JP2013013406A - Polynucleotide encoding linalool synthase and application of the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new gene, in more detail, polynucleotides encoding linalool synthase applicable to impart aroma to plants or modification of the aroma, and to provide applications of the same.SOLUTION: The polynucleotide includes a specific base sequence encoding a protein having linalool synthesizing activity. The introduction of the polynucleotides to plants imparts aroma to the plants, or modifies aroma of plants.

Description

  The present invention relates to a polynucleotide encoding linalool 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 scents have increased, and scents 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, there is a problem that the size of the flower tends to be smaller than that of an existing variety, and color variations are limited.

  On the other hand, the scent of flowers is a mixture of many types of aroma components (Non-Patent Document 1), and the aroma components are largely classified into aromatic compounds, terpenoids, aliphatic compounds, nitrogen-containing compounds, and the like. For example, the quality of the scent of a flower varies greatly depending on the proportion of the same fragrance component, as well as the difference in the fragrance component contained. In this way, various floral fragrances are born by blending various aromatic components. Among them, linalool is contained in many flowers having a sweet floral scent such as roses and lily of the valley.

  In addition, linalool, which is the main fragrance component of these plants, is not limited to use as a fragrance component of flowers. For example, flavors such as foods and alcoholic beverages made from these plants, and industrial products such as cosmetics and perfumes. Widely used.

  There are two known linalool biosynthetic pathways in the case of plants: the mevalonic acid (MVA) pathway present in the cytoplasm and the methylerythritol (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 basic structures of terpenoid aromatic compounds. The origin of a certain isoprene skeleton. On the other hand, in the methylerythritol pathway, IPP and DMAPP are synthesized from 3-phosphoglyceraldehyde and pyruvic acid via 2-methyl-erythritol-4-phosphate (MEP) generated through decarboxylation and isomerization. . And, by the action of geranyl pyrophosphate synthase (hereinafter referred to as GPPS), one molecule of IPP is added to DMAPP to form geranyl pyrophosphate (hereinafter referred to as GPP), and linalool synthase (hereinafter referred to as GPP). Monoterpene synthase such as LIS) acts to produce linalool.

The LIS gene has been obtained from a large number of plants so far as shown below. Examples of the LIS gene include, for example, Clarkia breweri- derived (S) -LIS gene (Non-patent document 2), Artemisia-derived (3R) -LIS gene (Non-patent document 3), Spruce-derived LIS gene (Non-patent document 4). , Lavender-derived (R) -LIS gene (Non-patent document 5), tomato-derived (R) -LIS gene (Non-patent document 6), snapdragon-derived NES / LIS genes 1 and 2 (Non-patent document 7), and salnaci-derived LIS Genes (Non-patent Document 8), Toranojiso-derived LIS gene (Non-patent Document 9), Matatabi-derived LIS gene (Non-patent Document 10), Sitka spruce-derived LIS gene (accession number HQ426164.1), Canadian spruce-derived LIS gene (accession number) HQ426151.1) and the like.

Dudareva N et al. Curr. Opin. Biotechnol. . 181-189, 2008 Plant Cell, 8, 1137-1148, 1996 Arch. Biochem, Biophys. , 372, 143-149, 1999 Plant Physiol. , 135, 1908-1927, 2004 Arch. Biochem. Biophys. , 465, 417-429, 2007 Plant Mol. biol. , 64, 251-263, 2007 Plant J, 55, 224-239, 2008 Funct. Plant Biol. , 37, 232-243, 2010 Phytochem, 71, 1068-1075, 2010 Funct. Plant Biol. , 37, 232-243, 2010

  As described above, cross breeding causes various problems, and thus it is considered useful to apply a gene recombination technique to impart or modify fragrance while maintaining excellent characteristics. Accordingly, an object of the present invention is to provide a novel gene, more specifically a polynucleotide encoding linalool 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 linalool synthesis activity (c) consisting of a base sequence having 90% or more identity to any of the base sequences of (a) above , A polynucleotide encoding a protein having linalool 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 linalool synthesis activity (e) SEQ ID NOs: 2, 4, 6, 8, A polynucleotide encoding a protein consisting of 0 or 12 amino acid sequences (f) consisting of 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) above , A polynucleotide encoding a protein having linalool synthesis activity (g) encoding a protein having an amino acid sequence having 90% or more identity to any amino acid sequence of (e) and having linalool synthesis activity Polynucleotide

  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 an amino acid sequence and having linalool synthesis activity (C) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of (A) and having linalool synthesis activity

  According to the polynucleotide of the present invention, linalool synthase can be expressed by genetic engineering techniques. For this reason, for example, by producing the transformant by introducing the polynucleotide of the present invention into a plant, linalool synthase is expressed in the transformant, thereby synthesizing linalool which is 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.

FIG. 1 is a gas chromatogram of components produced by a recombinant protein derived from FA25 cDNA in Example 3 of the present invention. FIG. 2 is a gas chromatogram of components produced by a recombinant protein derived from FA26 cDNA in Example 3 of the present invention. FIG. 3 is a gas chromatogram of components produced by the recombinant protein derived from SS19 cDNA in Example 3 of the present invention. FIG. 4 is a gas chromatogram of components produced by the recombinant protein derived from FA25 cDNA in Example 4 of the present invention. FIG. 5 is a gas chromatogram of components produced by a recombinant protein derived from FA26 cDNA in Example 4 of the present invention. FIG. 6 is a gas chromatogram of components produced by the recombinant protein derived from SS19 cDNA in Example 4 of the present invention. FIG. 7 is a gas chromatogram of components produced by a recombinant protein derived from FA26 cDNA in Example 5 of the present invention. FIG. 8 is a gas chromatogram of components produced by a recombinant protein derived from FA26 cDNA in Example 5 of the present invention.

<Polynucleotide>
As described above, 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 linalool synthesis activity (c) consisting of a base sequence having 90% or more identity to any of the base sequences of (a) above , A polynucleotide encoding a protein having linalool 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 linalool synthesis activity (e) SEQ ID NOs: 2, 4, 6, 8, A polynucleotide encoding a protein consisting of 0 or 12 amino acid sequences (f) consisting of 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) above , A polynucleotide encoding a protein having linalool synthesis activity (g) encoding a protein having an amino acid sequence having 90% or more identity to any amino acid sequence of (e) and having linalool synthesis activity Polynucleotide

  These polynucleotides can be obtained from, for example, freesia and sweet pea as described later. The linalool synthase genes obtained so far are all derived from dicotyledonous plants, and linalool synthase genes derived from monocotyledonous plants have been clarified for the first time by the present invention.

  As described above, the polynucleotide of the present invention encodes a protein having linalool synthesis activity. Therefore, for example, a protein having linalool synthesis activity can be produced by expressing the 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. Moreover, protein can be expressed from the polynucleotide, and linalool can be synthesized by the linalool synthesis activity of the expressed protein. The linalool can be synthesized, for example, by adding it to a reaction system containing the expressed protein as a substrate, and introducing the polynucleotide into the non-human host. Linalool synthesis by the expressed and expressed protein can also be performed. By performing the linalool synthesis in the non-human host, for example, the non-human host can be changed from non-aromatic to fragrant, enhanced fragrance, and fragrance can be modified. 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, linalool synthesis activity is hereinafter referred to as LIS activity. Since the polynucleotide of the present invention is a polynucleotide encoding a protein having LIS activity, it can also be represented as a linalool synthase gene (hereinafter also referred to as “LIS gene”). The protein encoded by the polynucleotide of the present invention is the protein of the present invention described later, and has LIS activity, and therefore can also be represented as linalool synthase (hereinafter also referred to as “LIS” or “LIS protein”). .

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

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

  Linalool has an asymmetric carbon at the 3-position, and there is a set of optical isomers of (3S) -linalool and (3R) -linalool. (3S) -linalool has, for example, petitgrain and lavender-like fragrances, and (3R) -linalool has, for example, floral, lavender and woody-like fragrances. In particular, (3R) -linalool is known to be fragrant even in a trace amount because the odor threshold is about 80 times lower than (3S) -linalool. For this reason, in the present invention, the linalool synthesis activity may be, for example, either (3R) -linalool synthesis activity or (3S) -linalool synthesis activity, or both, and preferably (3R) -linalool synthesis. Active.

  The protein encoded by the polynucleotide of the present invention may have, for example, only LIS 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.

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 1779 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 592 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 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.

FA25 gene (s +: including signal peptide coding sequence)
1779 bp (SEQ ID NO: 1)
ATGGCTCTCTTGCCGTGTCTTCCATCTCAGTTTCCATGTTCTCCAGTAACAGGCTTCGTGCGATTGCCTCTTCTCTCTTCCCGTAGGTCATCCAAAGGAGTTCAAAGCTCTAACTATCGTTTTCGCTGTTGTATCAACACTGGAACTTCAGTGTCTCAGCCTTTGCGACGGGCGGCAAGATTGTGGTGAGGCATGA
FA25 protein (s +: including signal peptide)
592aa (SEQ ID NO: 2)
MALLPCLPSQFPCSPVTGFVRLPLLSSRRSSKGVQSSNYRFRCCINTGTSVSQPLRRAASYPQNIWDDSYIQALNCGY MGDEQVNEIRKLKEEVGQLFSDSKEILYQIELIDELQQLGVAYHFQDEIKDKLSTIFCSLEKTSLFMENDLKATSLVFRLLREHGFHASADIFNNFRENKGNFKSCLKNDMEGMINLYEASFFAVEGENQLDEARVFATEHLRHLSESLVEASLRERVAHALELPLHFRMSRLHTRWFIDWYEKKVDKNSNLCRLAKLDFNFVQNIYKRELKELSRWWTNLGLGQKLSFARDRLVENYLFVIGWAFEPKLWQNREAMTMANCLVTTLDDIYDVYGSLDELELFTDAVNRWDAEAIEQLPDYMKTCIMALFNTTNLTANKIMYSKGVNIIPQLRRSWADLCKAYLVEAKWYHSGYMPTLEEYLDTAWISISGPVVLTQAYCTSENITDEALKCYNFYPDVVRQSSMISRLWNDLATSTAEMERGDVPKSIQCYMHEKGVSEEVAREHIRDMIVSISKKFDYDCISNSSIAESLKSVALDVHRMSQCVYQYEDGYGEQGHQKREQVISLLFEPIPL

FA25 gene (s-: not including coding sequence of signal peptide)
1545 bp (SEQ ID NO: 3)

FA25 protein (s-: not including signal peptide)
514aa (SEQ ID NO: 4)
MGDEQVNEIRKLKEEVGQLFSDSKEILYQIELIDELQQLGVAYHFQDEIKDKLSTIFCSLEKTSLFMENDLKATSLVFRLLREHGFHASADIFNNFRENKGNFKSCLKNDMEGMINLYEASFFAVEGENQLDEARVFATEHLRHLSESLVEASLRERVAHALELPLHFRMSRLHTRWFIDWYEKKVDKNSNLCRLAKLDFNFVQNIYKRELKELSRWWTNLGLGQKLSFARDRLVENYLFVIGWAFEPKLWQNREAMTMANCLVTTLDDIYDVYGSLDELELFTDAVNRWDAEAIEQLPDYMKTCIMALFNTTNLTANKIMYSKGVNIIPQLRRSWADLCKAYLVEAKWYHSGYMPTLEEYLDTAWISISGPVVLTQAYCTSENITDEALKCYNFYPDVVRQSSMISRLWNDLATSTAEMERGDVPKSIQCYMHEKGVSEEVAREHIRDMIVSISKKFDYDCISNSSIAESLKSVALDVHRMSQCVYQYEDGYGEQGHQKREQVISLLFEPIPL

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 1779 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 592 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: 7 represents the full-length coding sequence of the mature protein excluding the coding sequence of the signal peptide in 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.

FA26 gene (s +: including signal peptide coding sequence)
1779 bp (SEQ ID NO: 5)
ATGGCTTTCTTGCCGTGTCTTCCATCTCAGTTTTCATGTTCTCCAGTAACGGGCTTCGTCCGATTGCCTCTTCTCTCTACCCGTAGGTCATCCCAAGGAGTTCAAAGCTCTAACTATCGTTTTCACTGTTGTATCAACACTGGAACTTCAGTGTCTCAGCCTTTGCGACGGACGGCACATCGTGG
FA26 protein (s +: including signal peptide)
592aa (SEQ ID NO: 6)
MAFLPCLPSQFSCSPVTGFVRLPLLSTRRSSQGVQSSNYRFHCCINTGTSVSQPLRRTASYPQNIWDDSYIQALNCGY MGDEQVNEIRKLKEDVRQLFSDSKEILYQIELIDELQQLGVAYHFQDEIKDKLSTIFCSLEKTSLFMDNDLKATSLIFRLLREHGFHASADIFNNFIENKGNFKSCLKDDIEGMINLYEASFFAMEGENQLDEARVFATEHLRQLSESLVEASLRERVAHALELPLHFRMSRLHTRWFIDWYEKKVDKNSNLCRLAKLDFNFVQNIYKRELKELSRWWTNLGLGQKLSFARDRLVENYLFVIGWAFEPKLSQNREAMAMANCLVTTLDDIYDVYGSLDELELFTDAVNRWDAEAIEQLPDYMKICFMALFNTTNLTAYKIMYSKGVNIIPQLRRSWADLCKAYLVEAKWYHNGYMPTLEEYLDTAWISISGPVVLTQAYCTNGNITDEALKCYNFYPDVVRQSSMISRLWNDLATSTAEMERGDVPKSIQCYMHEKGVSEEVAREHIRDIIVSISKKFDYDCISNSSIAESLKSVALDVHRMSQCVYQNEDGYGEQGNEKRKQVISLLIEPIPL

FA26 gene (s-: not including coding sequence of signal peptide)
1545 bp (SEQ ID NO: 7)

FA26 protein (s-: signal peptide not included)
514aa (SEQ ID NO: 8)
MGDEQVNEIRKLKEDVRQLFSDSKEILYQIELIDELQQLGVAYHFQDEIKDKLSTIFCSLEKTSLFMDNDLKATSLIFRLLREHGFHASADIFNNFIENKGNFKSCLKDDIEGMINLYEASFFAMEGENQLDEARVFATEHLRQLSESLVEASLRERVAHALELPLHFRMSRLHTRWFIDWYEKKVDKNSNLCRLAKLDFNFVQNIYKRELKELSRWWTNLGLGQKLSFARDRLVENYLFVIGWAFEPKLSQNREAMAMANCLVTTLDDIYDVYGSLDELELFTDAVNRWDAEAIEQLPDYMKICFMALFNTTNLTAYKIMYSKGVNIIPQLRRSWADLCKAYLVEAKWYHNGYMPTLEEYLDTAWISISGPVVLTQAYCTNGNITDEALKCYNFYPDVVRQSSMISRLWNDLATSTAEMERGDVPKSIQCYMHEKGVSEEVAREHIRDIIVSISKKFDYDCISNSSIAESLKSVALDVHRMSQCVYQNEDGYGEQGNEKRKQVISLLIEPIPL

In the above (a), the polynucleotide comprising the nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 11 is obtained from, for example, Sweetpy ( Lathyrus odoratus ), and specifically from cultivar Sweet snow ( Lathyrus odoratus cv. Sweet Snow). can get. 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 252 is the signal peptide coding sequence, and positions 253 to 1692 are the full-length code of the mature protein. Is an array. The amino acid sequence of the protein encoded by the nucleotide sequence of SEQ ID NO: 9 is represented by SEQ ID NO: 10, the underlined region at positions 1 to 84 is the amino acid sequence of the signal peptide, and positions 85 to 563 are the mature protein. Full-length amino acid sequence. The signal peptide is present, for example, as a chloroplast transit (cTP) sequence in the sweet pea. The base 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 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.

SS19 gene (s +: including signal peptide coding sequence)
1692 bp (SEQ ID NO: 9)
ATGGCTGTTACTTCTCAACTACGAGTTTCATATCTCACAAACTCAATTCCTCGTTGCAACTCTTGGACTTCTACGTATCACTCACGGAATTTGTCGCTTAAAACTTCAAATTCAAGGATGGTTCATATTAAGTCCATTGCTTTTAATGATAAGTTGCCACCTTTACAGAAGACGGATGTGATGATGAATCCG
SS19 protein (s +: including signal peptide)
563aa (SEQ ID NO: 10)
MAVTSQLRVSYLTNSIPRCNSWTSTYHSRNLSLKTSNSRMVHIKSIAFNDKLPPLQKVDKTRSLEQVKRRSRDSLLNSSDPIET MKMIDSIQGLGISHHLEDEVNIQLERICDWDASTNLFATSLQFRLLRHNGWPTCSDIFRDFLDKSGNFKESLAKDIFGMLSLYEASYLGTEDEEILKKAMQFSKDHLNESIPHLSPEVGKHIDRALTLPKHLRMARLEARNYMDEYRHASKQIPALWELAKLDNDMIQSLHQTELREICRWWKELGLVEKLSFARDRPTECFLWTVGIFPEPCYTNCRIELTKTICILDVIDDIFDNYGTLDELILFTNAIKRWDLDSMEQLPEYMKICYMALYNTTNEISYKIQTVHGINGLSYLKRTWMDMFDAYLEEAKWFNSGYVPSFRTYLDNGTISVGSCMAMVHATFLIGNGLSKETISMMRPYPRLFTCTGEILRLWDDLGTSTEERERGDNASSIECLMEENNILDENESRKHIRLLIRNLWRELNGLAMNKTMPLSVVKSSLNMARTAQVIYQHGDDQSTFTVDDYVQTLIFSSLPTSH

SS19 gene (s-: not including coding sequence of signal peptide)
1440 bp (SEQ ID NO: 11)

SS19 protein (s-: signal peptide not included)
479aa (SEQ ID NO: 12)
MKMIDSIQGLGISHHLEDEVNIQLERICDWDASTNLFATSLQFRLLRHNGWPTCSDIFRDFLDKSGNFKESLAKDIFGMLSLYEASYLGTEDEEILKKAMQFSKDHLNESIPHLSPEVGKHIDRALTLPKHLRMARLEARNYMDEYRHASKQIPALWELAKLDNDMIQSLHQTELREICRWWKELGLVEKLSFARDRPTECFLWTVGIFPEPCYTNCRIELTKTICILDVIDDIFDNYGTLDELILFTNAIKRWDLDSMEQLPEYMKICYMALYNTTNEISYKIQTVHGINGLSYLKRTWMDMFDAYLEEAKWFNSGYVPSFRTYLDNGTISVGSCMAMVHATFLIGNGLSKETISMMRPYPRLFTCTGEILRLWDDLGTSTEERERGDNASSIECLMEENNILDENESRKHIRLLIRNLWRELNGLAMNKTMPLSVVKSSLNMARTAQVIYQHGDDQSTFTVDDYVQTLIFSSLPTSH

  In the above (b), “1 or several” may be within a range in which the protein encoded by the polynucleotide of (b) has the LIS 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. Addition includes, for example, the meaning of insertion into a sequence. In the present invention, the numerical range of the number of bases and the like discloses, for example, all positive integers belonging to the range. That is, for example, the description “1 to 3” means all disclosures of “1, 2, and 3 bases” (hereinafter the same).

  In the above (c), the “identity” may be, for example, a range in which the protein encoded by the polynucleotide of (c) has the LIS activity. The identity is 90% or more, preferably 95% or more, 96% or more, 97% or more, more preferably 98% or more, and further preferably 99% or more. The identity can be calculated with default parameters using analysis software such as BLAST and FASTA (hereinafter the same).

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.

  The polynucleotide (e) may be any nucleotide sequence in which the protein encoded by the polynucleotide (e) has the LIS 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. Specifically, 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.

  In the above (f), “one or several” amino acids may be within a range in which the protein encoded by the polynucleotide of (f) has the LIS 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.

  In the above (g), the “identity” regarding the amino acid sequence may be, for example, within a range in which the protein encoded by the polynucleotide of (g) has the LIS activity. The identity is 90% or more, preferably 95% or more, 96% or more, 97% 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 FASTA, 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 LIS protein of the present invention encoded by the polynucleotide, the production of an expression vector that expresses the LIS protein, and the character that expresses the LIS protein. For example, the production of a transformant and the synthesis of linalool by the linalool synthesis activity of the LIS protein. When the LIS 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 host, for example, the production of the LIS protein of the invention, the production of a transformant expressing the LIS protein of the invention, and the synthesis of linalool by expressing the LIS protein of the invention. It is possible to produce a transformant to be used. The host is not particularly limited, and can be appropriately selected depending on, for example, the purpose of use of the polynucleotide of the present invention described above. 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 modified to be aromatic, or the fragrance derived from linalool can be further enhanced. Moreover, it is possible to synthesize linalool as a fragrance compound different from the fragrance compound originally possessed by the non-human host before introduction, and to change to a different fragrance, or to change to a different fragrance by further synthesis of linalool. 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 plant is not particularly limited, and examples thereof include roses, scorpionaceae plants (such as snapdragons), solanaceous plants (such as petunias), rosaceae plants (such as roses), orchidaceae plants (such as orchids). , Iridaceae plants (freesia, iris, gladiolus, etc.), liliaceae plants (lily, etc.), waxy plants (pelargonium, geranium, etc.), camellia plants (camellia, tea tree, etc.) and the like. Among these, carnation and torenia are preferable.

When the LIS 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, or cultured cells thereof 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 LIS 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- Examples thereof include a dextran method, 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 LIS 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 plants or parts thereof, microorganisms, animal cells, insect cells, or cultured cells thereof, 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. When transforming 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 a regulatory sequence that regulates, for example, the expression of the polynucleotide and the LIS 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 LIS protein of the present invention encoded thereby can be functionally regulated. 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, a DEAE-dextran method, 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. 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 or a part thereof, preferably a part of the plant, more preferably the tissue or the cell, and still more preferably. 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 LIS 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 LIS protein of this invention. Since the LIS protein of the present invention produced by the transformant exhibits linalool synthesis activity, it can be used for linalool synthesis as described above. The linalool can be synthesized, for example, by adding the LIS protein recovered from the transformant to a reaction solution containing a substrate. In the transformant, the expression of the LIS protein and the LIS protein can be synthesized. The linalool synthesis by can also be performed. By performing the linalool synthesis 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.

  In the transformant of the present invention, the LIS protein of the present invention having LIS activity is preferably expressed based on the introduced polynucleotide of the present invention. Moreover, the LIS 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 linalool 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 called 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 as in the case of the transformant, and examples thereof include 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. It is preferable that the progeny of the present invention obtains the same properties as the transformant of the present invention. The progeny of the present invention may be, for example, a plant or 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 linalool by expressing the protein of the present invention having LIS 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.

  It is preferable that the modification method of the present invention 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 introducing 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 LIS 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.

<LIS 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. (C) a protein having an LIS activity and comprising an amino acid sequence having 90% or more identity to the amino acid sequence of (A).

  As described above, the protein of the present invention is a protein having LIS activity, and can also be referred to as LIS or LIS protein. The description of the polynucleotide of the present invention can be cited for the LIS protein of the present invention unless otherwise indicated. The LIS protein of the present invention only needs to have LIS 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), “1 or several” may be within a range in which the protein having the amino acid sequence of the (B) has the LIS 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, a range in which the protein comprising the amino acid sequence of (C) has the LIS activity. The identity is, for example, 90% or more, preferably 95% or more, 96% or more, 97% 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 FASTA.

  The LIS protein of the present invention can be used for the synthesis of linalool that can be used as a fragrance for industrial products such as foods, alcoholic beverages, cosmetics and perfumes.

<Method for producing LIS protein>
The method for producing a protein of the present invention is a method for producing a protein having linalool synthesis activity, and synthesizes a protein having linalool 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 LIS 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 is preferably a microorganism, an animal cell, an insect cell, or a cultured cell thereof. 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 LIS protein. The method for purifying the LIS protein is not particularly limited, and can be performed, for example, by salting out or various column chromatography.

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

  The production method of the present invention is characterized by using the LIS protein of the present invention, and the other steps and conditions are not limited at all. Examples of the linalool obtained by the production method of the present invention include (3R) -linalool and (3S) -linalool, and preferably (3R) -linalool.

  In the production method of the present invention, as the LIS protein, for example, a LIS 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 LIS protein can be obtained, for example, by the method for producing a LIS protein of the present invention. The LIS protein may be, for example, a non-purified protein, but is preferably a purified protein.

  In the latter case, the production method of the present invention preferably includes, for example, a protein synthesis step for synthesizing the LIS protein, and the synthesized LIS protein is used in the linalool synthesis step. The protein synthesis step is not particularly limited, and examples thereof include a step of synthesizing a protein by the method for producing a LIS 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 linalool synthesis activity encoded by the polynucleotide. At this time, the LIS protein synthesized in the non-human host can be isolated and used in the linalool synthesis step. For example, the protein synthesis step and the linalool synthesis step may be performed in the non-human host. That is, the protein synthesis step is a step of synthesizing the LIS protein in the non-human host, and the linalool synthesis step synthesizes linalool in the non-human host by the linalool synthesis activity of the synthesized LIS protein. It may be a step of.

  The linalool thus obtained can be used, for example, as a fragrance for industrial products such as foods, alcoholic beverages, cosmetics and perfumes.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

[Example 1]
LIS gene cDNA was cloned from monocotyledonous freedia petals and dicotyledonous sweet pea petals.

(1) Screening 1
Total RNA was obtained from about 2 to 3 g of fresh petals of freesia (variety aladdin) and sweet pea (variety sweet snow) by RNeasy Plant Mini Kit (QIAGEN) by the method recommended by the manufacturer, and Oligotex-dT30 (super ) PolyA + RNA added with polyA was purified using mRNA Purification Kit (Takara). A cDNA library was constructed from the polyA + RNA using Uni-Zap XR Vector Kit (Stratagene) and Gigapack III Gold-4 RXN (Agilent technology). The obtained libraries were 7.2 × 10 ⁴ plaque forming unit and 7.1 × 10 ⁴ plaque forming unit, respectively.

  Then, screening was performed using the cDNA library with a known lavender-derived (R) -linalool synthase (LaLIS) cDNA (Arch. Biochem, Biophys. 465, 2007) as a probe.

The probe was produced as follows. First, the oligonucleotide of the full length region of LaLIS cDNA was amplified by PCR and labeled with the DIG labeling system (Roche Diagnostics) according to the conditions recommended by the manufacturer. For PCR, 10 ng of a plasmid containing LaLIS cDNA was used as a template, and 0.2 μmol / L of each of the following two types of oligonucleotides was used as a primer. The PCR conditions were 95 ° C for 2 minutes, 95 ° C for 10 seconds, 55 ° C for 30 seconds, and 72 ° C for 2 minutes for a total of 35 cycles. Treated for minutes. The reaction product after PCR was purified by Quick Spin Columns (Roche), and the purified product was used as a screening probe.
(Primer)
LaLISDIG-F (SEQ ID NO: 13)
5'-ATGTCGATCAATATCAACATGCCTGC-3 '
LaLISDIG-R (SEQ ID NO: 14)
5'-TCATGCGTACGGCTCGAACAG-3 '

  Screening of the cDNA library was performed using a non-radio system DNA detection kit (Roche). Hybridization was performed overnight at 37 ° C. in 5 × SSC containing 0.02% SDS and 30% formamide, and the filter was washed 2 × 30 minutes at 65 ° C. using 2 × SSC, 1% SDS. I went twice. About 240,000 plaques were screened, and 45 positive signals were obtained from the freesia cDNA library and 41 positive signals from the Sweetpy cDNA library. However, as a result of analyzing the nucleotide sequence by the primer walking method using a synthetic oligonucleotide primer, a clone having significant homology with the LaLIS gene was not obtained.

(2) Screening 2 (Freesia-derived LIS gene cDNA)
In the above (1), it was revealed that the cDNA of the free-dia LIS gene could not be cloned by screening using homology based on the sequence of the known LIS gene. Therefore, the following degenerate primer was prepared. Primer combinations are (i) LISdpF1 and LISdpR1, (ii) LISdpF2 and LISdpR1, (iii) LISdpF3 and LISdpR1, (iv) LISdpF1 and LISdpR2, (v) LISdpF2 and LISdpL2, p It was. In the sequence below, “I” is inosine.
(Primer)
LISdpF1 (SEQ ID NO: 15)
5'-GA (C / T) GA (C / T) GTITA (C / T) GA (C / T) AT (A / C / T) -3 '
LISdpF2 (SEQ ID NO: 16)
5'-GA (C / T) GA (C / T) GTITA (C / T) GA (C / T) GTITA (C / T) -3 '
LISdpF3 (SEQ ID NO: 17)
5'-GA (C / T) GA (C / T) AT (A / C / T) TT (C / T) GTITA (C / T) -3 '
LISdpR1 (SEQ ID NO: 18)
5'-IGTICCIA (A / G) (A / G) TC (A / G) TCIGG-3 '
LISdpR2 (SEQ ID NO: 19)
5'-ICCIA (A / G) (A / G) TC (A / G) TCCCA-3 '

  Total RNA was extracted from petals of freesia (variety Aladdin) using RNeasy Plant Mini Kit (QIAGEN), 1 μg of which was extracted using SuperScript First-Strand System for RT-PCR (Invitrogen). CDNA was synthesized by the method recommended by J. Next, using 1 μl of the synthesized cDNA as a template, two kinds of degenerate primers of the above combination (each 0.2 μmol / L) and Takara Ex Taq polymerase (Takara) 1.25 U, and using the method recommended by the manufacturer The PCR reaction was performed with a total of 50 μl of the reaction solution. PCR conditions were 94 ° C for 5 minutes, 94 ° C for 30 seconds, 40 ° C for 1 minute, 72 ° C for 1 minute, 1 cycle at 72 ° C, and repeated for a total of 40 cycles, then held at 72 ° C for 7 minutes did.

  After the PCR reaction, the reaction solution was separated by agarose gel electrophoresis. As a result, an amplified fragment of about 0.5 kb was obtained for the freesia in the reaction solution used for the (v) LISdpF2 and LISdpR2 primers. The amplified fragment was recovered and subcloned into the pCR2.1 TOPO vector (Invitrogen) using the TOPO TA cloning kit (Invitrogen) by the method recommended by the manufacturer. This plasmid was designated as pCR2.1-FAF2R2 # 38, and its nucleotide sequence was determined.

  In order to obtain the full-length cDNA of the clone FAF2R2 # 38, screening was performed using the cDNA library derived from the freedia of (1) above, using the clone as a probe.

The probe was produced as follows. First, the oligonucleotide of the full length region of the clone was amplified by PCR and labeled with the DIG labeling system (Roche Diagnostics) according to the conditions recommended by the manufacturer. For PCR, 10 ng of a plasmid containing 10 ng of each of the cloned cDNAs was used as a template, and 0.2 μmol / L of each of the two types of oligonucleotides shown below was used as a primer. The PCR conditions were 95 ° C for 2 minutes, 95 ° C for 10 seconds, 55 ° C for 30 seconds and 72 ° C for 2 minutes for a total of 35 cycles, and finally 72 ° C for 7 minutes. Processed. The reaction product after PCR was purified by Quick Spin Columns (Roche), and the purified product was used as a screening probe.
(Primer)
DIG-FA # 38F (SEQ ID NO: 20)
5'-GATGATGTGTATGATGTGTATGGCTCCTT-3 '
DIG-FA # 38R (SEQ ID NO: 21)
5'-TGCCGAGGTCGTCCCATAACC-3 '

  Screening of the cDNA library was performed using a non-radio system DNA detection kit (Roche). Hybridization was performed overnight at 37 ° C. in 5 × SSC containing 0.02% SDS and 30% formamide, and the membrane was washed with 2 × SSC and 1% SDS for 30 minutes at 65 ° C. Washed for 30 minutes at 65 ° C. using 5 × SSC and 1% SDS. About 240,000 plaques were screened. The protein was expressed for the obtained positive clone by the same method as in Example 2 described later, and the target LIS synthesis activity was confirmed. As a result, the LIS gene cDNA consisting of the base sequence of SEQ ID NO: 1 (FA25 cDNA) and sequence A LIS gene cDNA (FA26 cDNA) consisting of the nucleotide sequence of No. 5 was obtained.

(3) Screening 3 (Suity-derived LIS gene cDNA)
As described above, in the above (1), it was revealed that the cDNA of the sweet pea LIS gene could not be cloned by the screening using the homology based on the sequence of the known LIS gene. Therefore, the same degenerate primer as in (2) was prepared.

  In the same manner as in (2) above, total RNA was extracted from petals of sweet pea (variety sweet snow), and cDNA synthesis, PCR reaction, and electrophoresis were performed. As a result, an amplified fragment of about 0.5 kb was obtained for the sweet pea in the reaction solution used for the (vi) LISdpF3 and LISdpR3 primers, unlike the freesia of (2). This amplified fragment was recovered and subcloned into the pCR2.1 TOPO vector (Invitrogen) using the TOPO TA cloning kit (Invitrogen) by the method recommended by the manufacturer. This plasmid was designated as pCR2.1-SSSS4F3R2-06 and pCR2.1-SSSS4F3R2-38. Then, the base sequences of the obtained clones SSSt4F3R2 # 06 and SSSt4F3R2 # 38 were determined.

In order to obtain the full-length cDNAs of SSSt4F3R2 # 06 and SSSt4F3R2 # 38, screening was performed using the cDNA library derived from the sweet pea of (1) above using the clone as a probe. The screening method was the same as (2) except that the following primers were used.
(Primer)
DIG-SSst4 # 38F (SEQ ID NO: 22)
5'-GATGATATTTTCGTGTATGGAAAATTTGATG-3 '
DIG-SSst4 # 38R (SEQ ID NO: 23)
5'-GCCTATGGGACGACCTCGGC-3 '
DIG-SSst4 # 06F (SEQ ID NO: 24)
5'-GACGATATTTTTGTGTACGGCACATTAGAT-3 '
DIG-SSst4 # 06R (SEQ ID NO: 25)
5'-GCCGAGGTCGTCCCATAATCG-3 '

  About 240,000 plaques were screened. By the same method as in Example 2 described later, proteins were expressed for the obtained positive clones, and the target LIS synthesis activity was confirmed. As a result, LIS gene cDNA (SS19 cDNA) consisting of the base sequence of SEQ ID NO: 9 was obtained.

[Example 2]
Freesia and Sweetpy LIS gene cDNAs were introduced into E. coli to express recombinant LIS protein. And linalool synthesis activity was confirmed using geranyl pyrophosphate (GPP) as a substrate.

(1) Construction of Expression Vector An expression vector pSPB5025 for expressing FA25 cDNA (s +) consisting of the nucleotide 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 expression was performed using the 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, in order to introduce the Nde I recognition sequence 5 ′ of the start codon and the Xho I recognition sequence 3 ′ of the stop codon with respect to the coding sequence of the FA25 cDNA (s +) shown in SEQ ID NO: 1, PCR reaction was performed. The PCR reaction uses a PCR reaction solution having the following composition containing the following two primers InF-pET-FA25full-F and InF-pET-FA25full-R, and is 98 seconds at 98 ° C. and 15 seconds at 65 ° C. The reaction for 1 minute and 30 seconds at 72 ° C. was defined as one cycle, and a total of 25 cycles were performed.
(Primer for FA25)
InF-pET-FA25full-F (SEQ ID NO: 26)
5'-CGCGGCAGCCATATGGCTCTCTTGCCGTGT-3 '
InF-pET-FA25full-R (SEQ ID NO: 27)
5'-AGCCGGATCCTCGAGTTAGAGGGGAATGGGTTCA-3 '
(PCR reaction solution)
pSPB5025 100pg
1x PrimeSTAR buffer (Takara)
0.2mmol / L dNTPs (Takara)
Each primer 0.2μmol / L
PrimeSTAR HS DNA polymerase (Takara) 1.25U
Total volume 25μl

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. 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 added to this reaction solution. The total volume was adjusted to 10 μl. And after making it 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).

And plasmid DNA was extracted from the obtained 4 types of transformants, and the whole base sequence was determined by the primer walking method by a synthetic oligonucleotide primer. As a result, it has the coding sequence of the FA25 cDNA (s +) shown in SEQ ID NO: 1 in which an Nde I recognition sequence is introduced 5 'to the start codon and an Xho I recognition sequence is introduced 3' to the termination codon. The clone was designated as expression vector pSPB5025.

Expression vector pSPB5026 for expressing FA26 cDNA (s +) comprising the nucleotide sequence of SEQ ID NO: 5 and SS19 cDNA (s +) comprising the nucleotide sequence of SEQ ID NO: 9 including the signal peptide coding sequence by the same method as above. (FA26 cDNA) and pSPB5046 (SS19 cDNA) were constructed respectively. As primers for introducing the Nde I recognition sequence on the 5 ′ side of the start codon and the Xho I recognition sequence on the 3 ′ side of the stop codon, the following primers were used for each cDNA.
(Primer for FA26)
InF-pET-FA26full-F (SEQ ID NO: 28)
5'-CGCGGCAGCCATATGGCTTTCTTGCCGTGT-3 '
InF-pET-FA26full-R (SEQ ID NO: 29)
5'-AGCCGGATCCTCGAGTTAGAGGGGAATGGGTTCAATAAGT-3 '
(Primer for SS19)
InF-pET-SS19full-F (SEQ ID NO: 30)
5'-CGCGGCAGCCATATGGCTGTTACTTCTCAACTAC-3 '
InF-pET-SS19full-R (SEQ ID NO: 31)
5'-AGCCGGATCCTCGAGTTAATGGCTAGTGGGTAATGAT-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, a clone having a coding sequence of the target cDNA into which an Nde I recognition sequence was introduced 5 ′ of the start codon and an Xho I recognition sequence 3 ′ of the stop codon was introduced into the expression vector pSPB5026. (FA26 cDNA), pSPB5046 (SS19 cDNA).

(2) Induction of expression of recombinant LIS protein Expression of recombinant LIS protein was induced by the following method using each transformant obtained in (1) above. Induction of protein expression was carried out using an Overnight Express Automation System 1 (Novagen).

  The transformed strain of (1) was prepared by using OnEx Solution 1 (Novagen) 40 μl, OnEx Solution 2 (Novagen) 100 μl, OnEx Solution 3 (Novagen) 2 μl, ampicillin 50 μg / ml and LB containing 0.05% glucose. The culture was performed with 5 ml of medium at 37 ° C. with shaking for 4 hours. Then, the whole amount of the above 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.

  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 substitution with 10% glycerol], it was used in the following enzyme reaction.

(3) Activity measurement of LIS protein Reagent [10 μmol / L GPP, 1 mmol / L MgCl ₂ , 0.5 mmol / L MnCl ₂ , 0.1 mmol / L Na ₂ WO ₄ , 0.05 mmol / L was added to the crude enzyme solution. NaF] was added to adjust the total volume of the reaction solution to 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, and the generated component volatilized in the headspace was collected by the collector. .

Next, the scavenger was recovered, and 2 ml of dichloromethane was added to the scavenger, and then volatile components were extracted and recovered by ultrasonic treatment. Volatile components extracted into 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. Under the following conditions, linalool was detected at a retention time of 32.8 minutes, and it was confirmed by a standard that a characteristic fragment ion of m / z 154, 121, 93, 71 was detected in the MS spectrum. .
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 peak of linalool was observed only at a retention time of 32.8 minutes, and only linalool was detected as a product component. From these results, it was confirmed that the various LIS genes exhibited linalool synthesis activity.

[Example 3]
Recombinant expressed from the recombinant LIS protein expressing LIS cDNA derived from freesia and sweet pea in Example 2 and the linalool synthase ((R) -LIS) cDNA derived from known lavender ( Lavandula angustifolia ) About linalool synthase, the linalool synthesis activity was compared using each crude enzyme protein.

  In the same manner as in Example 2, FA25 cDNA and FA26 cDNA were introduced into Escherichia coli as LIS genes derived from freesia, SS19 cDNA was introduced as LIS gene derived from sweet pea, and recombinant LIS protein was expressed, respectively. An enzyme solution was prepared.

On the other hand, an expression vector pSPB5045 containing the sequence of the (R) -LIS cDNA of SEQ ID NO: 32 was constructed as an expression vector for expressing the (R) -LIS gene derived from lavender. The amino acid sequence of the (R) -LIS protein encoded by the (R) -LIS cDNA is shown in SEQ ID NO: 33. The above procedure was carried out except that the Nde I recognition sequence was introduced 5 'to the start codon of the cDNA and the Xho I recognition sequence was introduced 3' to the stop codon. The expression vector was constructed in the same manner as in Example 2. Then, in the same manner as in Example 2, a transformant was obtained, recombinant protein expression was induced, and a crude enzyme solution was prepared.

(R) -LIS cDNA
1695 bp (SEQ ID NO: 32)

(R) -LIS protein 564aa (SEQ ID NO: 33)
MSININMPAAAVLRPFRCSQLHVDETRRSGNYRPSAWDSNYIQSLNSQYKEKKCLTRLEGLIEQVKELKGTKMEAVQQLELIDDSQNLGLSYYFQDKIKHILNLIYNDHKYFYDSEAEGMDLYFTALGFRLFRQHGFKVSQEVFDRFKNENGTYFKHDDTKGLLQLYEASFLVREGEETLEQAREFATKSLQRKLDEDGDGIDANIESWIRHSLEIPLHWRAQRLEARWFLDAYARRPDMNPVIFELAKLNFNIVQATQQEELKALSRWWSSLGLAEKLPFVRDRLVESYFWAIPLFEPHQYGYQRKVATKIITLITSLDDVYDIYGTLDELQLFTNLFERWDNASIGRLPEYLQLFYFAIHNFVSEVAYDILKEKGFTSIVYLQRSWVDLLKGYLKEAKWYNSGYTPSLEEYFDNAFMTIGAPPVLSQAYFTLGSSMEKPIIESMYEYDNILRVSGMLVRLPDDLGTSSFEMERGDVPKSVQLYMKETNATEEEAVEHVRFLNREAWKKMNTAEAAGDSPLVSDVVAVAANLGRAAQFMYFDGDGNQSSLQQWIVSMLFEPYA

  And activity measurement was performed like the said Example 2 using each crude enzyme liquid. In addition, the protein usage-amount of the said crude enzyme in the reaction liquid (3 ml) of activity measurement was 5 to 5.5 mg.

  These results are shown in FIGS. 1 to 3 are gas chromatograms of product components, respectively, FIG. 1 is a result of FA25 cDNA-derived recombinant protein (FA25), and FIG. 2 is a result of FA26 cDNA-derived recombinant protein (FA26). FIG. 3 shows the result (SS19) of the recombinant protein derived from the SS19 cDNA child, together with the result of the recombinant protein derived from the lavender (R) -LIS cDNA (LaLIS). As shown in FIG. 1 to FIG. 3, the recombinant protein derived from FA25 cDNA and FA26 cDNA has a higher amount of linalool and is significantly stronger than the known recombinant protein derived from (R) -LIS cDNA. Showed activity. Moreover, the recombinant protein derived from SS19 cDNA was produced in the same amount as the known recombinant protein derived from (R) -LIS cDNA, and showed the same activity.

[Example 4]
Linalool has an asymmetric carbon at the 3-position and there is a set of optical isomers of (3S) -linalool and (3R) -linalool. Therefore, it was confirmed which optical isomer was linalool produced by the LIS protein of the present invention.

The extract obtained after the reaction obtained in Example 2 was analyzed by GC-FID. The analysis conditions were as follows. Under the following conditions, it was confirmed with a sample that (3S) -linalool was detected at a retention time of 23.8 minutes and (3R) -linalool was detected at a retention time of 24.0 minutes.
Capillary column: InertCap CHIRAMIX
(30m × 0.25mm × 0.25μm, GL Sciences Inc.)
Carrier gas: He
Temperature condition: Hold at 50 ° C. for 1 minute, then heat up to 180 ° C. at 3 ° C./min and finally hold at 180 ° C. for 1 minute Inlet temperature: 230 ° C.
Detector temperature: 250 ° C
Sample: 1 μl injection in split mode 10: 1

  These results are shown in FIGS. 4 to 6 are gas chromatograms of the produced components, respectively. FIG. 4 shows the result of FA25 cDNA-derived recombinant protein (FA25), and FIG. 5 shows the result of FA26 cDNA-derived recombinant protein (FA26). FIG. 6 shows the results of SS19 cDNA-derived recombinant protein (SS19), and also shows the results of (3R) -linalool and (3S) -linalool preparations (STD) as controls. As shown in FIGS. 4 to 6, in the extract using any recombinant protein, a signal having a retention time identical to (3R) -linalool was obtained. From these results, it was confirmed that the freesia-derived FA25 protein and FA26 protein, and the sweet pea-derived SS19 protein all had (3R) -linalool synthesis activity.

[Example 5]
Purification of Recombinant LIS Protein Expressing Freesia-Derived LIS cDNA and Recombinant Linalool Synthase Expressing Linalool Synthase ((R) -LIS) cDNA Derived from Known Lavender ( Lavandula angustifolia ) The linalool synthesis activity was compared using enzyme proteins.

(1) Expression induction of recombinant LIS protein In the same manner as in Example 2 and Example 3, FA26 cDNA as a freesia-derived LIS gene and (R) -LIS cDNA derived from lavender were introduced into Escherichia coli, respectively. . Using these transformants, expression of recombinant LIS protein was induced by the following method. Induction of protein expression was carried out using an Overnight Express Automation System 1 (Novagen).

  The transformed strain was cultured with shaking at 37 ° C. for 4 hours in 10 ml of LB medium containing 50 μg / ml of ampicillin and 0.05% glucose. Then, 2 ml 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 16 ° C. for 24 hours. Five of each were prepared, and a total of 500 ml was cultured. The obtained culture solution was centrifuged (5000 × g, 5 minutes, 4 ° C.) to collect E. coli. The microbial cells were added to 10 ml of 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 microbial cells. Suspended. The suspension was sonicated to disrupt the E. coli, then centrifuged (15,000 rpm, 15 minutes, 4 ° C.), and further filtered through a 0.45 μm MILLEX-HP filter (MILLIPORE). The obtained supernatant was used as a crude enzyme solution.

  Next, the crude enzyme was purified by His Tag purification using Bio-Scale Mini Proficiency IMAC Cartridges 1 ml (BIO RAD) using Profinia Protein Purification System (BIO RAD), and then Bio-Scale Mini P6 Desalting with Desalting Cartridges 10 ml (BIO RAD).

  Next, the purified enzyme solution after desalting was subjected to buffer solution [20 mmol / L MOPSO buffer solution (pH 7.0), 1 mmol / L DTT, 0. 10 mL using Econo-Pac 10DG Desalting Columns (BIO RAD). After replacement with 1 mmol / L APMSF, 10% glycerol], it was used in the following enzyme reaction.

(2) Activity measurement of LIS protein Reagent [10 μmol / L GPP, 1 mmol / L MgCl ₂ , 0.5 mmol / L MnCl ₂ , 0.1 mmol / L Na ₂ WO ₄ , 0.05 mmol / L was added to the purified enzyme solution. NaF] was added to adjust the total volume of the reaction solution to 3 ml. The amount of purified protein used was 0.3 mg. And this reaction liquid was made to react at 30 degreeC for 3 hours or 10 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, and the product components volatilized in the headspace were collected by the collector. .

Next, the collection agent was collected, and after adding 2 ml of dichloromethane to the collection agent, 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. Under the following conditions, linalool was detected at a retention time of 32.8 minutes, and it was confirmed by a standard that a characteristic fragment ion of m / z 154, 121, 93, 71 was detected in the MS spectrum. .
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

  These results are shown in FIG. FIG. 7 is a gas chromatogram of the product components, and shows the result of the recombinant protein derived from FA26 cDNA (FA26) and the result of the recombinant protein derived from lavender (R) -LIS cDNA (LaLIS). As shown in FIG. 7, when reacted for 3 hours, the recombinant protein derived from FA26 cDNA produced approximately 7.2 linalool than the known recombinant protein derived from lavender (R) -LIS cDNA. It was twice as many and showed significantly stronger activity. Furthermore, as shown in FIG. 8, when reacted for 10 hours, the amount of linalool produced by the recombinant protein derived from FA26 cDNA was about 30.8 of the amount of linalool produced by the recombinant protein derived from lavender (R) -LIS cDNA. It was twice. On the other hand, the amount of linalool produced by the recombinant protein derived from lavender (R) -LIS cDNA reacted for 10 hours was almost the same as when reacted for 3 hours. From this, it became clear that the recombinant protein derived from FA26 cDNA has higher stability and higher reactivity than the recombinant protein derived from lavender (R) -LIS cDNA. It should be noted that linalool activity could not be confirmed in E. coli into which the empty vector pET15b into which no cDNA had been inserted was introduced (pET15b in FIGS. 7 and 8).

[Example 6]
LIS gene cDNAs derived from freesia and sweet pea were introduced into binary vectors, respectively, and transformed into torenia. And the linalool synthetic activity in the obtained recombinant was confirmed. Further, as a control, transformation and linalool synthesis activity were confirmed in the same manner for cDNAs of known lavender (R) -LIS genes.

1. Construction of various binary vectors (1) Construction of pSPB5083 (El ₂ 35Sp :: FA25 :: Nost) A binary vector pSPB5083 for expressing the FA25 gene in plants was constructed by the following method.

In order to introduce a Kpn I recognition sequence 5 ′ to the start codon and a Sal I recognition sequence 3 ′ to the stop codon, respectively, with respect to the FA25 cDNA (s +) coding sequence shown in SEQ ID NO: 1, went. The PCR reaction uses a PCR reaction solution having the following composition containing the following two types of primers trFA25FW and trFA / nos_RV. A total of 25 cycles were performed.
(Primer for FA25)
trFA25_FW (SEQ ID NO: 34)
5'-ATTCGGAGAGGTACCATGGCTCTCTTGCCGTGT-3 '
trFA25 / nos_RV (SEQ ID NO: 35)
5'-GAAATTCGGGTCGACTTAGAGGGGAATGGGTTCAAA-3 '
(PCR reaction solution)
pSPB5025 10pg
1x PrimeSTAR MasterMix (Takara)
0.2mmol / L dNTPs (Takara)
Each primer 0.2μmol / L
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. To this reaction solution, 1 μl of 1 × In-Fusion Reaction buffer (Clontech), 1 μl of In-Fusion Enzyme (Clontech), 100 ng of pSPB3593 excluding the LaLIS gene, and 10 μl of a total volume of Cloning Enhanced Treated PCR Insert were added to 10 μl. . The pSB3593 is on pUCPP (WO2005 / _017147), a vector with El 2 35S promoter, lavender from linalool synthase (LaLIS) gene and Nos terminator by digestion with Kpn I and Sal I, the LaLIS gene Excluded. And after making it react at 37 degreeC for 15 minutes, it was made to react at 50 degreeC for 15 minutes. A total amount of 2.5 μl of this mixed solution was transformed into Competent high DH5α (TOYOBO).

And plasmid DNA was extracted from the obtained 4 types of transformants, and the whole base sequence was determined by the primer walking method by a synthetic oligonucleotide primer. As a result, a clone having the coding sequence of FA25 cDNA (s +) shown in SEQ ID NO: 1 in which a Kpn I recognition sequence was introduced 5 'to the start codon and a Sal I recognition sequence was introduced 3' to the termination codon was obtained. It was. This clone was designated as pSPB5079.

Next, pBINPLUS was digested with Pac I, dephosphorylated, and a DNA fragment of about 2.9 kb obtained by digesting pSPB5079 with Pac I and Dra I was ligated thereto. The obtained plasmid was an expression vector having a cauliflower mosaic virus-derived El ₂ 35S promoter, a freesia-derived FA25 gene, and an Agrobacterium-derived Nos terminator on pBINPLUS, and was designated as pSPB5083.

(2) Construction of pSPB5082 (El ₂ 35Sp :: FA25 :: HSPt) A binary vector pSPB5082 for expressing the FA25 gene in a plant was constructed by the following method.

A PCR reaction was performed to introduce a Kpn I recognition sequence 5 ′ of the start codon with respect to the coding sequence of FA25 cDNA (s +) shown in SEQ ID NO: 1. In this PCR reaction, a PCR reaction solution having the following composition containing two kinds of primers trFA25_FW and trFA25 / HSP_RV shown below was used. Further, for high expression of the target gene product in the plant body, a PCR reaction was performed in order to introduce an Eco RI recognition sequence 3 ′ of the stop codon with respect to the Arabidopsis thaliana-derived HSP terminator sequence. In this PCR reaction, a PCR reaction solution having the following composition containing the following two types of primers HSP / FA25_FW and TrHSPt_RV2 was used. In each of these PCRs, a reaction of 98 ° C. for 10 seconds, 65 ° C. for 15 seconds, and 72 ° C. for 1 minute 30 seconds was performed for a total of 25 cycles.
(Primer for FA25)
trFA25 FW (SEQ ID NO: 34)
5'-ATTCGGAGAGGTACCATGGCTCTCTTGCCGTGT-3 '
trFA25 / HSP_RV (SEQ ID NO: 36)
5'-ATCTTCATCTTCATATTAGAGGGGAATGGGTTCAAAAAG-3 '
(Primer for HSP terminator)
HSP / FA25_FW (SEQ ID NO: 37)
5'-CCCATTCCCCTCTAATATGAAGATGAAGAT-3 '
TrHSPt_RV2 (SEQ ID NO: 38)
5'-GTTAATTAAGAATTCCTTATCTTTAATCATATTCCATAGT-3 '
(PCR reaction solution)
pSPB5025 or pRI201-AN-GUS 10pg
1x PrimeSTAR MasterMix (Takara)
0.2mmol / L dNTPs (Takara)
Each primer 0.2μmol / L
Total volume 20μl

About 5 μl of each of the obtained PCR products, 2 μl of Cloning Enhancer was added, reacted at 37 ° C. for 15 minutes, and further reacted at 80 ° C. for 15 minutes. 1 μl of 1 × In-Fusion Reaction buffer (Clontech), 1 μl of In-Fusion Enzyme (Clontech), 100 ng of pSPB3593 excluding LaLIS gene, and 2 μl of Cloning Enhancing-Treated PCR Insert were added to the reaction solution. It was adjusted. The pSB3593 is the same as above (1), by digesting with Kpn I and Sal I, except LaLIS gene. And after making it react at 37 degreeC for 15 minutes, it was made to react at 50 degreeC for 15 minutes. A total amount of 2.5 μl of this mixed solution was transformed into Competent high DH5 DH5α (TOYOBO).

And plasmid DNA was extracted from the obtained 4 types of transformants, and the whole base sequence was determined by the primer walking method by a synthetic oligonucleotide primer. As a result, the FA25 cDNA shown in SEQ ID NO: 1, wherein a Kpn I recognition sequence was introduced 5 'to the start codon of the FA25 cDNA (s +) sequence and an Eco RI recognition sequence was introduced 3' to the start codon of the HSP terminator sequence. A clone with the coding sequence of (s +) and the HSP terminator sequence was obtained. This clone was designated as pSPB5078.

Next, pBINPLUS was digested with Pac I, dephosphorylated, and a DNA fragment of about 2.9 kb obtained by digesting pSPB5078 with Pac I and Dra I was ligated thereto. The obtained plasmid was an expression vector having a cauliflower mosaic virus-derived El ₂ 35S promoter, a freesia-derived FA25 gene, and an Arabidopsis-derived HSP terminator on pBINPLUS, and was designated as pSPB5082.

(3) Construction of pSPB5091 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: FA25 :: Nost) In plants, the FA25 gene and AmGPPS.SSU, AmGPPS.LSU A binary vector pSPB5091 for expressing a gene was constructed by the following method.

  First, a small subunit (AmGPPS.SSU) gene and a large subunit (AmGPPS.LSU) gene were obtained for Snapdragon-derived geranyl pyrophosphate synthase.

  Total RNA was extracted from flowers of Snapdragon (variety Maryland True Pink, Murakami Seed Co., Ltd.) using RNeasy Plant Mini Kit (QIAGEN). CDNA was synthesized from 1 μg of the total RNA using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) by the method recommended by the manufacturer. Next, 1 μl of the synthesized cDNA was used as a template, and PCR was performed using a primer and PrimeSTAR HS DNA polymerase (Takara) 1.25 U in a total of 25 μl of reaction solution according to the method recommended by the manufacturer. For amplification of the small subunit gene, two types of small subunit amplification primers AmGPPS • SSU-F2 and AmGPPS • SSU-R2 prepared based on the known sequence (AmGPPS.SSU; GenBank Accession No. AY34686.1) Were used at a concentration of 0.2 μmol / L. For amplification of the large subunit gene, two types of large subunit amplification primers AmGPPS / LSU-F and AmGPPS / LSU prepared based on the known sequence (AmGPPS.LSU; GenBank Accession No. AY5344867.1) are used. -R was used at a concentration of 0.2 μmol / L each. PCR was repeated for 30 cycles in total of 98 ° C for 10 seconds, 55 ° C for 15 seconds, and 72 ° C for 2 minutes, and then held at 72 ° C for 4 minutes.

The obtained PCR products were separated by agarose gel electrophoresis. Then, bands of about 0.95 kb and 1.3 kb were respectively recovered and subcloned into the pCR4 Blunt TOPO vector (Invitrogen) using the Zero Blunt TOPO PCR cloning kit (Invitrogen) by the method recommended by the manufacturer. When the nucleotide sequence of the obtained plasmid was determined, the nucleotide sequence (SEQ ID NO: 53) encoding the amino acid sequence of AmGPPS.SSU (SEQ ID NO: 54) and the nucleotide sequence encoding the amino acid sequence of AmGPPS.LSU (SEQ ID NO: 56) were determined. A plasmid containing (SEQ ID NO: 55) was obtained. The former plasmid was designated as pSPB3506, and the latter plasmid was designated as pSPB1400. AmGPPS.SSU is registered in, for example, GenBank AY534686.1, and AmGPPS.LSU is registered in, for example, GenBank AY5344867.1.
AmGPPS • SSU-F2 (SEQ ID NO: 39)
5'-AAATGGCCCACGGCCTCAC-3 '
AmGPPS • SSU-R2 (SEQ ID NO: 40)
5'-CATCAGGGTTGTAGGTCTAATTACCCCT-3 '
AmGPPS · LSU-F (SEQ ID NO: 41)
5'-TCAACAACAATGAGCCTGGTAAATC-3 '
AmGPPS · LSU-R (SEQ ID NO: 42)
5'-TCACAAATGAAGTTGTGTTATACATGACAT-3 '

AmGPPS.SSU gene 894 bp (SEQ ID NO: 51)

AmGPPS.SSU protein 297aa (SEQ ID NO: 52)
MAHGLTHFNTKSGLFPSITKSKTTRPSTRPVILAMTRTQTYRATIESDIESYLKKAIPIRAPESVFEPMHHLTFAAPRTSASALCVAACELVGGDRSDAMAAAAAVHLMHVAAYTHENLPLTDGPMSKSEIQHKFDPNIELLTGDGIIPFGLELMARSMDPTRNNPDRILRAIIELTRVMGSEGIVEGQYHELGLNQLNDLELIEYVCKKKEGTLHACGAACGAILGGCDEDKIEKLRRFGLYVGTVQGLLGKNRSGFEGRIKELKELAVKELESFGGEKIELIRGVFELEHSLAGV

AmGPPS.LSU gene 1119 bp (SEQ ID NO: 53)

AmGPPS.LSU protein 372aa (SEQ ID NO: 54)
MSLVNPITTWSTTTTSKSPKNVQTTTRSRSIILPHKISLFPSNPKSKSKTHLRFSISSILTKNPQESSQKTSKDPTFTLDFKTYMLEKASSVNKALEQAVLLKEPLKIHESMRYSLLAGGKRVRPMLCIAACELVGGLESTAMPSACAVEMIHTMSLIHDDLPCMDNDDLRRGKPTNHKIYGEDVAVLAGDALLAFSFEHVAKSTKGVSSDRIVRVIGELAKCIGSEGLVAGQVVDISSEGMTEVGLEHLEFIHVHKTAALLEASVVLGAIVGGADDEDVEKLRKFARCIGLLFQVVDDILDVTKSSQELGKTAGKDLVADKTTYPKLLGIEKSREFAEKLNREAQEQLEGFDSVKAAPLIALANYIAYRDN

Next, pSPB1477 having a Mac1 promoter, a carnation-derived S12A2 gene, and a mas terminator was prepared on pUCAA (see WO2004 / 018682). The pSPB1477 is digested with Xba I and Kpn I to remove the carnation-derived S12A2 gene, and the pSPB3506 of (3) is digested with Xba I and Kpn I (about 0.95 kb DNA fragment) AmGPPS.SSU gene) was ligated. The obtained plasmid was a plasmid having the Mac1 promoter, AmGPPS.SSU gene, and mas terminator on its pUCAA, and was designated as pSPB3513.

Furthermore, an about 1.3 kb DNA fragment (AmGPPS.LSU gene) obtained by digesting the pSPB1400 with Xba I and Kpn I was ligated to pSPB2311 (see WO2007 / 049816) digested with Xba I and Kpn I. The obtained plasmid was a plasmid having a Mac1 promoter, AmGPPS.LSU gene and mas terminator on its pBINPLUS, and was designated as pSPB3514. Next, the pSPB3513 was digested with Asc I, and the resulting DNA fragment of about 3 kb was ligated to the Asc I site of the pSPB3514 . This plasmid was designated as pSPB3533.

After digestion the pSPB3533 with Pac I, and dephosphorylated, to which was ligated the DNA fragment of about 2.9kb obtained by digesting the pSPB3560 with Pac I. The pSPB3560 is a plasmid having an El ₂ 35S promoter, a Clakia-derived LIS gene, and a Nos terminator on pUCPP (WO2005 / 015147). The plasmid obtained by the ligation of the DNA fragments was a plasmid having AmGPPS.SSU gene, AmGPPS.LSU gene, and Clakia-derived LIS gene on its pBINPLUS, and was designated as pSPB3562.

Next, the pSPB1840 added with Sgf I and Fse I in the multiple cloning site of pBINPLUS, was digested with Asc I and Pac I. Then, after digesting pSPB3562 with Asc I, a DNA fragment of about 9.5 kb obtained by partial digestion with Pac I was ligated to the digest of pBINPLUS. This plasmid was designated as pSPB5007.

Finally, the pSPB5007 was digested with Pac I, dephosphorylated, and a DNA fragment of about 2.9 kb obtained by digesting pSPB5079 of (1) with Pac I and Dra I was ligated thereto. The obtained plasmid was an expression vector having AmGPPS.SSU gene, AmGPPS.LSU gene and freesia-derived FA25 gene on pBINPLUS, and was designated as pSPB5091.

(4) Construction of pSPB5090 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: FA25 :: HSPt) In plants, the FA25 gene and AmGPPS.SSU, AmGPPS.LSU A binary vector pSPB5090 for expressing a gene was constructed by the following method.

The pSPB5007 of (3) was digested with Pac I, dephosphorylated, and a DNA fragment of about 2.9 kb obtained by digesting pSPB5078 of (2) with Pac I and Dra I was ligated thereto. The obtained plasmid was an expression vector having a freesia-derived FA25 gene, a snapdragon-derived AmGPPS.SSU gene and an AmGPPS.LSU gene on pBINPLUS, and was designated as pSPB5090.

(5) Construction of pSPB5074 (El ₂ 35Sp :: FA26 :: Nost) A binary vector pSPB5074 for expressing the FA26 gene in plants was constructed by the following method.

In order to introduce a Kpn I recognition sequence 5 ′ to the start codon and a Sal I recognition sequence 3 ′ to the stop codon with respect to the coding sequence of FA26 cDNA (s +) shown in SEQ ID NO: 5, PCR reaction was performed. It was. The PCR reaction uses a PCR reaction solution having the following composition containing the following two types of primers trFA26_FW2 and trFA26_RV-nos2, and is a reaction at 98 ° C. for 10 seconds, 65 ° C. for 15 seconds, and 72 ° C. for 1 minute and 30 seconds. A total of 25 cycles were performed.
(Primer for FA26)
trFA26_FW2 (SEQ ID NO: 43)
5'-CATTCGGAGAGGTACCATGGCTTTCTTGCCGTGTC-3 '
trFA26_RV-nos2 (SEQ ID NO: 44)
5'-GAAATTCGGGTCGACTTAGAGGGGAATGGGTTCAATAA-3 '
(PCR reaction solution)
pSPB5026 10pg
1x PrimeSTAR MasterMix (Takara)
0.2mmol / L dNTPs (Takara)
Each primer 0.2μmol / L
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. To this reaction solution, 1 μl of 1 × In-Fusion Reaction buffer (Clontech), 1 μl of In-Fusion Enzyme (Clontech), pSPB3593 100 ng excluding the LaLIS gene, and 2 μl of total volume of Cloning Enhancing-Treated PCR Insert were added to 10 μl. It was adjusted. The pSPB3593 is the same as above (1), by digesting with Kpn I and Sal I, except LaLIS gene. And after making it react at 37 degreeC for 15 minutes, it was made to react at 50 degreeC for 15 minutes. A total amount of 2.5 μl of this mixed solution was transformed into Competent high DH5α (TOYOBO).

And plasmid DNA was extracted from the obtained 4 types of transformants, and the whole base sequence was determined by the primer walking method by a synthetic oligonucleotide primer. As a result, the FA26 cDNA (s +) coding sequence shown in SEQ ID NO: 5 was introduced, in which a Kpn I recognition sequence was introduced 5 ′ of the start codon and a Sal I recognition sequence was introduced 3 ′ of the stop codon. A clone with the sequence was obtained. This clone was designated as pSPB5070.

Next, pSPB5001 was digested with Pac I, dephosphorylated, and a DNA fragment of about 2.9 kb obtained by digesting pSPB5070 with Pac I and Dra I was ligated thereto. The pSPB5001 is a plasmid having an El ₂ 35S promoter, a lavender-derived LIS gene, and a Nos terminator on pBINPLUS. By ligating the DNA fragment to the pSPB5001, a plasmid having the cauliflower mosaic virus-derived El ₂ 35S promoter, the freesia-derived FA26 gene and the Agrobacterium-derived Nos terminator was obtained on the pBINPLUS. This was designated as pSPB5074.

(6) Construction of pSPB5073 (El ₂ 35Sp :: FA26 :: HSPt) In plants, a binary vector pSPB5073 for expressing the FA26 gene was constructed by the following method.

A PCR reaction was performed to introduce a Kpn I recognition sequence 5 ′ of the start codon with respect to the coding sequence of FA26 cDNA (s +) shown in SEQ ID NO: 5. For this PCR reaction, a PCR reaction solution having the following composition containing the following two types of primers trFA26_FW2 and trFA26_RV-hsp was used. Further, for high expression of the target gene product in the plant body, a PCR reaction was performed in order to introduce an Eco RI recognition sequence 3 ′ of the stop codon with respect to the Arabidopsis thaliana-derived HSP terminator sequence. For this PCR reaction, a PCR reaction solution having the following composition containing each of the following two types of primers trHSPt_FW and trHSPt_RV2 was used. Each of these pCRs was 25 cycles in total, with a reaction of 98 ° C. for 10 seconds, 65 ° C. for 15 seconds, and 72 ° C. for 1 minute and 30 seconds as one cycle.
(Primer for FA26)
trFA26_FW2 (SEQ ID NO: 43)
5'-CATTCGGAGAGGTACCATGGCTTTCTTGCCGTGTC-3 '
trFA26_RV-hsp (SEQ ID NO: 45)
5'-TCTTCATATTAGAGGGGAATGGGTTCAATAAG-3 '
(Primer for HSP terminator)
trHSPt_FW (SEQ ID NO: 46)
5'-CCCTCTAATATGAAGATGAAGATGAAATATTT-3 '
trHSPt_RV2 (SEQ ID NO: 38)
5'-GTTAATTAAGAATTCCTTATCTTTAATCATATTCCATAGT-3 '
(PCR reaction solution)
pSPB5026 or pRI201-AN-GUS 10pg
1x PrimeSTAR MasterMix (Takara)
0.2mmol / L dNTPs (Takara)
Each primer 0.2μmol / L
Total volume 20μl

2 μl of Cloning Enhancer was added to 5 μl of each obtained PCR product, reacted at 37 ° C. for 15 minutes, and further reacted at 80 ° C. for 15 minutes. To this reaction solution, 1 × In-Fusion Reaction buffer (Clontech), In-Fusion Enzyme (Clontech) 1 μl, pSPB3593 100 ng excluding the LaLIS gene, Cloning Enhancer-Treated PCR Insert, 2 μl each in total Adjusted. The pSB3593 is the same as above (1), by digesting with Kpn I and Sal I, except LaLIS gene. And after making it react at 37 degreeC for 15 minutes, it was made to react at 50 degreeC for 15 minutes. A total amount of 2.5 μl of this mixed solution was transformed into Competent high DH5α (TOYOBO).

And plasmid DNA was extracted from the obtained 4 types of transformants, and the whole base sequence was determined by the primer walking method by a synthetic oligonucleotide primer. As a result, a Kpn I recognition sequence was introduced 5 ′ of the start codon of the FA26 cDNA (s +) sequence, and an Eco RI recognition sequence was introduced 3 ′ of the start codon of the HSP terminator sequence. A clone having the coding sequence of cDNA (s +) and the HSP terminator sequence was obtained. This clone was designated as pSPB5069.

Next, in the same manner as in (5) above, pSPB5001 is digested with Pac I, dephosphorylated, and then pSPB5069 is digested with Pac I and Dra I to obtain a DNA fragment of about 2.9 kb. Were concatenated. The probe thus obtained was a plasmid having a cauliflower mosaic virus-derived El ₂ 35S promoter, a freesia-derived FA26 gene and an Arabidopsis-derived HSP terminator on pBINPLUS of the sPB5001, and was designated as pSPB5073.

(7) Construction of pSPB5075 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: FA26 :: Nost) In plants, the FA26 gene and AmGPPS.SSU, AmGPPS.LSU A binary vector pSPB5075 for expressing a gene was constructed by the following method.

The pSPB5007 of (3) was digested with Pac I, dephosphorylated, and a DNA fragment of about 2.9 kb obtained by digesting pSPB5070 of (5) with Pac I and Dra I was ligated thereto. The obtained plasmid was an expression vector having free FA-derived FA26 gene, snapdragon-derived AmGPPS.SSU gene and AmGPPS.LSU gene on pBINPLUS, and was designated as pSPB5075.

(8) Construction of pSPB5072 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: FA26 :: HSPt) In plants, the FA26 gene and AmGPPS.SSU, AmGPPS.LSU A binary vector pSPB5072 for expressing a gene was constructed by the following method.

The pSPB5007 of (3) was digested with Pac I, dephosphorylated, and a DNA fragment of about 2.9 kb obtained by digesting pSPB5069 of (6) with Pac I and Dra I was ligated thereto. The obtained plasmid was an expression vector having free FA-derived FA26 gene, snapdragon-derived AmGPPS.SSU gene and AmGPPS.LSU gene on pBINPLUS, and was designated as pSPB5072.

(9) Construction of binary vector pSPB5084 (El ₂ 35S :: SS19 :: Nost) In plants, a binary vector pSPB5084 for expressing the SS19 gene was constructed by the following method.

In order to introduce a Kpn I recognition sequence 5 ′ to the start codon and a Sal I recognition sequence 3 ′ to the stop codon with respect to the coding sequence of SS19 cDNA (s +) shown in SEQ ID NO: 9, a PCR reaction was performed. It was. The PCR reaction uses a PCR reaction solution having the following composition containing the following two types of primers trSS19_FW and trSS19 / nos_RV. A total of 25 cycles were performed.
(Primer for SS19)
trSS19_FW (SEQ ID NO: 47)
5'-ATTCGGAGAGGTACCATGGCTGTTACTTCTCAACTAC-3 '
trSS19 / nos_RV (SEQ ID NO: 48)
5'-GAAATTCGGGTCGACTTAATGGCTAGTGGGTAAT-3 '
(PCR reaction solution)
pSPB5046 10pg
1x PrimeSTAR MasterMix (Takara)
0.2mmol / L dNTPs (Takara)
Each primer 0.2μmol / L
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. 1 μl of 1 × In-Fusion Reaction buffer (Clontech), 1 μl of In-Fusion Enzyme (Clontech), 100 ng of pSPB3593 excluding LaLIS gene, and 2 μl of Cloning Enhancing-Treated PCR Insert were added to the reaction solution. It was adjusted. The pSPB3593 is the same as above (1), by digesting with Kpn I and Sal I, except LaLIS gene. And after making it react at 37 degreeC for 15 minutes, it was made to react at 50 degreeC for 15 minutes. A total amount of 2.5 μl of this mixed solution was transformed into Competent high DH5α (TOYOBO).

And plasmid DNA was extracted from the obtained 4 types of transformants, and the whole base sequence was determined by the primer walking method by a synthetic oligonucleotide primer. As a result, the SS19 cDNA (s +) sequence shown in SEQ ID NO: 9 was introduced with a Kpn I recognition sequence 5 'to the start codon and a Sal I recognition sequence 3' to the termination codon, respectively. A clone with the coding sequence of cDNA (s +) was obtained. This clone was designated as pSPB5081.

Next, pBINPLUS was digested with Pac I, dephosphorylated, and a DNA fragment of about 2.8 kb obtained by digesting pSPB5081 with Pac I and Sca I was ligated thereto. The obtained plasmid was an expression vector having a cauliflower mosaic virus-derived El ₂ 35S promoter, a sweet pea-derived SS19 gene and an Agrobacterium-derived Nos terminator on pBINPLUS, and was designated as pSPB5084.

(10) Construction of pSPB5097 (El ₂ 35S :: SS19 :: HSPt) In plants, a binary vector pSPB5097 for expressing the SS19 gene was constructed by the following method.

In order to introduce a Kpn I recognition sequence 5 ′ to the start codon with respect to the coding sequence of SS19 cDNA (s +) shown in SEQ ID NO: 9, PCR reaction was performed. In this PCR reaction, a PCR reaction solution having the following composition containing each of the following two types of primers trSS19_FW and trSS19 / HSP_RV was used. Further, for high expression of the target gene product in the plant body, PCR reaction was performed to introduce an Eco RI recognition sequence 3 ′ of the stop codon with respect to the Arabidopsis thaliana-derived HSP terminator sequence. In this PCR reaction, a PCR reaction solution having the following composition containing each of the following two types of primers HSP / SS19_FW and TrHSPt_RV2 was used. Each of these PCR reactions was carried out for a total of 25 cycles, with one cycle consisting of 98 ° C. for 10 seconds, 65 ° C. for 15 seconds, and 72 ° C. for 1 minute 30 seconds.
(Primer for SS19)
trSS19_FW (SEQ ID NO: 47)
5'-ATTCGGAGAGGTACCATGGCTGTTACTTCTCAACTAC-3 '
trSS19 / HSP_RV (SEQ ID NO: 49)
5'-ATCTTCATCTTCATATTAATGGCTAGTGGGTAATGATGAG-3 '
(Primer for HSP terminator)
HSP / SS19_FW (SEQ ID NO: 50)
5'-CCCACTAGCCATTAATATGAAGATGAAGAT-3 '
TrHSPt_RV2 (SEQ ID NO: 38)
5'-GTTAATTAAGAATTCCTTATCTTTAATCATATTCCATAGT-3 '
(PCR reaction solution)
pSPB5046 or pRI201-AN-GUS 10pg
1x PrimeSTAR MasterMix (Takara)
0.2mmol / L dNTPs (Takara)
Each primer 0.2μmol / L
Total volume 20μl

2 μl of Cloning Enhancer was added to 5 μl of each obtained PCR product, reacted at 37 ° C. for 15 minutes, and further reacted at 80 ° C. for 15 minutes. To this reaction solution, 1 μl of 1 × In-Fusion Reaction buffer (Clontech), 1 μl of In-Fusion Enzyme (Clontech), 100 ng of pSPB3593 excluding the LaLIS gene, and 10 μl of Cloning Enhancer-Treated PCR Insert, 2 μl each in total. It was adjusted. The pSPB3593 is the same as above (1), by digesting with Kpn I and Sal I, except LaLIS gene. And after making it react at 37 degreeC for 15 minutes, it was made to react at 50 degreeC for 15 minutes. A total amount of 2.5 μl of this mixed solution was transformed into Competent high DH5α (TOYOBO).

And plasmid DNA was extracted from the obtained 4 types of transformants, and the whole base sequence was determined by the primer walking method by a synthetic oligonucleotide primer. As a result, the SS19 cDNA shown in SEQ ID NO: 9 was introduced, in which a Kpn I recognition sequence was introduced 5 ′ of the start codon of the SS19 cDNA (s +) sequence and an Eco RI recognition sequence was introduced 3 ′ of the start codon of the HSP terminator sequence, respectively. A clone was obtained which had the coding sequence of s +) and the HSP terminator sequence. This clone was designated as pSPB5080.

Next, after digesting pBINPLUS with Pac I, dephosphorylation treatment was performed, and a DNA fragment of about 2.8 kb obtained by digesting pSPB5080 with Pac I and Sca I was ligated and derived from cauliflower mosaic virus on pBINPLUS. An expression vector pSPB5097 having a Cauliflower mosaic virus-derived El ₂ 35S promoter, a sweet pea-derived SS19 gene, and an Arabidopsis thaliana-derived HSP terminator was obtained.

(11) Construction of binary vector pSPB5619 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: SS19 :: Nost) In plants, the SS19 gene and AmGPPS.SSU, AmGPPS A binary vector pSPB5619 for expressing the LSU gene was constructed by the following method.

The pSPB5007 prepared in (3) is digested with Pac I, dephosphorylated, and the pSPB5081 prepared in (9) is digested with Pac I and Sac I and then a DNA fragment of about 2.8 kb is ligated. did. The obtained plasmid was an expression vector having a sweet pea-derived SS19 gene, a snapdragon-derived AmGPPS.SSU gene and an AmGPPS.LSU gene on pBINPLUS, and was designated as pSPB5619.

(12) Construction of binary vector pSPB5098 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: SS19 :: HSPt) In plants, the SS19 gene and AmGPPS.SSU, AmGPPS A binary vector pSPB5098 for expressing the LSU gene was constructed by the following method.

After digesting pSPB5007 prepared in (3) with Pac I, dephosphorylation, and ligating the approximately 2.8 kb DNA fragment obtained by digesting pSPB5080 prepared in (10) with Pac I and Sca I. did. The obtained plasmid was an expression vector having a sweet pea-derived SS19 gene, a snapdragon-derived AmGPPS.SSU gene and an AmGPPS.LSU gene on pBINPLUS, and was designated as pSPB5098.

(13) Construction of pSPB5001 (El ₂ 35S :: LaLIS :: Nost) A binary vector pSPB5001 for expressing a lavender-derived LIS (LaLIS) gene in a plant was constructed by the following method.

The pSPB3560 of the (3) except for Kurakia from LIS gene by digestion with Kpn I and Sal I. On the other hand, was digested pSPB3591 with Kpn I and Sal I, to obtain a DNA fragment of about 2.8kb with LaLIS gene. Then, the DNA fragment was ligated to pSPB3560 excluding the Krachia-derived LIS gene. The resulting plasmid, on _PUCPP, a plasmid with El 2 35S promoter, LaLIS gene, the Nos terminator, was PSPB3593.

Next, pBINPLUS was digested with Pac I, dephosphorylated, and a DNA fragment of about 1.7 kb obtained by digesting pSPB3593 with Pac I was ligated thereto. The obtained plasmid was an expression vector having the cauliflower mosaic virus-derived El ₂ 35S promoter, the lavender-derived LIS gene, and the Agrobacterium-derived Nos terminator on pBINPLUS, and was designated as pSPB5001.

(14) Construction of pSPB5003 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: LaLIS :: Nost) In plants, the LaLIS gene and AmGPPS.SSU, AmGPPS.LSU A binary vector pSPB5003 for expressing a gene was constructed by the following method.

PSPB1477 having a Mac1 promoter, a carnation-derived S12A2 gene, and a mas terminator on pUCAA (see WO2004 / 018682) as in (3) above was used. The pSPB1477 is digested with Xba I and Kpn I to remove the carnation-derived S12A2 gene, and the pSPB3506 of (3) is digested with Xba I and Kpn I (about 0.95 kb DNA fragment) AmGPPS / SSU gene) was ligated. The obtained plasmid was a plasmid having a Mac1 promoter, AmGPPS.SSU gene, and mas terminator on its pUCAA, and was designated as pSPB3513.

Further, pSPB2311 obtained by digesting pSPB1400 of (3) above with Xba I and Kpn I (AmGPPS.LSU gene) digested with Xba I and Kpn I (see WO2007 / 049816) Connected. The obtained plasmid was a plasmid having a Mac1 promoter, AmGPPS.LSU gene, and mas terminator on its pBINPLUS, and was designated as pSPB3514.

The pSPB3514 was digested with Asc I, dephosphorylated, and a DNA fragment of about 3 kb obtained by digesting the pSPB3513 of (3) with Asc I was ligated thereto . The obtained plasmid was designated as pSPB3533.

After digestion the pSPB3533 with Pac I, and dephosphorylated, to which was ligated the DNA fragment of about 2.8kb obtained by digesting the pSPB3593 with Pac I of the (13). The obtained plasmid was an expression vector having a lavender-derived LIS gene, a snapdragon-derived AmGPPS.SSU gene and an AmGPPS.LSU gene on pBINPLUS, and was designated as pSPB5003.

2. Production of Transformation Torenia Transformation was performed using a leaf piece of Torenia cultivar Summer Wave Blue (SWB) based on the method described in International Publication WO2009 / 072542. The obtained transformants were given the following strain names for each introduced expression vector.
pSPB5091 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: FA25 :: Nost): TT175
pSPB5090 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: FA25 :: HSPt): TT174
pSPB5074 (El ₂ 35Sp :: FA26 :: Nost): TT166
pSPB5073 (El ₂ 35Sp :: FA26 :: HSPt): TT165
pSPB5075 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: FA26 :: Nost): TT164
pSPB5072 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: FA26 :: HSPt): TT162
pSPB5001 (El ₂ 35Sp :: LaLIS :: Nost): TT144
pSPB5003 (Mac1p :: AmGPPS.SSU :: Nost; Mac1p :: AmGPPS.LSU :: Nost; El ₂ 35Sp :: LaLIS :: Nost): TT143

3. Measurement of the amount of linalool synthesis in each recombinant 5 flowers bloomed from the recombinant obtained in “2.” above were randomly selected. On each petal, one headspace component MonoTrap DCC18 (GL Sciences Inc.) per flower, that is, a total of five collectors per individual, is installed in the headspace. Volatilized product components were collected with the scavenger for 24 hours.

  On the next day, five collection agents were collected from each individual, 2 ml of dichloromethane was added to the collection agent, and then volatile components were extracted and collected by ultrasonic treatment. Volatile components extracted in dichloromethane were concentrated to dryness and redissolved in 100 μl of dichloromethane. This solution was analyzed by GC-MS as an extract of the product component. The analysis conditions were the same as the GC-MS in Example 2 (3).

These results are shown in Table 1 below. The table below shows the results and average values for each of the five flowers for each recombinant. As shown in Table 1 below, in the recombinants TT144 and TT143 into which a known lavender-derived linalool synthase gene was introduced, no synthesis of linalool was observed. On the other hand, in each of the recombinants into which the freesia-derived LIS gene was introduced, a peak was observed at a retention time of 32.8 minutes, and linalool was detected as a product component. On the other hand, linalool was not detected from the host SWB. From these results, it was confirmed that the freesia-derived LIS gene exhibits linalool synthesis activity in the plant body.

  As described above, according to the polynucleotide of the present invention, linalool synthase can be expressed by genetic engineering techniques. Therefore, for example, by introducing the polynucleotide of the present invention into a plant to produce a transformant, linalool synthase is expressed in the transformant, thereby synthesizing linalool, which is 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 (25)

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 linalool synthesis activity (c) consisting of a base sequence having 90% or more identity to any of the base sequences of (a) above , A polynucleotide encoding a protein having linalool 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 linalool synthesis activity (e) SEQ ID NOs: 2, 4, 6, 8, A polynucleotide encoding a protein consisting of 0 or 12 amino acid sequences (f) consisting of 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) above , A polynucleotide encoding a protein having linalool synthesis activity (g) encoding a protein having an amino acid sequence having 90% or more identity to any amino acid sequence of (e) and having linalool synthesis activity Polynucleotide The polynucleotide according to claim 1, wherein the linalool synthesis activity is (3R) -linalool synthesis activity. A linalool 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 modification method according to claim 10, wherein the non-human host is a plant or a part thereof. The modification method according to claim 11, wherein the plant is carnation or torenia. The modification method according to any one of claims 10 to 12, further comprising a step of growing the non-human host into which the polynucleotide or the expression vector has been introduced. 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 13. 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 an amino acid sequence and having linalool synthesis activity (C) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of (A) and having linalool synthesis activity The protein according to claim 16, wherein the linalool synthesis activity is (3R) -linalool synthesis activity. A method for producing a protein having linalool synthesis activity, comprising a protein synthesis step of synthesizing a protein having linalool synthesis activity encoded by the polynucleotide by expressing the polynucleotide according to claim 1 or 2. . The production method according to claim 18, wherein the protein synthesis step is 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 is introduced. . The production method according to claim 19, wherein in the protein synthesis step, the non-human host is a non-human host into which the expression vector according to claim 4 has been introduced. The production method according to claim 18, wherein the protein synthesis step is a step of expressing the polynucleotide in a cell-free protein synthesis system. A method for producing linalool, comprising the step of synthesizing linalool using the protein according to claim 16 or 17 and synthesizing linalool by the linalool synthesizing activity of the protein. The production method according to claim 22, wherein the linalool is (3R) -linalool. Furthermore, the method for producing a protein according to any one of claims 18 to 21, further comprising a protein synthesis step of synthesizing the protein,
The production method according to claim 22 or 23, wherein the synthesized protein is used in the linalool 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 exhibits the linalool synthesis activity encoded by the polynucleotide. A step of synthesizing a protein having
25. The production method according to claim 24, wherein the linalool synthesis step is a step of synthesizing linalool by the linalool synthesis activity of the protein in the non-human host.

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