JP2003245080A - Targeting peptide of protein transportation to mitochondria derived from ucp gene, and dna encoding the peptide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simultaneously transport two proteins to mitochondria. <P>SOLUTION: The protein transportation system to the mitochondria uses the D2 region of wheaten UCP which is discovered to have very strong protein transportation properties. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention belongs to the field of genetic engineering, and particularly relates to intracellular protein transport in higher organisms. Furthermore, the present invention relates to polypeptides that transport proteins to mitochondria, especially in plant cells.

[0002]

2. Description of the Related Art Mitochondria are respiratory and energy generating organs that characterize eukaryotic cells and have a size of 0.5 μm.
It is a front and back organelle. In mitochondria, there are two types of membranes, an inner membrane and an outer membrane, which can be divided into a matrix region inside the inner membrane and an intermembrane region between the inner and outer membranes. A large number of transport proteins are present in the outer membrane and penetrate the lipid bilayer to form a large hydrophilic channel. Therefore, the outer membrane is free to pass small molecules of about 10,000 daltons or less, including proteins. The intima, on the other hand, is impermeable. The main functions of mitochondria are the matrix and the inner membrane (molecular biology of cells, Bruce Alberts et al.).

There are many proteins in mitochondria. However, very few proteins are encoded on the mitochondrial genome, and most of them are encoded by the nuclear genome. At the N-terminus of most nuclear genome-encoded mitochondrial proteins, there is a targeting sequence that is not required for mitochondrial protein function and only determines mitochondrial transport (Schatz and Dobberstein).
(1996) Science 271: 1519-1526).

The protein translated in the cytoplasm as a precursor with a targeting sequence is transported to the mitochondria, whereafter the targeting sequence is enzymatically cleaved to the intact form. However, some mitochondrial proteins encoded by the nuclear genome do not have a clear N-terminal targeting sequence, but are correctly transported to mitochondria. Mitochondrial uncoupling protein (Uncoupling pro
Tein (UCP) is one of the nuclear genome-encoded mitochondrial proteins that do not have targeting sequences.

[0005] UCP is a membrane protein existing in the inner mitochondrial membrane, and is believed to eliminate the proton concentration gradient in the inner membrane caused by respiration without synthesizing ATP and generate heat (K1ause (1991). ) Inter. J. Bioc
hem. 23: 791-801). 4 in mammals so far
Different UCP isoforms have been identified and detailed studies have been conducted on each isoform (Bouill
aud et al. (1985) Proc. Natl. Acad. Sci. USA 82:44
5-448, F1eur et al. (1997) Nat. Genet. 15: 269-272,
Boss et al. (1997) FEBS Lett. 408: 39-42, Mao et a
L. (1999) FEBSLett. 443: 326-330). On the other hand, in plants, UCP gene has been isolated in potato, Arabidopsis thaliana, Zanthoxylum and wheat (Laloi et al.
(1997) Nature 398: 135-136, Maia et al. (1998) FEBS
Lett. 429: 403-406, Ito (1999) Plant Sci. 149: 167-1
73, Murayama and Handa (2000) Mol. Gen. Genet. 26
4: 112-118). UCP is one of the mitochondrial carrier proteins, and has a structure in which three domains are connected in parallel, with the sequence commonly found in the two transmembrane regions and the mitochondrial carrier protein sandwiched between them as the basic unit (domain). Hold (Pa1mieri (19
94) FEBS Lett. 346: 48-54).

[0006] UCP, like many mitochondrial proteins, has its information encoded in the nuclear genome, translated in the cytoplasm and then transported to mitochondria.
It is reported that one-third of the N-terminal side has regions involved in mitochondrial transport and insertion into mitochondrial membrane (Li
u et al. (1988) J. Cell Biol. 107: 503-509), but it is unknown which site in the protein is involved in mitochondrial transport.

[0007]

[Problems to be Solved by the Invention] Since mitochondria play a very important role in respiration and energy production as an organelle in cells, gene modification targets such as increase in respiration efficiency and modification of energy efficiency As important as. However, since the protein transport signal to mitochondria has not been fully elucidated, a system for transporting the target protein to mitochondria after expression has not been established. Furthermore, in the case of enzyme subunits, it is often desirable that each subunit be transported to the vicinity. Therefore, it has been strongly desired to develop a system that simultaneously transports the two subunits into the mitochondria, respectively.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have found that the wheat UCP gene (Murayama and Handa (2000) Mol. Gen. Genet.
4: 112-118) to construct wheat UCP protein 3
For each of the three domains, a green fluorescent protein (GFP) at the C-terminal side or a fusion protein having a red fluorescent protein (DsRed) at the N-terminal side and GFP at the C-terminal side was expressed in yeast, and the wheat UCP 3 The transport ability of two domains to mitochondria was verified. This allows UC
It was found that the D2 region of P has a very strong ability to transport to mitochondria, and a substance transport system to mitochondria using the above D2 region was developed to complete the present invention.

Here, the D2 region means a portion (including a transmembrane region) sandwiched between the third and fourth transmembrane regions from the amino-terminal side of UCP, for example, SEQ ID NO: 2 Alternatively, in the sequence of 4, the N-terminal is located within the range of 90 to 105 and the C-terminal is included within the range of 191 to 204. What is. Also, as long as it has the ability to transport to mitochondria, for each of the D2 regions defined above,
Furthermore, one to several amino acids may be deleted, added, or substituted. The D2 region of the present invention is preferably a plant UCP D2 region, more preferably a wheat UCP D2 region.
The D2 region of UCP of monocotyledonous plants including the region can be mentioned.

According to the present invention, any protein to be transported to mitochondria can be introduced into mitochondria by fusing to the N-terminal or C-terminal of UCP. For example, a gene involved in cytoplasmic male sterility can be introduced. Such a male-sterile gene introduced into a plant includes atp9 (Proc. Natl. Acad. Sc
i. USA 90: 2370-2374), orf239 (Proc. Natl. Acad. S
ci. USA 93: 11763-11768), T-urf13 (Proc. Natl. Aca
d. Sci. USA 92: 1167-1171). Furthermore, according to the present invention, a protein involved in the respiratory system such as ATP synthase can be introduced into mitochondria.

Further, since the D2 region of the present invention is located at the intermediate portion of UCP, it is fused with the protein fused to the C-terminal side and N-terminal.
Both of the proteins fused to the terminal side can be transported, which is an excellent feature not found in conventional transport proteins. Taking advantage of the property of simultaneously transporting these different proteins into mitochondria, in the present invention, heterodimeric enzymes in mitochondria such as maleate dehydrogenase, ornithine acetyltransferase involved in respiratory system, and maturation of proteins in mitochondria are used. Proteins such as mitochondrial processing peptidase, inner membrane protease I and the like, which are involved in Escherichia coli, can be genetically modified and introduced into mitochondria to improve their functions.

[Application to Genetically Recombinant Plant] When the targeting (signal) sequence of the present invention is used for plant improvement, a target gene other than the wheat UCP protein used for plant improvement is targeted by the targeting sequence. The recombinant construct bound to can be incorporated into an expression vector and introduced into a plant cell, and the gene-introduced cell can be regenerated into a plant.

[Construction of Expression Vector] Expression of foreign genes in plants has already been established by various attempts. The promoter used for the expression system of the coding sequence is translatable in an appropriate host tissue.
It has sufficient transcriptional activity to express mRNA,
Examples thereof include the cauliflower mosaic virus-derived 35S promoter, T-DNA octopine synthase (OCS) promoter, alcohol dehydrogenase derived from monocotyledonous plants such as corn, and promoters of two genes encoding Adh1 and Adh2.

Enhancers or enhancer-like components can be included in the promoter construct. For example, the one found in the 35S promoter (Odell e
al. (1988) Plant Mol. Biol. 10: 263-272), and an enhancer from the opine gene (Fromm et al. (1989) Plant Cell 1: 977-984). In addition, a polyadenylation signal can also be included.

As a method for introducing the vector having the targeting sequence of the present invention into plant cells, transformation vectors based on Ti and Ri plasmids of Agrobacterium species, and binary types of these vectors can be used. Direct incorporation of foreign DNA constructs, electroporation techniques (Fromm et al. (1985) Proc. Natl. Aca
d. Sci. USA 82: 5824-2828), or particle gun method with microparticles coated with nucleic acid construct (Kline et al.
(1987) Nature 327: 70) can also be used. Furthermore, the gene-introduced plant cells can be regenerated into plants by a well-known method after appropriately growing.

Since the targeting sequence for transporting to mitochondria of the present invention shows homology with animal sequences, it functions similarly in other higher organism species such as animals or yeast. . In fact, as described below, it has been confirmed to function in yeast. Therefore, the mitochondrial transport targeting peptide of the present invention can also be used when transporting non-mitochondrial proteins or foreign proteins to animals or yeast.

[0017]

[Example] In order to confirm that the targeting sequence of the present invention indeed transports the polypeptide to mitochondria, GFP or G was added to each domain of the wheat UCP protein.
The fusion protein in which FP and DsRed are ligated in yeast (in vi
vo). As a result, the localization of the fusion protein in the yeast cell could be visually observed under a fluorescence microscope, and the transport ability of each domain in vivo could be verified. In addition, the wheat UCP gene isolated by the present inventors was added to pYE
When expressed in yeast using the S2.1 / WhUCP1b :: V5-His plasmid construct, Western analysis of mitochondrial fractions revealed that the fusion protein was transported to mitochondria.

The present embodiment does not limit the present invention. Even in yeast different from the wheat from which the targeting sequence was derived, it was confirmed that the targeting sequence has a function of transporting the substance to the mitochondria.As a result, higher organisms in general can use the sequence to transport a polypeptide to the mitochondria. It will be obvious to those skilled in the art.

[Example 1] (1) Preparation of plant material Wheat (Triticum aestivum L. cultivar Value vskay
a) was cultivated at a daytime temperature of 20 ° C and a nighttime temperature of 15 ° C with an illumination time of 15 hours.

(2) Isolation of wheat UCP gene A primer for isolating full-length cDNA encoding wheat UCP was synthesized. After germination, total RN extracted from plants at the bileaflet stage
First strand cDNA was synthesized from 1 μg of A. Based on the conserved amino acid sequences found in the first and sixth transmembrane regions of UCP, which are conserved in various mammals and plants in order to amplify most of the wheat UCP coding region. Two degenerate primers, Uni-5 'and Uni-
3'was used. The 3'end and 5'end were determined by RACE-PCR.

The PCR product was ligated into pCR2.1-TOPO using the TOPO TA cloning kit (Invitrogen). DNA sequencing was performed using TaKaRa PCR using the 7-diaza-dGTP thermosequenase fluorescent-labeled primer cycle sequence kit (Amersham Biosciences).
I went with Thermo Cycler MP. The obtained wheat UCP-encoding genes, WhUCP1a and WhUCP1b, are as shown in SEQ ID NOs: 1 and 3.

(3) Construction of an expression vector containing a domain in the gene of WhUCP1b cDNA derived from wheat Using the isolated WhUCPlb cDNA (DDBJ Acc. No. AB042429) clone (Murayama and Handa (2000)), FIG. The six constructs shown in Figure 4 are yeast expression vector pYES2.
1 (Invitrogen) was cloned. The plasmid construct was prepared according to the following procedure.

[0023]

[Table 1]

WhUCP1b amino acid sequence 1 to 104, 105 to 19
1, 192-277 are defined as domain 1, domain 2, and domain 3 (hereinafter abbreviated as D1, D2, D3, respectively),
A region corresponding to each domain was amplified by PCR from the WhUCP1b cDNA clone using the primer pairs of primers 1-2, 3-4, and 5-6 shown in Table 1. At that time, S for each primer
The restriction enzyme sequence of alI or NcoI was inserted. The amplified DNA fragment was digested with restriction enzymes SalI and NcoI, and the resulting SalI / NcoI fragment of each domain was used as a GFP plasmid construct (pTH-2 / GFP) (transferred from Professor Kobayashi of Shizuoka Prefectural University) (Chiu et al. (1996) Curr. Biol. 6: 325-330)
Cloned into SalI / NcoI site of pTH-2 / D1 :: GFP,
Plasmid constructs of pTH-2 / D2 :: GFP and pTH-2 / D3 :: GFP were obtained. Furthermore, these plasmid DNAs were digested with restriction enzymes SalI and NotI, and the excised fragments were digested with pBlu.
escriptII SK + plasmid vector (Stratagene, USA)
Subcloned into the SalI / NotI site of (pSK / D1 :: G
FP, pSK / D2 :: GFP, pSK / D3 :: GFP). Amplification is performed by using the plasmid construct, pSK / D1 :: GFP, pSK / D2 :: GFP, and pSK / D3 :: GFP as templates, and using each of the primer pairs of primers 1, 3, and 5 and primer 7. Done DNA
The fragment was cloned into the yeast expression vector pYES2.1, pYB
S2.1 / D1 :: GFP, pYES2.1 / D2 :: GFP, and pYES2.1 / D3 :: GFP plasmid constructs were obtained.

On the other hand, using the DsRed plasmid construct (pDsRed, Clontech) as a template, primer 8,
PCR was performed using 9 and the resulting PCR product was ApaI and SalI.
Cleaved with pSK / D1 :: GFP, pSK / D2 :: GFP, pSK / D3 :: GFP and cloned into the ApaI / SalI site of the plasmid construct (pSK / DSR :: D1 :: GFP, pSK / DSR :: D
2 :: GFP, pSK / DSR :: D3 :: GFP). Plasmid construct, pSK / DSR :: D1 :: GFP, pSK / DSR :: D2 :: GFP, pSK / DSR :: D
PCR is performed using a primer pair of primers 7 and 8 with 3 :: GFP as a template, and the obtained PCR product is cloned into a pYES2.1 vector, pYES2.1 / DSR :: D1 :: GFP, pYES.
2.1 / DSR :: D2 :: GFP and pYES2.1 / DSR :: D3 :: GFP plasmid constructs were obtained.

(4) Transformation of yeast and induction of protein expression The GAL1 promoter in the pYES2.1 plasmid vector used in this experiment represses transcription in the presence of glucose, and galactose instead of glucose is added as a carbon source. Is a promoter that induces transcription (West et al.
a1. (1984) Mol. Cell Biol. 4: 2467-2478, Giniger et.
al. (1985) Cell 40: 767-774). Therefore, after culturing yeast in a selective medium (SD-URA, Bio 101) in the presence of glucose, an induction medium using galactose as a carbon source (0.67% yeast nitrogen base without amino acids wi
th ammonium su1fate, 2% raffinose, 0.01% adenine,
arginine, cysteine, 1eucine, 1isine, threonine, an
d tryptophan, 0.005% aspartic acid, histidine, iso
1eucine, methionine, pheny1a1anine, pro1ine, serin
e, tyrosine, and va1ine) to induce the expression of the fusion protein. First, pYES2 created above.
1 / D1-3 :: GFP, pYES2.1 / DSR :: D1-3 :: GFP plasmid construct in yeast (INVSC1 strain, Invitrogen)
Introduced into the ScEasyComp transformation kit (Invitrogen) to select medium (SDA-URA,
The transformants were selected on Bio 101). Next, the obtained transformant was cultured overnight in 50 ml of SD-URA liquid medium, and then the value of OD600 was measured. 50 from the value of OD600
The amount at which the value of OD600 was 0.4 in m1 was calculated, and the suspension was collected by centrifugation to suspend the cells in 50 ml of induction medium for 24 hours of induction.

(5) Observation of yeast under fluorescence microscope After induction for 24 hours, for DSR :: D1 to 3 :: GFP, the bacteria collected by centrifugation were washed with sterilized water and collected again,
Staining was performed for 10 minutes with 4,6-diamidino-2-phenylindole (DAPI), which specifically stains DNA, and observation was performed under a fluorescence microscope. On the other hand, for D1-3 :: GFP, mitotracker red (molecular probe) was added (final concentration 200 n
M) After culturing for 1 hour, the cells were collected by centrifugation and washed with sterile water, and the cells were observed. Moreover, when observing yeast with a fluorescence microscope, GFP fluorescence is GFP (R) -BP (EX480 / 40,
DM505, BA535 / 50) filter, DsRed fluorescence is G-2A (EX5
10-560, DM575, BA590) filter, DAPI fluorescence is UV-1
It was observed through an A (EX365 / 10, DM400, BA400) filter.

(6) Results As a result of expressing a fusion protein of each domain and GFP,
GFP fluorescence was observed demonstrating the expression of the fusion protein in all domains (Fig. 2-B, D, F). In addition, it was possible to identify mitochondria in yeast cells as granules by staining with mitotracker (Fig. 2-A,
C, E). Although the fluorescence of GFP can be confirmed by the expression of D1 :: GFP and D3 :: GFP, the whole cell or a part of the cell is only vaguely shining, and it is clear when the mitochondria shine. No granular fluorescence could be confirmed (Fig. 2-B, F). Therefore, D1
It was suggested that D3 and D3 do not have or have a very low mitochondrial transport capacity. On the other hand, in the expression of D2 :: GFP, some yeast cells showed fluorescence similar to those of the other two domains, but many cells showing clear granular GFP fluorescence were observed (Fig. 2-D). Also observed GF
The P fluorescent granules corresponded to mitochondrial granules stained by MitoTracker, which revealed that the D2 :: GFP fusion protein was transported to the mitochondria.

On the other hand, when a fusion protein in which GFP and DsRed were linked to each domain was expressed, granular fluorescence was recognized in yeast cells in D1 and D3 as in the case of expressing the fusion protein of GFP only. I couldn't do it. However, in D2, even when a fusion protein in which the domain was sandwiched between two types of proteins, DsRed and GFP, was expressed, mitochondria and granular granular fluorescence were observed (Fig. 3-A, B). In yeast stained with DAPI that specifically stains DNA, granular fluorescence was observed due to staining of the mitochondrial genome (Fig. 3-C). Since GFP and DsRed are expressed as a single fusion protein having D2 in the middle, the granular fluorescence seen in the GFP image and the granular fluorescence seen in the DsRed image matched. In addition, some of the granular fluorescence observed in the GFP image and the DsRed image was consistent with the fluorescence of mitochondrial granules seen in the DAPI image. Therefore, it was revealed that DSR :: D2 :: GFP, like D2 :: GFP, was also transported to mitochondria. GFP image and DsR
Some of the granular fluorescence observed in the ed image was DAPI
It was not observed in the image, which was thought to be due to its failure to detect due to insufficient DAPI staining.

In this example, as a result of expressing a fusion protein in which a fluorescent protein was added to the three domains constituting the wheat UCP protein in yeast, it was revealed that the D2 region has the ability to transport to mitochondria. .

[0031]

EFFECT OF THE INVENTION Since the D2 region is located in the center of the UCP protein, and in this experiment as well, the fusion protein having a structure in which GFP and DsRed were added to both ends was transported to mitochondria. , It was found that the mitochondrial transport capacity is high and that the transport capacity is not lost at any position in the protein. Therefore, by using the D2 region as a targeting sequence to mitochondria, it became possible to simultaneously transport two completely different proteins to mitochondria. Also, by using this method, a protein having an important function at the N-terminus can be transported to mitochondria without modifying the N-terminus.

[0032]

[Sequence list] SEQUENCE LISTING <110> National Agricaltual Research Organization <120> Targeting peptide for protein transport to Mitochondria and DNA co ding therefor <130> P01-0740 <140> <141> <160> 13 <170> PatentIn Ver. 2.1 <210> 1 <211> 974 <212> DNA <213> Triticum aestivum L. <223> WhUCP1a <400> 1 cttctaccct tctttccctc tgctcgccat cgaccgaacc acagccgccg ccgcttcccc 60 ggggataaaa tggcgacggc ctcttccttc gccgccgtct tcatcagcag cgccatcgcc 120 tcctgcttcg ctgaggtgtg caccattcct ctggacacag ccaaggtgcg tcttcagctg 180 caaaagaaaa cagctgctgg gcctgcaggt acagtaggaa tgctgggcac aatgatgtcg 240 attgcaaggg aggaaggcgt caccgcactt tggaagggca tcatccctgg ctttcatcgc 300 cagtgcctct atggcggcct ccgtgtcggc ttgtatgagc ctgtcaaagc cttatttgtg 360 tttgtaggtg atgccacttt aatgaacaag attcttgccg ctcttacaac tggtgtcata 420 gcgattgctg tcgcaaatcc aactgatctt gtcaaagtga gattgcaagc agatggaaaa 480 tctactgccg tcaagaggca ctattctgga gcccttaatg cgtatgccac catagtcaga 540 caggaaggta tcggagcttt gtggactggc cttggtccta atatggcacg aaatgctttg 600 attaatgccg ccgagttggc cagctacgac caatttaaac agatgtttct aggtcttcct 660 gggtttacag ataatgttta tactcatctt ttagctggac tcggtgccgg tatttttgct 720 gtttgcattg gatctccagt ggatgtggtg aaatcaagaa tgatgggcga ttcaacatac 780 agaagtacat ttgattgttt cgccaagaca ttaaaaaatg atggacttgc tgctttctac 840 aaggggttta ttgcaaactt ttgtcgagtt gggtcatgga atgtgataat gttcttaact 900 ttggaacagg ttagaagctt ctttcagtaa ggattatata tgaaatctgc gctgcaaggt 960 ttcatggaac aagc 974 <210> 2 <211> 286 <212> PRT <213> Triticum aestivum L. <223> WhUCP1a <400> 2 Met Ala Thr Ala Ser Ser Phe Ala Ala Val Phe Ile Ser Ser Ala Ile   1 5 10 15 Ala Ser Cys Phe Ala Glu Val Cys Thr Ile Pro Leu Asp Thr Ala Lys              20 25 30 Val Arg Leu Gln Leu Gln Lys Lys Thr Ala Ala Gly Pro Ala Gly Thr          35 40 45 Val Gly Met Leu Gly Thr Met Met Ser Ile Ala Arg Glu Glu Gly Val      50 55 60 Thr Ala Leu Trp Lys Gly Ile Ile Pro Gly Phe His Arg Gln Cys Leu  65 70 75 80 Tyr Gly Gly Leu Arg Val Gly Leu Tyr Glu Pro Val Lys Ala Leu Phe                  85 90 95 Val Phe Val Gly Asp Ala Thr Leu Met Asn Lys Ile Leu Ala Ala Leu             100 105 110 Thr Thr Gly Val Ile Ala Ile Ala Val Ala Asn Pro Thr Asp Leu Val         115 120 125 Lys Val Arg Leu Gln Ala Asp Gly Lys Ser Thr Ala Val Lys Arg His     130 135 140 Tyr Ser Gly Ala Leu Asn Ala Tyr Ala Thr Ile Val Arg Gln Glu Gly 145 150 155 160 Ile Gly Ala Leu Trp Thr Gly Leu Gly Pro Asn Met Ala Arg Asn Ala                 165 170 175 Leu Ile Asn Ala Ala Glu Leu Ala Ser Tyr Asp Gln Phe Lys Gln Met             180 185 190 Phe Leu Gly Leu Pro Gly Phe Thr Asp Asn Val Tyr Thr His Leu Leu         195 200 205 Ala Gly Leu Gly Ala Gly Ile Phe Ala Val Cys Ile Gly Ser Pro Val     210 215 220 Asp Val Val Lys Ser Arg Met Met Gly Asp Ser Thr Tyr Arg Ser Thr 225 230 235 240 Phe Asp Cys Phe Ala Lys Thr Leu Lys Asn Asp Gly Leu Ala Ala Phe                 245 250 255 Tyr Lys Gly Phe Ile Ala Asn Phe Cys Arg Val Gly Ser Trp Asn Val             260 265 270 Ile Met Phe Leu Thr Leu Glu Gln Val Arg Ser Phe Phe Gln         275 280 285 <210> 3 <211> 971 <212> DNA <213> Triticum aestivum L. <223> WhUCP1b <400> 3 cttctaccct tctttccctc tactcgccat cgaccgcaca gccgccgccg cttccccggg 60 gataaaatgg cgacggcctc ttccttcgcc gccgtcttca tcagcagcgc catcgcctcc 120 tgcttcgctg aggtgtgcac cattcctctg gacacagcca aggtgcgtct tcagctgcaa 180 aagaaaacag ctgctgggcc tgcagctaca gtaggaatgc tgggcacaat gatgtcgatt 240 gcaagggagg aaggcgtcag cgcactttgg aagggcatca tccctggctt tcaccgccag 300 tgcctctatg gcggcctccg tgtcggcttg tatgagcctg tcaaagcctt atttgtgttt 360 gtaggtgatg ccactttaat gaacaagatt cttgccgctc ttacaactgg tgtcatagcg 420 attgctgtcg caaatccaac tgatcttgtc aaagtgagat tgcaagcaga tggaaaatct 480 actgctgtca agaggcacta ttctggagcc cttaatgcgt atgccaccat agtcagacag 540 gaaggtatcg gagctttgtg gactggcctt ggtcctaata tggcacgaaa tgctttgatt 600 aatgccgccg agttggccag ctatgaccaa tttaaacaga tgtttctagg tcttcctggg 660 tttacagata atgtttatac tcatcttttg gctggactcg gtgccggtat ttttgctgtt 720 tgcattggat ctccagtgga tgtggtgaaa tcaagaatga tgggcgattc aacatacaga 780 agtacatttg attgtttcgc caagacatta aaaaatgatg gacttgctgc tttctacaag 840 gggtttattg caaacttttg tcgagttggg tcatggaatg tcataatgtt cttaactttg 900 gaacaggtta gaagattctt tcagtaagga ttatatatga actctgcgct gcaaggtttc 960 atggaacaag c 971 <210> 4 <211> 286 <212> PRT <213> Triticum aestivum L. <223> WhUCP1b <400> 4 Met Ala Thr Ala Ser Ser Phe Ala Ala Val Phe Ile Ser Ser Ala Ile   1 5 10 15 Ala Ser Cys Phe Ala Glu Val Cys Thr Ile Pro Leu Asp Thr Ala Lys              20 25 30 Val Arg Leu Gln Leu Gln Lys Lys Thr Ala Ala Gly Pro Ala Ala Thr          35 40 45 Val Gly Met Leu Gly Thr Met Met Ser Ile Ala Arg Glu Glu Gly Val      50 55 60 Ser Ala Leu Trp Lys Gly Ile Ile Pro Gly Phe His Arg Gln Cys Leu  65 70 75 80 Tyr Gly Gly Leu Arg Val Gly Leu Tyr Glu Pro Val Lys Ala Leu Phe                  85 90 95 Val Phe Val Gly Asp Ala Thr Leu Met Asn Lys Ile Leu Ala Ala Leu             100 105 110 Thr Thr Gly Val Ile Ala Ile Ala Val Ala Asn Pro Thr Asp Leu Val         115 120 125 Lys Val Arg Leu Gln Ala Asp Gly Lys Ser Thr Ala Val Lys Arg His     130 135 140 Tyr Ser Gly Ala Leu Asn Ala Tyr Ala Thr Ile Val Arg Gln Glu Gly 145 150 155 160 Ile Gly Ala Leu Trp Thr Gly Leu Gly Pro Asn Met Ala Arg Asn Ala                 165 170 175 Leu Ile Asn Ala Ala Glu Leu Ala Ser Tyr Asp Gln Phe Lys Gln Met             180 185 190 Phe Leu Gly Leu Pro Gly Phe Thr Asp Asn Val Tyr Thr His Leu Leu         195 200 205 Ala Gly Leu Gly Ala Gly Ile Phe Ala Val Cys Ile Gly Ser Pro Val     210 215 220 Asp Val Val Lys Ser Arg Met Met Gly Asp Ser Thr Tyr Arg Ser Thr 225 230 235 240 Phe Asp Cys Phe Ala Lys Thr Leu Lys Asn Asp Gly Leu Ala Ala Phe                 245 250 255 Tyr Lys Gly Phe Ile Ala Asn Phe Cys Arg Val Gly Ser Trp Asn Val             260 265 270 Ile Met Phe Leu Thr Leu Glu Gln Val Arg Arg Phe Phe Gln         275 280 285 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 1 <400> 5 ttccccgtcg acaaaatggc gacggc 26 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 2 <400> 6 cttgtccatg gaagtggcat cacctac 27 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 3 <400> 7 gtaggtgtcg acactttaat gaacaag 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 4 <400> 8 ctagaaccat gggtttaaat tggtcgt 27 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 5 <400> 9 ccagcgtcga ccaatttaaa cagatg 26 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 6 <400> 10 ctgttccatg gttaagaaca ttatcac 27 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 7 <400> 11 gcggccgctt tacttgtaca gc 22 <210> 12 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 8 <400> 12 gcctgcaggt cgactctaga ggggccccgg gta 33 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer 9 <400> 13 cggccgctaa aggaacagat ggtcgactcc ctc 33

[0033]

[Sequence list free text] SEQ ID Nos. 1 and 2 are WhUCP1
SEQ ID Nos. 3 and 4 are for WhUCP1b. The nucleic acids of SEQ ID NOs: 5 to 13 are the primers of primer numbers 1 to 9, respectively.

[Brief description of drawings]

FIG. 1 Structure of plasmid construct

[Fig. 2] Fluorescence photograph and staining photograph of yeast into which the GFP gene has been introduced.
The mito tracker dyeing | staining image and the GFP fluorescence image of yeast which expressed P, D2 :: GFP, D3 :: GFP are shown. Mitotracker stained image of yeast expressing D1-D3 :: GFP fusion protein (left side A,
In the C, E) and GFP fluorescence images (right side B, D, F), the granular fluorescence found in yeast indicates mitochondria.

FIG. 3: Fluorescence photograph of yeast introduced with DsRed and GFP bound to D2 region (A) GFP fluorescence image of yeast expressing DSR :: D2 :: GFP (B) DsRed fluorescence image (C) DAPI staining Image arrows indicate the fluorescent granules that are the same in the GFP fluorescent image, the DsRed fluorescent image, and the DAPI stained image.

Continued front page    F term (reference) 2B030 AA02 AB03 CA17 CA19 CB02                       CD03 CD07 CD09 CG01                 4B024 AA08 BA63 BA79 CA04 DA12

Claims (8)

[Claims] 1. A DNA encoding a targeting peptide capable of simultaneously transporting two proteins to mitochondria. 2. A DNA encoding a polypeptide containing the following (a) or (b), which is a targeting peptide that transports a protein to mitochondria. (A) From the 105th residue to the 1st residue of the sequence shown in SEQ ID NO: 2 or 4.
A polypeptide consisting of 91 residues. (B) 105th to 191st of the sequence shown in SEQ ID NO: 2 or 4
A polypeptide having one to several amino acids deleted, substituted, or added to the sequence consisting of the th amino acid and having a targeting activity capable of transporting a protein to mitochondria. 3. A vector comprising a construct in which the DNA encoding any polypeptide other than native wheat UCP is ligated to the 3′-end and / or the 5′-end of the DNA according to claim 1 or 2. 4. A method for transporting a polypeptide to mitochondria using the vector according to claim 3. 5. A plant transformed with the vector according to claim 3 and having an arbitrary polypeptide introduced into mitochondria. 6. A vector obtained by recombining a male sterility gene in the vector according to claim 3. 7. A vector in which a respiratory subunit is recombined in the vector according to claim 3. 8. A non-human animal transformed with the vector of claim 3.