WO2010122849A1 - デルフィニジンを花弁に含有するキク植物を生産する方法 - Google Patents
デルフィニジンを花弁に含有するキク植物を生産する方法 Download PDFInfo
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- WO2010122849A1 WO2010122849A1 PCT/JP2010/053904 JP2010053904W WO2010122849A1 WO 2010122849 A1 WO2010122849 A1 WO 2010122849A1 JP 2010053904 W JP2010053904 W JP 2010053904W WO 2010122849 A1 WO2010122849 A1 WO 2010122849A1
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- WIPO (PCT)
- Prior art keywords
- chrysanthemum
- seq
- gene
- delphinidin
- hydroxylase
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- C12N15/09—Recombinant DNA-technology
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
- A01H6/1424—Chrysanthemum
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- C12N15/09—Recombinant DNA-technology
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
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Definitions
- the present invention relates to a method for producing chrysanthemum plants containing delphinidin in petals using a transcriptional regulatory region of chrysanthemum-derived flavanone 3-hydroxylase (F3H) gene, a nucleic acid of the regulatory region, an expression vector containing the nucleic acid, or an expression
- F3H chrysanthemum-derived flavanone 3-hydroxylase
- the present invention relates to a chrysanthemum plant, its progeny or its vegetative growth body, or a part or tissue thereof, particularly petals and cut flowers, into which the cassette and the regulatory region have been introduced.
- a new trait can be imparted to a plant by expressing a useful gene in the target plant.
- Genetically modified plants produced in this way have already been cultivated in a wide range. Regulation of gene expression is mainly controlled at the transcriptional stage, and transcriptional regulation is most important in regulating gene expression. That is, it is important for producing an industrially useful genetically modified plant that transcription is performed at an appropriate time and in an appropriate tissue with an appropriate strength. Transcription is often controlled by a DNA sequence 5 'to the translation region. A region on DNA that determines the transcription start site of a gene and directly regulates its frequency is called a promoter.
- the promoter may be present several tens of bp 5 ′ of the start codon and often includes a sequence such as a TATA box. Furthermore, on the 5 'side, there are cis elements to which various transcription regulatory factors bind, and their presence controls the timing of transcription, the tissue in which transcription is performed, and the strength of transcription. Transcriptional regulators are classified into many families by amino acid sequences. For example, a Myb type transcriptional regulatory factor and a bHLH (basic helix loop helix) type transcriptional regulatory factor are well-known families. Actually, the transcription control region and the promoter are often used in the same meaning.
- Anthocyanins the main component of flower color, are members of secondary metabolites collectively referred to as flavonoids.
- the color of anthocyanins depends on its structure. That is, when the number of hydroxyl groups on the B ring of anthocyanidin, which is an anthocyanin chromophore, increases, the color becomes blue.
- aromatic acyl groups coumaroyl groups, caffeoyl groups, etc.
- the color of anthocyanins turns blue (that is, the absorption maximum shifts to longer wavelengths) and the stability of anthocyanins increases. Is known (see Non-Patent Document 1 below).
- Enzymes involved in anthocyanin biosynthesis and genes encoding the enzymes have been well studied (see Non-Patent Document 1). For example, enzyme genes that catalyze the reaction of transferring an aromatic acyl group to anthocyanins are obtained from gentian, lavender, petunia, and the like (hereinafter, see Patent Document 1 and Patent Document 2).
- Promoters responsible for gene transcription in plants are so-called constitutive promoters that function in any tissue or any stage of development, and function only in specific organs and tissues. There are organ- and tissue-specific promoters, and time-specific promoters that are expressed only at specific times in the developmental stage. Constitutive promoters are often used as promoters for expressing useful genes in transgenic plants.
- constitutive promoters cauliflower mosaic virus 35S promoter (hereinafter sometimes referred to as CaMV35S promoter), a promoter constructed based on the promoter (refer to Non-Patent Document 3 below), Mac1 promoter (hereinafter referred to as the following).
- CaMV35S promoter cauliflower mosaic virus 35S promoter
- Non-Patent Document 3 a promoter constructed based on the promoter
- Mac1 promoter hereinafter referred to as the following.
- tissue / organ-specific or time-specific manner in plants, many genes are expressed in a tissue / organ-specific or time-specific manner. This suggests that it is necessary for plants to express genes in a tissue / organ-specific or time-specific manner.
- tissue / organ-specific or time-specific transcriptional regulatory regions There is an example of genetic recombination of plants using such tissue / organ-specific or time-specific transcriptional regulatory regions.
- proteins are accumulated in seeds using a seed-specific transcriptional regulatory region.
- plants can blossom in a variety of colors, but there are few species that can blossom in all colors because of the genetic limitations of the species. For example, varieties that bloom blue and purple flowers in roses and carnations do not exist naturally. This is because roses and carnations do not have the flavonoid 3 ', 5'-hydroxylase gene required to synthesize the anthocyanidin called delphinidin, which is synthesized by many blue and purple flowers. By introducing flavonoid 3 ', 5'-hydroxylase genes such as petunia and pansy which are blue and purple flowers to bloom into these species, the color of the flowers in these species can be made blue did it.
- the transcriptional regulatory region of the chalcone synthase gene derived from snapdragon or petunia is used to transcribe the flavonoid 3 ′, 5′-hydroxylase gene derived from different species.
- plasmids pCGP485 and pCGP653 plasmids containing constitutive transcriptional regulatory regions described in Patent Document 3 below
- plasmid pCGP628 Mac1 promoter
- the plasmid pSPB130 described in Patent Document 4 below including the CaMV35S promoter to which the El2 enhancer has been added.
- Non-Patent Document 5 below. Therefore, several promoters have been used to change the color of flowers, but in order to change the color of other flowers, useful promoters according to the host plant and purpose are required.
- chrysanthemum plants account for about 30% of the wholesale price (summary of the results of the 2007 survey of wholesale flowering in agriculture, forestry and fisheries statistics) in Japan, and about 9% of roses. It is also an important work compared to about 7% of carnations. Chrysanthemum has flower colors such as white, yellow, orange, red, peach and purple red, but existing varieties and closely related wild species do not have blue flowers such as purple and blue. Therefore, blue breeding is regarded as one of the goals to stimulate new demand. Chrysanthemum flower color is expressed by a combination of anthocyanins and carotenoids.
- Anthocyanins can express a variety of colors depending on the structure of anthocyanidins, which are the basic skeleton, and modifications by sugars and organic acids.
- the anthocyanins responsible for chrysanthemum are two types of cyanidin modified with glucose and malonic acid (cyanidin 3-O- (6 "-O-monomalonyl- ⁇ -glucopyranoside and cyanidin 3-O- (3 “, 6” -O-dimalonyl- ⁇ -glucopyranoside) (see Non-Patent Document 6), and the structure thereof is relatively simple (see FIG. 1).
- chrysanthemum is the most important flower in Japan, but it has a high polyploidy called hexaploid and has a large genome size, so in addition to low transformation efficiency, Silencing (inactivation) may occur, and it is not easy to obtain transgenic chrysanthemum that stably expresses the transgene.
- GUS ⁇ -glucuronidase
- Non-patent Document 10 an example in which the mutated ethylene receptor gene is expressed in chrysanthemum to extend the flower life (hereinafter referred to as Non-patent Document 10), and an example in which the expression of chrysanthemum AGAMOUS gene is suppressed and the shape of the flower is changed (hereinafter referred to as non-patent document 10).
- non-patent document 11 an example in which the expression of a foreign gene in chrysanthemum is increased using a translation enhancer (see patent document 7) of tobacco alcohol dehydrogenase (see non-patent document 12).
- Non-Patent Document 13 a report that suppresses the gene of chalcone synthase (CHS) by the co-suppression method and makes the pink color white
- CCD4a carotenoid-degrading enzyme
- Patent Document 5 Non-Patent Document 15
- delphinidin produced by the action of the introduced F3′5′H gene accumulates in the lingual petals, and there is no report that blue chrysanthemum was produced.
- delphinidin production is not observed even when F3′5′H is expressed by the CaMV35S promoter (see Non-Patent Document 15).
- Non-Patent Document 7 examples of promoters that can efficiently and stably express foreign genes in chrysanthemum with chrysanthemum include potato Lhca3.St.1 promoter (hereinafter referred to as Non-Patent Document 16), chrysanthemum UEP1 promoter (hereinafter referred to as Non-Patent Document 17), The tobacco EF1 ⁇ promoter (see Patent Document 6 and Non-Patent Document 9) has been developed. However, there is no report on chrysanthemum flower color modification by overexpression of foreign genes using these promoters.
- the problem to be solved by the present invention is a method for producing chrysanthemum plants containing delphinidin in petals using the transcriptional regulatory region of chrysanthemum-derived flavanone 3-hydroxylase (F3H) gene, and the regulatory region was introduced.
- F3H chrysanthemum-derived flavanone 3-hydroxylase
- the inventors of the present application have made extensive studies to solve the above-mentioned problems, and as a result of repeated experiments, the flavonoid 3 ′, 5′-hydroxylation using the transcriptional regulatory region of chrysanthemum-derived flavanone 3-hydroxylase (F3H).
- F3H chrysanthemum-derived flavanone 3-hydroxylase
- a nucleic acid comprising the base sequence represented by SEQ ID NO: 34 or SEQ ID NO: 87 (2) A chrysanthemum-derived flavanone 3-hydroxylase (F3H) gene can function as a transcriptional regulatory region, and has one or several nucleotide sequences relative to the nucleotide sequence shown in SEQ ID NO: 34 or 87 A nucleic acid comprising a base sequence modified by addition, deletion and / or substitution of (3) A nucleic acid that can function as a transcriptional regulatory region of chrysanthemum-derived flavanone 3-hydroxylase (F3H) gene and has a base sequence complementary to the base sequence shown in SEQ ID NO: 34 or 87 And (4) a chrysanthemum-derived flavanone 3-hydroxylase (F3H) gene that can function as a transcriptional regulatory region, and is SEQ ID NO: 34 or SEQ ID NO: A nucleic acid having at least 90% sequence identity to the base sequence shown in 87; A flachrysanthemum-derived
- the flavonoid 3 ′, 5′-hydroxylase (F3′5′H) when expressed in chrysanthemum using the transcriptional regulatory region of chrysanthemum-derived flavanone 3-hydroxylase (F3H), More delphinidin accumulates in petals, the color of the flower changes towards blue, and the addition of a translation enhancer from tobacco alcohol dehydrogenase results in more delphinidin Accumulated, it turned out that the color of the flower changes towards blue.
- flavonoids 3 ′ and 5 ′ are obtained by the 5 ′ region of a chrysanthemum flavanone 3-hydroxylase (F3H) gene (hereinafter also referred to as “CmF3Hpro” or “chrysF3H 5 ′”).
- F3Hpro chrysF3H 5 ′
- chrysF3H 5 ′ chrysanthemum flavanone 3-hydroxylase
- the content of delphinidin in the petals is preferably 25% by mass or more of the total amount of anthocyanidins, and the petal color is modified in the blue direction.
- the present invention also relates to chrysanthemum plants or their progeny or vegetative growths thereof or parts or tissues produced by the method or containing CmF3Hpro.
- the part or tissue is preferably a petal or cut flower.
- the “expression cassette” refers to a DNA fragment in which a promoter and a terminator are linked to an arbitrary nucleic acid.
- the F3'5'H gene is expressed in chrysanthemum petals, and the enzyme protein is synthesized and functions to synthesize and accumulate delphinidin-based anthocyanins, thereby producing blue-colored flower-colored petals. Chrysanthemum can be created.
- Rose CHSpro rose chalcone synthase (CHS) gene promoter
- R. rugosa DFRpro Humanus dihydroflavonol-4-reductase (DFR) ) Gene promoter
- Examples of the transcription control region according to the present invention include a nucleic acid having the base sequence shown in SEQ ID NO: 34 or 87. However, several (1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) base additions, deletions and / or deletions in the nucleic acid consisting of the base sequence shown in SEQ ID NO: 34 or 87 Alternatively, a promoter composed of a base sequence modified by substitution is considered to maintain the same activity as the original promoter. Therefore, as long as it functions as a transcriptional regulatory region of chrysanthemum-derived flavanone 3-hydroxylase (F3H) gene, the transcriptional regulatory region according to the present invention is one or several relative to the nucleotide sequence shown in SEQ ID NO: 34 or SEQ ID NO: 87. It can also be a nucleic acid consisting of a base sequence modified by addition, deletion and / or substitution of a single base sequence.
- F3H chrysanthemum-derived flavanone 3-hydroxylase
- the transcriptional regulatory region according to the present invention can further function as a transcriptional regulatory region of chrysanthemum-derived flavanone 3-hydroxylase (F3H) gene, and is higher than the nucleotide sequence shown in SEQ ID NO: 34 or SEQ ID NO: 87.
- nucleic acids can be hybridized under stringency conditions with a polynucleotide containing the base sequence shown in SEQ ID NO: 34 or 87, preferably about 70% or more, and more than the base sequence shown in SEQ ID NO: 34 Preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, and most preferably about 99% of the nucleic acid having the base sequence identity.
- stringency conditions are hybridization conditions that are easily determined by those skilled in the art, and are generally determined empirically depending on probe length, washing temperature, and salt concentration. In general, the longer the probe, the higher the temperature for proper annealing, and the shorter the probe, the lower the temperature. Hybridization generally depends on the ability of denatured DNA to reanneal when the complementary strand is present in an environment close to its melting point but lower. Specifically, for example, as a low stringency condition, the filter may be washed in a 5 ⁇ SSC, 0.1% SDS solution at a temperature of 37 ° C. to 42 ° C. in a filter washing step after hybridization. Can be mentioned. Examples of high stringency conditions include washing in 65 ° C., 0.1 ⁇ SSC and 0.1% SDS in the washing step. By making the stringency conditions higher, polynucleotides having high homology or identity can be obtained.
- the flavonoid 3 ′, 5′-hydroxylase (F3′5′H) is preferably derived from campanula, cineraria (cinellaria), verbena, and pansy # 40.
- the translation enhancer is preferably directly linked to the start codon of the F3′5′H gene in the gene cassette of the expression vector.
- the delphinidin content in the petals is preferably 25% by mass or more of the total amount of anthocyanidins.
- the present invention is a chrysanthemum plant or its progeny or its vegetative growth body, or a part or tissue thereof produced (produced) by the method of the present invention or introduced with the nucleic acid, preferably a petal or cut flower.
- the present invention also relates to a processed product (cut flower processed product) using the cut flower.
- the cut flower processed product includes, but is not limited to, a pressed flower using the cut flower, a preserved flower, a dried flower, a resin sealed product, and the like.
- Reference Examples 1 to 9 are examples using promoters other than the 5 ′ region (CmF3Hpro) of the gene encoding chrysanthemum flavanone 3-hydroxylase (F3H), while Examples 1 to 10 is an example relating to the 5 ′ region (CmF3Hpro) of a gene encoding chrysanthemum flavanone 3-hydroxylase (F3H).
- Reference Example 1 Expression of F3 ′, 5′H gene by tobacco EF1a promoter About 1.2 kb of DNA containing tobacco EF1 ⁇ promoter sequence by digesting pBIEF1 ⁇ described in Patent Document 6 with restriction enzymes HindIII and BamHI A fragment was obtained. This DNA fragment was inserted into the 5 ′ side of the iris DFR cDNA of pSPB909 described in Patent Document 4 to obtain plasmid pSFL339. Similarly, a plasmid pSFL340 in which a petunia DFR cDNA (described in International Publication WO 96/36716) was inserted instead of the iris DFR cDNA was also constructed.
- pSPB575 a fragment of pansy F3'5'H gene BP40 excised by partial degradation of BamHI and XhoI from pCGP1961 described in Patent Document 4
- the inserted plasmid was designated as pSPB575.
- the promoter portion of this plasmid was replaced with the tobacco EF1 ⁇ promoter described above to obtain pSFL338.
- AscI-digested pSFL339 a fragment containing iris DFR cDNA was inserted into this AscI site.
- the obtained plasmid was designated as pSFL346.
- This plasmid pSFL346 is designed to express the bungee F3′5′H and the iris DFR gene in plants under the control of the tobacco EF1 ⁇ promoter.
- Plasmid pLHF8 containing lavender F3'5'HcDNA is described in International Publication No. WO 04/20637.
- a plasmid pSPB2772 was obtained by ligating a DNA fragment obtained by digesting this plasmid with BamHI and XhoI and a DNA fragment having a large molecular weight among the DNA fragments of pSPB176 obtained by digesting with BamHI and SalI.
- F3′5′H cDNA derived from lavender is linked to the CaMV35S promoter to which the El2 enhancer is added. This promoter part was replaced with the above-mentioned tobacco EF1 ⁇ promoter using HindIII and BamHI to obtain plasmid pSPB2778.
- pSPB2780 A fragment containing Petunia DFR cDNA in pSFL340 digested with AscI was inserted into the AscI site.
- the obtained plasmid was designated as pSPB2780.
- This plasmid pSPB2780 is designed to express lavender F3′5′H and petunia DFR gene in plants under the control of tobacco EF1 ⁇ promoter.
- the promoter part of the plasmid pSPB748 (F3'5'H cDNA derived from butterfly is linked to the CaMV35S promoter with the El2 enhancer) described in Plant Biotechnol 23, 5-11 (2006) was used using HindIII and BamHI
- the plasmid pSPB2777 was obtained by replacing the above-mentioned tobacco EF1 ⁇ promoter.
- a fragment containing Petunia DFR cDNA in pSFL340 digested with AscI was inserted into the AscI site.
- the obtained plasmid was designated as pSPB2779.
- This plasmid pSPB2779 is designed to express the F3'5'H of fowl and the DFR gene of petunia in plants under the control of the tobacco EF1 ⁇ promoter.
- the above plasmids pSFL346, pSPB2780, and pSPB2779 were introduced into Agrobacterium, and introduced into chrysanthemum variety 94-765 using this transformed Agrobacterium. Analysis of the anthocyanidins in the petals of transformed chrysanthemum did not detect delphinidin.
- a cDNA library was prepared using the ZAP-cDNA (registered trademark) Library Construction Kit (Stratagene, product number 200450) according to the method recommended by the manufacturer. .
- This cDNA library is recommended by the company using Sturgeon F3'5'H cDNA (Clitoria ternatea) (see Plant Biotechnology 23, 5-11 (2006)) labeled with the DIG system (Roche Applied Science) Was screened.
- the resulting phages showing 48 signals were purified. From these phages, plasmids were obtained by in vivo excision by the method recommended by the manufacturer (Stratagene).
- Ci5a18F1 SEQ ID NO: 81
- Ci5a18R1 SEQ ID NO: 82
- plaques were finally obtained from the obtained 17 plaques, and these were converted into plasmids by in vivo excision.
- DNA base sequences were determined, they contained the same sequence.
- a clone designated as gCi01-pBluestar was used for the subsequent experiments.
- the gCi01-pBluestar clone base sequence is shown in SEQ ID NO: 79. This sequence was expected to contain a 5′-untranslated region, a translated region, and a 3′-untranslated region containing sequences with promoter activity of Cineraria F3′5′H.
- SEQ ID NO: 80 An approximately 5.7 kb DNA fragment (SEQ ID NO: 80) cut out from gCi01-pBluestar with PvuI and EcoRV was blunt-ended using DNA blunting kit (TAKARA). This DNA fragment was cloned into the SmaI site of pBinPLUS and named pSPB3130. This binary vector had an nptII gene that could be used for selection by kanamycin in the T-DNA region.
- PSPB3130 was introduced into chrysanthemum variety 94-765 by the Agrobacterium method. The petals were analyzed for anthocyanidins in the transformed chrysanthemum, but no delphinidin was detected and the color of the flowers did not change.
- delphinidin production using the rose chalcone synthase gene promoter pSPB3325 (rose CHSpro :: pansy # 18 + rose CHSp :: chrysanthemum F3'H IR) described in the ninth from the top in Table 1 can be mentioned.
- delphinidin production was up to 3.6%.
- Reference Example 4 Delphinidin production using pansy F3'5'H gene promoter (1) Cloning of lysoanthocyanin 3-acyltransferase chromosomal gene
- the chromosomal DNA was prepared from the former leaves by the reported method (see Plant Mol Biol. December 1997; 35 (6): 915-27). This chromosomal DNA was partially decomposed with Sau3AI (manufactured by TOYOBO), and a fraction containing a DNA fragment of 10 to 15 kb was collected by a sucrose density gradient method.
- a chromosomal DNA library was prepared by inserting this fragment into the BamHI site of EMBL3 (Promega), which is a kind of lambda phage vector, using a known method.
- the obtained library was screened using pSAT208 (Plant Cell Physiol. April 2000; 41 (4): 495-502) which is a cDNA of anthocyanin 3-acyltransferase derived from perilla as a probe.
- the screening of the library was based on the method already reported (see Plant Cell Physiol. July 1996; 37 (5): 711-6).
- the plaque hybridized with the probe was purified and cultured, and DNA was prepared from the obtained phage.
- the obtained plasmid was designated as pSPB513.
- the perilla-derived DNA sequence contained in this plasmid was determined by the primer walking method.
- the base sequence is shown in SEQ ID NO: 4.
- This sequence has a region highly homologous to pSAT208, which is an anthocyanin 3-acyl group cDNA.
- the amino acid sequence of the protein encoded by this region (SEQ ID NO: 6) is 19 amino acids compared to the amino acid sequence encoded by pSAT208. A substitution of amino acid residues and a deletion of two amino acids were seen, and no intron was observed.
- sequence of the region showing high homology with pSAT208 contained a sequence of 3438 bp upstream of ATG considered to be the start codon and a sequence of 2052 bp downstream of TAA considered to be the stop codon.
- ORF open reading frame
- the plasmid pSPB567 described in Patent Document 4 (the cauliflower mosaic 35S promoter to which the El2 enhancer has been added is linked to the 3 ′ side of the pansy-derived flavonoid 3 ′, 5′-hydroxylase gene, and further to the 3 ′ side of the plasmid pSPB567.
- the palin synthase terminator was digested with PacI, and a DNA fragment containing the pansy flavonoid 3 ′, 5′-hydroxylase gene was cloned into the PacI site of pBin +.
- the plasmid in which the cauliflower mosaic 35S promoter to which the El2 enhancer was added was present near the AscI site of pBin + was designated as pSPB575.
- This plasmid was digested with HindIII and BamHI, and a DNA fragment obtained by digesting a DNA fragment of about 1.1 kb containing the transcriptional regulatory region of the above-mentioned soyanthocyanin 3-acyltransferase with HindIII and BamHI was inserted into this plasmid.
- the obtained plasmid was designated as pSFL205.
- PSFL205 was digested with HindIII and SacI to recover a DNA fragment of about 100 bp. This DNA fragment was ligated with the approximately 4 kb DNA fragment obtained by digesting pSPB513 with SacI and XbaI and the plasmid pBin + digested with HindIII and XbaI (see Transgenic Research 4, 288-290, ⁇ 1995).
- pSPB3311 was obtained.
- This plasmid pSPB3311 is a binary vector comprising the nucleotide sequence shown in SEQ ID NO: 2, and comprises the transcriptional regulatory region of the thysoanthocyanin 3-acyltransferase gene, the translated region of the gene, and the 3 ′ untranslated region. Including.
- reaction solution was diluted 10 times with water.
- 0.5 ⁇ l was used as a template
- 5 pmol of primer C2 (5′-CGTTAGAACGCGTAATACGACTCACTATAGGGAGA-3 ′, SEQ ID NO: 11, part of HindIII cassette sequence included in Kit)
- 5 pmol of primer BP40-i7 (5 ′ -Reaction with 25 ⁇ l of a reaction solution containing -GACCATACTTCTTAGCGAGTTTGGC-3 ', SEQ ID NO: 12) was performed at 98 ° C for 5 minutes, and then the reaction at 98 ° C for 20 seconds and 68 ° C for 15 minutes was repeated 30 times.
- the obtained DNA fragment was introduced into plasmid pCR2.1 (Invitrogen).
- plasmid pCR2.1 Invitrogen
- the nucleotide sequence of the inserted DNA was determined, there was a portion where the sequence did not match the cDNA sequence of BP # 40. This is probably because an error occurred during PCR.
- the following operation was performed.
- 50 pmol primer BP40-i7 SEQ ID NO: 12
- 50 pmol primer BP40 were used using 200 ng pansy chromosomal DNA as a template.
- PCR was performed in 25 ⁇ l of the reaction solution using pro-F (5′-ACTCAAACAAGCATCTCGCCATAGG-3 ′, SEQ ID NO: 13, sequence in 5′-untranslated region of BP # 40 gene). After treatment at 98 ° C. for 5 minutes, the reaction consisting of 98 ° C. for 20 seconds and 68 ° C. for 15 minutes was repeated 30 times. The amplified DNA fragment was inserted into pCR2.1. This DNA fragment contained an approximately 2.1 kb 5′-untranslated region and a 200 bp translated region. This plasmid was designated as pSFL614. The nucleotide sequence of the plasmid pSFL614 sequence is shown in SEQ ID NO: 14.
- the approximately 2.1 kb 5′-untranslated region (BP40pro, SEQ ID NO: 15) contained in pSFL614 was used to transcribe the BP # 40 gene. At this time, the BamHI site was changed to NheI.
- 50 pmol of primer BP40pro-HindIII-F 5'-AAG CTT GTG ATC GAC ATC TCT CTC C-3 ', SEQ ID NO: 16
- 50 pmol of primer BP40pro-NheI-R 5'- CGA GGC TAG CTA AAC ACT TAT-3 ', SEQ ID NO: 17
- Ex-Taq DNA polymerase was added, and the reaction was held at 25 ° C for 5 minutes at 98 ° C, followed by 25 reactions at 98 ° C for 20 seconds and 68 ° C for 15 minutes Repeated.
- the amplified DNA fragment was cloned into pCR2.1. It was determined by confirming the base sequence that there was no error due to PCR in this sequence. This plasmid was digested with HindIII and NheI to obtain a 470 bp DNA fragment. This DNA fragment was designated as fragment A.
- the larger fragment of DNA fragments generated by digesting plasmid pSPB567 described in Patent Document 4 with BamHI and HindIII was recovered, and the above-mentioned fragment A and fragment B were ligated to obtain pSFL620.
- pSFL620 After digesting pSFL620 with PacI, a DNA fragment of about 3.2 kb was recovered. This DNA fragment was inserted into the PacI site of pBin +.
- the obtained plasmid was designated as pSPB3317.
- a fragment obtained by digesting pSPB3311 with AscI and XbaI was introduced into the AscI and XbaI sites of pSPB3317, and the resulting plasmid was designated as pSPB3323.
- Anthocyanidins extracted by the following method were analyzed. Tongue petals were crushed after freezing, and 50-100 mg of crushed petals were extracted with 500 ⁇ L of 1% hydrochloric acid methanol, and 500 ⁇ L of 4N hydrochloric acid (HCl) was added to this extract and mixed. Decomposition was performed. After cooling the decomposed solution, 1 ml of 0.05 M trifluoroacetic acid (TFA) was added and mixed. Next, this solution was added to Sep-Pak C18 (Millipore) to adsorb the hydrolysis product. Sep-Pak C18 was washed with 80% acetonitrile (MeCN) in advance and equilibrated with 0.05M TFA.
- TFA trifluoroacetic acid
- the hydrolyzate adsorbed on Sep-Pak C18 was washed with 0.05M TFA, and further washed with 20% MeCN and 0.05M TFA, the hydrolyzate was eluted with 80% MeCN and 0.05M TFA to obtain an analysis sample. .
- the analysis sample was analyzed under the following conditions using high performance liquid chromatography.
- the column was Inertsil ODS-2 (particle size 5 ⁇ m, 4.6 ⁇ 250 mm, GL Science), the flow rate was 0.8 ml / min, the mobile phase contained 1.5% phosphoric acid, 5% acetic acid, 6.25% acetonitrile to 20% acetic acid, After a 20% linear gradient of 25% acetonitrile, isocratic elution was performed for 5 minutes with 25% acetonitrile containing 1.5% phosphoric acid and 20% acetic acid.
- an Agilent 1100 series diode array detector (GL Science) was used to detect the wavelength region from 250 nm to 600 nm, and the abundance ratio of each anthocyanidin was determined from the area of absorbance at 530 nm.
- delphinidin was detected in 0.9%, 0.8%, 1.4%, and 0.6% of the total anthocyanidins in the analysis lines 1300-3, 1300-4, 1300-5, and 1300-6, respectively. It was. This suggests that the BP # 40 transcriptional regulatory region of pansy was responsible for transcription of BP # 40, but did not change the flower color.
- chromosomal DNA library of Hermanus was obtained from ⁇ BlueSTAR TM Xho I Half-Site Arms Kit (Novagen, http://www.merckbiosciences.com/product/69242). And produced as follows. Chromosomal DNA was prepared from young leaves of Hermanus using Nucleon Phytopure [Registered] (TEPNEL Life Sciences). About 100 ⁇ g of chromosomal DNA was partially digested with the restriction enzyme Sau3AI. This DNA fragment was partially filled in with DNA polymerase I Klenow fragment (TOYOBO) in the presence of dGTP and dATP, and fractionated by sucrose density gradient centrifugation.
- DNA polymerase I Klenow fragment TOYOBO
- DNA was recovered to a size of about 13 kb and concentrated by ethanol precipitation. About 180 ng of DNA was ligated with 1 ⁇ L of ⁇ BlueSTAR TM Xho I Half-Site Arms Kit at 4 ° C. for 15 hours, and then subjected to in vitro packaging reaction to obtain a chromosome library.
- This library was screened using cultivated rose DFR cDNA (Plant and Cell Physiology, 36, 1023-1031, 1995) to obtain plaques indicating a signal. From this plaque, in vivo was excised using the method recommended by the manufacturer (Novagen) to obtain plasmid pSFK710. This plasmid contained a DNA sequence that closely matched the aforementioned DFR cDNA of cultivated roses.
- PCR was performed with 50 ⁇ L of the reaction solution. PCR reaction conditions were as follows: 94 ° C for 5 minutes, followed by 30 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 30 seconds, and further maintained at 72 ° C for 7 minutes. As a result, an approximately 350 bp DNA fragment A was obtained.
- each 25 pmol of primers DFRproNheIF (5'-ACACGCTAGCATAAGTCTGTTG-3 ', SEQ ID NO: 22) and DFRproBamHI-R (5'-GCTTGGGGATCCATCTTAGG-3', SEQ ID NO: 23)
- enzyme ExTaq DNA Polymerase (TOYOBO ) PCR was performed with 50 ⁇ L of the reaction solution using a sputum.
- the reaction conditions for PCR were 94 ° C for 5 minutes, followed by 30 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 30 seconds, and further maintained at 72 ° C for 7 minutes. As a result, a 600 bp DNA fragment B was obtained.
- Reference Example 6 Production of Delphinidin in Chrysanthemum Using the Hermanus F3H Promoter
- the Hermanus chromosomal DNA library prepared in Reference Example 5 was screened by Torenia flavanone 3-hydroxylase (F3H) cDNA (NCBI No. AB211958) A plaque showing was obtained.
- One plaque was converted into a plasmid in the same manner as in Reference Example 5. This was digested with the restriction enzyme SpeI, and a 2.6 kb DNA fragment was recovered and subcloned into the SpeI site of pBluescript SKII- (STRATAGENE) to obtain plasmid pSPB804.
- This plasmid had a base sequence showing homology to F3H.
- primer RrF3H-F 5′-AAGCTTCTAGTTAGACAAAAAGCTA-3 ′, SEQ ID NO: 24
- primer RrF3H 5′-GGATCCTCTCTTGATATTTCCGTTC-3 ′, SEQ ID NO: 25
- Ex-Taq DNA Polymerase TOYOBA
- the reaction conditions for PCR were 94 ° C for 5 minutes, followed by 30 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 72 ° C for 30 seconds, and further maintained at 72 ° C for 7 minutes.
- the obtained DNA fragment was inserted into pCR-TOPO (INVITROGEN) to obtain plasmid pSPB811. From this plasmid, an approximately 1.2 kb F3H 5′-untranslated region can be recovered using HindIII and BamHI. As described in Reference Example 5, the promoter portion of pSPB567 was replaced with an approximately 1.2 kb F3H 5′-untranslated region using HindIII and BamHI, and plasmid pSFL814 (R.
- Reference Example 8 Preparation of pBIN19 rose CHSpro :: ADH-Pansie F3'5'H # 18 :: NOSter ABI KpnI Forward (5'-CGGTACCGTCTATTTAACTCAGTATTC-3 ', SEQ ID NO: 32) and GUS19R (5 A DNA fragment amplified by PCR using '-TTTCTACAGGACGTAACATAAGGGA-3', SEQ ID NO: 33) as a primer was digested with KpnI and SmaI to obtain an approximately 110 bp tobacco ADH-5'UTR DNA fragment.
- This DNA fragment was linked to the DNA fragment of the binary vector obtained by digesting pBRBP18 (with pBIN19 and rose CHSpro :: pansy F3'5'H # 18 :: NOSter expression cassette) with KpnI and SmaI.
- pBIN19 rose CHSpro :: ADH-pansy F3 ′, 5′H # 18 :: NOSter was obtained.
- this plasmid there is a 38b spacer between tobacco ADH-5'UTR and pansy F3'5'H # 18. This plasmid was introduced into Agrobacterium tumefaciens EHA105 strain.
- Reference Example 9 Preparation of pBI121-Rose CHSpro :: ADHNF-Pansy F3'5'H # 40 :: NOSter No. 85) and NS-RhCHSpro3k-Rv (5′-AAAGCTAGCACTAGTCATCTCGGAGAAGGGTCG-3 ′, SEQ ID NO: 86) were used as primers, and a DNA fragment obtained by PCR using Pyrobest Polymerase (Takara) was digested with HindIII and NheI.
- PBI121-RhCHSp-GUS-NOSt was a binary vector obtained by ligating pBI121 ADHNF with a fragment of a binary vector obtained by digesting with HindIII and XbaI.
- the 5′H # 40 DNA fragment was ligated to obtain pBI121-rose CHSpro :: ADHNF-pansy F3′5′H # 40 :: NOSter and transformed into Agrobacterium tumefaciens EHA105 strain. Using this transformed Agrobacterium, 19 recombinant chrysanthemum varieties derived from chrysanthemum cultivar 94-765 were obtained, but no individual was detected for delphinidin.
- Example 1 Cloning of chrysanthemum flavanone 3-hydroxylase gene promoter region F3H promoter region obtained by screening genomic library using chrysanthemum flavanone 3-hydroxylase gene (F3H) cDNA fragment PBluescript SK - gF3H9 (Ogano et al.
- HANS-F3Hpro1k-Fd (5'-CCAAGCTTGGCGCGCCGCGGCCGCATTTAAAT TTACAAAACCATGTGCAAGAATG- 3 ′, SEQ ID NO: 36, the underlined portion is a sequence that anneals to DNA containing the F3H promoter region) and SNM-F3Hpro-Rv (5′-ACTAGTGCTAGCACGCG TTTTTTATTTTTTCTTCACACACTTG- 3 ′, SEQ ID NO: 37, the underlined portion is the F3H promoter region
- a DNA fragment amplified by PCR using a primer containing a DNA containing a DNA containing DNA as a primer was cloned into pCR2.1 (Invitrogen) to obtain pCR HANS-CmF3Hp1k-NS.
- PCR was performed using HANS-F3Hpro1k-Fd and NSM-F3Hpro-Rv (5'-GCTAGCACTAGTACGCG TTTTTTATTTTTTCTTCACACACTTG- 3 ', SEQ ID NO: 38, the sequence that anneals to the DNA containing the F3H promoter region) as primers.
- the amplified DNA fragment was cloned into pCR2.1 to obtain pCR HANS-CmF3Hp1k-SN.
- HANS-F3Hpro500-Fd (5'-CCAAGCTTGGCGCGCCGCGGCCGCATTTAAAT TACTGTTCGAACCTACAAAGG- 3 ', SEQ ID NO: 83, underlined is a sequence that anneals to DNA containing the F3H promoter region), MX-F3Hpro-Rv 5'-TTTCTAGAACGCGT TTTTTATTTTTTCTTCACACACTTG -3 ', SEQ ID NO: 84, underlined portion is a sequence that anneals to DNA containing the F3H promoter region) as a primer, and a DNA fragment amplified by PCR was cloned into pCR2.1 and pCR HANS -CmF3Hpro500-X was obtained.
- Example 2 Preparation of pBI121 chrysanthemum F3Hpro1k :: ADHNF-campanula F3'5'H :: NOSter Translation sequence of F3'5'HcDNA (accession number D14590) of Campanula registered in DNA database GenBank Based on the above, two kinds of primers, CamF1 (5′-GTGAAGCCACCATGTCTATAG-3 ′, SEQ ID NO: 49) and CamR1 (5′-GCATTTGCCTAGACAGTGTAAG-3 ′, SEQ ID NO: 50) were synthesized.
- CamF1 (5′-GTGAAGCCACCATGTCTATAG-3 ′, SEQ ID NO: 49
- CamR1 5′-GCATTTGCCTAGACAGTGTAAG-3 ′, SEQ ID NO: 50
- RNA was extracted from the petal of a commercially available campanula using RNeasy Mini Plant Kit (QIAGEN), and 1st strand DNA was synthesized using RT-PCR kit. Using this 1st strand DNA as a template, PCR was performed using primers. The obtained DNA fragment was cloned into pCR-TOPO II. Among these, the base sequence of clone # 4 (referred to as pSPB2561) was determined to be SEQ ID NO: 51.
- a vector in which tobacco ADH-5′UTR 94bp and F3′5′H gene were linked was constructed as follows (FIG. 4). The same applies to the examples described later.
- Two types of DNA fragments were mixed and used as a template, and PCR was performed using XbaI-
- chrysanthemum varieties derived from chrysanthemum variety 94-765 were obtained. Delphinidin was detected in 30 petals, and the delphinidin content reached 80.5%.
- pSPB3738 was constructed from pBI121 chrysanthemum F3Hpro1k :: ADHNF-campanula F3′5′H :: NOSter. This plasmid was introduced into Agrobacterium tumefaciens AGL0 strain and used to transform chrysanthemum cultivar Sei Taitan (Seikoen). Among the 26 recombinant chrysanthemum obtained, 6 lines showed a change in flower color, and delphinidin could be detected by thin layer chromatography.
- Example 3 pIG121-Hm- chrysanthemum F3Hpro1k :: ADHNF- lisianthus F3'5'H :: Noster Preparation pBluescript SK - was cloned into the lisianthus F3'5'H gene (EgF3'5'H, GenBank AB078957), and After digestion with XhoI, blunting with Blunting High (TOYOBO), pIG121-obtained by digesting with XbaI and EcoICRI an about 1.9 kb EgF3′5′H DNA fragment obtained by digestion with XbaI PIG121-Hm 35S :: EgF3′5′H was obtained by ligation to a fragment of the Hm binary vector.
- ADH-EgF3'5'H-Fd (5'-CAAGAAAAATAAATGGCTGTTGGAAATGGCGTT-3 ', SEQ ID NO: 40) and HpaI-EgF3'5'H-Rv (5' -GTTAACGCTGAGCCTAGTGCC-3 ', SEQ ID NO: 41) as a primer, a DNA fragment amplified by PCR, and pBI221 ADH-221 (Satoh J.
- This pIG121-Hm 35S ADHNF-EgF3'5'H digested with HindIII and XbaI, about 1.2 kb EgF3'5'H DNA fragment and about 15 kb binary vector DNA fragment, or pCR A DNA fragment obtained by digesting HANS-CmF3Hp1k-MNS with HindIII and SpeI was ligated to obtain pIG121-Hm chrysanthemum F3Hpro1k :: ADHNF-Eustoma F3'5'H :: NOSter.
- This plasmid was introduced into Agrobacterium tumefaciens EHA105 strain.
- Example 4 pBI121 Chrysanthemum F3Hpro1k :: ADHNF- Lobelia F3'5'H :: Preparation pBluescript of Noster SK - of lobelia from petals cloned into F3'5'H gene (LeF3'5'H1, GenBank AB221077 and LeF3 '5'H4, GenBank AB221078) was digested with KpnI, blunted with Blunting High (TOYOBO), and further digested with XbaI to obtain an approximately 1.9 kb EgF3'5'H DNA fragment, PIG121-Hm 35S :: LeF3′5′H1 and pIG121-Hm 35S :: LeF3′5′H4 were obtained by ligation to a fragment of the pIG121-Hm binary vector obtained by digestion with EcoICRI.
- ADH-LeF3'5'H-Fd (5'-CAAGAAAAATAAATGGACGCGACAWACATTGC-3 ', SEQ ID NO: 45) and HpaI- A DNA fragment amplified by PCR using LeF3′5′H-Rv (5′-GTTAACATCTCGGGCAGCACC-3 ′, SEQ ID NO: 46) as a primer, and XbaI-ADH-Fd (SEQ ID NO: 42) using pBI221 ADH-221 as a template.
- LeF3'5'H-ADH-Rv (5'-TGTCGCGTCCATTTATTTTTCTTGATTTCCTTCAC-3 ', SEQ ID NO: 47) were used as a template by mixing two types of DNA fragments amplified with primers, and XbaI-ADH-Fd (SEQ ID NO: 42) and HpaI-LeF3′5′H-Rv (5′-GTTAACATCTCGGGCAGCACC-3 ′, SEQ ID NO: 48) as a primer by PCR, and tobacco ADH-5′UTR 94bp is LeF3′5′H1 or LeF3′5 DNA fragments directly linked to the start codon of 'H4 were obtained.
- DNA fragments are each TA cloned into pCR2.1, and then digested with XbaI and HpaI to obtain an approximately 800b DNA fragment, pIG121-Hm 35S :: LeF3'5'H1 or pIG121-Hm 35S :: LeF3 PIG121-Hm 35S :: ADHNF-LeF3'5'H1 and pIG121-Hm 35S :: ADHNF-LeF3 by ligating the binary vector fragments obtained by digesting '5'H4 with XbaI and HpaI, respectively. '5' H4 was obtained. Approx.
- ADHNF-LeF3'5'H1 NOSter DNA fragment obtained by digesting these binary vectors with XbaI and EcoRV, and binary vector obtained by digesting pBI121 HANS-CmF3Hp1k-S with SpeI and EcoICRI By ligating the fragments, pBI121 chrysanthemum F3Hpro1k pro :: ADHNF-Robelia F3′5′H1 :: NOSter and pBI121 chrysanthemum F3Hpro1k pro :: ADHNF-Robelia F3′5′H4 :: NOSter were obtained.
- Example 5 Preparation of pBINPLUS chrysanthemum F3Hpro1k :: ADHNF-pansy F3'5'H # 40 :: NOSter About 1.1 kb chrysanthemum F3H promoter DNA fragment obtained by digesting pCR HANS-CmF3Hp1k-BclI with AscI and BclI PBinPLUS Chrysanthemum F3Hpro :: ADHNF-Pansy F3'5'H # 40 :: NOSTer is digested with AscI and BamHI to ligate the binary vector fragment, pBinPLUS Chrysanthemum F3Hpro1k :: ADHNF-Pansy F3 I got '5'H # 40 :: NOSter.
- This plasmid was introduced into Agrobacterium tumefaciens EHA105 strain. Using this transformed Agrobacterium, 6 recombinant chrysanthemum varieties derived from chrysanthemum variety 94-765 were obtained. Among them, delphinidin was detected in 4 petals, and the delphinidin content reached 26.8%.
- Example 6 Production of pBI121 chrysanthemum F3Hpro1k :: ADHNF-Cinerealia F3'5'H :: NOSter and introduction into chrysanthemum CENALARIA F3'5'H (pSPB2774) obtained in Reference Example 2 was used as a template for ADH-ScF3 ' PCR was performed using 5'H-Fd (5'-CAAGAAAAATAAATGAGCATTCTAACCCTAATC-3 ', SEQ ID NO: 57) and NdeI-ScF3'5'H-Rv (5'-CATATGTTTAGCTCCAGAATTTGG-3', SEQ ID NO: 58) as primers.
- 5'H-Fd 5'-CAAGAAAAATAAATGAGCATTCTAACCCTAATC-3 ', SEQ ID NO: 57
- NdeI-ScF3'5'H-Rv 5'-CATATGTTTAGCTCCAGAATTTGG-3', SEQ ID
- Two types of DNA fragments amplified in step 1 were mixed and used as a template.
- XbaI-ADH-Fd (SEQ ID NO: 42) and NdeI-ScF3'5'H-Rv (5'-CATATGTTTAGCTCCAGAATTTGG-3 ', SEQ ID NO: 60) were used.
- a DNA fragment in which tobacco ADH-5′UTR 94 bp was directly linked to the initiation codon of Cinearia F3′5′H was obtained by PCR using the primer. After TA cloning this DNA fragment into pCR2.1, the DNA fragment obtained by digesting with XbaI and NdeI and the vector fragment obtained by digesting pSPB2774 with XbaI and NdeI were ligated to pBluescript Sk - ADHNF -Obtained Cineraria F3'5'H.
- pBluescript Sk - ADHNF-Cinearia F3'5'H was digested with XbaI and XhoI and the approximately 1.7 kb DNA fragment was ligated with the vector fragment obtained by digesting pCR2.1 with XbaI and XhoI. As a result, pCR2.1 ADHNF-Cinearia F3′5′H was obtained.
- Example 7 pBI121 Chrysanthemum F3Hpro1k :: ADHNF- Gentiana F3'5'H :: Preparation of Noster pBluescript SK - a cloned into the gentian F3'5'H (described plasmid pG48, WO2004 / 020637) as a template ADH- DNA fragment amplified by PCR using Gentian-Fd (5'-CAAGAAAAATAAATGTCACCCATTTACACCACCC-3 ', SEQ ID NO: 61) and SalI-GentianF3'5'H-Rv (5'-GTCGACGCTATTGCTAAGCC-3', SEQ ID NO: 62) as primers DNA fragments amplified by PCR using pBI221 ADH-221 as template and XbaI-ADH-Fd (SEQ ID NO: 42) and Gentian-ADH-Rv (5'-AATGGGTGACATTTATTTTTCTTGATTTCCTTCAC-3
- Example 8 pBI121 Chrysanthemum F3Hpro1k :: ADHNF- verbena F3'5'H :: Preparation pBluescript of Noster SK - cloned into the verbena F3'5'H (pHVF7, Plant Biotechonology 23, 5-11, 2006, DNA Database ADH-Verbena-Fd (5'-CAAGAAAAATAAATGACGTTTTCAGAGCTTATAAAC-3 ', SEQ ID NO: 65) and NcoI-VerbenaF3'5'H-Rv (5'-CCATGGAGTAAATCAGCATCTC-3', SEQ ID NO: 66) As a primer, a DNA fragment amplified by PCR, and XbaI-ADH-Fd (SEQ ID NO: 42) and Verbena-ADH-Rv (5'-TGAAAACGTCATTTATTTTTCTTGATTTCCTTCAC-3 ', SEQ ID NO: 67) using pBI2
- pBluescript SK - ADHNF-Verbena F3'5'H was digested with XbaI and XhoI and the 1.8 kb DNA fragment was ligated with the vector fragment obtained by digesting pCR2.1 with XbaI and XhoI.
- PCR2.1 ADHNF-Verbena F3′5′H was obtained.
- Example 9 Preparation of pBI121 Chrysanthemum F3Hpro1k :: ADHNF-Blue Qualcomm F3'5'H :: NOSter
- a Uni-ZAP XR Vector kit (STRATAGENE) is used as a type of Qualcomm by the method recommended by the manufacturer (
- a cDNA library was prepared using mRNA obtained from anther of Antirrhinum kelloggii. This library was screened by the method described in Reference Example 2, and plasmids pSPB3145 and pSPB3146 containing two types of F3'5'HcDNA # 1 (SEQ ID NO: 69) and F3'5'HcDNA # 12 (SEQ ID NO: 71), respectively.
- ADH-AkF3'5'H-Fd (5'-CAAGAAAAATAAATGCAGATAATAATTCCGGTCC-3 ', SEQ ID NO: 73) and NsiI-AkF3'5'H-Rv (5'-ATGCATGTCCTCTAACATGTATC-3', SEQ ID NO: 74) as a primer and a DNA fragment amplified by PCR
- XBI-ADH-Fd SEQ ID NO: 42
- AkF3'5'H-ADH-Rv (5'-TATTATCTGCATTTATTTTTCTTGATTTCCTTCAC-3 'using pBI221 ADH-221 as a template , SEQ ID NO: 75) was used as a template by mixing two types of DNA fragments amplified by PCR using primers, and XbaI-ADH-Fd (SEQ ID NO: 42) and NsiI-AkF3
- This DNA fragment is TA-cloned into pCR2.1 and then digested with XbaI and NsiI.
- the vector fragments obtained by digesting 5′H # 12) with XbaI and NsiI were respectively ligated to obtain pBluescript SK - ADHNF-AkF3′5′H # 1 and # 12.
- pBluescript SK - ADHNF-AkF3'5'H # 1 and # 12 are digested with XbaI and XhoI, about 700b of DNA fragment, and pCR2.1 is digested with XbaI and XhoI. Were linked to obtain pCR2.1 ADHNF-AkF3′5′H # 1 and # 12.
- pBI121 chrysanthemum F3Hpro1k :: ADHNF-AkF3′5′H # 1 :: NOSter and pBI121 chrysanthemum F3Hpro1k :: ADHNF-AkF3′5′H # 12 :: NOSter were obtained.
- These plasmids were introduced into Agrobacterium tumefaciens EHA105 strain.
- Example 10 Preparation of pBI121-Chrysanthemum F3Hpro500 :: ADHNF-Cinearia F3'5'H :: NOSter DNA fragment of binary vector obtained by digesting pBI121 HANS-CmF3Hp500-X obtained in Example 1 with XbaI and EcoICRI PBI121-Chrysanthemum ligated with the DNA fragment of ADHNF-Cinearia F3'5'H obtained by digesting pCR2.1 ADHNF-Cinearia F3'5'H obtained in Example 6 with XbaI and EcoRV.
- F3Hpro500 ADHNF-Cinearia
- F3′5′H NOSter was obtained and transformed into Agrobacterium tumefaciens EHA105 strain. Using this transformed Agrobacterium, 7 recombinant chrysanthemums derived from chrysanthemum cultivar Ohira were obtained. Of these, delphinidin was detected in 5 strains, and the delphinidin content reached 25.5%.
- flavonoid 3 ′, 5′-hydroxylase (F3′5′H) is expressed in chrysanthemum using the transcriptional regulatory region of chrysanthemum-derived flavanone 3-hydroxylase (F3H), and delphinidin is expressed in petals.
- F3H chrysanthemum-derived flavanone 3-hydroxylase
- delphinidin is expressed in petals.
- Chrysanthemum has flower colors such as white, yellow, orange, red, peach and purple red, but existing varieties and closely related wild species do not have blue flowers such as purple and blue.
- the blue chrysanthemum produced by the method leads to new demand.
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Abstract
Description
アントシアニンの生合成に関わる酵素や該酵素をコードする遺伝子はよく研究されている(同非特許文献1参照)。例えば、アントシアニンに芳香族アシル基を転移する反応を触媒する酵素遺伝子はリンドウ、ラベンダー、ペチュニアなどから得られている(以下、特許文献1、特許文献2参照)。赤ジソの葉に蓄積されるアントシアニン(マロニルシソニン、3-O-(6-O-(E)-p-coumaroyl-β-D-glucopyranosyl)-5-O-(6-O-malonyl-β-D-glucopyranosyl)-cyanidin)(以下、非特許文献2参照)の合成に関わる酵素遺伝子は、ヒドロキシシナモイルCoA:アントシアニン3グルコシド-芳香族アシル基転移酵素(3AT)の遺伝子{以下、単に「シソアントシアニン3-アシル基転移酵素(3AT)遺伝子」ともいう。}を始め既に報告されている(特許文献1参照)。さらにアントシアニンの生合成酵素の遺伝子の転写制御(調節)についても知見が得られている。これらの遺伝子の開始コドンの5'側に位置する転写調節領域の中には、Myb型転写調節因子、bHLH型転写調節因子が結合するシスエレメント配列がある。Myb型転写調節因子とbHLH型転写調節因子がペチュニア、トウモロコシ、シソなどのアントシアニンの合成を制御していることも知られている(非特許文献1参照)。
したがって、いくつかのプロモーターが花の色を変えるために使われてきたが、さらに別の花の色を変えるために宿主植物と目的に応じた有用なプロモーターが求められている。
したがって、青色系の育種は、新たな需要を喚起するための目標の一つとされている。キクの花色は、アントシアニンとカロテノイドの組み合わせにより発現している。アントシアニンは基本骨格であるアントシアニジンの構造の違いや、糖や有機酸による修飾の違いなどにより、多様な色を発現できる。しかし、キクの花色を担うアントシアニンは、シアニジンの3位がグルコースとマロン酸により修飾された二種類(cyanidin 3-O-(6"-O-monomalonyl-β-glucopyranosideとcyanidin 3-O-(3",6"-O-dimalonyl-β-glucopyranoside)であることが知られている(以下、非特許文献6参照)。また、それらの構造は比較的単純である(図1参照)。これにより、キクのアントシアニンによる発色の幅が極めて小さい要因になっている。ところで、青色系の発色は、主にアントシアニンによるものであるが、キクには、鍵となる酵素であるフラボノイド3',5'-水酸化酵素(F3'5'H)をコードする遺伝子が存在しないため、青を発色するデルフィニジン系アントシアニンが生合成されない(図1参照)。そこで、キクの花弁に蓄積するアントシアニン系色素を改変して、青色系の花色をもつキクの作出を可能にするために、遺伝子工学的手法を用いたキクのアントシアニンの発現制御技術の開発が望まれている。
すなわち、本発明は、以下の通りである。
(1)配列番号34又は配列番号87に示す塩基配列を含む核酸、
(2)キク由来フラバノン3-水酸化酵素(F3H)遺伝子の転写調節領域として機能することができ、かつ、配列番号34又は配列番号87に示す塩基配列に対して1個又は数個の塩基配列の付加、欠失及び/又は置換により修飾された塩基配列を含む核酸、
(3)キク由来フラバノン3-水酸化酵素(F3H)遺伝子の転写調節領域として機能することができ、かつ、配列番号34又は配列番号87に示す塩基配列に対して相補的な塩基配列から成る核酸と高ストリンジェンシー条件下でハイブリダイズすることができる核酸、及び
(4)キク由来フラバノン3-水酸化酵素(F3H)遺伝子の転写調節領域として機能することができ、かつ、配列番号34又は配列番号87に示す塩基配列に対して少なくとも90%の配列同一性を有する核酸、
から成る群から選ばれる核酸を転写調節領域として用いて、フラボノイド3',5'-水酸化酵素(F3'5'H)をキク植物において発現させて、その花弁にデルフィニジンを含むキク植物を生産する方法。
[8]配列[7]に記載の切り花を用いた切り花加工品。
本明細書中、「発現カセット」とは、任意の核酸にプロモーターとターミネーターを連結したDNA断片をいう。
本発明は、上記切り花を用いた加工品(切り花加工品)にも関する。ここで、切り花加工品としては、当該切り花を用いた押し花、プリザーブドフラワー、ドライフラワー、樹脂密封品などを含むが、これに限定されるものではない。
分子生物学的手法は特に断らない限り、Molecular Cloning(Sambrook and Russell, 2001)に依った。
特許文献6に記載されたpBIEF1αを、制限酵素HindIIIとBamHIで消化することにより、タバコEF1αのプロモーター配列を含む約1.2kbのDNA断片を得た。このDNA断片を、特許文献4に記載されたpSPB909のアイリスDFR cDNAの5'側に挿入することによりプラスミドpSFL339を得た。同様に、アイリスDFR cDNAの代わりにペチュニアDFR cDNA(国際公開公報WO 96/36716に記載)を挿入したプラスミドpSFL340も構築した。
青いサイネリアセネッティー(サントリーフラワーズ社)の蕾の花弁から定法に基づいてRNAを抽出した。このRNAから調製したpoly-A + RNAを用いて、ZAP-cDNA(登録商標)Library Construction Kit(ストラタジーン社、製品番号200450)を用いて製造者の推奨する方法に従って、cDNAライブラリーを作製した。このcDNAライブラリーを、DIGシステム(Roche Applied Science社)で標識したチョウマメF3'5'H cDNA(Clitoria ternatea)(Plant Biotechnology 23, 5-11 (2006)参照)を用いて、同社が推奨する方法でスクリーニングを行った。得られた48個のシグナルを示すファージを純化した。これらのファージから製造者(ストラタジーン社)の推奨する方法でin vivo excisionによりプラスミドを得た。
国際特許出願PCT/AU03/01111に記載されているバラ由来のカルコン合成酵素プロモーターにパンジー由来のF3'5'HBP#18遺伝子を連結したバイナリーベクターを構築し、pBRBP18とした。参考例1及び2に記載したように、このバイナリーベクターが含む遺伝子をキク品種キク品種94-765に導入した。形質転換キクの花弁のアントシアニジンを分析したところ、全アントシアニジンに対し最高5.4%のデルフィニジンが検出されたが、花の色に変化は見られなかった。
また、バラカルコン合成酵素遺伝子プロモーターを用いたデルフィニジン生産の例として、表1中、上から9番目に記載するpSPB3325(バラCHSpro::パンジー#18+バラCHSp::キクF3'H IR)が挙げられ、この例では、デルフィニジン生産は最高3.6%であった。
(1)シソアントシアニン3-アシル基転移酵素染色体遺伝子のクローニング
シソにはアントシアニンを葉に蓄積する赤い変種と蓄積しない緑色の変種が知られている。前者の葉から、その染色体DNAを、報告されている方法(Plant Mol Biol. December 1997; 35(6):915-27参照)で調製した。この染色体DNAをSau3AI(TOYOBO社製)で部分分解し、ショ糖密度勾配法により10~15kbのDNA断片を含む画分を回収した。この断片を、公知の方法を用いてラムダファージベクターの一種であるEMBL3(Promega社製)のBamHI部位に挿入することにより、染色体DNAライブラリーを作製した。得られたライブラリーをシソに由来するアントシアニン3-アシル基転移酵素のcDNAであるpSAT208(Plant Cell Physiol. April 2000; 41(4):495-502参照)をプローブとして用いてスクリーニングした。ライブラリーのスクリーニングは、既に報告されている方法(Plant Cell Physiol. July 1996; 37(5):711-6参照)に拠った。プローブとハイブリダイズしたプラークを純化後、培養し、得られたファージからDNAを調製した。
上記で得たDNA10μgをXbaIで消化し、0.7%アガロースゲルで分離した後、ハイボンドN(アマシャム社製)にブロットした。この膜を上記と同じようにハイブリダイズしたところ、約6.8kbのDNA断片がプローブとハイブリダイズすることがわかった。同じDNA20μgをXbaIで消化し、0.7%アガロースゲルで分離した後、約6.8kbのDNA断片を、ジーンクリーンを用いて精製し、XbaIで消化したpBluescriptSKII-と連結した。得られたプラスミドをpSPB513とした。このプラスミドに含まれるシソ由来のDNA配列をプライマーウォーキング法により決定した。その塩基配列を配列番号4に示す。この配列にはアントシアニン3-アシル基cDNAであるpSAT208と高い相同性を示す領域があり、この領域がコードする蛋白質のアミノ酸配列(配列番号6)はpSAT208のコードするアミノ酸配列と比べて19個のアミノ酸残基の置換と2個のアミノ酸の欠失が見られ、イントロンは観察されなかった。また、上記pSAT208と高い相同性を示す領域の配列は、その開始コドンと考えられるATGの上流に3438bpの配列と、その終止コドンと考えられるTAAの下流に2052bpの配列を含んでいた。上記3438bpの配列内には、アントシアニン3-アシル基転移酵素ではない別のオープンリーディングフレーム(ORF、配列番号5)が存在した。この部分を除く、シソアントシアニン3-アシル基転移酵素遺伝子の転写調節領域を増幅するために、以下の実験を行った。
1ngのpSPB513を鋳型として2種のプライマー(5’-AAGCTTAACTATTATGATCCCACAGAG-3’(配列番号7、下線部はHindIIIの認識配列)、5’-GGATCCGGCGGTGTTGAACGTAGC-3’(配列番号8、下線部はBamHIの認識配列)を用いて、PCR(95℃1分間保持後、52℃1分間、72℃2分間、95℃1分間からなる反応を25サイクル)を行なった。増幅された約1.1kbのDNA断片をHindIIIとBamHIで消化した。
パンジーのフラボノイド3',5'-水酸化酵素遺伝子BP#40(WO 04/020637参照)の転写調節領域をTaKaRa LA PCRTM in vitro Cloning Kitを用いて、以下のように増幅した。
パンジーの葉からDNA easy plant kit (QIAGEN社)を用いて染色体DNAを調製した。3μgの染色体DNAを制限酵素HindIIIにより消化した。消化したDNAをHindIII末端DNA(TaKaRa LA PCRTM in vitro Cloning Kitに含まれる)と、Ligation High (TaKaRa)を用いて、16℃で40分間反応させ、連結した。反応液4μlを10μlの水で薄め、連結したDNAを94℃10分間の処理により変性させた後、氷中で冷却した。5pmolのプライマーC1 (5’-GTACATATTGTCGTTAGAACGCGTAATACGACTCA-3’、配列番号9、HindIIIカセット配列の一部の配列でKitに含まれる)と5pmolのプライマーBP40-i5 (5’-AGGTGCATGATCGGACCATACTTC-3’、配列番号10、BP#40の翻訳領域の相補鎖に相当する)を加え、キットのプロトコールに従い、25μlの反応液で、98℃20秒間、68℃で15分間の反応を30回繰り返した。反応液を水で10倍に希釈した。このうち、0.5μlを鋳型として、5pmolのプライマーC2(5’-CGTTAGAACGCGTAATACGACTCACTATAGGGAGA-3’、配列番号11、HindIIIカセット配列の一部の配列でKitに含まれる)と5pmolのプライマー BP40-i7 (5’-GACCATACTTCTTAGCGAGTTTGGC-3’、配列番号12)を含む25μlの反応液で、98℃で5分間反応後、98℃で20秒間、68℃で15分間の反応を30回繰り返した。
約2kbのBP#40の5'-非翻訳領域と200bpの翻訳領域を増幅するために、200ngのパンジーの染色体DNAを鋳型として、50pmolのプライマー BP40-i7 (配列番号12)と50pmolのプライマーBP40 pro-F (5’-ACTCAAACAAGCATCTCGCCATAGG-3’、配列番号13、BP#40遺伝子の5'-非翻訳領域にある配列)を用いて25μlの反応液でPCRを行なった。98℃5分間処理した後、98℃20秒間と68℃15分間からなる反応を30回繰り返した。増幅されたDNA断片をpCR2.1に挿入した。このDNA断片には約2.1kbの5’-非翻訳領域と200bpの翻訳領域が含まれていた。このプラスミドをpSFL614とした。プラスミドpSFL614配列の塩基配列を配列番号14に示す。
pSFL620をPacIで消化後、約3.2kbのDNA断片を回収した。このDNA断片をpBin+のPacI部位に挿入した。得られたプラスミドをpSPB3317とした。上述のpSPB3311をAscIとXbaIで消化して得られる断片を、pSPB3317のAscIとXbaI部位に導入し、得られたプラスミドをpSPB3323とした。
上記(4)で調製したpSPB3323をアグロバクテリウムに導入し、このアグロバクテリウムを用いてキク品種94-765(精興園、未発売)を公知の方法により形質転換した。6系統の形質転換系統を取得した。
分析サンプルは、高速液体クロマトグラフィーを用いて以下の条件で分析した。カラムはInertsil ODS-2(粒径5μm, 4.6×250mm, ジーエルサイエンス)を用い、流速0.8ml/minで、移動相は1.5%リン酸を含む、5%酢酸、6.25%アセトニトリルから20%酢酸、25%アセトニトリルの直線濃度勾配20分間の後、1.5%リン酸および20%酢酸を含む25%アセトニトリルで5分間のイソクラティック溶出を行った。検出はAgilent 1100 シリーズ ダイオードアレイ検出器(ジーエルサイエンス)を用い、250nmから600nmの波長領域を検出し、530nmの吸光度の面積により各アントシアニジンの存在比を求めた。
分析の結果、形質転換体である分析系統1300-3、1300-4、1300-5、及び1300-6において、それぞれ、デルフィニジンが全アントシアニジンの0.9%、0.8%、1.4%、及び0.6%検出された。これは、パンジーのBP#40転写調節領域がBP#40の転写を司ったことを示唆するが、花色の変化にはいたらなかった。
ハマナスの染色体DNAライブラリーを、λBlueSTARTM Xho I Half-Site Arms Kit (Novagen, http://www.merckbiosciences.com/product/69242)を用いて、以下のように作製した。ハマナスの若い葉から、Nucleon Phytopure [Registered] (TEPNEL Life Sciences)を用いて染色体DNAを調製した。約100μgの染色体DNAを制限酵素Sau3AIで部分消化した。
このDNA断片をdGTP とdATPの存在下においてDNA polymerase I Klenow fragment (TOYOBO)で部分的にフィルインをし、ショ糖密度勾配遠心により分画した。約13kbの大きさにDNAを回収し、エタノール沈殿により濃縮した。約180ngのDNAを1μL の λBlueSTARTM Xho I Half-Site Arms Kitと4℃で15時間連結した後、in vitro packaging反応を行ない、染色体ライブラリーを得た。
この形質転換アグロバクテリウムを用いて、品種94-765から組換えキクを得た。花弁でアントシアニジン全量のうち0.6%程度のデルフィジンを生産している系統があった。
また、ハマナスDFRプロモーターを用いた他の参考例は、表1中、上から2番目(pSPB3316(ハマナスDFRpro::パンジー#40+バラANSpro::トレニア5GT、デルフィニジン生産系統無し)と5番目(pSPB3325(ハマナスDFRpro::パンジー#40+リンドウ3'GTpro::トレニアMT、デルフィニジン最高0.9%)がある。いずれも花色の変化には至らなかった。
参考例5で作製したハマナス染色体DNAライブラリーをトレニアのフラバノン3-水酸化酵素(F3H) cDNA (NCBI No. AB211958)によりスクリーニングし、シグナルを示すプラークを得た。そのうち1プラークについて参考例5と同様にプラスミドに変換した。これを制限酵素SpeIで消化し、2.6kbのDNA断片を回収し、pBluescript SKII- (STRATAGENE)のSpeI部位にサブクローングすることにより、プラスミドpSPB804を得た。このプラスミドはF3Hに相同性を示す塩基配列を有していた。
この形質転換アグロバクテリウムを用いて、品種94-765から組換えキクを3系統得たが、花弁でデルフィニジン生産が見られた系統はなかった(表1参照)。
pSFL814(参考例6)を鋳型に、ADH-BP40-Fd(5'-CAAGAAAAATAAATGGCAATTCTAGTCACCGAC-3'、配列番号26)とNcoI-BP40-Rv(5'-CTCGAGCGTACGTGAGCATC-3'、配列番号27)をプライマーに用いてPCRで増幅したDNA断片、及びpBI221 ADH-221を鋳型にBamHI-ADH-Fd(5'-CGCGGATCCGTCTATTTAACTCAGTATTC-3'、配列番号28)とBP40-ADH-Rv(5'-TAGAATTGCCATTTATTTTTCTTGATTTCCTTCAC-3'、配列番号29)をプライマーに用いてPCRで増幅したDNA断片を混合して鋳型に用い、BamHI-ADH-Fd(5'-CGCGGATCCGTCTATTTAACTCAGTATTC-3'、配列番号30)とNcoI-BP40-Rv(5'-CTCGAGCGTACGTGAGCATC-3'、配列番号31)をプライマーに用いて、PCRにより、タバコADH-5'UTR 94bpがパンジーF3'5'H#40の開始コドンに直結したDNA断片を得た。
この形質転換アグロバクテリウムを用いて、キク品種94-765由来の得られた形質転換体4系統のうちでデルフィニジンが検出された個体はなかった(表1参照)。
pBI221 ADH221を鋳型に、ADH KpnI Forward (5'-CGGTACCGTCTATTTAACTCAGTATTC-3'、配列番号32)とGUS19R(5'-TTTCTACAGGACGTAACATAAGGGA-3'、配列番号33)をプライマーに用いてPCRで増幅したDNA断片を、KpnIとSmaIで消化して約110bpのタバコADH-5'UTR DNA断片を得た。このDNA断片と、pBRBP18(pBIN19上にバラCHSpro::パンジーF3'5'H#18::NOSterの発現カセットを入れたもの)をKpnIとSmaIで消化して得られるバイナリーベクターのDNA断片を連結して、pBIN19バラCHSpro::ADH-パンジーF3',5'H#18::NOSterを得た。このプラスミドでは、タバコADH-5'UTRとパンジーF3'5'H#18の間に38bのスペーサーが存在する。このプラスミドをAgrobacterium tumefaciens EHA105株に導入した。
この形質転換アグロバクテリウムを用いて、キク品種94-765由来の組換えキク30系統を得た。このうち5系統の花弁でデルフィニジンが検出され、デルフィニジン含有率は1.9%に達した。しかし、花色の変化は見られなかった。
pBRBP18(参考例3)を鋳型に、HAPS-RhCHSpro3k-Fd(5'-CCAAGCTTGGCGCGCCTTAATTAAATTTAAATCAGCAAGAGTTGAAGAAATAG-3'、配列番号85)とNS-RhCHSpro3k-Rv(5'-AAAGCTAGCACTAGTCATCTCGGAGAAGGGTCG-3'、配列番号86)をプライマーとして用い、Pyrobest Polymerase(Takara)を用いたPCRにより得られたDNA断片を、HindIIIとNheIで消化し、pBI121 ADHNFをHindIIIとXbaIで消化して得られるバイナリーベクターの断片と連結することにより得られたバイナリーベクターをpBI121-RhCHSp-GUS-NOStとした。
pBI121-RhCHSp-GUS-NOStをSpeIおよびEcoICRIで消化して得られるバイナリーベクターの断片に、実施例10で得られたpCR-ADHBP40-SpeSacをSpeI及びEcoICRIで消化して得られるADHNF-パンジーF3'5'H #40 DNA断片を、連結して、pBI121-バラCHSpro::ADHNF-パンジーF3'5'H#40::NOSterを得て、Agrobacterium tumefaciens EHA105株に形質転換した。
この形質転換アグロバクテリウムを用いて、キク品種94-765由来の組換えキク19系統を得たが、デルフィニジンが検出された個体はなかった。
キクのフラバノン3-水酸化酵素遺伝子(F3H)cDNA断片を用いたゲノミックライブラリのスクリーニングにより得られた、F3Hのプロモーター領域を含むDNA断片をサブクローニングして得られたpBluescript SK- gF3H9(菅野ら(2001)園学雑70(別2)193、配列番号35)を鋳型に、HANS-F3Hpro1k-Fd (5'-CCAAGCTTGGCGCGCCGCGGCCGCATTTAAATTTACAAAACCATGTGCAAGAATG-3'、配列番号36、下線部はF3Hのプロモーター領域を含むDNAとアニールする配列)と、SNM-F3Hpro-Rv(5'-ACTAGTGCTAGCACGCGTTTTTTATTTTTTCTTCACACACTTG-3'、配列番号37、下線部はF3Hのプロモーター領域を含むDNAとアニールする配列)をプライマーに用いてPCRにより増幅したDNA断片をpCR2.1(Invitrogen)にクローニングして、pCR HANS-CmF3Hp1k-NSを得た。また、HANS-F3Hpro1k-Fdと、NSM-F3Hpro-Rv(5'-GCTAGCACTAGTACGCGTTTTTTATTTTTTCTTCACACACTTG-3'、配列番号38、下線部はF3Hのプロモーター領域を含むDNAとアニールする配列)をプライマーに用いてPCRにより増幅したDNA断片をpCR2.1にクローニングして、pCR HANS-CmF3Hp1k-SNを得た。HANS-F3Hpro1k-FdとBclI-CmF3Hp-Rv(5'-TTTTGATCATTTTTTATTTTTTCTTCACACAGTG-3'、配列番号39、下線部はF3Hのプロモーター領域を含むDNAとアニールする配列)をプライマーに用いてPCRにより増幅したDNA断片をpCR2.1にクローニングしてpCR HANS-CmF3Hp1k-BclIを得た。さらに、pBI121 ADHNF(Satoh J. et al., (2004) J Biosci Bioengineer)をHindIII及びXbaIで消化して得られるバイナリーベクター断片とpCR HANS-CmF3Hp1k-SNをHindIII及びNheIで消化して得られる約1.1kbのキクF3HプロモーターDNA断片を連結することにより、pBI121 HANS-CmF3Hp1k-Sを得た。
pBluescript SK- gF3H9を鋳型に、HANS-F3Hpro500-Fd(5'-CCAAGCTTGGCGCGCCGCGGCCGCATTTAAATTACTGTTCGAACCTACAAAGG-3'、配列番号83、下線部はF3Hのプロモーター領域を含むDNAとアニールする配列)と、MX-F3Hpro-Rv(5'-TTTCTAGAACGCGTTTTTTATTTTTTCTTCACACACTTG-3'、配列番号84、下線部はF3Hのプロモーター領域を含むDNAとアニールする配列)をプライマーとして用いてPCRにより増幅したDNA断片をpCR2.1にクローニングして、pCR HANS-CmF3Hpro500-Xを得た。また、pBI121 ADHNFをHindIIIとXbaIで消化して得られるバイナリーベクターの断片と、pCR HANS-CmF3Hpro500-XをHindIII及びXbaIで消化して得られる約500bpのキクF3HプロモーターDNA断片を連結することにより、pBI121 HANS-CmF3Hp500-Xを得た。
DNAデータベースGenBankに登録されているカンパニュラ(Campanula medium)のF3'5'HcDNA(アクセション番号D14590)の翻訳配列に基づいて2種のプライマーCamF1 (5’-GTGAAGCCACCATGTCTATAG-3’、配列番号49)とCamR1 (5’-GCATTTGCCTAGACAGTGTAAG-3'、配列番号50)を合成した。市販のカンパニュラの蕾の花弁からRNeasy Mini Plant Kit (QIAGEN社)を用いてRNAを抽出し、RT-PCRキットを用いて1st strand DNAを合成した。この1st strand DNAを鋳型に、プライマーを用いてPCRを実施した。得られたDNA断片をpCR-TOPO IIにクローニングした。この内、クローン#4(pSPB2561とした)の塩基配列を配列番号51と決定した。
pSPB2561を鋳型に、ADH-Campa-Fd(5'-CAAGAAAAATAAATGTCTATAGACATAACCATTC-3'、配列番号53)とHpaI-Campa-Rv(5'-GTTAACATCTCTGGCACCACC-3'、配列番号54)をプライマーに用いてPCRで増幅したDNA断片、並びにpBI221 ADH-221を鋳型に、XbaI-ADH-Fd(配列番号42)とCampa-ADH-Rv(5'-GTCTATAGACATTTATTTTTCTTGATTTCCTTCAC-3'、配列番号55)をプライマーに用いてPCRで増幅したDNA断片の二種類を混合して鋳型に用い、XbaI-ADH-Fd(配列番号42)とHpaI-Campa-Rv(5'-GTTAACATCTCTGGCACCACC-3'、配列番号56)をプライマーに用いてPCRにより、タバコADH-5'UTR 94bpがカンパニュラF3'5'Hの開始コドンに直結したDNA断片を得た。このDNA断片をpCR2.1にTAクローニングした後にXbaIとHpaIで消化して得られる約650bの断片と、pSPB2561をXbaIとHpaIで消化して得られるベクター断片とを連結してpCR ADHNF-Campanula F3'5'Hを得た。
この形質転換アグロバクテリウムを用いて、キク品種94-765由来の組換えキク48系統を得た。このうち30系統の花弁でデルフィニジンが検出され、デルフィニジン含有率は80.5%に達した。
pBI121キクF3Hpro1k::ADHNF-カンパニュラF3'5'H::NOSterからpSPB3738を構築した。このプラスミドをAgrobacterium tumefaciens AGL0株に導入し、これを用いてキク品種Sei Taitan(精興園)を形質転換した。得られた26系統の組換えキクのうち6系統で花色の変化が見られ、薄層クロマトグラフィーにより、デルフィニジンを検出できた。
pBluescript SK-にクローニングしたトルコギキョウF3'5'H遺伝子(EgF3'5'H, GenBank AB078957)を、XhoIで消化した後に、Blunting High (TOYOBO)で平滑末端化し、さらに、XbaIで消化して得られた約1.9kbのEgF3'5'H DNA断片を、XbaI及びEcoICRIで消化して得られるpIG121-Hmバイナリーベクターの断片に連結することにより、pIG121-Hm 35S::EgF3'5'Hを得た。
次に、pBluescript SK- EgF3'5'Hを鋳型に、ADH-EgF3'5'H-Fd(5'-CAAGAAAAATAAATGGCTGTTGGAAATGGCGTT-3'、配列番号40)とHpaI-EgF3'5'H-Rv(5'-GTTAACGCTGAGCCTAGTGCC-3'、配列番号41)をプライマーに用いてPCRにより増幅したDNA断片、並びにpBI221 ADH-221(Satoh J. et al., (2004) J Biosci Bioengineer)を鋳型に、XbaI-ADH-Fd(5'-ACGCGTTCTAGAGTCTATTTAACTCAGTATTC-3'、配列番号42)及びEgF3'5'H-ADH-Rv(5'-TCCAACAGCCATTTATTTTTCTTGATTTCCTTCAC-3'、配列番号43)をプライマーに用いてPCRにより増幅したDNA断片の二種類を混合して鋳型に用い、XbaI-ADH-Fd(配列番号42)及びHpaI-EgF3'5'H-Rv(5'-GTTAACGCTGAGCCTAGTGCC-3'、配列番号44)をプライマーに用いてPCRにより、タバコADH-5'UTR 94bp(Satoh J. et al., (2004) J Biosci Bioengineer)がEgF3'5'Hの開始コドンに直結したDNA断片を得た。このDNA断片をpCR2.1にTAクローニングした後に、XbaI及びHpaIで消化して得られる約1.3kbのDNA断片と、pIG121-Hm 35S::EgF3'5'HをXbaI及びHpaIで消化して得られるバイナリーベクターの断片を連結することにより、pIG121-Hm 35S::ADHNF-EgF3'5'Hを得た。このpIG121-Hm 35S::ADHNF-EgF3'5'HをHindIII及びXbaIで消化して得られる約1.2kbのEgF3'5'HのDNA断片と約15kbのバイナリーベクターのDNA断片、さたにpCR HANS-CmF3Hp1k-MNSをHindIII及びSpeIで消化して得られるDNA断片を連結し、pIG121-Hm キクF3Hpro1k::ADHNF-トルコギキョウF3'5'H::NOSterを得た。このプラスミドをAgrobacterium tumefaciens EHA105株に導入した。
この形質転換アグロバクテリウムを用いて、キク品種94-765由来の組換えキク5系統を得た。このうち1系統の花弁でデルフィニジンが検出され、デルフィニジン含有率は4.4%であった。
pBluescript SK-にクローニングしたロベリアの花弁由来のF3'5'H遺伝子(LeF3'5'H1, GenBank AB221077及びLeF3'5'H4, GenBank AB221078)をKpnIで消化した後に、Blunting High (TOYOBO)で平滑末端化し、さらに、XbaIで消化して得られた約1.9kbのEgF3'5'H DNA断片を、XbaI及びEcoICRIで消化して得られるpIG121-Hmバイナリーベクターの断片に連結することにより、pIG121-Hm 35S::LeF3'5'H1及びpIG121-Hm 35S::LeF3'5'H4を得た。
pBI121-キクF3Hpro1k::ADHNF-ロベリアF3'5'H1::NOSterを形質転換したアグロバクテリウムを用いて、キク品種'94-765'由来の組換えキク12系統を得たが、デルフィニジンを含有する個体は得られなかった。また、pBI121-キクF3Hpro1k::ADHNF-ロベリアF3'5'H4::NOSterを形質転換したアグロバクテリウムを用いて、キク品種'94-765'由来の組換えキク34系統を得たが、デルフィニジンを含有する個体は得られなかった。
pCR HANS-CmF3Hp1k-BclIをAscI及びBclIで消化することにより得られる約1.1kbのキクF3HプロモーターDNA断片と、pBinPLUS ハマナスF3Hpro::ADHNF-パンジーF3'5'H#40::NOSTerをAscIとBamHIで消化することにより得られるバイナリーベクターの断片を連結することにより、pBinPLUSキクF3Hpro1k::ADHNF-パンジーF3'5'H#40::NOSterを得た。このプラスミドをAgrobacterium tumefaciens EHA105株に導入した。
この形質転換アグロバクテリウムを用いて、キク品種94-765由来の組換えキク6系統を得た。このうち4系統の花弁でデルフィニジンが検出され、デルフィニジン含有率は26.8%に達した。
参考例2で得たシネラリアF3'5'H(pSPB2774)を鋳型に、ADH-ScF3'5'H-Fd(5'-CAAGAAAAATAAATGAGCATTCTAACCCTAATC-3'、配列番号57)とNdeI-ScF3'5'H-Rv(5'-CATATGTTTAGCTCCAGAATTTGG-3'、配列番号58)をプライマーに用いてPCRで増幅したDNA断片、並びにpBI221 ADH-221を鋳型にXbaI-ADH-Fd(配列番号42)とScF3'5'H-ADH-Rv(5'-TAGAATGCTCATTTATTTTTCTTGATTTCCTTCAC-3'、配列番号59)をプライマーに用いてPCRで増幅したDNA断片の二種類を混合して鋳型に用い、XbaI-ADH-Fd(配列番号42)とNdeI-ScF3'5'H-Rv(5'-CATATGTTTAGCTCCAGAATTTGG-3'、配列番号60)をプライマーに用いてPCRにより、タバコADH-5'UTR 94bpがシネラリアF3'5'Hの開始コドンに直結したDNA断片を得た。このDNA断片をpCR2.1にTAクローニングした後に、XbaIとNdeIで消化して得られたDNA断片と、pSPB2774をXbaIとNdeIで消化して得られたベクター断片とを連結してpBluescript Sk- ADHNF-シネラリアF3'5'Hを得た。
この形質転換アグロバクテリウムを用いて、キク品種94-765由来の組換えキ50系統を得た。このうち37系統の花弁でデルフィニジンが検出され、デルフィニジン含有率は36.2%に達した。
pBluescript SK-にクローニングされたリンドウF3'5'H(プラスミドpG48、WO2004/020637に記載)を鋳型にADH-Gentian-Fd(5'-CAAGAAAAATAAATGTCACCCATTTACACCACCC-3'、配列番号61)とSalI-GentianF3'5'H-Rv(5'-GTCGACGCTATTGCTAAGCC-3'、配列番号62)をプライマーに用いてPCRで増幅したDNA断片、並びにpBI221 ADH-221を鋳型にXbaI-ADH-Fd(配列番号42)とGentian-ADH-Rv(5'-AATGGGTGACATTTATTTTTCTTGATTTCCTTCAC-3'、配列番号63)をプライマーに用いてPCRで増幅したDNA断片の二種類を混合して鋳型に用い、XbaI-ADH-Fd(配列番号42)とSalI-GentianF3'5'H-Rv(5'-GTCGACGCTATTGCTAAGCC-3'、配列番号64)をプライマーに用いたPCRにより、タバコADH-5'UTR 94bpがリンドウF3'5'Hの開始コドンに直結したDNA断片を得た。このDNA断片をpCR2.1にTAクローニングした後に、XbaIとSalIで消化して得られる約400bのDNA断片と、pG48をXbaIとSalIで消化して得られるベクター断片とを連結してpBluescript SK- ADHNF-リンドウF3'5'Hを得た。
この形質転換アグロバクテリウムを用いて、キク品種94-765由来の組換えキク21系統を得たが、デルフィニジンを含有する個体は得られなかった。
pBluescript SK-にクローニングされたバーベナF3'5'H(pHVF7、Plant Biotechonology 23, 5-11, 2006, DNAデータベースアクセッション番号ABA234898)を鋳型に、ADH-Verbena-Fd(5'-CAAGAAAAATAAATGACGTTTTCAGAGCTTATAAAC-3'、配列番号65)とNcoI-VerbenaF3'5'H-Rv(5'-CCATGGAGTAAATCAGCATCTC-3'、配列番号66)をプライマーとして用いてPCRで増幅したDNA断片、並びにpBI221 ADH-221を鋳型にXbaI-ADH-Fd(配列番号42)とVerbena-ADH-Rv(5'-TGAAAACGTCATTTATTTTTCTTGATTTCCTTCAC-3'、配列番号67)をプライマーとして用いてPCRで増幅したDNA断片の二種類を混合して鋳型に用い、XbaI-ADH-Fd(配列番号42)とNcoI-VerbenaF3'5'H-Rv(5'-CCATGGAGTAAATCAGCATCTC-3'、配列番号68)をプライマーとして用いたPCRにより、タバコADH-5'UTR 94bpがバーベナF3'5'Hの開始コドンに直結したDNA断片を得た。このDNA断片をpCR2.1にTAクローニングした後に、XbaIとNcoIで消化して得られる約700bのDNA断片と、pHVF7をXbaIとNcoIで消化して得られるベクター断片を連結してpBluescript SK- ADHNF-バーベナF3'5'Hを得た。
この形質転換アグロバクテリウムを用いて、キク品種94-765由来の組換えキク17系統を得た。このうち11系統の花弁でデルフィニジンが検出され、デルフィニジン含有率は最高28.4%であった。
Uni-ZAP XR Vector kit (STRATAGENE社)を用いて製造者の推奨する方法により、キンギョソウの一種である(Antirrhinum kelloggii、青キンギョソウ)の蕾から得たmRNAを用いて、cDNAライブラリーを作製した。このライブラリーを参考例2に記載した方法でスクリーニングし、2種類のF3'5'HcDNA#1(配列番号69)とF3'5'HcDNA#12(配列番号71)をそれぞれ含むプラスミドpSPB3145とpSPB3146を得た。
この形質転換アグロバクテリウムを用いて、キク品種94-765由来の組換えキク1系統を得た。この系統の花弁でデルフィニジンが検出され、デルフィニジン含有率は2.9%に達した。
実施例1で得られたpBI121 HANS-CmF3Hp500-XをXbaIとEcoICRIで消化し得られるバイナリーベクターのDNA断片と、実施例6で得られたpCR2.1 ADHNF-シネラリアF3'5'HをXbaIとEcoRVで消化して得られる、ADHNF-シネラリアF3'5'HのDNA断とを連結してpBI121-キクF3Hpro500::ADHNF-シネラリアF3'5'H::NOSterを得て、Agrobacterium tumefaciens EHA105株に形質転換した。
この形質転換アグロバクテリウムを用いて、キク品種大平由来の組換えキク7系統を得た。このうち5系統でデルフィニジンが検出され、デルフィニジン含有率は25.5%に達した。
Claims (8)
- 以下の:
(1)配列番号34又は配列番号87に示す塩基配列を含む核酸、
(2)キク由来フラバノン3-水酸化酵素(F3H)遺伝子の転写調節領域として機能することができ、かつ、配列番号34又は配列番号87に示す塩基配列に対して1個又は数個の塩基配列の付加、欠失及び/又は置換により修飾された塩基配列を含む核酸、
(3)キク由来フラバノン3-水酸化酵素(F3H)遺伝子の転写調節領域として機能することができ、かつ、配列番号34又は配列番号87に示す塩基配列に対して相補的な塩基配列からなる核酸と高ストリンジェンシー条件下でハイブリダイズすることができる核酸、及び
(4)キク由来フラバノン3-水酸化酵素(F3H)遺伝子の転写調節領域として機能することができ、かつ、配列番号34又は配列番号87に示す塩基配列に対して少なくとも90%の配列同一性を有する核酸、
から成る群から選ばれる核酸を転写調節領域として用いて、フラボノイド3',5'-水酸化酵素(F3'5'H)をキク植物において発現させて、その花弁にデルフィニジンを含むキク植物を生産する方法。 - 前記フラボノイド3',5'-水酸化酵素(F3'5'H)が、カンパニュラ、サイネリア(シネラリア)、バーベナ、及びパンジー#40由来である、請求項1に記載の方法。
- 前記転写調節領域に加え、タバコのアルコールデヒドロゲナーゼ由来の翻訳エンハンサーをさらに用いる、請求項1に記載の方法。
- 前記翻訳エンハンサーが前記F3'5'H遺伝子の開始コドンに直結されている発現ベクター又は発現カセットを用いる、請求項3に記載の方法。
- 前記花弁中のデルフィニジンの含有率が、アントシアニジンの合計量の25質量%以上である、請求項1~4のいずれか1項に記載の方法。
- 請求項1項に記載の核酸を含むか又は請求項1に記載の方法により生産された、キク植物若しくはその子孫若しくはその栄養増殖体又はその部分若しくは組織。
- 切り花である、請求項6に記載のキク植物若しくはその子孫若しくはその栄養増殖体又はその部分若しくは組織。
- 請求項7に記載の切り花を用いた切り花加工品。
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CN102485897A (zh) * | 2010-12-06 | 2012-06-06 | 华中农业大学 | 利用棉花基因GbF3H改变花瓣颜色 |
WO2012096307A1 (ja) | 2011-01-14 | 2012-07-19 | サントリーホールディングス株式会社 | 新規糖転移酵素遺伝子及びその使用 |
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WO2013157502A1 (ja) | 2012-04-16 | 2013-10-24 | サントリーホールディングス株式会社 | 新規カンパニュラフラボノイド3',5'-水酸化酵素遺伝子及びその使用 |
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JP2020150880A (ja) * | 2019-03-20 | 2020-09-24 | 北海道三井化学株式会社 | イチイ由来のプロモーター及びその用途 |
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Also Published As
Publication number | Publication date |
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CN102421903B (zh) | 2014-04-16 |
EP2423312A1 (en) | 2012-02-29 |
KR20120068758A (ko) | 2012-06-27 |
US20120096589A1 (en) | 2012-04-19 |
CA2759258C (en) | 2016-05-17 |
JPWO2010122849A1 (ja) | 2012-10-25 |
CA2759258A1 (en) | 2010-10-28 |
CN102421903A (zh) | 2012-04-18 |
JP5697040B2 (ja) | 2015-04-08 |
EP2423312B1 (en) | 2016-10-19 |
CO6460696A2 (es) | 2012-06-15 |
EP2423312A4 (en) | 2013-02-20 |
US9074215B2 (en) | 2015-07-07 |
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