JP5753052B2 - Polynucleotide encoding terpineol synthase and uses thereof - Google Patents

Description

  The present invention relates to a polynucleotide encoding terpineol synthase, and further relates to uses such as a transformant, a method for modifying the fragrance of a plant, and a method for producing a fragrance modified plant.

  In flowers, fragrance is one of the charms of flowers along with color and shape. In particular, many wild species have fragrances, but in the process of breeding, color, shape, disease resistance, shelf life, etc. were given priority, resulting in a reduction in the original fragrance, Many of them have lost their scent. There are individual differences in the way scents are perceived and preferences, and since the variation in scents due to cultivation is large, handling is particularly difficult compared to colors and shapes. For this reason, to date, few attempts have been made to breed aromatic varieties compared to color and shape.

  However, in recent years, with the diversification of needs, understanding and consumption of fragrances have increased, and fragrances are regarded as one of the great attractions in fresh flowers. In addition, fragrance has an important role for human sensitivity. Under such circumstances, a garden plant exhibiting excellent aromaticity in addition to color, shape and shelf life is expected to have high commercial value, and its development is desired. So far, aromatic varieties have been developed by cross breeding for camellia, sweet pea, cyclamen, and the like. In this case, a wild scent or a relatively close variety is usually used for one parent, but these wild species or varieties that are close to the wild species generally have smaller flowers and flower colors than horticultural species. limited. For this reason, in the obtained hybrid, the size of the flower tends to be smaller than that of existing varieties, and there is a problem that color variations are limited.

  On the other hand, the scent of flowers is a mixture of various types of fragrance components (Non-Patent Document 1), and the fragrance components are large, such as aromatic compounds, terpenoids (monoterpenes, etc.), aliphatic compounds, nitrogen-containing compounds, etc. being classified. For example, in the case of flowers, not only the difference in the aroma components contained, but even if the aroma components contained are the same, the quality of the aroma varies greatly depending on the ratio. In this way, various floral fragrances are born by blending various aromatic components.

  Terpineol is a kind of naturally occurring monoterpene, and is also a component of essential oils of laurels, rosemary, anise, marjoram, etc., plants such as Akigiri and Juniper, and turpentine oil. As the terpineol, four types of isomers, α, β, γ, and δ-terpineol, which are different in the position of a double bond from a hydroxy group are known. Terpineol usually exists as a mixture of these in nature, and the main component is α-terpineol. The terpineol has a fragrance similar to lilac and is used as a fragrance. For example, it is used as an additive in cosmetics, soaps and the like. The terpineol is used as a plasticizer for hot melt adhesives, for example. The terpineol is also known to have an insect repellent effect.

  There are two known terpineol biosynthetic pathways in the case of plants: the mevalonic acid (MVA) pathway present in the cytoplasm and the methylerythritol phosphate (MEP) pathway present in plastids. In the mevalonate pathway, monoterpene is synthesized via mevalonic acid produced by the reaction of acetyl CoA with the other two molecules of acetyl CoA and reduction. Specifically, mevalonic acid is decarboxylated into isopentenyl pyrophosphate (hereinafter referred to as “IPP”) and dimethylallyl pyrophosphate (hereinafter referred to as “DMAPP”), and these two are terpenoid aromatic compounds. This is the origin of the isoprene skeleton, which is the basic structure of. On the other hand, in the methylerythritol phosphate pathway, IPP and DMAPP are synthesized from 3-phosphoglyceraldehyde and pyruvic acid via 2-methyl-erythritol-4-phosphate (MEP) generated through decarboxylation and isomerization. Is done. And, by the action of geranyl pyrophosphate synthase (hereinafter referred to as “GPPS”), a single molecule of IPP is added to DMAPP to form geranyl pyrophosphate (hereinafter referred to as “GPP”). Monoterpene synthase such as terpineol synthase (hereinafter referred to as “TES”) acts on this GPP to produce terpineol (Non-patent Document 2).

The TES gene has been obtained from several plants so far. Specifically, for example, there are a TES gene derived from Vitis vinifera (grape) (Non-patent Document 3) and a TES gene derived from Magnolia grandiflora (Non-patent Document 4).

Dudareva N et al. Curr. Opin. Biotechnol. . 181-189, 2008 Plant terpenoid synthase: Molecular biology and phylogenetic analysis, Jorg Bohlmann Gilbert Meyer-Gauen Rodney Croteau, Proc. Natl. Acad. Sci. (95) 4126-4133 Identification of Vitis vinifera (-)-α-terpineol synthase by in silico screening of full length cDNA ESTs and functional characterization of the recombination. (2004) Diane M. et al. Martine, Jorg Bohlmann, Phytochemistry (65) 1223-1229 Biochemical and genomic charac- terization of terpene synthesis in Magnolia grandiflora. (2008) Plant Physiol. (147) 1017-1033

  As described above, according to cross breeding, various problems occur, and it is considered useful to apply a gene recombination technique in order to impart or modify fragrance while maintaining excellent traits. Accordingly, an object of the present invention is to provide a novel gene, more specifically a polynucleotide encoding terpineol synthase, and its use, which can be used for imparting fragrance to a plant or modifying fragrance.

In order to achieve the above object, the polynucleotide of the present invention is at least one polynucleotide selected from the group consisting of the following (a) to (g).
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11; (b) in the nucleotide sequence of any one of (a), one or several bases are deleted, substituted and A polynucleotide encoding a protein having terpineol synthesizing activity (c) consisting of a base sequence having 90% or more identity to any of the base sequences of (a) , A polynucleotide encoding a protein having terpineol synthesis activity (d) a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the base sequences of (a) above A polynucleotide encoding a protein having terpineol synthesis activity (e) SEQ ID NO: 2, A polynucleotide encoding a protein comprising the amino acid sequence of 6, 8, 8, 10 or 12 (f) In the amino acid sequence of any one of (e), one or several amino acids are deleted, substituted and / or added. (G) a polynucleotide encoding a protein having terpineol synthesizing activity (g) comprising an amino acid sequence having 90% or more identity to any amino acid sequence of (e) above, and comprising terpineol synthesizing activity A polynucleotide encoding a protein having

  The expression vector of the present invention comprises the polynucleotide of the present invention.

  The transformant of the present invention is a non-human host into which the polynucleotide of the present invention or the expression vector of the present invention has been introduced.

  The offspring, vegetative propagation body, organ, tissue or cell of the transformant of the present invention is characterized by having the same properties as the transformant of the present invention.

  The processed product of the present invention is a processed product of the transformant of the present invention or a progeny, a vegetative propagation body, an organ, a tissue, or a cell of the transformant.

  The method for modifying fragrance of the present invention comprises a step of introducing the polynucleotide of the present invention or the expression vector of the present invention into a non-human host.

  The method for producing an aromatic non-human host of the present invention comprises a step of modifying the aromaticity of a non-human host by the method for modifying an aromatic property of the present invention.

The protein of the present invention is at least one protein selected from the group consisting of the following (A) to (C).
(A) A protein comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12 (B) In the amino acid sequence of (A), one or several amino acids are deleted, substituted and / or added. A protein having a terpineol synthesizing activity (C) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of (A) and having a terpineol synthesizing activity

  According to the polynucleotide of the present invention, terpineol synthase can be expressed by genetic engineering techniques. For this reason, for example, by introducing the polynucleotide of the present invention into a plant to produce a transformant, terpineol synthase is expressed in the transformant, thereby synthesizing terpineol as an aroma component. It becomes possible. For this reason, according to the present invention, for example, since it is possible to impart fragrance to a plant or modify its fragrance, it is possible to provide a horticultural plant with a high commercial value in cultivation of horticultural plants such as fresh flowers, Very useful.

<Polynucleotide>
As described above, the polynucleotide of the present invention is at least one polynucleotide selected from the group consisting of (a) to (g).

These polynucleotides can be obtained from freesia, for example, as described later. The aforementioned Vitis vinifera (grape) -derived TES gene and Magnolia grandiflora (Taisamboku) -derived TES gene obtained so far are all derived from dicotyledonous plants, and terpineol synthase genes derived from monocotyledonous plants are as follows: This has been revealed for the first time by the present invention.

  As described above, the polynucleotide of the present invention encodes a protein having terpineol synthesis activity. For this reason, for example, a protein having terpineol synthesis activity can be produced by expressing a protein from the polynucleotide. Expression of the protein may be performed, for example, by introduction into a non-human host or in a so-called cell-free protein synthesis system. Further, by expressing a protein from the polynucleotide, terpineol can be synthesized by the terpineol synthesis activity of the expressed protein. The terpineol can be synthesized, for example, by adding the expressed protein to a reaction system containing a substrate, or by introducing the polynucleotide into the non-human host and expressing the protein in the non-human host. And can be synthesized in the non-human host by the protein. By performing the terpineol synthesis in the non-human host, for example, the non-human host can be changed from non-aromatic to fragrant, enhanced fragrance, and scented. When the aromaticity is modified by the polynucleotide, the non-human host is preferably, for example, a plant or a part thereof, as described later.

  In the present invention, hereinafter, terpineol synthesis activity is referred to as TES activity. Since the polynucleotide of the present invention is a polynucleotide encoding a protein having TES activity, it can also be represented as a terpineol synthase gene (hereinafter also referred to as “TES gene”). The protein encoded by the polynucleotide of the present invention is the protein of the present invention described later, and has TES activity, and can also be represented as terpineol synthase (hereinafter also referred to as “TES” or “TES protein”). .

  The TES activity is an activity that catalyzes the production of terpineol from a substrate. The substrate is not particularly limited, and examples thereof include geranyl pyrophosphate (GPP).

  The terpineol produced by the TES activity can be converted into, for example, a glycoside, an aldehyde, a ketone or an ester by the action of another enzyme or automatic chemical conversion.

  Terpineol has four types of isomers, α-terpineol, β-terpineol, γ-terpineol, and δ-terpineol, which are different from each other in the position of the hydroxy group and the double bond. The terpineol has an aroma similar to lilac. Terpineol usually exists as a mixture of these in nature, and the main component is α-terpineol. In the present invention, the terpineol synthesis activity may be, for example, any of the synthesis activities of α-terpineol, β-terpineol, γ-terpineol, and δ-terpineol, and may be the synthesis activity of two or more isomers, Preferably, it is alpha-terpineol synthetic activity.

  The protein encoded by the polynucleotide of the present invention may have, for example, only TES activity or may have other catalytic functions.

  The polynucleotide of the present invention may be, for example, a polynucleotide comprising a base sequence complementary to the polynucleotides (a) to (g). The polynucleotide of the present invention may be, for example, a single strand or a double strand. In the latter case, for example, the sense strand includes any of the polynucleotides (a) to (g), and the antisense strand includes a polynucleotide having a base sequence complementary to the polynucleotide.

  The polynucleotide of the present invention may be, for example, DNA composed of deoxyribonucleotides or RNA composed of ribonucleotides. In the latter case, for example, RNA having a base sequence corresponding to the base sequence of DNA, specifically, a base sequence in which base T is replaced with base U can be mentioned.

Said (a) is a polynucleotide which consists of a base sequence of sequence number 1, 3, 5, 7, 9, or 11. In the above (a), the polynucleotide consisting of the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 3 is obtained from, for example, Freesia ( Freesia refracta ), and specifically from cultivar Aladdin ( Freesia refracta cv. Aladdin). . The base sequence of SEQ ID NO: 1 is a full-length cDNA sequence encoding a protein containing a signal peptide, the underlined region at positions 1 to 234 is the signal peptide coding sequence, and positions 235 to 1785 are the full-length code of the mature protein. Is an array. The amino acid sequence of the protein encoded by the base sequence of SEQ ID NO: 1 is represented by SEQ ID NO: 2, the underlined region at positions 1 to 78 is the amino acid sequence of the signal peptide, and positions 79 to 594 are the mature protein. Full-length amino acid sequence. The signal peptide is present, for example, as a chloroplast transit peptide (cTP) sequence in the freesia. The base sequence of SEQ ID NO: 3 represents the full-length coding sequence of the mature protein excluding the coding sequence of the signal peptide in the base sequence of SEQ ID NO: 1. The amino acid sequence of the protein encoded by the base sequence of SEQ ID NO: 3 is represented by SEQ ID NO: 4.
FAfn01 gene (s +: including signal peptide coding sequence)
1785 bp (SEQ ID NO: 1)
ATGGCGTGTCTTCCATTCCACTACACGACTTACAGTCGATCACCTGCCGGAATATTGTTCCGATCGTCTCTGCCTTCTTCTCACTGTAGAGCACGCAGAAGCAGAAGCAATGAAAGTGCAAACCGCATTCGGTGTTGCAACAACACTCAAATTTCTCAGCCTTTACGGCGGACGGCGAACTATCCTGAGAGCATCATTTCATG
FAfn01 protein (s +: including signal peptide)
594aa (SEQ ID NO: 2)
MACLPFHYTTYSRSPAGILFRSSLPSSHCRARRSRSNESANRIRCCNNTQISQPLRRTANYPPTIWENSYIQELNTDY MEDEETIEIGKLKEYVMTRLIISNSDQIELIDTLQQLGVAYHFQEEIQNILATIFCSIKKIIPTIHNDIYATALLFRLLREKGFHVSTNIFNNFKEEGGTFKACLKNDIKGMLSLYEASFLAVEGENELDEARLFATECLKHTMENSLSLEPSMKERIVHALELPLHWRMSRLHSRWFIDQYEKDEKMNPTLLRLAKLDFNFVQTIYKRELKELSRWWSNLDLLGDKLGFARDRLVENYLWTVGSAFEPKFWQSREALTKANCLITTIDDIYDVYGTLDELVLFTDVVDRWDVNAIEQLPDYMKTCLLALFNTTNDTAYKILNLKGVIIIPQLKKVWADLCKAYLVEAKWYHSGYMPTLEEYLDNGWISISGHVALAHAFCTSEDITYRALQCYNQLSPNLLFHSSVIVRLVDDLATSTAELERGDVPKAIQCYMKQNRVSEEVAREKIKEMIVSTWEKLNGDLIATSSVVESFQSVALNFPRMAQCIYQYGDGYGDPTQKTKDQIVSLLIQPVSL
FAfn01 gene (s-: not including signal peptide coding sequence)
1551 bp (SEQ ID NO: 3)

FAfn01 protein (s-: signal peptide not included)
516aa (SEQ ID NO: 4)
MEDEETIEIGKLKEYVMTRLIISNSDQIELIDTLQQLGVAYHFQEEIQNILATIFCSIKKIIPTIHNDIYATALLFRLLREKGFHVSTNIFNNFKEEGGTFKACLKNDIKGMLSLYEASFLAVEGENELDEARLFATECLKHTMENSLSLEPSMKERIVHALELPLHWRMSRLHSRWFIDQYEKDEKMNPTLLRLAKLDFNFVQTIYKRELKELSRWWSNLDLLGDKLGFARDRLVENYLWTVGSAFEPKFWQSREALTKANCLITTIDDIYDVYGTLDELVLFTDVVDRWDVNAIEQLPDYMKTCLLALFNTTNDTAYKILNLKGVIIIPQLKKVWADLCKAYLVEAKWYHSGYMPTLEEYLDNGWISISGHVALAHAFCTSEDITYRALQCYNQLSPNLLFHSSVIVRLVDDLATSTAELERGDVPKAIQCYMKQNRVSEEVAREKIKEMIVSTWEKLNGDLIATSSVVESFQSVALNFPRMAQCIYQYGDGYGDPTQKTKDQIVSLLIQPVSL

In the above (a), the polynucleotide comprising the nucleotide sequences of SEQ ID NO: 5 and SEQ ID NO: 7 is obtained from, for example, Freesia ( Freesia refracta ), and specifically from cultivar Aladdin ( Freesia refracta cv. Aladdin). . The base sequence of SEQ ID NO: 5 is a full-length cDNA sequence encoding a protein containing a signal peptide, the underlined region at positions 1 to 234 is the signal peptide coding sequence, and positions 235 to 1788 are the full-length code of the mature protein. Is an array. The amino acid sequence of the protein encoded by the base sequence of SEQ ID NO: 5 is represented by SEQ ID NO: 6, the underlined region at positions 1 to 78 is the amino acid sequence of the signal peptide, and positions 79 to 595 are the mature protein. Full-length amino acid sequence. The signal peptide is present, for example, as a chloroplast transit peptide (cTP) sequence in the freesia. The nucleotide sequence of SEQ ID NO: 7 represents the full-length coding sequence of the mature protein excluding the coding sequence of the signal peptide in the nucleotide sequence of SEQ ID NO: 5. The amino acid sequence of the protein encoded by the base sequence of SEQ ID NO: 7 is represented by SEQ ID NO: 8.
FAfn16 gene (s +: including signal peptide coding sequence)
1788 bp (SEQ ID NO: 5)
ATGGCGTGTCTTCCATTCCACTACACGACTTACAGTCGATCACCTGCCGGAATATTGTTCCGATCGTCTCTGCCTTCTTCTCACTGTAGAGCACGCAGAAGCAGAAGCAATGAAAGTGCAAACCGCATTCGGTGTTGCAACAACACTCAAATTTCTCAGCCTTTACGGCGGACGGCGAACTATCCTGAGAGCATCATTTCATG
FAfn16 protein (s +: including signal peptide)
595aa (SEQ ID NO: 6)
MACLPFHYTTYSRSPAGILFRSSLPSSHCRARRSRSNESANRIRCCNNTQISQPLRRTANYPPTIWENSYIQELNTDY MQEDEETIEIGKLKEYVMTRLIISNSDQIELIDTLQQLGVAYHFQEEIQNILATIFCSIKKIIPTIHNDIYATALLFRLLREKGFHVSTNIFNNFKEEGGTFKACLKNDIKGMLSLYEASFLAVEGENELDEARLFATECLKHTMENSLSLEPSMKERIVHALELPLHWRMSRLHSRWFIDQYEKDEKMNPTLLRLAKLDFNFVQTIYKRELKELSRWWSNLDLLGDKLGFARDRLVENYLWTVGSAFEPKFWQSREALTKANCLITTIDDIYDVYGTLDELVLFTDVVDRWDVNAIEQLPDYMKTCLLALFNTTNDTAYKILNLKGVIIIPQLKKVWADLCKAYLVEAKWYHSGYMPTLEEYLDNGWISISGHVALAHAFCTSEDITYRALQCYNQLSPNLLFHSSVIVRLVDDLATSTAELERGDVPKAIQCYMKQNRVSEEVAREKIKEMIVSTWEKLNGDLIATSSVVESFQSVALNFPRMAQCIYQYGDGYGDPTQKTKDQIVSLLIQPVSL
FAfn16 gene (s-: not including signal peptide coding sequence)
1554 bp (SEQ ID NO: 7)

FAfn16 protein (s-: signal peptide not included)
517aa (SEQ ID NO: 8)
MQEDEETIEIGKLKEYVMTRLIISNSDQIELIDTLQQLGVAYHFQEEIQNILATIFCSIKKIIPTIHNDIYATALLFRLLREKGFHVSTNIFNNFKEEGGTFKACLKNDIKGMLSLYEASFLAVEGENELDEARLFATECLKHTMENSLSLEPSMKERIVHALELPLHWRMSRLHSRWFIDQYEKDEKMNPTLLRLAKLDFNFVQTIYKRELKELSRWWSNLDLLGDKLGFARDRLVENYLWTVGSAFEPKFWQSREALTKANCLITTIDDIYDVYGTLDELVLFTDVVDRWDVNAIEQLPDYMKTCLLALFNTTNDTAYKILNLKGVIIIPQLKKVWADLCKAYLVEAKWYHSGYMPTLEEYLDNGWISISGHVALAHAFCTSEDITYRALQCYNQLSPNLLFHSSVIVRLVDDLATSTAELERGDVPKAIQCYMKQNRVSEEVAREKIKEMIVSTWEKLNGDLIATSSVVESFQSVALNFPRMAQCIYQYGDGYGDPTQKTKDQIVSLLIQPVSL

In the above (a), the polynucleotide consisting of the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 11 is obtained from, for example, Freesia ( Freesia refracta ), and specifically from cultivar Aladdin ( Freesia refracta cv. Aladdin). . The nucleotide sequence of SEQ ID NO: 9 is a full-length cDNA sequence encoding a protein containing a signal peptide, the underlined region at positions 1 to 234 is the signal peptide coding sequence, and positions 235 to 1788 are the full-length code of the mature protein. Is an array. The amino acid sequence of the protein encoded by the base sequence of SEQ ID NO: 9 is represented by SEQ ID NO: 10, the underlined region at positions 1 to 78 is the amino acid sequence of the signal peptide, and positions 79 to 595 are the mature protein. Full-length amino acid sequence. The signal peptide is present, for example, as a chloroplast transit peptide (cTP) sequence in the freesia. The nucleotide sequence of SEQ ID NO: 11 represents the full-length coding sequence of the mature protein excluding the coding sequence of the signal peptide in the nucleotide sequence of SEQ ID NO: 9. The amino acid sequence of the protein encoded by the base sequence of SEQ ID NO: 11 is represented by SEQ ID NO: 12.
FAfn27 gene (s +: including signal peptide coding sequence)
1788 bp (SEQ ID NO: 9)
ATGGCGTGTCTTCCATTCCACTACACGACTTACAGTCGATCACCTGCCGGAATATTGTTCCGATCGTCTCTGCCTTCTTCTCACTGTAGAGCACGCAGAAGCAGAAGCAATGAAAGTGCAAACCGCATTCGGTGTTGCAACAACACTCAAATTTCTCAGCCTTTACGGCGGACGGCGAACTATCCTGAGAGCATCATTTCATG
FAfn27 protein (s +: including signal peptide)
595aa (SEQ ID NO: 10)
MACLPFHYTTYSRSPAGILFRSSLPSSHCRARRSRSNESANRIRCCNNTQISQPLRRTANYPPTIWENSYIQELNTDY MQEDEETIEIGKLKEYVMTRLIISNSDQIELIDTLQQLGVAYHFQEEIQNILATIFCSIKKIIPTIHNDIYATALLFRLLREKGFHVSTNIFNNFKEEGGTFKACLKNDIKGMLSLYEASFLAVEGENELDEARLFATECLKHTMENSLSLEPSMKERIVHALELPLHWRMSRLHSRWFIDQYEKDEKMNPTLLRLAKLDFNFVQTIYKRELKELSRWWSNLDLLGDKLGFARGRLVENYLWTVGSAFEPKFWQSREALTKANCLITTIDDIYDVYGTLDELVLFTDVVDRWDVNAIEQLPDYMKTCLLALFNTTNDTAYKILNLKGVIIIPQLKKVWADLCKAYLVEAKWYHSGYMPTLEEYLDNGWISISGHVALAHAFCTSEDITYRALQCYNQLSPNLLFHSSVIVRLVDDLATSTAELERGDVPKAIQCYMKQNRVSEEVAREKIKEMIVSTWEKLNGDLIATSSVVESFQSVALNFPRMAQCIYQYGDGYGDPTQKTKDQIVSLLIQPVSL
FAfn27 gene (s-: not including signal peptide coding sequence)
1554 bp (SEQ ID NO: 11)

FAfn27 protein (s-: signal peptide not included)
517aa (SEQ ID NO: 12)
MQEDEETIEIGKLKEYVMTRLIISNSDQIELIDTLQQLGVAYHFQEEIQNILATIFCSIKKIIPTIHNDIYATALLFRLLREKGFHVSTNIFNNFKEEGGTFKACLKNDIKGMLSLYEASFLAVEGENELDEARLFATECLKHTMENSLSLEPSMKERIVHALELPLHWRMSRLHSRWFIDQYEKDEKMNPTLLRLAKLDFNFVQTIYKRELKELSRWWSNLDLLGDKLGFARGRLVENYLWTVGSAFEPKFWQSREALTKANCLITTIDDIYDVYGTLDELVLFTDVVDRWDVNAIEQLPDYMKTCLLALFNTTNDTAYKILNLKGVIIIPQLKKVWADLCKAYLVEAKWYHSGYMPTLEEYLDNGWISISGHVALAHAFCTSEDITYRALQCYNQLSPNLLFHSSVIVRLVDDLATSTAELERGDVPKAIQCYMKQNRVSEEVAREKIKEMIVSTWEKLNGDLIATSSVVESFQSVALNFPRMAQCIYQYGDGYGDPTQKTKDQIVSLLIQPVSL

  (B) is a polynucleotide encoding a protein having a terpineol synthesizing activity, comprising a base sequence in which one or several bases have been deleted, substituted and / or added in any of the base sequences of (a). It is a nucleotide. In (b), “one or several” may be within a range in which the protein encoded by the polynucleotide of (b) has the TES activity. In the base sequence of any of the above (a), the number of bases is, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 9, further preferably 1 to 5, and particularly preferably 1 to 5. Three, most preferably one or two, may be deleted, substituted and / or added.

  (C) is a polynucleotide encoding a protein having a terpineol synthesizing activity, comprising a nucleotide sequence having 90% or more identity to any of the nucleotide sequences of (a). In the above (c), the “identity” may be, for example, a range in which the protein encoded by the polynucleotide in (c) has the TES activity. The identity is preferably 95% or more, more preferably 98% or more, and further preferably 99% or more. The identity can be calculated from default parameters using analysis software such as BLAST and FAST (hereinafter the same).

(D) is a protein having a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to the polynucleotide of any one of (a) and having terpineol synthesis activity Is a polynucleotide encoding. In (d), the “hybridizable polynucleotide” is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide in (a). The hybridization can be detected by, for example, various hybridization assays. The hybridization assay is not particularly limited, and examples thereof include known methods such as Southern hybridization assay, colony hybridization assay, plaque hybridization assay and the like. Hybridization assays, for example, Zanburuku (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) " [(Cold Spring Harbor Laboratory Press (1989 ) ] and the like It is also possible to adopt the method described in.

In (d) above, the “stringent conditions” may be, for example, any of low stringent conditions, moderate stringent conditions, and highly stringent conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 32 ° C. “Medium stringent conditions” are, for example, 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. The degree of stringency can be set by those skilled in the art by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, time, and the like. "Stringent conditions" are, for example, Zanburuku previously described (Sambrook) et al., Eds., "Molecular Cloning: A Laboratory Manual 2nd Edition (Molecular Cloning:. A Laboratory Manual 2 nd Ed) " [(Cold Spring Harbor Laboratory Press (1989)] and the like can also be employed.

  Said (e) is a polynucleotide which codes the protein which consists of an amino acid sequence of sequence number 2, 4, 6, 8, 10 or 12. The polynucleotide (e) may be a nucleotide sequence in which the protein encoded by the polynucleotide (e) has the TES activity, for example. The nucleotide sequence of the polynucleotide (e) can be designed, for example, by substituting the corresponding codon based on the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12. As a specific example, the base sequence of the polynucleotide encoding the protein consisting of the amino acid sequence of SEQ ID NO: 2 or 4 is, for example, the base sequence of SEQ ID NO: 1 or 3 in (a) above. Examples of the base sequence of the polynucleotide encoding the protein consisting of the amino acid sequence of SEQ ID NO: 6 or 8 include the base sequence of SEQ ID NO: 5 or 7 in (a) above. Examples of the base sequence of the polynucleotide encoding the protein consisting of the amino acid sequence of SEQ ID NO: 10 or 12 include the base sequence of SEQ ID NO: 9 or 11 in (a) above.

  The above (f) is an amino acid sequence in which one or several amino acids are deleted, substituted, and / or added in any one of the amino acid sequences of (e), and is a polyprotein encoding a protein having terpineol synthesis activity. It is a nucleotide. In (f) above, “one or several” amino acids may be within a range in which the protein encoded by the polynucleotide of (f) has the TES activity. In any one of the amino acid sequences of (e), the number of amino acids is, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 9, still more preferably 1 to 5, particularly preferably 1 to 1. Three, most preferably one or two, may be deleted, substituted and / or added.

  Said (g) is a polynucleotide which codes for the protein which consists of an amino acid sequence which has 90% or more identity with respect to any amino acid sequence of said (e), and has terpineol synthetic activity. In the above (g), the “identity” with respect to the amino acid sequence may be, for example, within a range in which the protein encoded by the polynucleotide of (g) has the TES activity. The identity is preferably 95% or more, more preferably 98% or more, and further preferably 99% or more. The identity can be calculated from default parameters using analysis software such as BLAST and FAST, as described above.

  The polynucleotide of the present invention can be produced, for example, by a known genetic engineering technique or synthetic technique.

  The purpose of use of the polynucleotide of the present invention is not particularly limited. For example, the production of the TES protein of the present invention encoded by the polynucleotide, the production of a transformant expressing the TES protein, and the terpineol synthesis of the TES protein It can be used for the synthesis of terpineol by activity. When the TES protein of the present invention is produced from the polynucleotide of the present invention, the polynucleotide may be used, for example, in a cell-free protein synthesis system or introduced into the non-human host. .

  The method for using the polynucleotide of the present invention is not particularly limited, and for example, it is preferably introduced into a non-human host (hereinafter also referred to as “host”). By introducing the polynucleotide into the non-human host, for example, production of the TES protein of the present invention, production of a transformant expressing the TES protein of the present invention, expression of the TES protein of the present invention, and terpineol It is possible to produce a transformant that synthesizes. The host is not particularly limited, and can be appropriately selected depending on the purpose of use of the polynucleotide of the present invention described above, for example. Examples of the host include plant bodies or parts thereof, microorganisms, animal cells, insect cells, and cultured cells thereof.

  In the present invention, “plant” means a plant individual showing the whole plant, and “plant part” is a part of the plant individual, for example, organ, tissue, cell, etc. Good. Examples of the organ include petals, corolla, flowers, leaves, seeds, fruits, stems (underground stems), roots, and baskets. Examples of the stem include bulbs such as bulbs, tubers, corms, rhizomes, liners, and the like, and examples of the roots include tuberous roots and transverse running roots. The tissue is, for example, a part of the organ. The plant part may be, for example, one kind of organ, tissue, and / or cell, or two or more kinds of organ, tissue, and / or cell.

  When the fragrance of the non-human host is modified by the polynucleotide of the present invention, the non-human host is preferably, for example, the plant or a part thereof. The type of the plant body and the type of the plant body part are not particularly limited. By introducing the polynucleotide of the present invention into the non-human host, for example, the non-aromatic property of the non-human host can be changed to aromatic, or the aromaticity derived from terpineol can be further enhanced. . It is also possible to synthesize terpineol as an aromatic compound different from the aromatic compound originally possessed by the non-human host before introduction and to change it to a different aromaticity, or to change it to a different aromaticity by further synthesis of terpineol. The transformant obtained by introducing the polynucleotide of the present invention into the non-human host is the transformant of the present invention described later, and is also referred to as an aromatic modified transformant. Further, when the non-human host is a plant or a part thereof, for example, it is also referred to as an aromatic modified plant.

  Plants subject to aromatic modification are not particularly limited, and examples thereof include solanaceous plants (for example, Calibrachoa, Neelenbergia, etc.), urchinaceae plants (carnation, gypsophila, etc.), asteraceae plants (Asteraceae, Gerbera, sunflower, Daisy, etc.), primaceae plants (cyclamen, etc.), gentian plants (Eustoma grandiflorum, gentian, etc.), scorpionaceae plants (Trennia, etc.), diatomaceous plants (Kalanchoe), lily family plants (tulips, etc.), convolvulaceae plants (morning glory) , Momijigigao, Yorugao, etc.), Hydrangeae plants (Hydrangea etc.), Cucurbitaceae plants (Yellow goose etc.), Coleaceae plants (Forsythia etc.) and the like. The aromatic modified plant is not particularly limited, and for example, roses, cynomolgus family plants (eg Snapdragon), solanaceous plants (eg petunia etc.), rosaceae plants (eg rose etc.), orchids (orchids etc.) ), Iridaceae plants (freesia, iris, gladiolus, etc.), liliaceae plants (lily, etc.), waxy plants (pelargonium, geranium, etc.), camellia plants (camellia, tea tree etc.) and the like. Carnation and torenia are preferred.

When the TES protein of the present invention is produced by the polynucleotide of the present invention, the non-human host is not particularly limited. The non-human host is the same as described above, but among them, microorganisms, animal cells, insect cells, cultured cells thereof and the like are preferable. Examples of the microorganism include prokaryotes and eukaryotes. The prokaryotes, for example, E. coli (Escherichia coli) Escherichia genus such as Bacillus subtilis (Bacillus subtilis) Bacillus such as, Pseudomonas such as Pseudomonas putida (Pseudomonas putida), Rhizobium meliloti (Rhizobium meliloti), such as Examples include bacteria of the genus Rhizobium. Examples of the eukaryote include yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe . Examples of the animal cells include COS cells and CHO cells, and examples of the insect cells include Sf9 and Sf21. For example, the TES protein of the present invention can be produced by introducing the polynucleotide of the present invention into the non-human host. The transformant obtained by introducing the polynucleotide of the present invention into the non-human host is the transformant of the present invention as described above.

  The method for introducing the polynucleotide of the present invention into the host is not particularly limited, and can be performed by a known method. The introduction method can be appropriately set depending on, for example, the type of the host.

  The introduction method includes, for example, a method using Agrobacterium, a method using a gene gun such as a particle gun, a calcium phosphate method, a polyethylene glycol method, a lipofection method using liposome, an electroporation method, an ultrasonic nucleic acid introduction method DEAE-dextran. And a direct injection method using a micro glass tube, a hydrodynamic method, a cationic liposome method, a method using an introduction aid, and the like. Examples of the liposome include lipofectamine and cationic liposome, and examples of the introduction aid include atelocollagen, nanoparticles, and polymers. In the case where the host is a plant or a part thereof, for example, among them, the method using Agrobacterium is preferable. The polynucleotide of the present invention may be introduced into the host by, for example, the expression vector of the present invention as described later.

  When the host is a plant or a part thereof, the introduction of the polynucleotide may be performed, for example, in either the plant or a part thereof, preferably a part of the plant, more preferably The tissue or cell, more preferably a cell. Examples of the tissue include a section cut out from the plant body, and specifically, a section such as a leaf, a stem, and a root. Examples of the cells include cells collected from the plant or tissue thereof, cultured cells of the cells, protoplasts, and callus.

<Expression vector>
As described above, the expression vector of the present invention comprises the polynucleotide of the present invention. According to the expression vector of the present invention, for example, the polynucleotide of the present invention can be easily introduced into the host, and the expression of the polynucleotide of the present invention in the host can be easily controlled.

  The purpose and method of use of the expression vector of the present invention are not particularly limited, and are the same as those of the aforementioned polynucleotide of the present invention.

  The expression vector of the present invention may contain, for example, the polynucleotide of the present invention so that the TES protein of the present invention encoded by the polynucleotide of the present invention can be expressed in the host. The configuration is not limited at all. The host is not particularly limited, and can be appropriately selected according to the purpose of use of the polynucleotide of the present invention as described above. Examples of the host include plant bodies or parts thereof, microorganisms, animal cells, insect cells, cultured cells thereof and the like, as in the description of the polynucleotide of the present invention.

  The expression vector of the present invention can be prepared, for example, by inserting the polynucleotide of the present invention into a backbone vector (hereinafter also referred to as “basic vector”). The type of the vector is not particularly limited, and can be appropriately determined according to the type of the host. When the vector is transformed using the Agrobacterium method, for example, a binary vector is preferable, and examples thereof include pBI121, pPZP202, pBINPLUS, and pBIN19. When transforming bacteria such as E. coli, examples of the vector include a pET vector (Merck), a pCold vector (Takara Bio Inc.), a PQE vector (QIAGEN) and the like. In the case of transformation into eukaryotes such as yeast, examples of the vector include pYE22m, and commercially available yeast expression vectors such as pYES (Invitrogen) and pESC (Stratagene) can also be used. it can.

  The expression vector of the present invention preferably has, for example, a regulatory sequence that regulates the expression of the polynucleotide and the expression of the TES protein of the present invention encoded by the polynucleotide. Examples of the regulatory sequence include a promoter, terminator, enhancer, polyadenylation signal sequence, origin of replication sequence (ori) and the like. The origin of the promoter is not particularly limited, and examples thereof include cytomegalovirus (CMV), rous sarcoma virus (RSV), simian virus-40 (SV-40), muscle β-actin promoter, herpes simplex virus (HSV) and the like. can give. In addition to this, for example, a tissue-specific promoter such as a thymidine kinase promoter, a regulatory promoter such as a growth hormone regulatory promoter, a promoter under the control of the lac operon sequence, an inducible such as a zinc-inducible metallothionein promoter Promoters and the like. In the expression vector of the present invention, the arrangement of the regulatory sequences is not particularly limited. In the expression vector of the present invention, the regulatory sequence may be arranged so that, for example, the expression of the polynucleotide of the present invention and the expression of the TES protein of the present invention encoded thereby can be functionally regulated. The regulatory sequence can be arranged based on a known method. As the regulatory sequence, for example, a sequence provided in advance in the basic vector may be used, the regulatory sequence may be further inserted into the basic vector, and the regulatory sequence provided in the basic vector It may be replaced with the regulatory sequence.

  The expression vector of the present invention may further have a selection marker coding sequence, for example. Examples of the selection marker include drug resistance markers, fluorescent protein markers, enzyme markers, cell surface receptor markers, and the like.

  The method for introducing the expression vector of the present invention into the host is not particularly limited, and can be performed by a known method. The introduction method can be appropriately determined according to, for example, the type of host, the type of expression vector, and the like.

  The introduction method is, for example, the same as the description of the polynucleotide of the present invention, a method involving Agrobacterium, a method using a gene gun such as a particle gun, a calcium phosphate method, a polyethylene glycol method, a lipofection method using liposomes, Examples thereof include an electroporation method, an ultrasonic nucleic acid introduction method DEAE-dextran method, a direct injection method using a micro glass tube or the like, a hydrodynamic method, a cationic liposome method, and a method using an introduction aid. When the host is a plant or a part thereof, for example, among them, a method using Agrobacterium is preferable.

  The expression vector is introduced in the same manner as described for the polynucleotide of the present invention, for example. The host is, for example, as described above. When the host is a plant or a part thereof, for example, it may be performed on either the plant body or a part thereof, preferably a part of the plant body, more preferably the tissue or the cell, and still more preferably. It is a cell. Examples of the tissue include a section cut out from the plant body, and specifically, a section such as a leaf, a stem, and a root. Examples of the cells include cells collected from the plant or tissue thereof, cultured cells of the cells, protoplasts, and callus. The transformant obtained by introducing the expression vector of the present invention is the transformant of the present invention described later, and is also referred to as an aromatic modified transformant or an aromatic modified plant.

  The transformant obtained by introducing the polynucleotide or expression vector of the present invention may be further grown, for example. In the present invention, the “transformant” includes, for example, the meaning of a growth body in which these are grown. For example, when the transformant is obtained by introducing the polynucleotide of the present invention into the non-human host, the non-human host may be grown and further regenerated into a tissue, organ or individual. The cultured cells, tissues, organs or individuals thus obtained are included in the transformant as described above in the present invention. When the non-human host is the plant body or a part thereof, the transformant is preferably regenerated into a tissue, organ or plant individual.

<Transformants etc.>
The transformant of the present invention is a non-human host into which the polynucleotide of the present invention has been introduced. The transformant of the present invention may be a non-human host into which the expression vector of the present invention having the polynucleotide is introduced. Hereinafter, “introduction of the polynucleotide of the present invention” includes the meaning of “introduction of the expression vector of the present invention” unless otherwise specified.

  The transformant of the present invention may be a non-human host into which the polynucleotide of the present invention is introduced so that it can be expressed, and other configurations are not limited at all. “Polynucleotide is expressible” includes the meaning that the protein encoded by the polynucleotide can be expressed. The method for introducing the polynucleotide of the present invention and the expression vector of the present invention into the non-human host is not particularly limited, and the description of the polynucleotide of the present invention and the expression vector of the present invention can be cited.

  The method for using the transformant of the present invention is not particularly limited. As described above, the transformant of the present invention can express the TES protein of the present invention by expressing the polynucleotide of the present invention. For this reason, the said transformant can be used for manufacture of the TES protein of this invention. Since the TES protein of the present invention produced by the transformant exhibits terpineol synthesis activity, it can be used for terpineol synthesis as described above. The terpineol can be synthesized, for example, by adding the TES protein recovered from the transformant to a reaction solution containing a substrate, or expressing the TES protein in the transformant, It can also be synthesized by protein. By synthesizing the terpineol in the transformant, for example, the transformant can be changed from non-aromatic to aromatic, enhanced fragrance, and scented. Such a transformant can also be referred to as, for example, an aromatic modification transformant.

  The transformant of the present invention may be, for example, a grower obtained by introducing the polynucleotide of the present invention or the expression vector of the present invention into the non-human host and further growing it. As a specific example, when the transformant is obtained by introducing the polynucleotide of the present invention into the cell, for example, the cell may be grown and further regenerated into a tissue, organ or individual. The tissue, organ or individual thus obtained is included in the transformant as described above in the present invention.

  The transformant of the present invention preferably expresses the TES protein of the present invention having TES activity based on the introduced polynucleotide of the present invention. In addition, the TES protein of the present invention may be expressed based on the introduced polynucleotide of the present invention by, for example, further growing the transformant of the present invention.

  In the case of modifying aromaticity, the transformant is preferably, for example, a plant into which the polynucleotide of the present invention is introduced or a part thereof. The transformant can also be referred to as, for example, an aromatic modified plant. The transformant is more preferably a part of the plant, more preferably a tissue or a cell, and particularly preferably a cell. When the non-human host is the plant body or a part thereof, the transformant is preferably regenerated into a tissue, organ or plant individual.

  In the transformant of the present invention, the site where the terpineol is synthesized is not particularly limited. When the transformant is the plant body or a part thereof, examples of the synthesized site include petals, corolla, flowers, leaves, stems, roots, and the like.

  The transformant of the present invention can be further propagated, for example. At this time, the transformant of the present invention can be used as the propagation material. The propagation material is not particularly limited, and may be the whole or a part of the transformant, for example. When the transformant is the plant body or a part thereof, examples of the propagation material include seeds, fruits, shoots, stems such as tubers, roots such as tuberous roots, strains, callus, protoplasts, and the like.

  The method for breeding the transformant of the present invention is not particularly limited, and a known method can be adopted. The breeding method may be, for example, sexual reproduction or asexual reproduction, preferably asexual reproduction. The reproduction by asexual reproduction includes, for example, vegetative propagation and is also referred to as vegetative reproduction. The method of vegetative propagation is not particularly limited, and in the case of the plant body or a part thereof, for example, bud propagation, propagation by cuttings, subdividing from an organ to a plant individual, growth by callus, and the like can be mentioned. As the organ, for example, the leaves, stems, roots and the like as described above can be used.

  Hereinafter, a propagation obtained by vegetative propagation of the transformant is referred to as a vegetative propagation of the present invention. The vegetative propagation material of the present invention preferably has the same properties as the transformant of the present invention. The vegetative propagation material of the present invention is not particularly limited, for example, like the transformant, and includes, for example, a plant or a part thereof.

  In addition, when the transformant is sexually reproduced, for example, seeds and offspring such as a grown body grown from the seed can be obtained. The progeny of the present invention preferably has the same properties as the transformant of the present invention. The progeny of the present invention may be, for example, any of a plant and a part thereof, for example, like the transformant.

  The transformant of the present invention may be further processed, for example. The kind of the transformant to be processed is not particularly limited, and examples thereof include flowers, leaves, branches and the like. The processed product of the transformant is not particularly limited, and examples thereof include potpourri, dried flowers, dried flowers, preserved flowers, and resin-sealed products obtained by drying flowers, leaves, branches and the like. The processed product of the present invention may be, for example, a processed product of a progeny, a vegetative propagation body, an organ, a tissue, or a cell of the transformant.

<Method for modifying fragrance of non-human host and method for producing fragrance-modified non-human host>
As described above, the method for modifying the fragrance of the non-human host of the present invention includes the step of introducing the polynucleotide of the present invention into the non-human host. In addition, the method for modifying the aromaticity of a non-human host of the present invention may include a step of introducing the expression vector of the present invention containing the polynucleotide into the non-human host.

  According to the present invention, for example, the aforementioned transformant of the present invention can be obtained by introducing the polynucleotide or expression vector of the present invention into the non-human host. As described above, the transformant can synthesize terpineol by expressing the protein of the present invention having TES activity. For this reason, according to the modification method of the present invention, for example, the fragrance of the non-human host is changed from non-aromatic to fragrance as compared with the case where the fragrance is not introduced, and the fragrance is enhanced compared to the case where it is not introduced. It can be modified to a different fragrance from that which has not been introduced. As described above, the transformant of the present invention obtained by the modification method of the present invention can also be referred to as, for example, the aromatic modified transformant.

  In the present invention, the non-human host is not particularly limited and includes the above-mentioned ones. Among them, the plant body or a part thereof is preferable. When the non-human host is the plant body or a part thereof, the transformant can also be referred to as the aromatic modified plant body. The polynucleotide of the present invention may be introduced into any of plant bodies and parts thereof, for example, as described above, preferably a part of the plant body, more preferably tissues and cells. More preferably, it is a cell. The method for introducing the polynucleotide of the present invention is not limited at all, and the aforementioned method can be cited.

  The modification method of the present invention preferably further includes a step of growing the non-human host into which the polynucleotide has been introduced, that is, the transformant of the present invention after the introduction step. When the transformant obtained in the introducing step is the organ, tissue or cell, in this growth step, the organ is an individual, the tissue is an organ or individual, and the cell is a tissue, organ or individual. It is preferable to regenerate. The growth conditions are not particularly limited and can be appropriately set according to the type of the transformant. In the case where the transformant is, for example, a part of the plant body, specifically, the organ, the tissue or the cell, in this growth step, the organ is a plant individual, the tissue is an organ or plant individual, The cells are preferably regenerated into tissues, organs or plant individuals.

  As described above, the transformant preferably expresses the protein of the present invention having TES activity based on the introduced polynucleotide of the present invention. Moreover, the protein of the present invention may be expressed based on the introduced polynucleotide of the present invention by, for example, growing the transformant in the growing step.

  The site where the fragrance is modified is not particularly limited. When the transformant is a plant or a part thereof, for example, the whole plant may be used, or a petal, a corolla, a flower, a leaf, a stem, a root, or the like may be used.

  Next, the method for producing an aromatic modified non-human host of the present invention includes a step of modifying the aromaticity of the non-human host by the aromatic modifying method of the present invention as described above. To do. The production method of the present invention is characterized by carrying out the method for modifying aromaticity of the present invention, and other steps and conditions are not particularly limited. The production method of the present invention can refer to the method for modifying fragrance of the present invention, unless otherwise indicated.

  In the present invention, the modification of fragrance may be any of modification from non-fragrance to fragrance, enhancement of fragrance, modification of fragrance, and the like.

<TES protein>
As described above, the protein of the present invention is at least one protein selected from the group consisting of the following (A) to (C).
(A) A protein comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12 (B) In the amino acid sequence of (A), one or several amino acids are deleted, substituted and / or added. A protein having a terpineol synthesizing activity (C) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of (A) and having a terpineol synthesizing activity

  As described above, the protein of the present invention is a protein having TES activity, and can also be referred to as TES or TES protein. Unless otherwise indicated, the description of the polynucleotide of the present invention can be cited for the TES protein of the present invention. The TES protein of the present invention only needs to have TES activity as described above, and may be, for example, a polypeptide.

  In (A) above, the protein consisting of the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12 is as described above.

  In the (B), “one or several” may be within a range in which the protein comprising the amino acid sequence of the (B) has the TES activity. In any one of the amino acid sequences of (A), the number of amino acids is, for example, 1 to 20, preferably 1 to 10, more preferably 1 to 9, further preferably 1 to 5, and particularly preferably 1 to 5. Three, most preferably one or two, may be deleted, substituted and / or added.

  In (C) above, the “identity” may be, for example, within a range in which the protein comprising the amino acid sequence of (C) has the TES activity. The identity is 90% or more, preferably 95% or more, more preferably 98% or more, and further preferably 99% or more. The identity is the same as the identity of the base sequence, for example, and can be calculated with default parameters using analysis software such as BLAST and FAST.

  The TES protein of the present invention can be used, for example, for the synthesis of terpineol that can be used as a fragrance for industrial products such as foods, alcoholic beverages, cosmetics and perfumes, a plasticizer such as a hot melt adhesive, an insect repellent and the like.

<Method for producing TES protein>
The method for producing a protein of the present invention is a method for producing a protein having terpineol synthesis activity, and by synthesizing a protein having terpineol synthesis activity encoded by the polynucleotide by expressing the polynucleotide of the present invention. Including a process.

  The production method of the present invention is characterized in that the polynucleotide of the present invention is expressed to synthesize the TES protein of the present invention, and other methods and conditions are not limited at all. In the production method of the present invention, a known method can be employed for the expression of the polynucleotide of the present invention. For example, a host or a cell-free protein synthesis system may be used.

  Examples of the protein synthesis step include a step of expressing the polynucleotide in the non-human host by culturing the non-human host using a non-human host into which the polynucleotide has been introduced. The non-human host is, for example, the same as described above, and preferably a microorganism, an animal cell, an insect cell, a cultured cell thereof or the like. The method for introduction into the non-human host is not particularly limited, and the above description can be cited. The polynucleotide may be introduced by, for example, the expression vector of the present invention, and examples of the non-human host include a non-human host into which the expression vector has been introduced. The culture method is not particularly limited and can be appropriately set according to the type of the non-human host.

  Further, as described above, the protein synthesis step may be a step of expressing the polynucleotide in a cell-free protein synthesis system. In this case, the expression vector of the present invention may be used for the expression of the polynucleotide. The cell-free protein synthesis system can be performed by a known method using, for example, a cell extract, a buffer containing various components, and an expression vector into which the target polynucleotide has been introduced. Can be used.

  The production method of the present invention may further include, for example, a step of purifying the TES protein. The method for purifying the TES protein is not particularly limited, and can be performed, for example, by salting out or various column chromatography.

<Method for producing terpineol>
The method for producing terpineol of the present invention includes a terpineol synthesizing step using the TES protein of the present invention and synthesizing terpineol by the terpineol synthesizing activity of the TES protein.

  The production method of the present invention is characterized by using the TES protein of the present invention, and other steps and conditions are not limited at all. Examples of the terpineol obtained by the production method of the present invention include α-terpineol, β-terpineol, γ-terpineol, and δ-terpineol, and preferably α-terpineol.

  In the production method of the present invention, as the TES protein, for example, a TES protein produced in advance may be used, or may be synthesized by expression of the polynucleotide of the present invention.

  In the former case, the TES protein can be obtained, for example, by the method for producing a TES protein of the present invention. The TES protein may be, for example, a non-purified protein, but a purified protein is preferred.

  In the latter case, the production method of the present invention includes, for example, a protein synthesis step for synthesizing the TES protein, and it is preferable to use the synthesized TES protein in the terpineol synthesis step. The protein synthesis step is not particularly limited, and includes a step of synthesizing a protein by the method for producing a TES protein of the present invention. Specifically, for example, in the protein synthesis step, the non-human host introduced with the polynucleotide of the present invention is used, and the polynucleotide is expressed in the non-human host by culturing the non-human host. It is preferable to synthesize a protein having terpineol synthesis activity encoded by the polynucleotide. At this time, the TES protein synthesized in the non-human host can be isolated and used in the terpineol synthesis step. For example, the protein synthesis step and the terpineol synthesis step may be performed in the non-human host. That is, the protein synthesis step is a step of synthesizing the TES protein in the non-human host, and the terpineol synthesis step is performed by using the terpineol synthesis activity of the synthesized TES protein in the non-human host. It may be a process of synthesis.

  The terpineol thus obtained can be used, for example, as a fragrance for industrial products such as foods, alcoholic beverages, cosmetics and perfumes, a plasticizer such as a hot melt adhesive, an insect repellent and the like.

[Example 1]
From the monocotyledonous freesia petal, the TES gene cDNA was cloned.

  Total RNA was obtained from approximately 2 to 3 g of fresh petals of freesia (variety Aladdin) by RNeasy Plant Mini Kit (QIAGEN) by the method recommended by the manufacturer. A cDNA library was constructed from the total RNA using random primers. Proteins were synthesized from the cDNA library, and TES activity was confirmed by the same method as in Example 2 described later. As a result, cDNA of TES gene consisting of the base sequence of SEQ ID NO: 1 (FAfn01 cDNA), cDNA of TES gene consisting of the base sequence of SEQ ID NO: 5 (FAfn16 cDNA), and cDNA of TES gene consisting of the base sequence of SEQ ID NO: 9 (FAfn27 cDNA) could be obtained.

[Example 2]
The three types of freesia-derived TES gene cDNAs were each introduced into Escherichia coli to express recombinant TES protein. Then, terpineol synthesis activity was confirmed using geranyl pyrophosphate (GPP) as a substrate.

(1) Construction of Expression Vector An expression vector pSPB5065 for expressing FAfn01 cDNA (s +) consisting of the base sequence of SEQ ID NO: 1 containing the signal peptide coding sequence was constructed by the following method. The expression vector was constructed using In-Fusion Advantage PCR Cloning Kit (Clontech), and recombinant TES protein was expressed using pET expression system (Novagen).

E. coli protein expression vector pET15b (Novagen) was digested with restriction enzymes Nde I and Xho I, and then purified with GENECLEAN Turbo kit (Funakoshi Co., Ltd.).

Next, an Nde I recognition sequence is introduced 5 ′ of the start codon and an Xho I recognition sequence is introduced 3 ′ of the stop codon with respect to the coding sequence of the FAfn01 cDNA (s +) shown in SEQ ID NO: 1. Therefore, PCR was performed. For the PCR, a PCR reaction solution having the following composition containing the following two types of primers InF-pET-FAfn01-F and InF-pET-FAfn01-R was used. The PCR conditions were 98 ° C. for 10 seconds, 65 ° C. for 15 seconds, and 72 ° C. for 1 minute and 30 seconds for one cycle, for a total of 25 cycles.
(Primer for FAfn01)
InF-pET-FAfn01-F (SEQ ID NO: 13)
5'-CGCGGCAGCCATATGGAAGATGAGGAAACCAT-3 '
InF-pET-FAfn01-R (SEQ ID NO: 14)
5'-AGCCGGATCCTCGAGTTAGAGGGAAACAGGTTGTA-3 '
(PCR reaction solution)
pSPB5047 100pg
1x Prime STAR Max (Takara)
Each primer 0.2pmol / μl
Total volume 20μl

To 5 μl of the obtained PCR product, 2 μl of Cloning Enhancer was added, reacted at 37 ° C. for 15 minutes, and further reacted at 80 ° C. for 15 minutes. Into this reaction solution, 1 μl of 1 × In-Fusion Reaction buffer (Clontech), In-Fusion Enzyme (Clontech) 1 μl, the pET15b vector 100 ng treated with Nde I and Xho I, and Cloning Enhancer-Treated PCR 2 were mixed. The total volume was adjusted to 10 μl. And after making this liquid mixture react at 37 degreeC for 15 minutes, it was made to react at 50 degreeC for 15 minutes, and also TE40microliter was added. 3 μl of this mixture was transformed into Competent high DH5α (TOYOBO).

Then, plasmid DNA was extracted from the obtained four types of transformants, and the entire nucleotide sequence of the plasmid DNA was determined by a primer walking method using synthetic oligonucleotide primers. As a result, the plasmid DNA was encoded by the FAfn01 cDNA (s +) shown in SEQ ID NO: 1, in which an Nde I recognition sequence was introduced 5 'to the start codon and an Xho I recognition sequence was introduced 3' to the termination codon. Had a sequence. This plasmid DNA clone was designated as an expression vector pSPB5065.

Expression vector for expressing FAfn16 cDNA (s +) consisting of the nucleotide sequence of SEQ ID NO: 5 and FAfn27 cDNA (s +) consisting of the nucleotide sequence of SEQ ID NO: 9 including the signal peptide coding sequence by the same method as above pSPB5066 (FAfn16 cDNA) and pSPB5067 (FAfn27 cDNA) were constructed respectively. In addition, in PCR for introducing an Nde I recognition sequence on the 5 ′ side of the start codon and an Xho I recognition sequence on the 3 ′ side of the stop codon, two primers shown below are used as primers for both cDNAs. It was used.
(Primers for FAfn16 and FAfn27)
InF-pET-FAfn16 / 27-F (SEQ ID NO: 15)
5'-CGCGGCAGCCATATGCAGGAAGATGAGGAAA-3 '
InF-pET-FAfn16 / 27-R (SEQ ID NO: 16)
5'-AGCCGGATCCTCGAGTTAGAGGGAAACAGGTTGTA-3 '

Then, plasmid DNA was extracted from each of the obtained four types of transformants, and the entire base sequence was determined in the same manner as described above. As a result, the plasmid DNA was encoded by the FAfn16 cDNA (s +) shown in SEQ ID NO: 5 in which an Nde I recognition sequence was introduced 5 ′ of the start codon and an Xho I recognition sequence was introduced 3 ′ of the stop codon. And the coding sequence of the FAfn27 cDNA (s +) shown in SEQ ID NO: 9. These plasmid DNA clones were designated as an expression vector pSPB5066 (FAfn16 cDNA) and an expression vector pSPB5067 (FAfn27 cDNA).

(2) Induction of expression of recombinant TES protein Expression of each recombinant TES protein was induced by the following method using each transformant obtained in (1) above. The expression induction of the recombinant TES protein was performed using the Overnight Express Autoinduction System 1 (Novagen).

  LB medium containing OnEx Solution 1 (Novagen) 40 μl, OnEx Solution 2 (Novagen) 100 μl, OnEx Solution 3 (Novagen) 2 μl, ampicillin 50 μg / ml and 0.05% glucose In 5 ml, the cells were cultured with shaking at 37 ° C. for 4 hours. Then, the whole amount of the culture solution was added to 100 ml of LB medium containing OnEx Solution 1 2 ml, OnEx Solution 2 5 ml, OnEx Solution 3 100 μl, and ampicillin 50 μg / ml, and cultured with shaking at 20 ° C. overnight. The obtained culture solution was centrifuged (5000 × g, 5 minutes, 4 ° C.) to collect E. coli. The bacterial cells were added to 5 ml of a buffer solution [50 mmol / L MOPSO buffer (pH 7.0), 5 mmol / L DTT, 5 mmol / L sodium ascorbate, 0.1 mmol / L APMSF, 10% glycerol] per 1 g of the bacterial cells. Suspended. The suspension was sonicated to disrupt the E. coli and then centrifuged (15,000 rpm, 15 minutes, 4 ° C.), and the resulting supernatant was used as a crude enzyme solution containing recombinant TES protein. .

  Next, the crude enzyme solution was subjected to buffer solution [20 mmol / L MOPSO buffer (pH 7.0), 1 mmol / L DTT, 0.1 mmol / L APMSF using Econo-Pac 10DG Desalting Columns (BIO RAD). After replacement with 10% glycerol], it was used in the following enzyme reaction.

(3) Activity measurement of TES protein Reagent [10 μmol / L GPP, 1 mmol / L MgCl ₂ , 0.5 mmol / L MnCl ₂ , 0.1 mmol / L NaWO ₄ , 0.05 mmol / L NaF] is added to the crude enzyme solution. Was added to prepare a reaction solution having a total volume of 3 ml. And this reaction liquid was made to react at 30 degreeC for 3 hours. During the reaction, one MonoTrap DCC18 (GL Science Co., Ltd.) was suspended as a headspace component collector in the upper part of the container. The product components volatilized in the head space were collected with the scavenger.

Next, the collection agent was collected, and 2 ml of dichloromethane was added to the collection agent, and then volatile components were extracted and collected by ultrasonic treatment. The volatile components extracted in dichloromethane were concentrated to dryness and redissolved in 50 μl of dichloromethane. This solution was analyzed by GC-MS as an extract of the product component. The analysis conditions were as follows.
Capillary column: HP INNOWax
(60 m × 0.25 mm × 0.25 μm, Agilent)
Carrier gas: He
Temperature condition: held at 40 ° C. for 10 minutes, then raised to 240 ° C. at 5 ° C./min and finally held at 240 ° C. for 30 minutes Inlet temperature: 250 ° C.
Transfer line temperature: 230 ° C
Sample: 1 μl injection in splitless mode

  As a result, a main peak was observed in the extract of each product component at a retention time of 37.0 minutes. The GC-MS analysis result for this peak was searched by mass library (NIST and Wiley), and was found to agree with the mass fragment of α-terpineol. From these results, it was confirmed that each of the TES genes (FAfn01 gene, FAfn16 gene and FAfn27 gene) exhibited α-terpineol synthesis activity.

  As described above, according to the polynucleotide of the present invention, terpineol synthase can be expressed by a genetic engineering technique. For this reason, for example, by introducing the polynucleotide of the present invention into a plant to produce a transformant, terpineol synthase is expressed in the transformant, thereby synthesizing terpineol as an aroma component. It becomes possible. For this reason, according to the present invention, for example, it is possible to provide a plant with high commercial value in cultivation of a garden plant such as a fresh flower because it can impart aromaticity to the plant or modify its aromaticity. Therefore, it is extremely useful.

Claims (21)

At least one polynucleotide selected from the group consisting of the following (a) to (g).
(A) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9 or 11; (b) in the nucleotide sequence of any one of (a), one or several bases are deleted, substituted and A polynucleotide encoding a protein having terpineol synthesizing activity (c) consisting of a base sequence having 90% or more identity to any of the base sequences of (a) , A polynucleotide encoding a protein having terpineol synthesis activity (d) a base sequence complementary to a polynucleotide that hybridizes under stringent conditions to a polynucleotide comprising any one of the base sequences of (a) above A polynucleotide encoding a protein having terpineol synthesis activity (e) SEQ ID NO: 2, A polynucleotide encoding a protein comprising the amino acid sequence of 6, 8, 8, 10 or 12 (f) In the amino acid sequence of any one of (e), one or several amino acids are deleted, substituted and / or added. (G) a polynucleotide encoding a protein having terpineol synthesizing activity (g) comprising an amino acid sequence having 90% or more identity to any amino acid sequence of (e) above, and comprising terpineol synthesizing activity A polynucleotide encoding a protein having The polynucleotide according to claim 1, wherein the terpineol synthesizing activity is α-terpineol synthesizing activity. A terpineol synthase gene comprising the polynucleotide according to claim 1 or 2. An expression vector comprising the polynucleotide according to claim 1 or 2. A transformant, which is a non-human host into which the polynucleotide according to claim 1 or 2 or the expression vector according to claim 4 has been introduced. The transformant according to claim 5, wherein the non-human host is a plant or a part thereof. The transformant according to claim 6, wherein the plant is carnation or torenia. A progeny, a vegetative propagation body, an organ, a tissue, or a cell of the transformant having the same properties as the transformant according to any one of claims 5 to 7. The transformant according to any one of claims 5 to 8, or a processed product of the progeny, vegetative propagation body, organ, tissue, or cell of the transformant. A method for modifying the fragrance of a non-human host, comprising the step of introducing the polynucleotide of claim 1 or 2 or the expression vector of claim 4 into a non-human host. The aromatic modification method according to claim 10, wherein the non-human host is a plant or a part thereof. 12. The method for modifying aromaticity according to claim 11, wherein the plant is carnation or torenia. A method for producing an aromatic modified non-human host, comprising the step of modifying the aromaticity of a non-human host by the aromatic modification method according to any one of claims 10 to 12. A method for producing an aromatic modified non-human host, comprising the step of vegetatively breeding the transformant according to any one of claims 5 to 7. At least one protein selected from the group consisting of the following (A) to (C).
(A) A protein comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10 or 12 (B) In the amino acid sequence of (A), one or several amino acids are deleted, substituted and / or added. A protein having a terpineol synthesizing activity (C) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of (A) and having a terpineol synthesizing activity The protein according to claim 15, wherein the terpineol synthesizing activity is α-terpineol synthesizing activity. A method for producing a protein having terpineol synthesis activity, comprising a protein synthesis step of synthesizing a protein having terpineol synthesis activity encoded by the polynucleotide by expressing the polynucleotide according to claim 1 or 2. . A method for producing terpineol, comprising the step of synthesizing terpineol using the protein according to claim 15 or 16 and synthesizing terpineol by the terpineol synthesis activity of the protein. The manufacturing method according to claim 18, wherein the terpineol is α-terpineol. Furthermore, the protein production method according to claim 17, further comprising a protein synthesis step of synthesizing the protein,
The production method according to claim 18 or 19, wherein the synthesized protein is used in the terpineol synthesis step. The protein synthesizing step uses a non-human host into which the polynucleotide is introduced, and cultivates the non-human host to express the polynucleotide in the non-human host, and has a terpineol synthesis activity encoded by the polynucleotide. A step of synthesizing a protein having
21. The production method according to claim 20, wherein the terpineol synthesis step is a step of synthesizing terpineol by the terpineol synthesis activity of the protein in the non-human host.