section. . After hybridization, an anti-digoxigenin antibody was reacted and developed with NBT / BCIP. After staining, counterstaining was performed using Kernechtrot solution (Muto Chemical).

[Example 1] Isolation of PvTRET1 gene and structure of PvTRET1 protein The inventors of the present application have been annotating an EST database of Nemurian chironomid that was originally constructed. In the process, it was confirmed that there are multiple EST clones that encode proteins that are relatively similar to human glucose transporters and beet sugar transporter-like proteins. Sugar transporters are very variant and do not know which homologs are involved in trehalose transport. In fact, when searching the Drosophila melanogaster genome database, there are as many as 44. In addition, trehalose is an insect's blood sugar, and since there is little quantitative fluctuation, it seems that there is little fluctuation in the expression of the trehalose transporter itself, and it was easily predicted that it would be extremely difficult to narrow down from many homologs. This fact indicates that it is impossible to know which is a trehalose transporter only by high homology with a sugar transporter. On the other hand, it was considered that the expression of trehalose transporter was also induced because trehalose of Nemuri chironomid increased with drying. Therefore, among the annotated sugar transporters on the chironomid EST database, those exhibiting drought-induced changes in expression were considered as the first candidates for the trehalose transporter and were desired for the experiment. Then, when these clones were used as clues and the EST database was searched again with a BLASTN search, six EST clones were found. These EST clones formed one cluster including the clones used for the BLASTN search. Based on the base sequence of this cluster, a primer was prepared, and RT-PCR and RACE were performed using RNA isolated from Chironomidae that had been dried for 12 hours. As a result, a full-length cDNA of approximately 2.3 kbp was obtained. After successful isolation, this cDNA was named PvTRET1 (FIG. 1A). The base sequence of PvTRET1 is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2. PvTRET1 contained one ORF, and the protein translated from it was predicted to be 504 amino acid residues, 55 kDa. As a result of BlastX search, PvTRET1 was found to contain a sugar transporter motif (pfam00083: Sugar_tr) (FIG. 1B). As a result of predicting the structure of PvTRET1 using SOSUI (http://sosui.proteome.bio.tuat.ac.jp/), it was found that it was a 12-transmembrane protein, similar to the known sugar transporter family. Expected (Figure 1C). As a result of protein localization prediction using PSORT II (http://psort.ims.u-tokyo.ac.jp/), it is predicted that PvTRET1 protein is localized in the cell membrane with a probability of 87.0%. It was. From these results, it was suggested that the translation product of the PvTRET1 gene may be a membrane protein having an activity of promoting some membrane permeation of sugar (ie, sugar transport activity).

[Example 2] PvTRET1 gene expression variation is consistent with trehalose content change
A chironomid isolated from the PvTRET1 gene explosively synthesizes trehalose as it dries (Watanabe, M., Kikawada, T., Minagawa, N., Yukuhiro, F. and Okuda, T. (2002) J Exp Biol 205, 2799-802., Watanabe, M., Kikawada, T. and Okuda, T. (2003) J Exp Biol 206, 2281-6.). Changes in gene expression of PvTRET1 during this dry stimulation were examined. The expression level of the PvTRET1 gene increases with drying, reaches a plateau after 6 hours of drying, is almost constant up to 48 hours when the larvae completely dry, and shows a variation pattern very similar to that of trehalose. (Figure 2). This suggested that PvTRET1 may be a sugar transporter related to trehalose metabolism.

[Example 3] PvTRET1 was examined whether PvTRET1 had trehalose transport activity using an Xenopus oocyte expression system having trehalose transport activity. First, when oocytes expressing PvTRET1 were soaked in Bath modification buffer (MBS) containing 105 mM trehalose, intracellular trehalose increased. On the other hand, trehalose did not increase in oocytes injected with a small amount of water (FIG. 3A). In addition, the oocytes expressing PvTRET1 were soaked in MBS containing 105 mM of various sugars (trehalose, sucrose, maltose, lactose, methyl-α-glucopyranoside (MAG) and 2-deoxyglucose (2-DOG)). , Which sugar was permeated. As a result, PvTRET1 selectively permeated only trehalose, MAG and 2-DOG. Moreover, the trehalose permeation amount was about twice that of MAG and about 8 times that of 2-DOG (FIG. 3B). It is very interesting that sucrose and maltose, which are the same α-glucosides as trehalose, hardly permeated. On the other hand, the oocytes expressing human glucose transporter 1 (hGLUT1) used for control permeated only 2-DOG (FIG. 3B). These facts revealed that PvTRET1 is a sugar transporter having high specificity for trehalose, unlike the known glucose transporter.

[Example 4] The activity of PvTRET1 is broadly divided into the case where the transport performed by the transporter independent of H ⁺ ions is against the difference in concentration on both sides of the membrane and the case where it follows. The former is a transport method that uses an electrochemical gradient of ions (H ⁺ and Na ⁺ ) forced by an ATP-dependent pump to counter the concentration gradient and is called co-transport or secondary active transport . The latter is a transport method that transports only one molecule at a time according to a concentration gradient, and is called single transport or facilitated transport. For example, the yeast maltose-H ⁺ symporter (MAL11 / Agt1), which also has trehalose transport activity, is a typical example of the former. This MAL11 / Agt1 uses a concentration gradient of H ⁺ ions, so its activity is strongly pH-dependent. Optimal around pH 4 and almost no activity near neutral (Stambuk, BU, De Araujo PS, Panek, AD and Serrano, R. (1996) Eur J Biochem 237, 876-81.). Therefore, in order to know whether the trehalose transport activity of PvTRET1 is dependent on H ⁺ ions, the trehalose transport activity was examined using two types of buffers having different pHs. As a result, the activity of PvTRET1 was not changed at pH 4.2 or 7.6 (FIG. 4A). Therefore, since it was suggested that there was no influence of H ⁺ ion, it was considered that PvTRET1 is not at least an H ⁺ symporter like MAL11 / Agt1.

[Example 5] PvTRET1 is a transporter of facilitated transport type
To determine whether PvTRET1 is really a facilitated transporter, we investigated the activity of trehalose in the presence of an electrochemical gradient and an agent (ionophore and uncoupler) that interferes with ATP synthesis. As a result, membrane potential was disturbed by valinomycin (K ⁺ -ionophore) and nigericin (alkali metal ion-H ⁺ antiport ionophore), or mitochondrial oxidative phosphorylation and ATP synthesis by CCCP (H ⁺ -ionophore). Even when uncoupled, there was no change in the trehalose transport activity of PvTRET1 (FIG. 4B). This indicates that PvTRET1 is a transporter that does not depend on an electrochemical gradient or ATP, that is, a transporter of facilitated transport.

[Example 6] The transporter of the facilitated transport type that has no direction in trehalose transport of PvTRET1 transports substances depending on the concentration difference on both sides of the membrane. Reverse. Therefore, whether PvTRET1 is the same was examined. As a result, Xenopus oocytes expressing PvTRET1 were allowed to stand in a buffer containing 105 mM trehalose for 3 hours to fully incorporate trehalose and then replaced with a trehalose-free buffer. Then, the intracellular trehalose concentration decreased, and in the end, trehalose disappeared completely (Fig. 5). From this, it was clarified that the direction of trehalose transport of PvTRET1 changes according to the concentration difference inside and outside the membrane.

[Example 7] PvTRET1 also functions in mammalian cells
Whether PvTRET1 functions in cells other than Xenopus oocytes or not, and incorporates PvTRET1 gene into three established mammalian cell lines (CHO-K1, NIH / 3T3-3-4 and HuH-7) with different origins The vector was transfected and a trehalose transport assay was performed. As a result, despite the fact that there is no difference in gene transfer efficiency among all three types of cells (Figure 6A), the cells into which the PvTRET1 gene was introduced incorporated significantly more trehalose than the cells into which only the vector was introduced. (Figure 6B). Therefore, it was found that PvTRET1 exerts its function regardless of the type of cells to be expressed.

[Example 8] The facilitated transport type trehalose transporter (TRET1) was examined whether the facilitated transport type trehalose transporter widely present in insects is specific to Nemurium chironomid or universally present in insects. As a result of analysis using tBTASTn search, we identified a cDNA (or EST) encoding an amino acid sequence homologous to PvTRET1 from genome databases and EST databases of insects (Drosophila melanogaster, Gambier mosquito, honeybee and silkworm). . Based on the nucleotide sequence information of the obtained DNA, we succeeded in isolating cDNA from each insect using RT-PCR and RACE method. Each of these clones contained one ORF, and the protein encoded by the ORF showed high homology with PvTRET 1 (Table 3). From this, the Drosophila Drosophila facilitated transport type trehalose transporter (TRET1) is DmTRET1 (base sequence is shown in SEQ ID NO: 3 and the amino acid sequence is shown in SEQ ID NO: 4), and the Gambier mosquito TRET1 is AgTRET1 (base sequence is shown). SEQ ID NO: 5, the amino acid sequence is shown in SEQ ID NO: 6), the bee TRET1 is AmTRET1 (the base sequence is shown in SEQ ID NO: 7, the amino acid sequence is shown in SEQ ID NO: 8), and the silkworm TRET1 is BmTRET1 ( The nucleotide sequence was named SEQ ID NO: 9, and the amino acid sequence was named SEQ ID NO: 10). These insect TRET1 orthologs had interspecific differences in activity, but they certainly had the ability to transport trehalose (Fig. 7). From the above, it was found that TRET1 exists universally at least in insects.

[Example 9] TRET1 is involved in trehalose release from fat bodies
In order to investigate the physiological role of TRET1, in situ hybridization of the PvTRET1 gene was performed. As a result, strong PvTRET1 gene expression was confirmed only in the fat pad (FIG. 8). Since fat bodies are trehalose synthesizing organs in insects, TRET1 is thought to play a role in releasing trehalose synthesized in fat bodies into hemolymph.

[Example 10] PvTRET1 protein kinetics
The kinetic analysis of trehalose transport activity of PvTRET1 was performed. Xenopus oocytes expressing PvTRET1 were cultured in a buffer containing trehalose at an arbitrary concentration for 15 minutes at 15 ° C., and the incorporated trehalose was quantified. As a result, a straight line was shown on the Eadie-Hofstee plot, and it was found that the reaction rate changed by taking over the typical Michaelis-Menten reaction equation. In addition, PvTRET1 had a Km of 114.5 ± 27.9 mM and a Vmax of 7.84 ± 0.77 nmol / 15 min / oocyte with respect to trehalose by calculation based on nonlinear approximation using Prism ver4 (GraphPad). This indicates that PvTRET1 is a highly functional transporter that does not saturate even at very high concentrations of substrate.

It is a figure which shows the gene and protein structure of PvTRET1. (A) Cloning of PvTRET1 cDNA. PD1202M17f, PD3608G15f, PD3606D10f, PD1205C05f, PD1204L14f, and PD1202F04f are murmured EM clones. The PvTRET1 cDNA was 2,337 bp and had one translational reading frame (ORF) at positions 121 to 1,632b. (B) The translation product of PvTRET1 cDNA has a conserved domain of the sugar transporter. (C) Three-dimensional model of PvTRET1 protein. As a result of analysis using SOSUI, it was shown to be a 12-transmembrane protein. A typical N-type sugar chain addition site exists in the first loop. It is a figure which shows the expression fluctuation | variation of the PvTRET1 gene at the time of drying in Nemuri chironika. Japanese red chironomid was dried and total RNA was sampled at the times shown in the figure. 15 μg of RNA was electrophoresed, and Northern blot analysis was performed using the full length of PvTRET1 cDNA as a probe. One band was confirmed at a position of about 2.3 kb. EtBr shows an ethidium bromide stained image of 28S rRNA during electrophoresis. It is a figure which shows that PvTRET1 is a trehalose transporter. (A) Xenopus oocytes were injected with PvTRET1 cRNA or sterile ultrapure water and cultured for 96 hours to fully express the protein. Then, the fluctuation | variation of the quantity of the trehalose taken in by the oocyte when it culture | cultivates by MBS containing 105 mM trehalose is shown in a figure. One experiment was conducted using 4 oocytes. Each value is an average value ± standard error, n = 3. (B) Substrate selectivity of PvTRET1. The amount of sugar taken in when oocytes expressing PvTRET1 and human glucose transporter 1 (hGLUT1) are left in a buffer solution containing 105 mM sugar for 3 hours is shown. Tre: trehalose, Mal: maltose, Suc: sucrose, Lac: lactose, MAG: methyl-α-glucopyranoside, 2-DOG: 2-deoxyglucose. Values are the same as (A), average value ± standard error, n = 3. It is a figure which shows that PvTRET1 is a transporter of a facilitated transport type. (A) pH dependence of PvTRET1. The amount of trehalose taken up when oocytes in which PvTRET1 is expressed in two types of buffers containing trehalose with different pHs is left for 3 hours. (B) Effect of uncoupler and ionophore on PvTRET1 activity. The amount of trehalose incorporated in the presence of an uncoupler or ionophore is shown. The same letters (a and b) in the figure indicate no significant difference. (ANOVA-Tukey ’s multiple comparison test, p> 0.05, n = 3) It is a figure which shows that the trehalose transport of PvTRET1 has no directionality. PvTRET1-expressing oocytes were left in a buffer containing 105 mM trehalose for 3 hours to incorporate trehalose. Thereafter, the oocytes are transferred to a trehalose-free buffer, and the fluctuations in the amount of trehalose in the cells are shown. PvTRET1 is a diagram showing that it also functions in mammalian cells. When PvTRET1 expression vector (pPvTRET1-IRES2-AcGFP1) and expression vector without insert (pIRES2-AcGFP1) are introduced into three types of mammalian cells (CHO-K1, HuH-7 and NIH / 3T3-3-4) The introduction efficiency (A) and the amount of trehalose uptake (B) are shown. The same letters (a, b, c) in the figure indicate no significant difference. (ANOVA-Tukey ’s multiple comparison test, p> 0.05, n = 3) It is a figure which shows that the insect TRET1 ortholog has trehalose transport activity. Results of trehalose uptake assay using Xenopus laevis oocytes expressing insect TRET1 orthologs (PvTRET1: Nemurian chironomid, AgTRET1: Gambier medaka, DmTRET1: Drosophila melanogaster, AmTRET1: Apis mellifera, BmTRET1: Silkworm) The same letter (a, b) in the figure indicates no significant difference. (ANOVA-Tukey ’s multiple comparison test, p> 0.05) It is a figure which shows that TRET1 of an insect is expressed with a fat body. An in situ hybridization image using a riboprobe encoding PvTRET1 gene antisense strand (Antisense) and sense strand (Sense) is shown on a cross section of dried Chironomidae for 24 hours. FB: fat body, MG: midgut, Mu: muscle. It is a figure which shows the dynamics of the trehalose transport activity of PvTRET1 protein. Changes in the activity of PvTRET1 with respect to any trehalose concentration are shown in a Michaelis-Menten plot. The results are shown as mean ± standard error, and the experiment was repeated three times. The figure enclosed by the square is an Eadie-Hofstee plot. It is a figure which shows the alignment comparison of the amino acid sequence of insect TRET1. ClustalX was used for alignment analysis. Insect TRET1 sites are indicated with asterisks, amino acid sites with very similar properties are indicated with colons, and amino acid sites with somewhat similar properties are indicated with dots. Of these matched sequences, the position of the conserved region found only in insect TRET1 is shown in a box, compared to the region conserved between human GLUT1, 2, 3, 4 and 5. FIG. 10B is a diagram showing a continuation of FIG. 10-a.

Claims (20)

DNA according to any of the following (a) or (b);
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, 8, or 10 ;
(B) DNA comprising the coding region of the base sequence described in SEQ ID NO: 1, 7, or 9 . DNA according to any one of (a) to ( c ) below:
(A) comprising an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 22, wherein the amino acid sequence set forth in SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
( B ) comprising the amino acid sequence shown in SEQ ID NO: 25, wherein the amino acid sequence shown in SEQ ID NO: 8 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
( C ) comprising the amino acid sequence shown in SEQ ID NO: 26, wherein the amino acid sequence shown in SEQ ID NO: 10 has an amino acid sequence in which 1 or 5 amino acids are substituted, deleted, added, and / or inserted. A DNA that encodes a protein having trehalose transport activity. DNA according to any one of (a) to ( c ) below:
(A) comprising an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO: 27, wherein the amino acid sequence shown in SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
( B ) comprising the amino acid sequence of SEQ ID NO: 30, wherein the amino acid sequence of SEQ ID NO: 8 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
( C ) comprising the amino acid sequence of SEQ ID NO: 31, wherein the amino acid sequence of SEQ ID NO: 10 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. A DNA that encodes a protein having trehalose transport activity. A vector comprising the DNA according to any one of claims 1 to 3. The transformed cell which hold | maintains the DNA in any one of Claims 1-3, or the vector of Claim 4 so that expression is possible. The transformed cell according to claim 5, wherein the cell is derived from any one of Nemurus, honeybee, and silkworm . A transgenic non-human organism into which the DNA according to any one of claims 1 to 3 or the vector according to claim 4 has been introduced. The transgenic non-human organism according to claim 7, wherein the non-human organism is selected from insects or vertebrates. 9. The transgenic non-human organism according to claim 8, wherein the insect is selected from Nemurian medica, Apis mellifera or silkworm . SEQ ID NO: 11～20,34 oligonucleotide consisting of the nucleotide sequence of any one of. At least one primer set shown in any of (a) to (e) below;
(A) DNA containing the base sequence set forth in SEQ ID NO: 11 and DNA containing the base sequence set forth in SEQ ID NO: 12
(B) DNA containing the base sequence set forth in SEQ ID NO: 13 and DNA containing the base sequence set forth in SEQ ID NO: 14
(C) DNA containing the base sequence set forth in SEQ ID NO: 15 and DNA containing the base sequence set forth in SEQ ID NO: 16
(D) DNA containing the base sequence set forth in SEQ ID NO: 17 and DNA containing the base sequence set forth in SEQ ID NO: 18
(E) DNA containing the base sequence set forth in SEQ ID NO: 19 and DNA containing the base sequence set forth in SEQ ID NO: 20. A protein encoded by the DNA according to any one of claims 1 to 3. The method for producing a protein according to claim 12, wherein the transformed cell according to claim 5 or 6 is cultured, and the expression product of the DNA according to any one of claims 1 to 3 is collected. A drug for introducing trehalose into a cell, which comprises any one of the DNA according to any one of (1) to (12) below, the vector according to claim 4, or the protein according to claim 12 ;
(1) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10,
(2) DNA containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9;
(3) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 22, wherein the amino acid sequence set forth in SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(4) comprising the amino acid sequence of SEQ ID NO: 23, wherein the amino acid sequence of SEQ ID NO: 4 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(5) Comprising the amino acid sequence shown in SEQ ID NO: 24, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 6 are substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(6) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 25, wherein the amino acid sequence set forth in SEQ ID NO: 8 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(7) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 26, wherein the amino acid sequence set forth in SEQ ID NO: 10 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(8) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 27, wherein the amino acid sequence of SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(9) Comprising the amino acid sequence shown in SEQ ID NO: 28, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 4 are substituted, deleted, added, and / or inserted DNA encoding a protein having trehalose transport activity,
(10) comprising the amino acid sequence of SEQ ID NO: 29, wherein the amino acid sequence of SEQ ID NO: 6 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(11) comprising the amino acid sequence of SEQ ID NO: 30, wherein the amino acid sequence of SEQ ID NO: 8 is substituted, deleted, added, and / or inserted with 1 or 5 amino acids DNA encoding a protein having trehalose transport activity,
(12) Comprising the amino acid sequence shown in SEQ ID NO: 31 and consisting of an amino acid sequence in which amino acids within 1 or 5 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 10. A DNA encoding a protein having trehalose transport activity . A drug for increasing drought tolerance of a cell, comprising any one of the DNA according to any one of (1) to (12) below, the vector according to claim 4, or the protein according to claim 12. ;
(1) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10,
(2) DNA containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9;
(3) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 22, wherein the amino acid sequence set forth in SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(4) comprising the amino acid sequence of SEQ ID NO: 23, wherein the amino acid sequence of SEQ ID NO: 4 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(5) Comprising the amino acid sequence shown in SEQ ID NO: 24, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 6 are substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(6) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 25, wherein the amino acid sequence set forth in SEQ ID NO: 8 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(7) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 26, wherein the amino acid sequence set forth in SEQ ID NO: 10 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(8) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 27, wherein the amino acid sequence of SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(9) Comprising the amino acid sequence shown in SEQ ID NO: 28, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 4 are substituted, deleted, added, and / or inserted DNA encoding a protein having trehalose transport activity,
(10) comprising the amino acid sequence of SEQ ID NO: 29, wherein the amino acid sequence of SEQ ID NO: 6 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(11) comprising the amino acid sequence of SEQ ID NO: 30, wherein the amino acid sequence of SEQ ID NO: 8 is substituted, deleted, added, and / or inserted with 1 or 5 amino acids DNA encoding a protein having trehalose transport activity,
(12) Comprising the amino acid sequence shown in SEQ ID NO: 31 and consisting of an amino acid sequence in which amino acids within 1 or 5 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 10. A DNA encoding a protein having trehalose transport activity . A method for increasing the trehalose permeability of a cell, comprising the step of expressing the DNA according to any one of (1) to (12) below in any cell ;
(1) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10,
(2) DNA containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9;
(3) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 22, wherein the amino acid sequence set forth in SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(4) comprising the amino acid sequence of SEQ ID NO: 23, wherein the amino acid sequence of SEQ ID NO: 4 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(5) Comprising the amino acid sequence shown in SEQ ID NO: 24, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 6 are substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(6) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 25, wherein the amino acid sequence set forth in SEQ ID NO: 8 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(7) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 26, wherein the amino acid sequence set forth in SEQ ID NO: 10 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(8) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 27, wherein the amino acid sequence of SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(9) Comprising the amino acid sequence shown in SEQ ID NO: 28, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 4 are substituted, deleted, added, and / or inserted DNA encoding a protein having trehalose transport activity,
(10) comprising the amino acid sequence of SEQ ID NO: 29, wherein the amino acid sequence of SEQ ID NO: 6 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(11) comprising the amino acid sequence of SEQ ID NO: 30, wherein the amino acid sequence of SEQ ID NO: 8 is substituted, deleted, added, and / or inserted with 1 or 5 amino acids DNA encoding a protein having trehalose transport activity,
(12) Comprising the amino acid sequence shown in SEQ ID NO: 31 and consisting of an amino acid sequence in which amino acids within 1 or 5 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 10. A DNA encoding a protein having trehalose transport activity . A method for introducing trehalose into a cell, comprising the step of expressing the DNA according to any one of (1) to (12) below in any cell ;
(1) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10,
(2) DNA containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9;
(3) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 22, wherein the amino acid sequence set forth in SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(4) comprising the amino acid sequence of SEQ ID NO: 23, wherein the amino acid sequence of SEQ ID NO: 4 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(5) Comprising the amino acid sequence shown in SEQ ID NO: 24, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 6 are substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(6) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 25, wherein the amino acid sequence set forth in SEQ ID NO: 8 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(7) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 26, wherein the amino acid sequence set forth in SEQ ID NO: 10 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(8) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 27, wherein the amino acid sequence of SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(9) Comprising the amino acid sequence shown in SEQ ID NO: 28, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 4 are substituted, deleted, added, and / or inserted DNA encoding a protein having trehalose transport activity,
(10) comprising the amino acid sequence of SEQ ID NO: 29, wherein the amino acid sequence of SEQ ID NO: 6 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(11) comprising the amino acid sequence of SEQ ID NO: 30, wherein the amino acid sequence of SEQ ID NO: 8 is substituted, deleted, added, and / or inserted with 1 or 5 amino acids DNA encoding a protein having trehalose transport activity,
(12) Comprising the amino acid sequence shown in SEQ ID NO: 31 and consisting of an amino acid sequence in which amino acids within 1 or 5 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 10. A DNA encoding a protein having trehalose transport activity . A screening method for a compound that modulates trehalose transport activity, comprising the following steps (a) to (c):
(A) contacting the test compound with the transcription product of the DNA according to any one of (1) to (12 ) below ,
(B) a step of detecting the binding between the transcription product of the DNA according to any one of (1) to (12 ) below and a test compound,
(C) a step of selecting a test compound that binds to the transcription product of the DNA according to any one of (1) to (12 ) below ;
(1) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10,
(2) DNA containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9;
(3) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 22, wherein the amino acid sequence set forth in SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(4) comprising the amino acid sequence of SEQ ID NO: 23, wherein the amino acid sequence of SEQ ID NO: 4 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(5) Comprising the amino acid sequence shown in SEQ ID NO: 24, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 6 are substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(6) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 25, wherein the amino acid sequence set forth in SEQ ID NO: 8 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(7) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 26, wherein the amino acid sequence set forth in SEQ ID NO: 10 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(8) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 27, wherein the amino acid sequence of SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(9) Comprising the amino acid sequence shown in SEQ ID NO: 28, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 4 are substituted, deleted, added, and / or inserted DNA encoding a protein having trehalose transport activity,
(10) comprising the amino acid sequence of SEQ ID NO: 29, wherein the amino acid sequence of SEQ ID NO: 6 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(11) comprising the amino acid sequence of SEQ ID NO: 30, wherein the amino acid sequence of SEQ ID NO: 8 is substituted, deleted, added, and / or inserted with 1 or 5 amino acids DNA encoding a protein having trehalose transport activity,
(12) Comprising the amino acid sequence shown in SEQ ID NO: 31 and consisting of an amino acid sequence in which amino acids within 1 or 5 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 10. A DNA encoding a protein having trehalose transport activity . A screening method for a compound that modulates trehalose transport activity, comprising the following steps (a) to (c):
(A) contacting a test compound with a cell collected from an invertebrate,
(B) a step of measuring the expression level of the transcription product of the DNA according to any one of (1) to (12 ) below ,
(C) selecting a test compound that has increased or decreased the expression level of the transcript as compared to the case where the test compound is not contacted ;
(1) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10,
(2) DNA containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9;
(3) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 22, wherein the amino acid sequence set forth in SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(4) comprising the amino acid sequence of SEQ ID NO: 23, wherein the amino acid sequence of SEQ ID NO: 4 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(5) Comprising the amino acid sequence shown in SEQ ID NO: 24, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 6 are substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(6) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 25, wherein the amino acid sequence set forth in SEQ ID NO: 8 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(7) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 26, wherein the amino acid sequence set forth in SEQ ID NO: 10 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(8) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 27, wherein the amino acid sequence of SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(9) Comprising the amino acid sequence shown in SEQ ID NO: 28, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 4 are substituted, deleted, added, and / or inserted DNA encoding a protein having trehalose transport activity,
(10) comprising the amino acid sequence of SEQ ID NO: 29, wherein the amino acid sequence of SEQ ID NO: 6 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(11) comprising the amino acid sequence of SEQ ID NO: 30, wherein the amino acid sequence of SEQ ID NO: 8 is substituted, deleted, added, and / or inserted with 1 or 5 amino acids DNA encoding a protein having trehalose transport activity,
(12) Comprising the amino acid sequence shown in SEQ ID NO: 31 and consisting of an amino acid sequence in which amino acids within 1 or 5 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 10. A DNA encoding a protein having trehalose transport activity . A kit for use in the screening method according to claim 18 or 19, comprising at least one of the DNA according to any one of (1) to (12) below or the primer set according to claim 11 .
(1) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 2, 4, 6, 8, or 10,
(2) DNA containing the coding region of the base sequence described in SEQ ID NO: 1, 3, 5, 7, or 9;
(3) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 22, wherein the amino acid sequence set forth in SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(4) comprising the amino acid sequence of SEQ ID NO: 23, wherein the amino acid sequence of SEQ ID NO: 4 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(5) Comprising the amino acid sequence shown in SEQ ID NO: 24, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 6 are substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(6) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 25, wherein the amino acid sequence set forth in SEQ ID NO: 8 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(7) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 26, wherein the amino acid sequence set forth in SEQ ID NO: 10 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(8) Consists of an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO: 27, wherein the amino acid sequence of SEQ ID NO: 2 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(9) Comprising the amino acid sequence shown in SEQ ID NO: 28, consisting of an amino acid sequence in which 1 or 5 amino acids in the amino acid sequence shown in SEQ ID NO: 4 are substituted, deleted, added, and / or inserted DNA encoding a protein having trehalose transport activity,
(10) comprising the amino acid sequence of SEQ ID NO: 29, wherein the amino acid sequence of SEQ ID NO: 6 has 1 or 5 amino acids substituted, deleted, added, and / or inserted. DNA encoding a protein having trehalose transport activity,
(11) comprising the amino acid sequence of SEQ ID NO: 30, wherein the amino acid sequence of SEQ ID NO: 8 is substituted, deleted, added, and / or inserted with 1 or 5 amino acids DNA encoding a protein having trehalose transport activity,
(12) Comprising the amino acid sequence shown in SEQ ID NO: 31 and consisting of an amino acid sequence in which amino acids within 1 or 5 amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence shown in SEQ ID NO: 10. A DNA encoding a protein having trehalose transport activity .