JP2004121233A - Polynucleotide as a gene fragment involved in biosynthesis of isoquinoline alkaloid, cDNA library containing the polynucleotide, and full-length gene and protein involved in isoquinoline alkaloid biosynthesis isolated using the polynucleotide, isolation thereof Method and transformant thereof - Google Patents

Abstract

An object of the present invention is to provide an isoquinoline alkaloid biosynthesis-related gene fragment, a cDNA library containing the same at a high frequency, and a method for efficiently and comprehensively isolating an isoquinoline alkaloid biosynthesis-related gene using the same.
SOLUTION: After subcloning cDNA obtained from cultured ureber cells having high berberine productivity into a pDR196 vector, the 5 'base of 2016 randomly selected clones was determined. Of these, the sequence of 1014 clones for which 100 bases or more could be sequenced was subjected to homology search using tBLASTx in the NCBl database, and its gene function was estimated. As a result, it was confirmed that the polynucleotide consisting of the base sequence constituting the specific region was a gene fragment involved in alkaloid biosynthesis.
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Description

The present invention is one of the secondary metabolites of plants, a polynucleotide as a gene fragment involved in the biosynthesis of isoquinoline alkaloids having high utility as pharmaceuticals, and a cDNA library containing the polynucleotide, Further, the present invention relates to a full-length gene and protein involved in isoquinoline alkaloid biosynthesis isolated using the polynucleotide, a method for isolating the same, and a transformant into which the polynucleotide or the full-length gene has been introduced.

様 々 Various secondary metabolites produced by plant cells are used not only as spices and dyes but also as useful drugs. Although these plants are collected or cultivated from nature, many plants are growing slowly, and production methods using other methods such as cultured cells are being sought.

Alkaloids, which are one of the secondary metabolites of such plants, often show remarkable physiological activities in humans and animals when administered in small amounts. Among the alkaloids, there are many isoquinoline derivatives and isoquinoline-based alkaloids derived biosynthetically having strong physiological activities such as berberine, morphine, and papaverine, and are still used in many useful medicines. Since the first half of this isoquinoline alkaloid biosynthesis system is common to various compounds, an efficient method for biosynthesis of the isoquinoline alkaloid includes a method using a gene involved in the biosynthesis. In order to utilize these genes for biosynthesis of isoquinoline alkaloids, isolation and identification of the genes are indispensable.

As shown in FIG. 1, berberine, which is a benzylisoquinoline alkaloid, is biosynthesized from two molecules of tyrosine, an amino acid, by a 13-step enzymatic reaction. To date, biosynthetic enzymes have been purified from cells having high berberine productivity, and genes for each enzyme have been isolated and identified.

Specifically, coclaurin N-methyltransferase (CNMT) (see Non-Patent Document 1), N-methyl-coclaurin 3'-hydroxylase (see Non-Patent Document 2), using, for example, cultured cultured cells of berberine that produce high levels of berberine. ), Berberine bridge enzyme (BBE) (see Non-Patent Document 3), and cDNA such as scourelin 9-O-methyltransferase (SMT) (see Non-Patent Document 4).

As an example of the berberine biosynthesis-related gene, Patent Document 1 discloses a gene encoding norcoclaurin 6-O-methyltransferase (6OMT), and Patent Document 2 discloses (S) -3′-hydroxyl. A gene encoding -N-methylcoclaurine 4'-O-methyltransferase (4OMT) has been described.

Norcoclaurine synthase, the first isoquinoline alkaloid enzyme, catalyzes, for example, the condensation reaction between dopamine and 4-hydroxyphenylacetaldehyde, which is the initial reaction of the berberine biosynthesis pathway shown in FIG. The isolation and identification of the gene has long been desired because it is an important enzyme for the biosynthesis of isoquinoline alkaloids. However, research has been delayed due to the difficulty of protein purification, and in recent years, its purification has finally been reported (see Non-Patent Documents 6 and 7). However, the gene has not been isolated or identified.
JP-A-11-178577 (release date: July 6, 1999) JP-A-11-178570 (publication date: July 6, 1999) Choi, K.-B., Morishige, T., Shitan.N., Yazaki, K., and Sato, F., Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis japonica. J. Biol. Chem. ., 277: 830-835 (2002) Pauli HH, Kutchan TM. 1998.Molecular cloning and functional heterologous expression of two alleles encoding (S) -N-methylcoclaurine 3'-hydroxylase (CYP80B1), a new methyl jasmonate-inducible cytochrome-p450-dependent mono-oxygenase of benzylisoquinoline alkaline biosynthesis. Plant J. 13, 793-801 Dittrich H, Kutchan TM. 1991.Molecular cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenantrhidine alkaloids in the response of plants to pathogen attack.Proc. Natl. Acad. Sci. USA 88: 9969 -9973 Takeshita, N., Fujiwara, H., Mimura, H., Fitchen, JH, Yamada, Y. and Sato, F., 1995.Molecular cloning and characterization of S-adenosyl--methionine: scoulerine-9-O-methyltransferase from cultured cells of Coptis japonica.Plant Cell Physiol36 1, pp. 29-36 Lange, BM, Wildung MR, Stauber, EJ, Sanchez, C, Pouchnik, D, Croteau, R. 2000.Probing essential oil biosynthesis and secretion by functional evaluation of expressed sequence tags from mint glandular trichomes.Proc. Nat'l Acad Sci (USA) 97: 2934-2939 Samanani N, Facchini PJ., Purification and Characterization of Norcoclaurine: The first committed enzyme in benzylisoquinoline alkaloid biosynthesis in plants.J. Biol. Chem., Vol. 277, 33878-33883, 2002 Samanani N, Facchini PJ., Isolation and partial characterization of norcoclaurine synthase, the first committed step in benzylisoquinoline alkaloid biosynthesis, from opium poppy.Planta.2001 Oct; 213 (6): 898-906.

As described above, conventionally, in order to isolate a gene related to production of a useful substance such as the above-described alkaloid, a technique of first purifying an enzyme that is a translation product thereof and then isolating a target gene is used. Had adopted. However, the production amount of the enzyme in the plant cells is limited, and the purification of the enzyme is extremely complicated and requires time and labor. For this reason, for example, norcoclaurine synthase gene and the like have not been isolated and identified yet.

As one solution, exhaustive analysis of genes expressed in cells that accumulate metabolites was considered, but in reality, many cells have extremely low substance productivity, and such analysis is difficult. That is the current situation. Non-Patent Document 5 discloses that the ESTs in the essential oil glands of peppermint contain a large number of biosynthetic genes of monoterpene, an essential oil, in ESTs of these cells. However, it is not clear to what kind of cells such a technique can be applied.

The present invention has been made in view of the above-mentioned problems, and has a high efficiency of isoquinoline alkaloid biosynthesis-related gene fragments and cDNA libraries containing the same, and isoquinoline alkaloid biosynthesis-related genes using the same. It is an object of the present invention to provide a method capable of isolating the isoquinoline alkaloid efficiently using the isolated gene.

As described above, the present inventors have established a cell with high isoquinoline alkaloid productivity while pursuing much effort so far, purified the biosynthetic enzyme using the cell as a material, and obtained a gene for each enzyme. We have been isolated and identified. Analysis of the high-alkaloid-producing cells using the isolated genes has revealed that the biosynthetic activity of the selected high-producing cells is extremely promoted, which has been considered difficult in the past. It was suggested that biosynthesis-related genes could be isolated from the analysis of the genes expressed in the alkaloid-producing cells. As a result of intensive studies based on such a research background, it has been found that isoquinoline alkaloid biosynthesis-related genes can be isolated by using ESTs (expressed cDNA clones) isolated from cultured alkaloid-producing spinach cells. And completed the present invention.

That is, the polynucleotide according to the present invention is a polynucleotide consisting of any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 52.

The nucleotide sequences shown in SEQ ID NOS: 1 to 52 are sequenced by the present inventors as ESTs (expressed gene fragments) contained in a cDNA library obtained from a cultured cell of Coptis japonica in the family Ranunculaceae. It has been decided. Then, ESTs having these nucleotide sequences were suggested to be gene fragments involved in the biosynthesis of isoquinoline alkaloids from homology analysis based on the sequence information in the database. Therefore, the polynucleotide according to the present invention may be a gene fragment involved in the biosynthesis of an isoquinoline alkaloid, or may be derived from cultured spinach cells.

(4) The polynucleotide is a gene fragment involved in the biosynthesis of isoquinoline alkaloids such as berberine and morphine, which are useful as pharmaceuticals, as described above. Therefore, it can be used as a probe for easily and efficiently isolating a full-length gene (full-length cDNA) encoding an enzyme of the isoquinoline alkaloid biosynthesis system, which was conventionally difficult to isolate. When the polynucleotide is used as a probe, only a part of the nucleotide sequence shown in SEQ ID NOS: 1 to 52 may be used. In this case, it is preferable to use a sequence excluding polyadenylic acid (polyΔA).

ＣＤＮＡ The cDNA library according to the present invention is characterized by containing at least one of the polynucleotides having the nucleotide sequences shown in SEQ ID NOS: 1 to 52. The above-mentioned cDNA library is preferably derived from cultured spinach cells, and is preferably one which produces berberine at a high level among cultured spinach cells. According to this, it can be expected that the cDNA library contains a large number of ESTs of isoquinoline alkaloid synthesis-related genes.

と し て Specific examples of the above-mentioned cDNA library include those derived from 156-1SMT cultured spinach cells that produce berberine as described in Examples. It has been confirmed that the above-mentioned cDNA library derived from the 156-1 SMT strain contains a high proportion (about 4%) of alkaloid biosynthesis-related genes, and can be effectively used for identification of unknown alkaloid biosynthesis-related genes. it can. The above-mentioned cDNA library derived from the 156-1 SMT strain contains all polynucleotides consisting of the nucleotide sequences shown in SEQ ID NOS: 1 to 52.

However, the cDNA library according to the present invention is not limited thereto, and preferably contains a plurality of the above-mentioned polynucleotides, more preferably 1% or more of all the genes related to isoquinoline alkaloid biosynthesis such as the above-mentioned polynucleotides. Just fine. According to this, an unknown alkaloid synthesis-related gene may be further identified.

全長 In addition, a full-length gene involved in the biosynthesis of isoquinoline alkaloids can be isolated using the polynucleotide (gene fragment) contained in the polynucleotide or the cDNA library of the present invention. Therefore, the method for isolating a gene according to the present invention includes a method for isolating a full-length gene involved in biosynthesis of an isoquinoline alkaloid using the above-mentioned polynucleotide as a probe, or a method for isolating a gene (gene Fragment) as a probe to isolate a full-length gene involved in isoquinoline alkaloid biosynthesis. The “full-length gene” means a gene containing the entire nucleotide sequence (from the start codon to the stop codon) corresponding to the amino acid sequence of the protein encoded by the gene.

According to the above method, conventionally, the isoquinoline alkaloid biosynthetic enzyme is easily purified without isolating the gene encoding the enzyme after purifying the enzyme from a plant or the like. It can be carried out.

Furthermore, the present invention also includes a full-length gene which has any one of the bases shown in SEQ ID NOs: 1 to 52 or 55 and is involved in isoquinoline alkaloid biosynthesis. Specific examples of the full-length gene include those having the nucleotide sequence shown in SEQ ID NO: 53 or 55. The base sequence shown in SEQ ID NO: 53 is a full-length cDNA of berberine biosynthesis-related gene CjEST64 described below, and was isolated using EST contained in the above cDNA library as a probe. It has been confirmed that the full-length cDNA of CjEST64 is a gene encoding a protein having a columbamine 7-O-methyltransferase activity. In addition, it has been confirmed that the nucleotide sequence represented by SEQ ID NO: 55 is a gene encoding a protein having an activity as norcoclaurine synthase, as described later.

遺 伝 子 The gene according to the present invention is a gene encoding the following protein (a) or (b).

(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 54;

(B) an amino acid sequence represented by SEQ ID NO: 54, wherein one or more amino acids are substituted, deleted, inserted, and / or added, and have an activity as a columbamine 7-O-methyltransferase. Protein to have. As shown in the sequence listing, the gene consisting of the nucleotide sequence shown in SEQ ID NO: 53 has the nucleotide sequence at positions 57 to 1109 in the sequence as an open reading frame, and its translation product is as shown in ( Since the protein is a), it can be said that it is one of the above genes.

遺 伝 子 The gene according to the present invention is a gene encoding the following protein (c) or (d).

(C) a protein consisting of the amino acid sequence represented by SEQ ID NO: 56.

(D) In the amino acid sequence shown in SEQ ID NO: 56, one or more amino acids consist of an amino acid sequence in which substitution, deletion, insertion, and / or addition are performed, and the amino acid sequence as norcoclaurine synthase An active protein. The gene consisting of the nucleotide sequence shown in SEQ ID NO: 55 has the 22nd to 1077th nucleotide sequences in the sequence as an open reading frame as shown in the sequence listing, and the translation product is as described in the above ( Since it is the protein of c), it can be said that it is one of the above genes.

In addition, the above-mentioned gene is preferably derived from spinach.

タ ン パ ク 質 The protein according to the present invention is the following protein (a) or (b).

(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 54;

(B) an amino acid sequence represented by SEQ ID NO: 54, wherein one or more amino acids are substituted, deleted, inserted, and / or added, and have an activity as a columbamine 7-O-methyltransferase. Protein to have.

タ ン パ ク 質 The protein according to the present invention is the following protein (c) or (d).

(C) a protein consisting of the amino acid sequence represented by SEQ ID NO: 56.

(D) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 56, and which has an activity as a norcoclaurin synthase .

The protein is preferably derived from spinach.

ベ ク タ ー The vector according to the present invention has the polynucleotide or the full-length gene integrated therein. The above-described vector can be expressed in a host such as a plant or a microorganism by a known transformation method so that the integrated gene or gene fragment can be expressed in the host.

形 質 The transformant according to the present invention is a transformant into which the above vector has been introduced. More specifically, the transformant according to the present invention is a transformant into which an alkaloid biosynthesis-related gene or a gene fragment thereof has been introduced. Here, the expression "introduced bacter" means that the gene integrated in the vector is introduced into the host so that it can be expressed by a known genetic engineering technique (gene manipulation technique).

The transformant can express an alkaloid biosynthesis-related gene in its own body. Therefore, if an appropriate cell such as a plant or a microorganism is used as a host to produce a transformant into which the above-described vector containing a promoter capable of large-scale expression is introduced, as a result, a large amount of an alkaloid biosynthetic enzyme is produced. be able to.

Since the vector is a gene (or gene fragment) derived from an isoquinoline alkaloid-producing cell classified as a plant, the host in the transformant is preferably a plant, and a plant producing the isoquinoline alkaloid. Is more preferable. Plants producing this isoquinoline alkaloid include, for example, plants of the genus Ouren, poppy, and poppy.

The above plants include not only complete plants but also some of them, for example, leaves, seeds, tubers, cuttings and the like. Further, the above-mentioned plants include plant tissues, protoplasts, cells, calli, organs, plant seeds, germs, pollen, egg cells, zygotes, and other proliferative plants, which are derived from pre-transformed transgenic plants and their progeny. Materials; parts of plants including flowers, stems, fruits, leaves, roots, and the like; and the like.

本 The present invention also includes a method for producing an isoquinoline alkaloid using at least one of the above proteins and the above transformants. According to the above method, an isoquinoline alkaloid can be efficiently and conveniently synthesized.

As described above, the polynucleotide according to the present invention is a gene fragment consisting of each base sequence shown in SEQ ID NOS: 1 to 52 and involved in the biosynthesis of isoquinoline alkaloids. Further, the cDNA library according to the present invention contains the polynucleotide at a high ratio. Therefore, the polynucleotide or the gene fragment contained in the cDNA library is useful as a probe for isolating the full-length gene of the biosynthesis system of isoquinoline alkaloids in spinach or other plants, and this full-length gene is comprehensive. Can be expected.

If the biosynthetic genes of isoquinoline alkaloids, which are secondary metabolites of plants, are comprehensively isolated, means for industrially mass-producing isoquinoline alkaloids useful as pharmaceutical raw materials will be secured. It has the potential to make a significant contribution to industry.

Hereinafter, the present invention will be described more specifically as an embodiment of the present invention, but the present invention is not particularly limited to this description.

(1) Regarding the polynucleotide according to the present invention The polynucleotide according to the present invention is a polynucleotide consisting of any one of the 52 base sequences shown in SEQ ID NOS: 1 to 52. Polynucleotides comprising the nucleotide sequences shown in SEQ ID NOS: 1 to 52 can be used as a cDNA library (cDNA library according to the present invention) prepared by a conventionally known method from cultured spinach cells that highly produce berberine, an isoquinoline alkaloid. Included. The polynucleotide was estimated to be a gene involved in the berberine biosynthesis system as a result of a homologous search of the nucleotide sequence of 1014 clones randomly sequenced from ESTs in the cDNA library.

The cDNA library contains a large number of pathogen-infected inducible protein genes, while 13 clones of the alkaloid biosynthesis genes identified so far (about 1%, including genes whose related functions are presumed to be included). 45 clones (about 4%). This frequency was high enough to search for biosynthesis-related genes based on these genes.

Next, the respective polynucleotides consisting of the nucleotide sequences shown in SEQ ID NOs: 1 to 52 will be described in order with reference to FIGS. 2, 5, 6, and 8 to 59. FIGS. 5 and 6 are tables showing genes estimated as alkaloid synthesis-related genes among ESTs (gene fragments) sequenced in Examples described later. The table also includes alkaloid synthesis-related genes that have already been identified. 8 to 59 show the same base sequence as the base sequences shown in SEQ ID NOS: 1 to 52 in order, and at the top of each figure, a sequence ID (ID) for identifying each sequence is shown. SEQ.ID). FIG. 2 is a table showing genes showing homology to previously identified alkaloid synthesis-related genes among the ESTs sequenced in the Examples described later.

As shown in FIG. 8, the polynucleotide comprising the nucleotide sequence shown in SEQ ID NO: 1 has a sequence ID of 010425seq1a02 and a nucleotide sequence of 270 bp. The above-mentioned 010425seq1a02 corresponds to the EST shown in No. 16 in the tables of FIGS.

ポ リ As shown in FIG. 9, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 has a sequence ID of 010425seq1e07 and a 828 bp nucleotide sequence. The above-mentioned 010425seq1e07 corresponds to the EST shown in No. 24 in the tables of FIGS.

ポ リ As shown in FIG. 10, the polynucleotide having the nucleotide sequence of SEQ ID NO: 3 has a sequence ID of 010425seq1d03 and has a nucleotide sequence of 106 bp. Further, 010425seq1d03 corresponds to the EST shown in No. 32 in the tables of FIGS.

ポ リ As shown in FIG. 11, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4 has a sequence ID of 010512 seq2e12 and a 217 bp nucleotide sequence. The above 010512seq2e12 corresponds to the EST shown in No. 35 in the tables of FIGS.

ポ リ As shown in FIG. 12, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 5 has a sequence ID of 010512seq2f08 and a 548 bp nucleotide sequence. The above 010512seq2f08 corresponds to the EST shown in No. 41 in the tables of FIGS.

As shown in FIG. 13, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 6 has a sequence ID of 010512seq2e10 and has a 549 bp nucleotide sequence. The above-mentioned 010512seq2e10 corresponds to the EST shown in No. 42 in the tables of FIGS.

ポ リ As shown in FIG. 14, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 7 has a sequence ID of 010512seq2b10 and a nucleotide sequence of 265 bp. The above 010512seq2b10 corresponds to the EST shown in No. 11 in the tables of FIGS.

As shown in FIG. 15, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8 has a sequence ID of 010511 seq1c10 and a nucleotide sequence of 553 bp. The above-mentioned 010511seq1c10 corresponds to the EST shown in No. 1 in the tables of FIGS.

As shown in FIG. 16, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 9 has a sequence ID of 010511seq1h09 and has a 177 bp nucleotide sequence. The above-mentioned 010511seq1h09 corresponds to the EST shown in No. 6 in the tables of FIGS.

As shown in FIG. 17, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10 has a sequence ID of 010511seq1d11 and has a nucleotide sequence of 151 bp. Further, 010511seq1d11 corresponds to the EST shown in No. 14 in the tables of FIGS.

As shown in FIG. 17, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 10 has a sequence ID of 010511seq1d11 and has a nucleotide sequence of 151 bp. Further, 010511seq1d11 corresponds to the EST shown in No. 14 in the tables of FIGS.

As shown in FIG. 18, the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 11 has a sequence ID of 010511seq1c07 and a nucleotide sequence of 211 bp. The above-mentioned 010511seq1c07 corresponds to the EST shown in No. 38 in the tables of FIGS.

As shown in FIG. 19, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 12 has a sequence ID of 010516seq5g11 and has a nucleotide sequence of 659 bp. The above-mentioned 010516seq5g11 corresponds to the EST shown in No. 4 in the tables of FIGS.

As shown in FIG. 20, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 13 has a sequence ID of 010516seq5h09 and a nucleotide sequence of 407 bp. The above-mentioned 010516seq5h09 corresponds to EST shown in No. 8 in the tables of FIGS.

ポ リ As shown in FIG. 21, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 14 has a sequence ID of 010516seq5g04 and a nucleotide sequence of 420 bp. The above 010516seq5g04 corresponds to the EST shown in No. 17 in the tables of FIGS.

As shown in FIG. 22, the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 15 has a sequence ID of 010516seq5d01 and has a 644 bp nucleotide sequence. The above-mentioned 010516seq5d01 corresponds to the EST shown in No. 21 in the tables of FIGS.

ポ リ As shown in FIG. 23, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 16 has a sequence ID of 010516seq5b04 and a nucleotide sequence of 114 bp. The above-mentioned 010516seq5b04 corresponds to the EST shown in No. 34 in the tables of FIGS.

ポ リ As shown in FIG. 24, the polynucleotide having the nucleotide sequence of SEQ ID NO: 17 has a sequence ID of 010516seq5d10 and a nucleotide sequence of 570 bp. The above 010516seq5d10 corresponds to the EST shown in No. 37 in the tables of FIGS.

As shown in FIG. 25, the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 18 has a sequence ID of 010516seq5c09 and a 239 bp nucleotide sequence. In addition, the above-mentioned 010516seq5c09 corresponds to the EST shown in No. 44 in the tables of FIGS.

ポ リ As shown in FIG. 26, the polynucleotide having the nucleotide sequence of SEQ ID NO: 19 has a sequence ID of 010523seq2g11 and a nucleotide sequence of 458 bp. The above-mentioned 010523seq2g11 corresponds to the EST shown in No. 39 in the tables of FIGS.

As shown in FIG. 27, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 20 has a sequence ID of 010523seq2c09 and a nucleotide sequence of 128 bp. In addition, the above-mentioned 010523seq2c09 corresponds to the EST shown in No. 5 in the tables of FIGS.

ポ リ As shown in FIG. 28, the polynucleotide having the nucleotide sequence of SEQ ID NO: 21 has a sequence ID of 010523seq2b10 and a nucleotide sequence of 170 bp. The above-mentioned 010523seq2b10 corresponds to the EST shown in No. 9 in the tables of FIGS.

As shown in FIG. 29, the polynucleotide having the nucleotide sequence of SEQ ID NO: 22 has a sequence ID of 010523seq1c02 and a nucleotide sequence of 217 bp. Further, 010523seq1c02 corresponds to the EST shown in No. 13 in the tables of FIGS.

ポ リ As shown in FIG. 30, the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 23 has a sequence ID of 010523seq1g12 and a nucleotide sequence of 669 bp. The above-mentioned 010523seq1g12 corresponds to the EST shown in No. 2 in the tables of FIGS.

ポ リ As shown in FIG. 31, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 24 has a sequence ID of 010523seq1c08 and a nucleotide sequence of 553 bp. The above 010523seq1c08 corresponds to the EST shown in No. 26 in the tables of FIGS.

ポ リ As shown in FIG. 32, the polynucleotide having the nucleotide sequence of SEQ ID NO: 25 has a sequence ID of 010512seq2b07 and a nucleotide sequence of 123 bp. The above 010512seq2b07 corresponds to the EST shown in No. 15 in the tables of FIGS.

ポ リ As shown in FIG. 33, the polynucleotide having the nucleotide sequence of SEQ ID NO: 26 has a sequence ID of 010525seq1d03 and a nucleotide sequence of 312 bp. Further, 010525seq1d03 corresponds to the EST shown in No. 10 in the tables of FIGS.

ポ リ As shown in FIG. 34, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 27 has a sequence ID of 010502seq1e07 and a 596 bp nucleotide sequence. The above-mentioned 010502seq1e07 corresponds to the EST shown in No. 3 in the tables of FIGS.

As shown in FIG. 35, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 28 has a sequence ID of 010501seq2f10 and a nucleotide sequence of 479 bp. The above-mentioned 010501seq2f10 corresponds to the EST shown in No. 20 in the tables of FIGS.

ポ リ As shown in FIG. 36, the polynucleotide having the nucleotide sequence of SEQ ID NO: 29 has a sequence ID of 010518 seq4e10 and a nucleotide sequence of 578 bp. Further, 010518seq4e10 corresponds to the EST shown in No. 22 in the tables of FIGS.

ポ リ As shown in FIG. 37, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 30 has a sequence ID of 010518seq4c09 and has a 226 bp nucleotide sequence. The above 010518seq4c09 corresponds to the EST shown in No. 23 in the tables of FIGS.

ポ リ As shown in FIG. 38, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 31 has a sequence ID of 010518seq4d05 and has a 509 bp nucleotide sequence. The above 010518seq4d05 corresponds to the EST shown in No. 25 in the tables of FIGS.

ポ リ As shown in FIG. 39, the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 32 has a sequence ID of 010518seq4d08 and a nucleotide sequence of 294 bp. The above 010518seq4d08 corresponds to the EST shown in No. 30 in the tables of FIGS.

ポ リ As shown in FIG. 40, the polynucleotide having the nucleotide sequence of SEQ ID NO: 33 has a sequence ID of 010501seq2h11 and a nucleotide sequence of 376 bp. The above-mentioned 010501seq2h11 corresponds to the EST shown in No. 27 in the tables of FIGS.

ポ リ The polynucleotide consisting of SEQ ID NO: 34 and the nucleotide sequence shown in FIG. 41 has a sequence ID of 010511seq1g03 (shown as CjEST731 in FIG. 41) and has a 596 bp nucleotide sequence. The 010511seq1g03 corresponds to the EST indicated by No. 29 in the tables of FIGS. 5 and 6, and corresponds to the EST having the EST number 731 in the table of FIG.

ポ リ As shown in FIG. 42, the polynucleotide consisting of SEQ ID NO: 35 and the nucleotide sequence shown in FIG. 42 has an identification name of CjEST179 and a 523 bp nucleotide sequence. Further, the CjEST 179 corresponds to the EST with the EST number of 179 in the table of FIG.

ポ リ As shown in FIG. 43, the polynucleotide consisting of SEQ ID NO: 36 and the base sequence shown in FIG. 43 has an identification name of CjEST502 and has a base sequence of 393 bp. Further, the CjEST 502 corresponds to the EST having the EST number 502 in the table of FIG.

As shown in FIG. 44, the polynucleotide consisting of SEQ ID NO: 37 and the nucleotide sequence shown in FIG. 44 has an identification name of CjEST558 and has a 553 bp nucleotide sequence. Further, the CjEST558 corresponds to the EST having the EST number of 558 in the table of FIG.

ポ リ As shown in FIG. 45, the polynucleotide consisting of SEQ ID NO: 38 and the nucleotide sequence shown in FIG. 45 has an identification name of CjEST1013 and has a 385 bp nucleotide sequence. Further, the CjEST 1013 corresponds to the EST having the EST number of 385 in the table of FIG.

ポ リ As shown in FIG. 46, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 39 has a sequence ID of 010509seq2c01 and a nucleotide sequence of 665 bp. The above-mentioned 010509seq2c01 corresponds to the EST shown in No. 7 in the tables of FIGS.

ポ リ As shown in FIG. 47, the polynucleotide having the nucleotide sequence of SEQ ID NO: 40 has a sequence ID of 010501seq2e02 and a nucleotide sequence of 341 bp. The above-mentioned 010501seq2e02 corresponds to the EST shown in No. 19 in the tables of FIGS. 5 and 6, and this also corresponds to the EST shown in No. 10 in the table of FIG.

ポ リ As shown in FIG. 48, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 41 has a sequence ID of 010516seq5h08 and a nucleotide sequence of 595 bp. In addition, the above-mentioned 010516seq5h08 corresponds to the EST shown in No. 28 in the tables of FIGS. 5 and 6, and this also corresponds to the EST shown in No. 3 in the table of FIG.

ポ リ As shown in FIG. 49, the polynucleotide having the nucleotide sequence of SEQ ID NO: 42 has a sequence ID of 010516seq6g12 and a nucleotide sequence of 642 bp. The above-mentioned 010516seq6g12 corresponds to the EST shown in No. 33 in the tables of FIGS. 5 and 6, and this also corresponds to the EST shown in No. 2 in the table of FIG.

ポ リ As shown in FIG. 50, the polynucleotide having the nucleotide sequence of SEQ ID NO: 43 has a sequence ID of 010425 to 27f09 and has a 788 bp nucleotide sequence. The above 010425 to 27f09 correspond to the ESTs shown in No. 1 in the table of FIG.

ポ リ As shown in FIG. 51, the polynucleotide having the nucleotide sequence of SEQ ID NO: 44 has a sequence ID of 010425 to 27c07 and a 596 bp nucleotide sequence. Further, the above 010425 to 27c07 correspond to the EST shown in No. 36 in the tables of FIGS. 5 and 6, and this also corresponds to the EST shown in No. 6 in the table of FIG.

ポ リ As shown in FIG. 52, the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 45 has a sequence ID of 010502seq1h06 and a nucleotide sequence of 252 bp. The above-mentioned 010502seq1h06 corresponds to the EST shown in No. 4 in the table of FIG.

ポ リ As shown in FIG. 53, the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 46 has a sequence ID of 010511seq1b11 and a nucleotide sequence of 289 bp. The above-mentioned 010511seq1b11 corresponds to the EST shown in No. 4 in the table of FIG.

ポ リ As shown in FIG. 54, the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 47 has a sequence ID of 010511seq1F03 and a nucleotide sequence of 159 bp. The above-mentioned 010511seq1F03 corresponds to the EST shown in No. 4 in the table of FIG.

ポ リ As shown in FIG. 55, the polynucleotide having the nucleotide sequence of SEQ ID NO: 48 has a sequence ID of 010525 seq1F08 and a nucleotide sequence of 350 bp. The above-mentioned 010525seq1F08 corresponds to the EST shown in No. 4 in the table of FIG.

ポ リ As shown in FIG. 56, the polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 49 has a sequence ID of 010516seq5f03 and a nucleotide sequence of 314 bp. Further, the above 010516seq5f03 corresponds to the EST shown in No. 5 in the table of FIG.

As shown in FIG. 57, the polynucleotide having the nucleotide sequence of SEQ ID NO: 50 has a sequence ID of 010518seq3A06 and a nucleotide sequence of 591 bp. Further, 010518seq3A06 corresponds to the EST shown in No. 7 in the table of FIG.

ポ リ As shown in FIG. 58, the polynucleotide having the nucleotide sequence of SEQ ID NO: 51 has a sequence ID of 010501seq2f03 and a nucleotide sequence of 158 bp. Further, 010501seq2f03 corresponds to the EST shown in No. 9 in the table of FIG.

ポ リ As shown in FIG. 59, the polynucleotide having the nucleotide sequence of SEQ ID NO: 52 has a sequence ID of 010516seq5c04 and a nucleotide sequence of 607 bp. The above-mentioned 010516seq5c04 corresponds to the EST shown in No. 11 in the table of FIG.

Each polynucleotide consisting of the nucleotide sequences shown in SEQ ID NOs: 34 to 38 is a gene fragment considered as a transcription factor. Of these transcription factors, only those shown in SEQ ID NO: 34 are positive regulators, and all others are negative regulators. The polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 34 is presumed to be a homologue of the Cj-ring-H2 finger protein, which has not been known as a transcription factor in alkaloid biosynthesis systems. However, its use is expected to lead to comprehensive induction of biosynthetic genes.

(4) The method for obtaining the polynucleotide is not particularly limited, and a general method is employed. For example, the polynucleotide may be cut out from a genomic DNA or cDNA library of an organism having the polynucleotide with an appropriate restriction enzyme and purified. In addition, as an organism having the above-mentioned polynucleotide, there can be mentioned, for example, a cultured cell of Ouren. In addition, if the purification is performed using cultured cells having high berberine productivity, among the above cultured spinach cells, the target polynucleotide can be more reliably isolated. Alternatively, the polynucleotide may be isolated by performing an amplification reaction using genomic DNA or cDNA of the organism as a template using appropriate primers.

(2) Method for isolating full-length cDNA of isoquinoline alkaloid biosynthesis gene Subsequently, a method for isolating the full-length gene (full-length cDNA) using a polynucleotide as a gene fragment (EST) according to the present invention as a probe explain.

Since the polynucleotide according to the present invention is an EST of an isoquinoline alkaloid biosynthesis-related gene derived from cultured spinach cells, the polynucleotide is used to produce the isoquinoline alkaloid biosynthesis from spinach plants (including cultured spinach cells) or other plants. It is possible to clone a full-length cDNA of a synthesis-related gene. As the cloning method in this case, a conventionally known method can be used and is not particularly limited.

Specifically, in the case of a plant in which at least a part of the genome is stored in a database, a base sequence having homology may be searched from the database based on the base sequence of the polynucleotide. For example, a homology search of a base sequence by BLAST, which is a widely used homology search algorithm, can be suitably used.

植物 Further, in the case of a plant whose genome is not stored in a database, for example, a hybridization method using a conventionally known DNA library can be used. Specifically, a step of preparing a genomic library or a cDNA library from a target plant using an appropriate cloning vector, and performing hybridization using at least a part of the polynucleotide as a probe, Detecting a positive fragment from the rally to the probe. Thus, the polynucleotide according to the present invention is useful as a probe. The region used for the probe preferably contains a sequence specific to the gene of interest. The length of the polynucleotide used as the probe is preferably 100 bp or more.

By using the above-described method, berberine can be obtained from spinach and various other plants (for example, poppy and poppy plants such as poppy, Egosaku, Tsurugi-family plants such as Tamasakitsu-rafuji, and barberry-like barberry such as barberry). And full-length genes involved in isoquinoline-based alkaloid biosynthesis such as magnoflorin, morphine, papaverine, sanguinarine, coridaline, and cepharanthin can be easily isolated.

(3) Method for Producing Transformant The transformant according to the present invention was obtained by introducing the above-mentioned polynucleotide or a vector into which the full-length gene isolated by using the above-mentioned polynucleotide as a probe was incorporated into a host. Things. The host is not particularly limited, and examples thereof include microorganisms such as yeast and Escherichia coli, and plants and animals. However, since the gene (gene fragment) incorporated in the vector is derived from a cultured cell of spinach, which is a kind of plant, the host is preferably a plant. Plants that produce isoquinoline alkaloids, such as valerian, are particularly preferred. As described above, the category of the plant includes not only a plant as an individual but also an organ, a tissue, a cell, and the like of the plant.

Specific examples of the method for producing the above transformant include microinjection, electroporation, ballistic particle acceleration, protoplast transformation / regeneration, and Agrobacterium-mediated transformation. , But is not particularly limited. In addition, it is preferable that the above-mentioned transformation method is appropriately selected depending on the type of a host plant or the like (for example, monocotyledonous plants and dicotyledonous plants).

ベ ク タ ー Examples of vectors that can be used in the present invention include vectors that have been conventionally used for transformation of microorganisms, plants, plant cells, and the like. And, the above-mentioned vector is, in addition to the above-mentioned polynucleotide or the above-mentioned full-length gene, a constitutively-expressed or expression-regulated promoter for expressing a conventionally known gene, a drug-resistance gene which facilitates selection of transformants. , A replication origin for using the binary-vector system for using the Agrobacterium binary-vector system, and the like.

More specifically, when introduced into a microorganism such as Escherichia coli, a pBluescript Iisk vector or a pET vector can be used. When introduced into plant cells, pBI101, pBI121, and the like can be mentioned as suitable for introduction by Agrobacterium. When introducing into a plant cell using the electroporation method, the vector is not particularly limited. Examples of the drug resistance gene include an ampicillin resistance gene, a kanamycin resistance gene, and a hygromycin resistance gene. Examples of the promoter include a cauliflower mosaic virus-derived 35S promoter (constant expression type) and a promoter of heat shock inducible protein (expression control type). Examples of the replication origin include a replication origin derived from Ti or Ri plasmid.

(4) Alkaloid production using an isolated alkaloid biosynthesis gene The present inventors can molecularly convert the alkaloid biosynthesis activity of Anopheles officinalis using an alkaloid biosynthesis-related gene isolated and identified before the present invention. Or the ability to increase the biosynthesis activity of spinach (Sato F, Hashimoto T, Hachiya A, Tamura K, Choi KB, Morishige T, Fujimoto H, Yamada Y. 2001. Metabolic engineering of plant alkaloid biosynthesis. Proc Nat'l Acad Sci (USA) 98: 367-372). It also shows that expression in E. coli allows N-methylation of 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, which is a more artificial substrate.

From these results, the full-length genes involved in alkaloid biosynthesis were isolated using the above-described polynucleotide as a probe, and the above-mentioned transformants using plants and microorganisms as hosts were isolated from these isolated genes. By preparing, it is possible to easily convert / improve the secondary metabolic function of a plant and to perform substance conversion using a microorganism cell system. Furthermore, if the transformant is prepared by incorporating the gene into a vector containing a promoter that overexpresses these genes (for example, a cauliflower mosaic virus-derived 35S promoter, etc.), an alkaloid biosynthetic enzyme is mass-produced. be able to.

(5) The protein according to the present invention, the gene encoding the same, and its use The protein according to the present invention may be any of the above-mentioned proteins (a), (b), (c) and (d). Here, the protein (a) is a protein having a columbamine 7-O-methyltransferase activity, as shown in the examples described later, and the protein (b) is a mutant protein thereof.

タ ン パ ク 質 Further, the protein of (c) is a protein having norcoclaurine synthase activity as shown in Examples described later, and (d) can be said to be a mutant protein thereof.

Here, “one or more amino acid substitutions, deletions, insertions, and / or additions” refers to substitutions, deletions, insertions, and substitutions by known methods for producing mutant proteins such as site-directed mutagenesis. And / or means that substitutions, deletions, insertions, and / or additions are made to the extent that amino acids can be added. The term “mutation” as used herein mainly refers to a mutation artificially introduced by a known method for producing a mutant protein, but may also be a naturally occurring mutant protein isolated and purified.

The mutant protein may include a mutant protein having an abnormality in wild-type activity (referred to as a mutant protein having a loss of function). Such a loss-of-function mutant protein is useful in protein function analysis, for example, by comparing a structure with a wild-type protein to reveal a region essential for activity in the structure. In addition, the gene according to the present invention may include a gene encoding the above-described loss-of-function mutant protein. Such a gene is useful, for example, for producing a new transformant.

タ ン パ ク 質 In addition, the protein according to the present invention may include a known additional sequence such as HA or Flag at the end thereof, or may be a fusion protein in order to easily purify or detect the protein. Further, it may be subjected to various modifications such as N-glycosylation, or may form a multimer of dimer or more.

Furthermore, genes encoding the above proteins (a) to (d) are also included in the present invention. Examples of the gene according to the present invention include a gene having the nucleotide sequence shown in SEQ ID NO: 53 or SEQ ID NO: 55.

These proteins and genes are involved in isoquinoline alkaloid biosynthesis and are very useful in themselves. Furthermore, these proteins and genes encoding these proteins can be used for modifying an alkaloid biosynthesis system. In addition, by using the transformant according to the present invention or the above protein, isoquinoline alkaloids can be produced regardless of in vivo or in vitro. That is, this method for producing an isoquinoline alkaloid may use at least the above protein or transformant, and includes other specific methods, conditions, materials (substrates and the like), enzymes, buffers, pH, temperature, and reaction time. And the like can be appropriately set, and are not limited. For example, conventionally known enzymes involved in the biosynthesis of isoquinoline alkaloids other than the above-mentioned proteins, for example, cokraurin N-methyltransferase (CNMT) (see Non-Patent Document 1), N-methyl-coclaurin 3'-hydroxylase (Non-Patent Document) 2), berberine bridge enzyme (BBE) (see Non-Patent Document 3), scourelin 9-O-methyltransferase (SMT) (see Non-Patent Document 4), norcoclaurin 6-O-methyltransferase (6OMT) (Patent Literature 1), (S) -3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4OMT) (see Patent Literature 2), and the like may be used.

This makes it possible to efficiently produce isoquinoline alkaloids that can be used for dyes, spices, and useful pharmaceuticals.

Hereinafter, the present invention will be described in detail with reference to examples. Note that the present invention is not limited to the following description.

(1) Isolation of ESTs and comprehensive nucleotide sequencing 156 Berberine high-producing strains of cultured spinach (Coptis japonica) cells established in the Totipotent Control Mechanism Laboratory, Graduate School of Life Sciences, Kyoto University and maintained for many years From the -1 strain (selected strain), mRNA was extracted by the method described in the above-mentioned conventional literature (Choi, et al., 2001). In addition, the above-mentioned berberine high-producing strain 156-1 of spinach is disclosed in JP-A-11-178579, JP-A-11-178579, and non-patent literature: F. Sato, and Y. Tamada, (1984) High berberine-producing. Phytochemistry, 23 (2): 281-385, etc. can also be used to produce and select the cultures of Coptis japonica cells. Then, after subcloning the cDNA into the pDR196 vector, the 5 'base of the randomly selected 2016 clone was determined by Megabase system (Amersham Pharmacia).

Among them, the sequence of 1014 clones for which 100 bases or more could be sequenced was subjected to homology search using tBLASTx in the NCBl database to estimate the gene function. As a result, as shown in the table of FIG. 2, it was confirmed from the high degree of homology that 11 putative alkaloid biosynthesis genes identified so far were included. In the table of FIG. 2, in order from the left, the identification number (No.) of each gene, the function deduced from the EST sequence (origin of the deduced gene, EST number), the homology with the deduced gene, and the EST sequence The length indicates the number of ESTs isolated. Thus, in the cDNA library of cultured spinach cells obtained in this experiment, 13 clones corresponding to about 1% of 1014 ESTs that could be sequenced are alkaloid biosynthesis-related genes that have already been identified. It was estimated. In addition, when genes that have not been identified but whose related functions are presumed were included, 45 clones corresponding to about 4% of the 1014 ESTs were presumed to be alkaloid biosynthesis-related genes.

Thus, in the above-mentioned selected strain, the result was obtained that the expression of the alkaloid biosynthesis-related gene was expressed far more than the root, which is the alkaloid biosynthesis site, in the non-selected strain and in the individual of our spinach.

In addition, among the 1014 ESTs, ESTs whose functions were unknown or unidentified as a result of the homology search accounted for about 40%, and it was expected that alkaloid biosynthesis-related genes would be included in these ESTs.

(2) Selection of Berberine Biosynthesis-Related Genes Based on EST Macro Array After PCR-amplification of the clones used for EST analysis, Biodyne A ^™ (Pall) membrane was used at 0.38 mm intervals using a Biomek 2000 robot. The blot was used as a macro array. MRNAs extracted from cultured spinach cell lines having different alkaloid productivity (156-1SMT as a high-producing strain, CJY and CJ8 as low-producing strains) were reverse-transcribed to the macroarray using Superscript II reverse transcriptase (Gibco BRL). , 32P label was used as a probe to quantify the gene expression level in each cell.

For detection in the macroarray, first, 0.5% SDS treatment was performed at room temperature for 5 minutes to remove weakly bound DNA, and the cells were washed with milliQ water. Then, after prehybridization was performed at 60 ° C. for 5 to 6 hours using an ExpressHyb (Clontech) solution containing 100 μg / ml ssDNA, a probe was added, and hybridization was performed at 60 ° C. for at least 16 hours. Thereafter, 2 × SSC, 1% SDS treatment at 60 ° C. for 30 minutes was performed four times at 60 ° C. 0.1 × SSC and 0.5% SDS for 30 minutes and 2 × SSC treatment at room temperature were performed once and quantified using Phosphor imaging plates (Fujix BAS2000). Data was analyzed with ArrayVision ^™ (Imaging Research Inc., Canada).

(3) As a result of the macroarray analysis, as shown in the graph of FIG. 3, it was found that there was a large difference in biosynthetic gene expression between CJY with clearly low berberine productivity and 156-SMT with high productivity. That is, the expression ratio in two strains having different berberine productivity differs depending on each gene, and the alkaloid-related genes (SMT, 4'OMT, SAMsynthetase, 6OMT, etc.) identified so far are higher in berberine-high producing strains. The higher expression was confirmed from the graph of FIG. FIG. 3 is a graph showing the gene expression level of the berberine-high-producing 156-1SMT cell line (vertical axis) relative to the berberine-low-producing CJY cell line (horizontal axis).

These results suggested that cells with high expression of alkaloid biosynthesis-related genes were selected by cell selection, and this led to the expression of 156-1SMT. That is, it was presumed that a gene having a high expression ratio of 156-1SMT to CJY is highly likely to be involved in berberine biosynthesis.

In addition, FIG. 4 shows the results of investigating the expression level of the berberine high-producing 156-1SMT cell line (vertical axis) gene relative to the berberine low-producing CJ8 cell line (horizontal axis). Interestingly, as shown in FIG. 4, it was confirmed that the expression of the berberine biosynthesis-related gene was high even in CJ8 having low berberine productivity. This was considered as follows from the selection history of CJ8. That is, CJ8, like 156-1SMT, has been produced as a strain having high berberine productivity by cell selection, but its productivity has been reduced due to insufficient maintenance. Therefore, although many alkaloid biosynthesis genes are highly expressed, it was presumed that the overall productivity was reduced due to the reduced expression of specific genes. This is considered to be a reasonable guess because the expression of 4'OMT and SAM synthase in the biosynthetic system is lower than that of 156-1SMT. In each of the graphs of FIGS. 3 and 4, the expression ratios of alkaloid biosynthesis genes (SMT, 4′OMT, SAMsynthetase, 6OMT) and genes of unknown function (unknown) are indicated by arrows. Indicated by. Numerical values in parentheses are expression ratios.

Based on the above inference, it was determined that a gene with high expression in 156-1SMT and CJ8 and a low expression in CJY was likely to be a berberine biosynthesis-related gene. FIGS. 5 and 6 show a list of the berberine biosynthesis candidate genes. 5 and 6, the numbers (No.) for identifying the ESTs and the expression ratio between the berberine-producing strains (SMT (156-1SMT) / CJY, SMT (156- 1SMT) / CJ8, CJ8 / CJY), sequence ID (sequence id), EST sequence length (bp), gene bank accession number of homologous gene (GenBank Acc), and estimated function (Notes).

よ う As shown in FIG. 5 and FIG. ESTs 1 to 3 (corresponding to ESTs consisting of the nucleotide sequences shown in SEQ ID NOs: 8, 23 and 27 in order) are considered as candidate genes that suppress alkaloid biosynthesis. It was considered a candidate gene to promote. No. The ESTs shown in 12, 18, 28, 33, 36, 40, 43, 45, and 46 are of known genes that have been identified, and all of them show high values in SMT / CJY and CJ8 / CJY. Was. No. The EST shown in FIG. 19 showed homology to O-methyltransferase, but it was also found that SMT / CJY and CJ8 / CJY showed high values and were involved in the biosynthesis of isoquinoline alkaloids. it was thought. Note that this No. As for 19 ESTs, the full-length cDNA was isolated using the ESTs as a probe, as described later.

In addition, FIG. 7 shows one of the 1014 ESTs whose sequence was determined, which was presumed to function as a transcription factor. This was considered to be a regulator of secondary metabolism-related gene expression, as a result of examining the expression in each berberine-producing strain 156-1SMT, CJ8, and CJY. It is. In the table of FIG. 7, in order from the left, the EST number, the estimated genetic function and its origin, the homology, the EST sequence length, and the presence / absence of expression in each strain (156-1 SMT / CJ8 / CJY) (+: expression) Yes,-: no expression). From the results of observation of the presence or absence of expression between the strains, it was considered that only the gene of EST No. 731 was a positive regulator, and the other genes shown in FIG. 7 were negative regulators. The EST number 731 gene, which is regarded as a positive regulator, corresponds to the N0.29 gene (Ring-H2 finger protein RHC1a homolog) in FIG. 5 and is involved in the control of alkaloid biosynthesis as a transcription factor. It is expected. The gene represented by N0.29 is a gene having the nucleotide sequence shown in SEQ ID NO: 34 and FIG. In addition, among the genes considered to be negative regulators shown in FIG. 7, the gene of EST No. 179 is composed of the nucleotide sequence shown in SEQ ID NO: 35 and FIG. 42, and the gene of EST No. 502 is shown in SEQ ID NO: 36 and FIG. The EST No. 558 gene has the base sequence shown in SEQ ID NO: 37 and FIG. 44, and the EST No. 1013 gene has the base sequence shown in SEQ ID NO: 38 and FIG.

(3) Selection of Berberine-Related Genes by Northern Analysis Total RNA extracted from cells at 1 week of cultured cell lines with different productivity was electrophoresed on a gel containing formaldehyde, and blotted onto Biodyne A ^™ (Pall) membrane. . After amplifying the isolated biosynthetic gene cDNA by PCR, the expression in each cell was determined by a conventional method (Hibi N., Higashiguchi S., Hashimoto T., Yamada Y., 1994, Plant Cell 6: 723-735). The method was confirmed according to the method described in 1.

RNA extraction was performed according to the TIGR plant RNA extraction method using TRIzol (http://www.powow.com/akatlab/tigr.html).

(4) Gene Function Analysis Using Selected Candidate Genes CjEST64 function analysis was performed as one of the genes expected to be involved in berberine biosynthesis by macroarray analysis. This gene is designated as No. 5 in FIG. It is a gene containing the EST shown in No. 19, and since this EST showed homology to O-methyltransferase, it was expected to be involved in the biosynthesis of palmatin, an analog of berberine.

全長 To elucidate the function of this gene, full-length cDNA was isolated using Marathon cDNA cDNA Amplification Kit (Clontech) and its nucleotide sequence was determined. The determined base sequence is shown in SEQ ID NO: 53 and FIG.

The isolated cDNA was inserted into an expression vector: pET-21d vector (Novagen) and then introduced into E. coli. Then, the recombinant enzyme was expressed by a conventional method, and a reaction was carried out using columbamine as a substrate. As a result of analysis by LC-MS, it was clarified that columbamine 7-O-methyltransferase activity was clearly exhibited. That is, it was confirmed that the protein consisting of the amino acid sequence shown in SEQ ID NO: 54, which is a translation product of the above cDNA, exhibited the activity of columbamine 7-O-methyltransferase. This proved the validity of the gene selection by this method.

(5) Isolation and identification of norcoclaurine synthase In this method, the protein is not purified at all, the relevant genes are narrowed down by the methods described in the above (1) and (2), and a heterologous expression system is used. We have succeeded in isolating and identifying the norcoclaurine synthase gene.

Specifically, it is as follows. Initially, the expression level of the obtained nucleotide sequence of the relevant gene candidate was relatively low and could not be detected by the above-described macroarray, and therefore was not described in SEQ ID NOs: 1 to 52. . However, a preliminary homology search of the nucleotide sequence showed homology between the nucleotide sequence obtained as a candidate for the gene and the dioxygenase gene. From this, it was predicted that the nucleotide sequence obtained as the relevant gene candidate was related to the alkaloid biosynthesis system.

Therefore, the correlation between the base sequence obtained as a candidate for the gene and the alkaloid biosynthesis activity was confirmed by the Northern method described in the above section (3). Specifically, regarding the nucleotide sequence (base sequence having homology with the dioxygenase gene) obtained as a candidate gene, the berberine high-producing strain SMT strain (culture 1, 2, 3 weeks) and the low-producing strain CJY strain (Culture 1, 2 weeks), and the expression in the CJ8 strain (culture 1, 2, 3 weeks) with low productivity but high biosynthetic enzyme gene expression were examined by Northern analysis. The result is shown in FIG. FIG. 64 (a) shows a gel in which the amount of mRNA was examined by Northern analysis, and FIG. 64 (b) shows the result of a relative evaluation of the result based on the value of the CJY strain. In the figures, SMT1w indicates the first week of the culture of the SMT strain, SMT2w indicates the second week of the culture of the SMT strain, and SMT3w indicates the third week of the culture of the SMT strain. Hereinafter, the same applies to CJY1w, CJY2w, CJ81w, CJ82w, and CJ83w.

As shown in the figure, compared to the expression level of B. CJY, SMT1w was expressed about 11 times, SMT2w was expressed about 13 times, CJ81w was expressed about 6 times, and CJ82w was expressed about 8 times more. . From these results, it was confirmed that there was a good correlation between the expression level of the base sequence obtained as a candidate for the gene and the alkaloid productivity or the expression level of the berberine biosynthetic enzyme gene.

Therefore, in the same manner as in the method described in the above section (4), the full-length cDNA of the nucleotide sequence obtained as the relevant gene candidate was isolated and the nucleotide sequence was determined. The determined base sequence is shown in SEQ ID NO: 55 and FIG.

(4) Next, a homology search was again performed based on the nucleotide sequence of the obtained full-length cDNA. As a result, it was found that the full-length cDNA had homology with 2-oxoglutarate / Fe (II) -dependent dioxygenase. However, the function cannot be estimated only from this sequence information, and the estimated reaction characteristics and biosynthesis system were examined. And a crude enzyme solution was prepared.

(5-1) Expression in Escherichia coli and Preparation of Enzyme Solution First, in the Escherichia coli expression vector, pET-41a (+) (Novagen), a protein having homology to dioxygenase among the nucleotide sequence of the full-length cDNA is encoded. An NdeI-XhoI fragment (1056 bp) containing the region was incorporated. The resulting plasmid was transformed into Escherichia coli DH5α and cultured in LB medium. After preculture in 10 ml of LB medium, main culture was performed in 1 liter medium. After shaking culture at 16 ° C. and 150 rpm, when the OD600 reached 0.5, IPTG was added to a final concentration of 1 mM, and shaking culture was further performed at 16 ° C. for 48 hours.

The Escherichia coli transfected with the above vector was centrifuged at 1,500 g for 20 minutes at 4 ° C. and collected, and then 25 ml of extraction buffer (100 mM Tris-HCl (pH 8), 5 mM EDTA, 20 mM 2-mercaptoethanol, 10% glycerol). After sonication of the cells, the cells were centrifuged at 12,000 g for 20 minutes at 4 ° C, and the resulting supernatant was used as a soluble fraction. The supernatant was fractionated with 30-60% ammonium sulfate, desalted, and separated on a DEAE Sepharose Fast Flow column (2.5 cm × 16 cm) (Pharmacia). Specifically, first, after the non-adsorbed protein was eluted with the extraction buffer, the protein was eluted with a concentration gradient (500 ml) of 0 to 0.5 M NaCl.

After that, when each fraction was confirmed by SDS-PAGE, the target protein was contained in the non-adsorbed protein fraction, and this non-adsorbed protein fraction was used as a crude enzyme solution in the following experiments.

(5-2) norcoclaurine synthase reaction using a crude enzyme solution A norcoclaurine synthase reaction was performed using the above crude enzyme solution. The reaction liquid composition was as shown in Table 1 below, and the reaction was carried out at 37 ° C. for 30 minutes to 2 hours. After stopping the reaction by adding an equal amount of methanol, the mixture was centrifuged at 12,000 g and 4 ° C. for 5 minutes, and the supernatant was analyzed by HPLC.

The substrate, 4-HPAA (4-hydroxyphenylacetaldehyde), was prepared by oxidative deamination of tyramine with tyramine oxidase (reacted at 30 ° C. for 2 hours) as shown in Table 2 below. After the reaction, the mixture was boiled for 3 minutes to deactivate tyramine oxidase, and then used in the above reaction.

(5-3) Measurement of norcoclaurine synthase activity by HPLC For HPLC analysis, SCL-10A (programming device), LC-10AD (pump), SPD-M10A (optical detector), DGU-12A ( CLASS-VP using CTO-10A (sample injection device, column thermostat). As the column, TDSOH ODS-80TM was used, and the absorbance at 280 nm was measured at 40 ° C. at a flow rate of 0.25 ml / min. The mobile phase used was H ₂ O: acetonitrile: methanol (125: 25: 25), 1% acetic acid.

The results are shown in FIGS. 65 (a) and (b). (A) shows the result of HPLC analysis of the reaction solution alone, and (b) shows the result of HPLC analysis of the product after reaction with a crude Escherichia coli solution expressing a gene encoding a protein having homology to dioxygenase. As shown in the figure, in the case of the crude enzyme solution in which a gene encoding a protein having homology to dioxygenase was expressed, the peak showing a retention time of about 14 minutes increased with the elapse of the reaction time. . An increase in the peak was also observed when only the control E. coli extract or buffer was used, but the increase was larger than that in the case of the crude enzyme solution in which the gene encoding a protein having homology to dioxygenase was expressed. Obviously low. It is known that this peak increase is caused by a non-enzymatic reaction between dopamine as a substrate and 4-hydroxyphenylacetaldehyde.

Therefore, it is thought that by expressing a gene encoding a protein having homology to dioxygenase, norcoclaurine is generated by an enzymatic reaction. Therefore, a protein having homology to the above dioxygenase is considered to have norcoclaurine synthase activity.

(5-4) Identification of Reaction Product by LC-MS Next, identification by LC-MS was performed to confirm that the peak showing a retention time of about 14 minutes observed in HPLC analysis was norcoclaurine. Was. For the analysis, LCMA-2010A manufactured by SHIMADZU was used, and separation was performed at a flow rate of 0.25 ml / min using the column and mobile phase used in the above-mentioned HPLC analysis.

As a result, as shown in FIG. 66, the reaction product of the E. coli crude enzyme solution expressing the gene encoding the protein having homology to dioxygenase has an m / z value (mass spectrum) corresponding to norcoclaurine. It was found to show m / z 272.

From the results of the above HPLC and LC-MS analyses, the reaction product of the gene product of the gene encoding the protein having homology to the isolated dioxygenase is norcoclaurine, and this enzyme (gene product) has norcoclaurine synthase activity It became clear. For this reason, this enzyme is referred to as norcoclaurine synthase. The amino acid sequence of this enzyme is shown in SEQ ID NO: 56 and FIG.

(4) As described above, this method was able to identify the gene of norcoclaurine synthase which had not been identified so far. Since this gene is responsible for the initial reaction of isoquinoline alkaloids, it can be said that the identification of this gene has greatly opened the way for molecular breeding of isoquinoline alkaloid biosynthesis.

As described above, according to the present invention, isoquinoline alkaloid biosynthesis-related genes can be efficiently and comprehensively isolated. If the biosynthetic genes of isoquinoline alkaloids, which are secondary metabolites of plants, are comprehensively isolated, means for industrially mass-producing isoquinoline alkaloids useful as pharmaceutical raw materials will be secured, It has the potential to make a significant contribution to industry.

BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows the biosynthesis pathway of berberine. It is a table | surface which shows the gene which showed homology with the alkaloid synthesis gene identified until now as a result of the homology search of 1014EST sequenced in this Example. It is a graph which shows the berberine high productivity 156-1SMT cell line (vertical axis) gene expression level with respect to the berberine low productivity CJY cell line (horizontal axis). It is a graph which shows the berberine high productivity 156-1SMT cell line (vertical axis) gene expression level with respect to the berberine low productivity CJ8 cell line (horizontal axis). It is a table | surface which shows the gene judged as a berberine biosynthesis candidate gene among the ESTs sequenced in the present Example. FIG. 6 is a table showing genes determined as berberine biosynthesis candidate genes among ESTs sequenced in this example, and is a continuation of the table shown in FIG. 5. It is a table | surface which shows the gene considered as a factor considered as a transcription factor among the ESTs sequenced in the present Example. FIG. 2 is a view showing a base sequence shown in SEQ ID NO: 1. FIG. 3 is a view showing a base sequence shown in SEQ ID NO: 2. FIG. 3 is a view showing a base sequence shown in SEQ ID NO: 3. FIG. 4 is a view showing a base sequence shown in SEQ ID NO: 4. FIG. 7 is a view showing a base sequence shown in SEQ ID NO: 5. FIG. 9 is a view showing a base sequence shown in SEQ ID NO: 6. FIG. 7 is a view showing a base sequence shown in SEQ ID NO: 7. FIG. 9 is a view showing a base sequence shown in SEQ ID NO: 8. FIG. 9 is a view showing a base sequence shown in SEQ ID NO: 9. FIG. 9 is a view showing a base sequence shown in SEQ ID NO: 10. It is a figure which shows the base sequence shown by sequence number 11. FIG. 14 is a view showing a base sequence shown in SEQ ID NO: 12. FIG. 14 is a view showing a base sequence shown in SEQ ID NO: 13. FIG. 14 is a view showing a base sequence shown in SEQ ID NO: 14. It is a figure which shows the base sequence shown by sequence number 15. It is a figure which shows the base sequence shown by sequence number 16. It is a figure which shows the base sequence shown by sequence number 17. It is a figure which shows the base sequence shown by sequence number 18. FIG. 9 is a view showing a base sequence shown in SEQ ID NO: 19. FIG. 7 is a view showing a base sequence shown in SEQ ID NO: 20. It is a figure which shows the base sequence shown by sequence number 21. It is a figure which shows the base sequence shown by sequence number 22. It is a figure which shows the base sequence shown by sequence number 23. It is a figure which shows the base sequence shown by sequence number 24. It is a figure which shows the base sequence shown by sequence number 25. It is a figure which shows the base sequence shown by sequence number 26. It is a figure which shows the base sequence shown by sequence number 27. FIG. 27 is a view showing a base sequence shown in SEQ ID NO: 28. It is a figure which shows the base sequence shown by sequence number 29. FIG. 27 is a view showing a base sequence shown in SEQ ID NO: 30. It is a figure which shows the base sequence shown by sequence number 31. It is a figure which shows the base sequence shown by sequence number 32. It is a figure which shows the base sequence shown by sequence number 33. FIG. 34 is a view showing a base sequence shown in SEQ ID NO: 34. It is a figure which shows the base sequence shown by sequence number 35. It is a figure which shows the base sequence shown by sequence number 36. It is a figure which shows the base sequence shown by sequence number 37. FIG. 35 is a view showing a base sequence shown in SEQ ID NO: 38. FIG. 39 is a view showing a base sequence shown in SEQ ID NO: 39. FIG. 7 is a view showing a base sequence shown in SEQ ID NO: 40. FIG. 42 is a view showing a base sequence shown in SEQ ID NO: 41. FIG. 42 is a view showing a base sequence shown in SEQ ID NO: 42. FIG. 44 is a view showing a base sequence shown in SEQ ID NO: 43. FIG. 44 is a view showing a base sequence shown in SEQ ID NO: 44. It is a figure which shows the base sequence shown by sequence number 45. FIG. 34 is a view showing a base sequence shown in SEQ ID NO: 46. FIG. 47 is a view showing a base sequence shown in SEQ ID NO: 47. FIG. 29 is a view showing a base sequence shown in SEQ ID NO: 48. FIG. 49 is a view showing a base sequence shown in SEQ ID NO: 49. It is a figure which shows the base sequence shown by sequence number 50. It is a figure which shows the base sequence shown by sequence number 51. It is a figure which shows the base sequence shown by sequence number 52. It is a figure which shows the base sequence shown by sequence number 53. FIG. 34 is a view showing an amino acid sequence represented by SEQ ID NO: 54. FIG. 34 is a view showing a base sequence shown in SEQ ID NO: 55. FIG. 36 is a view showing an amino acid sequence represented by SEQ ID NO: 56. (A) shows high berberine-producing strain SMT (culture 1, 2, 3 weeks), low-producing strain CJY (culture 1, 2 weeks), and low productivity but high expression of biosynthetic enzyme gene It is a figure which shows the result of having investigated the expression of the norcoclaurine synthase gene in the CJ8 strain (culture 1,2,3 weeks) by the Northern analysis, (b) shows the value of the CJY strain based on the result of (a). It is a figure showing the result of relative evaluation as a basis. It is a figure which shows the measurement result by HPLC of norcoclaurine synthase activity. It is a figure showing the result of LC-MS analysis of the reaction product generated by norcoclaurine synthase reaction.

Claims (21)

(4) A polynucleotide comprising any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 52. 。 The polynucleotide according to claim 1, wherein the polynucleotide is a gene fragment involved in biosynthesis of an isoquinoline alkaloid. The polynucleotide according to claim 1 or 2, wherein the polynucleotide is derived from cultured cells of cucumber. (4) A cDNA library comprising at least one of the polynucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 to 52. (5) The cDNA library according to (4), which is derived from cultured cells of spinach. (6) The cDNA library according to (5), wherein the cultured spinach cells produce berberine at a high level. (4) A full-length gene comprising any one of the nucleotide sequences shown in SEQ ID NOs: 1 to 52 or 55 and involved in isoquinoline alkaloid biosynthesis. A gene encoding the following protein (a) or (b):
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 54;
(B) an amino acid sequence represented by SEQ ID NO: 54, wherein one or more amino acids are substituted, deleted, inserted, and / or added, and have an activity as a columbamine 7-O-methyltransferase. Protein to have. A gene encoding the following protein (c) or (d):
(C) a protein consisting of the amino acid sequence represented by SEQ ID NO: 56;
(D) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 56, and which has an activity as a norcoclaurin synthase . 遺 伝 子 The gene according to claim 8 or 9, wherein the gene is derived from spinach. (4) A full-length gene involved in the biosynthesis of an isoquinoline alkaloid having the nucleotide sequence shown in SEQ ID NO: 53 or 55. The following protein (a) or (b):
(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 54;
(B) an amino acid sequence represented by SEQ ID NO: 54, wherein one or more amino acids are substituted, deleted, inserted, and / or added, and have an activity as a columbamine 7-O-methyltransferase. Protein to have. The following protein (c) or (d):
(C) a protein consisting of the amino acid sequence represented by SEQ ID NO: 56;
(D) a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence represented by SEQ ID NO: 56, and which has an activity as a norcoclaurin synthase . タ ン パ ク 質 The protein according to claim 12 or 13, wherein the protein is derived from spinach. (10) A method for isolating a full-length gene involved in isoquinoline alkaloid biosynthesis using the polynucleotide according to any one of (1) to (3) or (9). (7) A method for isolating a gene, comprising using the cDNA library according to any one of (4) to (6). A vector into which the polynucleotide according to any one of claims 1 to 3 or the gene or the full-length gene according to any one of claims 7 to 10 is incorporated. [18] A transformant into which the vector according to claim 17 has been introduced. 19. The transformant according to claim 18, wherein the vector is introduced into a plant. 20. The transformant according to claim 19, wherein the plant is a plant that produces an isoquinoline alkaloid. A method for producing an isoquinoline alkaloid using at least one of the proteins according to claims 12 to 14 and the transformant according to claims 18 to 20.

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