CN112831519A - Method for preparing santalol by utilizing sweet wormwood herbs - Google Patents

Abstract

The application discloses a method for preparing santalol by utilizing artemisia apiacea, which comprises the step of introducing SaSSY and SaCYP736A167 into the artemisia apiacea, wherein the SaSSY comprises a nucleic acid sequence shown as SEQ ID No.1, and the SaCYP736A167 comprises a nucleic acid sequence shown as SEQ ID No. 2.

Description

Method for preparing santalol by utilizing sweet wormwood herbs

Technical Field

The invention relates to the technical field of biosynthesis, in particular to a method for preparing santalol by utilizing sweet wormwood herb.

Background

Plants have evolved a series of adaptive mechanisms, such as protection from biotic or abiotic stresses in the environment by synthesizing various metabolites, in order to evade external adverse stimuli and to cope with various stresses from the environment. These metabolites generally have biological activities beneficial to humans and thus have a very promising application and commercial value.

The sandalwood essential oil has low toxicity in animals, has no mutagenicity, is a well-recognized and safe food additive in Europe and America, and the recommended safe dose is 0.0074 mg/(kg. d). Besides being used in perfumes and cosmetics, the sandalwood essential oil and the main components thereof also have the effects of tranquilizing and allaying excitement, resisting inflammation and pain, resisting bacteria, viruses, oxidation and tumors. Santalol is derived from santalol (Santalum album), is a sesquiterpene compound with profound odour of the woody plant, and is a perfume ingredient of many perfumes, which has two isomers of α -santalol (tricyclic sesquiterpene alcohol) and β -santalol (bicyclic sesquiterpene alcohol). The santalol has sweet fragrance similar to sandalwood, is usually used directly in the form of mixture, and has certain sterilizing effect. (Z) -alpha-santalol and (Z) -beta-santalol are the two most main aroma components in sandalwood essential oil. The natural sandalwood resource is unreasonably cut and utilized, the natural resource tends to be endangered, the renewal time of the natural sandalwood forest is as long as 30 years or even longer, the use of various sandalwood as raw materials or auxiliary materials is severely limited due to the factors, the product price is continuously increased, the price of the sandalwood on the market at present is about 15 million and one hundred thousand yuan per ton, and the sandalwood is increased at the speed of 9 percent per year. TFS corporation pharmaceutical grade sandalwood oil is sold at a price of about $ 4500 per kilogram.

The leap in the synthesis, assembly and analysis of DNA based on recombinant DNA technology has revolutionized genetics and molecular biology over the past two decades. These technological advances have accelerated the emergence of synthetic biology as a new discipline. The goal of synthetic biology is to design and create new biosynthetic pathways, elements or entities based on the introduction of engineering principles. Artemisia annua L, also known as Artemisia annua L, is an annual herbaceous plant, a major source of the antimalarial drug Artemisinin (Artemisinin), which is capable of producing sesquiterpenes and diterpenoids, and therefore, those skilled in the art are devoted to exploring and establishing a method for producing santalol using Artemisia annua and preparing a new variety of Artemisia annua capable of producing santalol.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is to provide a method for the biosynthesis of santalol. The precursor farnesyl pyrophosphate (FPP) present in plants is catalyzed by SaSSY (Santalene synthase), SaCYP736a167 (cytochromes P450 mooxygenases, CYP450, Cytochrome P450 monooxygenase), finally forming a mixture of santalols, which contains the four main components of santalols: ((Z) -a-santalol, (Z) -b-santalol, (Z) -epi-b-santalol and (Z) -a-exo-bergamotol).

To achieve the above objects, the present application provides a method for preparing santalol using artemisia annua, which comprises introducing SaSSY and SaCYP736a167 into the artemisia annua.

In certain embodiments, the SaSSY comprises the nucleic acid sequence set forth as SEQ ID No. 1.

In certain embodiments, the SaCYP736a167 comprises the nucleic acid sequence set forth as SEQ ID No. 2.

In certain embodiments, the introducing comprises transforming the sweet wormwood herb with an agrobacterium tumefaciens mediated expression vector comprising SaSSY and SaCYP736a 167.

In certain embodiments, the agrobacterium tumefaciens is agrobacterium tumefaciens EHA 105.

In certain embodiments, the expression vector is plasmid pCAMBIA 1300.

In certain embodiments, the expression vector is transferred into the agrobacterium tumefaciens by freeze-thaw methods.

In certain embodiments, the nucleic acid sequence comprising SaSSY and SaCYP736a167, obtained by amplification using the primers shown in SEQ ID nos. 7 and 8, is ligated into the plasmid pCAMBIA 1300.

In certain embodiments, the santalol comprises α -santalol and/or β -santalol.

In another aspect, the present application also provides the use of SaSSY including a nucleic acid sequence as shown in SEQ ID No.1 and SaCYP736a167 including a nucleic acid sequence as shown in SEQ ID No.2 in the preparation of santalol using artemisia annua.

According to the method for preparing santalol by utilizing sweet wormwood, sweet wormwood is transformed through a recombinant expression vector, so that a sweet wormwood transformant capable of producing santalol is obtained, and through cultivation and detection of the sweet wormwood transformant, santalol alpha-santalol and beta-santalol can be synthesized, so that the method has important significance for large-scale production of santalol, expansion of related material sources and reduction of loss of natural santalin forest resources.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 shows a schematic diagram of the pCAMBIA1300-SaSSY-SaCYP736A167 plasmid constructed in the present application.

FIG. 2 shows the detection results of the target gene fragment in the pCAMBIA1300-SaSSY-SaCYP736A167 plasmid of the present application, wherein SY represents SaSSY and CYP represents SaCYP736A 167.

Fig. 3 shows a general ion flow diagram of a UPLC standard for santalol in the present application, santalol1 and santalol2 being α -santalol and β -santalol, respectively.

FIG. 4 shows a UPLC total ion flow diagram of transgenic Artemisia annua leaf extract, santalol1 and santalol2 are α -santalol and β -santalol, respectively, in the present application.

Detailed Description

The present invention will now be further described with reference to examples, which are intended to be illustrative only, and the present invention may be embodied in many different forms of embodiments, and the scope of the present invention is not limited to the embodiments set forth herein.

In the present application, SaSSY refers to the gene shown in GenBank: HQ343276.1 or a derivative that retains its function; SaCYP736A167 refers to the gene shown in GenBank: KU169302.1 or the derivative retaining its function.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the conditions recommended by the relevant Material manufacturers.

Example 1 construction of pCAMBIA1300-SaSSY-SaCYP736A167 vector

(1) The pBSK plasmid is used as a vector, and SaSSY (shown as SEQ ID NO.1 in sequence) and SaCYP736A167 (shown as SEQ ID NO.2 in sequence) are subjected to whole-gene synthesis in bioengineering companies.

(2) PCR was carried out using pBSK-SaSSY and pBSK-SaCYP736A167 as templates, respectively, with the following specific primers:

the forward primer SaSSY-5-BamH 1: 5'-CCGGATCCATGGATTCTTCCACCGCCACCGCCATGAC-3' (SEQ ID NO.3)

The reverse primer SaSSY-3-3ns-spe 1: 5'-CCACTAGTCTACTCCTCGCCGAGAGGAATAGGGTCG-3' (SEQ ID NO.4)

And

the forward primer SaCYP167F-BamH 1: 5'-CCGGATCCATGTCTCCGGCAACAGCCGTTATCC-3' (SEQ ID NO.5)

The reverse primer SaCYP167-3ns-spe 1: 5'-CCACTAGTGGACTCCAAGCGATAGGTT-3' (SEQ ID NO.6)

The total volume of the PCR reaction is 50 mu L, and the reaction system is as follows: 5 μ L of 10 XKODPlus Buffer, 5 μ L of dNTPs,2 μ L of MgSO₄1 μ L of forward primer, 1 μ L of reverse primer, 1 μ L of plasmid template, 1 μ L of KODPlus enzyme (purchased from TOYOBO Co., Ltd.)ddH of 34. mu.L₂The content of O is filled to 50 mu L.

After the PCR product is recovered and purified, the two fragments are respectively cut by BamH1 and Spe1 enzyme, and then are connected with pHB plasmid (the sequence is shown as SEQ ID NO. 13) through T4 connection to construct PHB-SaSSY and PHB-SaCYP736A167 plasmids.

(3) Using PHB-SaSSY and PHB-SaCYP736A167 plasmids as templates, respectively using specific primer sequences shown in the following to amplify the two expression cassettes of 35S-SaSSY-RBCterminator (SEQ ID NO.14) and 35S-SaCYP736A 167-RBCterminator (SEQ ID NO.15) by PCR (reaction system as step (2)):

forward primer 35 SsyIE: 5'-TATGACCATGATTACGAATTC-3' (SEQ ID NO.7)

Reverse primer rbcseie: 5'-TACCGAGCTCGAATTCATCGATTGATGCATGTTGTCAA-3' (SEQ ID NO.8)

And

forward primer 35s167 ISal: 5'-ATCCTCTAGAGTCGACTATGACCATGATTACGAATTC-3' (SEQ ID NO.9)

Reverse primer RBC167 ISal: 5'-ATGCCTGCAGGTCGACATCGATTGATGCATGTTGTCAA-3' (SEQ ID NO.10)

The two expression cassettes were ligated to pCAMBIA1300(access number: AF234296.1) using Clonexpress II One Step Cloning Kit (available from Novow Kinza) to construct pCAMBIA1300-SaSSY-SaCYP736A167 plasmid, the structure of which is shown in FIG. 1; the PCR method is utilized to detect the primer SaSSY-5-BamH1(SEQ ID NO.3), the primer SaSSY-3-3ns-spe1(SEQ ID NO.4), the primer SaCYP167F-BamH1(SEQ ID NO.5) and the primer SaCYP167-3ns-spe1(SEQ ID NO.6) to obtain the successfully constructed pCAMBIA1300-SaSSY-SaCYP736A167 plasmid, and the result of gel electrophoresis is shown in figure 2.

Example 2 Agrobacterium tumefaciens-mediated pCAMBIA1300-SaSSY-SaCYP736A167 plasmid genetic transformation of Artemisia annua to obtain transgenic Artemisia annua plants

1. Obtaining of Agrobacterium tumefaciens engineering bacteria containing pCAMBIA1300-SaSSY-SaCYP736A167 plasmid

The pCAMBIA1300-SaSSY-SaCYP736A167 expression vector of example 1 was transformed into Agrobacterium tumefaciens (EHA105, a commercially available biomaterial with the strain number Gambar1, available from CAMBIA, Australia) by freeze-thawing, as follows:

(1) melting 100 mu L of agrobacterium tumefaciens infected ice;

(2) placing 5 μ L of plasmid into competent cells;

(3) ice-bath for 5 minutes;

(4) quickly freezing for 5 minutes by using liquid nitrogen;

(5) heat shock at 37 ℃ for 5 minutes;

(6) ice-cooling for 5 minutes, adding 400 mu L of fresh liquid LB, and resuscitating for 1 hour at 28 ℃ and 200 rpm;

(7) 100 μ L of the bacterial suspension was plated (rifampicin 25mg/L, kanamycin 100m g/L).

The PCR verification result shows that the expression containing pCAMBIA1300-SaSSY-SaCYP736A167 is successfully constructed into the Agrobacterium tumefaciens strain.

2. Agrobacterium tumefaciens mediated transformation of artemisia apiacea

2.1. Pre-culture of explants

Soaking herba Artemisiae Annuae seed in 75% ethanol for 1min, soaking in 20% NaClO for 20min, washing with sterile water for 3-4 times, blotting surface water with sterile absorbent paper, inoculating in hormone-free MS (Murashige and Skoog,1962) solid culture medium, and culturing at 25 deg.C under 16h/8h (light/dark) light to obtain herba Artemisiae Annuae sterile seedling. When the seedling grows to about 5cm, cutting a sterile seedling leaf explant for transformation.

2.2. Co-culture of Agrobacterium with explants

Transferring the leaf explants to a co-culture medium (1/2MS + AS 100 mu mol/L), dropwise adding 1/2MS suspension containing activated agrobacterium tumefaciens engineering bacteria (the bacterial strain of the transfection pCAMBIA1300-SaSSY-SaCYP736A167 plant expression vector obtained in the step 1), fully contacting the explants with a bacterial solution, and performing dark culture at 28 ℃ for 3 d. Leaf explants were controlled by dripping 1/2MS liquid medium suspension of Agrobacterium tumefaciens without the target gene.

2.3. Selection of resistant regenerated plants

Transferring the sweet wormwood herb explants cultured for 3d in the co-culture process to a germination screening culture medium (MS +6-BA0.5mg/L + NAA0.05mg/L + Hyg 8.75mg/L + Cb 500mg/L), carrying out light culture at 25 ℃ for 16h/8h (light/dark), carrying out subculture once every two weeks, and obtaining Hyg-resistant cluster buds after 2-3 subcultures. Shearing off the well-grown resistant cluster buds, transferring the cluster buds to a rooting culture medium (1/2MS + Cb 125mg/L) for culturing until the cluster buds grow to root, thereby obtaining a Hyg resistant regeneration sweet wormwood plant.

3. PCR detection of transgenic southernwood plant

Transferring the transgenic plant into a soil matrix, culturing for two weeks, sampling, and extracting total DNA of the transgenic plant in small quantity by using a TPS method. Using the extracted DNA as a template, and using a specific detection primer SaSSY-5: 5'-ATGGATTCTTCCACCGCCACCGCCATGAC-3' (SEQ ID NO.11) and SaSSY-3: 5'-CTACTCCTCGCCGAGAGGAATAGGGTCG-3' (SEQ ID NO.12), and detecting positive plants in the transgenic plants by using a PCR method. The result shows that the designed PCR specific detection primer can be used for amplifying a specific DNA fragment. When non-transformed genomic DNA of Artemisia annua is used as a template, no fragment is amplified.

In this example, the pCAMBIA1300-SaSSY-SaCYP736a167 plant expression vector was transformed into agrobacterium tumefaciens EHA105 to obtain an agrobacterium tumefaciens strain containing the pCAMBIA1300-SaSSY-SaCYP736a167 expression vector for transforming artemisia annua, and the constructed agrobacterium tumefaciens strain was used to transform artemisia annua to obtain a transgenic artemisia annua plant confirmed by PCR detection.

Example 3 determination of the Santalol content in transgenic Artemisia annua by Waters Acquity UPLC/SQD2

UPLC-SQD2 Condition and System suitability and Standard solution preparation

1.1 liquid phase conditions

And (3) UPLC: waters Acquity UPLC; a chromatographic column: BEH C182.1 × 100mm 1.7 μm);

mobile phase: 0.1% aqueous formic acid (a) -acetonitrile (B), gradient elution, elution procedure: 0-5 min, 40% → 80% B; 5-7 min, 80% B; 7-7.1 min, 80% → 100% B; 7.1-8 min, 100% B; 8-8.1 min, 100% → 40% B; 8.1-10 min, 40% B.

The column temperature is 35 ℃; the flow rate is 0.3mL min-¹(ii) a The injection volume was 5. mu.L.

1.2 Mass Spectrometry conditions

Electrospray ion source (ESI +); the ion source temperature is 150 ℃; the desolventizing gas flow rate is 650 L.h < -1 >; the temperature of desolventizing gas is 350 ℃; capillary voltage 3.5 kV; the taper hole voltage is 50V; using single ion detection scan mode (ES +221.35m/z)

1.3 preparation of control

Accurately weighing 50 mu L of santalol standard substance (rho is 0.968g/mL) (purchased from Sigma company), placing the santalol standard substance into a10 mL volumetric flask, dissolving the santalol standard substance by using methanol and fixing the volume to a scale mark, shaking the santalol standard substance evenly, accurately weighing 500 mu L, placing the santalol standard substance into the 10mL volumetric flask, dissolving the santalol standard substance by using methanol and fixing the volume to the scale mark to prepare mother liquor of alpha-santalol 193.6 mu g/mL and beta-santalol 31.46 mu g/mL, sequentially diluting the mother liquor by 5 times, 25 times, 50 times, 100 times, 125 times and 250 times, respectively injecting 5 mu L of the reference substance solution under corresponding chromatographic conditions, recording a chromatogram and chromatographic parameters, and respectively carrying out regression analysis on the content (X mu g/mL) of the standard substance by using a peak area (Y). Through research, the α -santalol in the embodiment shows a good linear relationship in the range of 0.39-38.72 μ g/mL, and the regression equation is: y1 ═ 4E-06X1-0.0776, R2 ═ 1; the beta-santalol shows a good linear relation in the range of 0.06-6.29 mu g/mL, and the regression equation is as follows: Y2-4E-07X 2-0.1841, R2-0.9862.

1.4 preparation of samples and determination of the Santalol content

Placing fresh sweet wormwood leaf blades with the fresh weight of 200mg into a 2mL centrifuge tube containing 2 steel balls, quickly freezing by liquid nitrogen, crushing the leaves by using a grinder, adding 1mL of methanol, reversing, uniformly mixing, performing ultrasonic treatment for 10 minutes, centrifuging at 12000rpm for 5min, and placing 100uL of supernatant into a sample injection bottle for UPLC-SQD2 detection. The method comprises the steps of determining the content of santalol by adopting an ultra performance liquid chromatography-mass spectrometry method, enabling the sample injection volume to be 5 mu L, substituting a peak area into a linear regression equation to calculate the content (mu g/mL) of the santalol in a sample, and dividing the content by the wet weight (mg) of sweet wormwood leaves of the sample to calculate the content of the santalol in sweet wormwood plants.

The UPLC total ion flow diagram of the santalol standard product is shown in figure 3, and the UPLC total ion flow diagram of the transgenic artemisia apiacea leaf extract is shown in figure 4.

The results show that santalol is generated in the transgenic sweet wormwood in the embodiment, wherein the content of the alpha-santalol reaches 0.05 per mill of the wet weight of the sample; the content of beta-santalol reaches 0.016 thousandth of the wet weight of the sample.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Sequence listing

<110> Shanghai university of transportation

SUZHOU TANGJI BIOLOGICAL TECHNOLOGY Co.,Ltd.

<120> a method for preparing santalol by using sweet wormwood herb

<130> CN084-20006PICN

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 1686

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SaSSY nucleotide sequence

<400> 1

atgacagctc cattcattga tcctactgat catgtgaatc tcaaaactga tacggatgcc 60

tcagagaatc gaaggatggg aaattataaa cccagcattt ggaattatga ttttttacaa 120

tcacttgcaa ctcatcacaa tattgtggaa gagaggcatc taaagctagc tgagaagctg 180

aagggccaag tgaagtttat gtttggggca ccaatggagc cgttagcaaa gctggagctt 240

gtggatgtgg ttcaaaggct tgggctaaac cacctatttg agacagagat caaggaagcg 300

ctgtttagta tttacaagga tgggagcaat ggatggtggt ttggccacct tcatgcgaca 360

tctctccgat ttaggctgct acgacagtgt gggcttttta ttccccaaga tgtgtttaaa 420

acgttccaaa acaagactgg ggaatttgat atgaaacttt gtgacaacgt aaaagggctg 480

ctgagcttat atgaagcttc atacttggga tggaagggtg aaaacatcct agatgaagcc 540

aaggccttca ccaccaagtg cttgaaaagt gcatgggaaa atatatccga aaagtggtta 600

gccaaaagag tgaagcatgc attggctttg cctttgcatt ggagagtccc tcgaatcgaa 660

gctagatggt tcattgaggc atatgagcaa gaagcgaata tgaacccaac actactcaaa 720

ctcgcaaaat tagactttaa tatggtgcaa tcaattcatc agaaagagat tggggaatta 780

gcaaggtggt gggtgactac tggcttggat aagttagcct ttgccaggaa taatttactg 840

cagagctata tgtggagctg cgcgattgct tccgacccga agttcaaact tgctagagaa 900

actattgtcg aaatcggaag tgtactcaca gttgttgacg atggatatga cgtctatggt 960

tcaatcgacg aacttgatct ctacacaagc tccgttgaaa ggtggagctg tgtggaaatt 1020

gacaagttgc caaacacgtt aaaattaatt tttatgtcta tgttcaacaa gaccaatgag 1080

gttggccttc gagtccagca tgagcgaggc tacaatagca tccctacttt tatcaaagcg 1140

tgggttgaac agtgtaaatc ataccagaaa gaagcaagat ggttccacgg gggacacacg 1200

cctccattgg aagaatatag cttgaatgga cttgtttcca taggattccc tctcttgtta 1260

atcacgggct acgtggcaat cgctgagaac gaggctgcac tggataaagt gcaccccctt 1320

cctgatcttc tgcactactc ctccctcctt agtcgcctca tcaatgatat aggaacgtct 1380

ccggatgaga tggcaagagg cgataatctg aagtcaatcc attgttacat gaacgaaact 1440

ggggcttccg aggaagttgc tcgtgagcac ataaagggag taatcgagga gaattggaaa 1500

atactgaatc agtgctgctt tgatcaatct cagtttcagg agccttttat aaccttcaat 1560

ttgaactctg ttcgagggtc tcatttcttc tatgaatttg gggatggctt tggggtgacg 1620

gatagctgga caaaggttga tatgaagtcc gttttgatcg accctattcc tctcggcgag 1680

gagtag 1686

<210> 2

<211> 1503

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SaCYP736A167 nucleotide sequence

<400> 2

atgtctccgg caacagccgt tatcctcact ctcctcgtgg ccctagggct atccatcctt 60

ttgcggcggc gccaaaaaag aaataatcta cctcccggtc cacccgcttt accgatcatc 120

ggaaacatcc acatattggg gacccttcct caccagagcc tctacaactt ggccaagaag 180

tatggtccca tcatgtcaat gaggctgggg ctcgtgccgg ctgttgtgat atcctctccg 240

gaggccgccg agctcgtcct caagacccac gatatcgttt tcgccagccg gcccagactc 300

caagttgcgg actacttcca ttacgggaca aagggcgtca tcctgacgga gtatggtaca 360

tattggcgca acatgcgaag gctgtgcacc gtgaagcttc tcaacacggt gaaaatcgat 420

tctttcgcag ggacaaggaa gaaggaggtg gcatcgttcg tgcagtccct taaggaggct 480

tcggtggcac acaaaatggt gaatttgagc gcgagggtgg cgaacgtcat tgaaaacatg 540

gtgtgcctta tggtgatcgg gcgaagtagc gatgagaggt ttaagctaaa ggaggtcatc 600

caggaggcag cgcagttggc gggagctttc aatatagggg attatgttcc attccttatg 660

ccccttgacc tacagggatt aactcggcgc ataaagtcag gaagtaaagc tttcgacgac 720

atcttggaag tcataatcga cgagcacgtg caagacatta aggaccatga tgatgaacaa 780

catggagact tcattgatgt gttgctggca atgatgaaca agcccatgga ttcgcgggag 840

ggtcttagta tcattgaccg aacaaacatc aaagcgatcc tagtggacat gattggagct 900

gcaatggaca cttcaacaag tggcgtcgag tgggcgattt cagagctcat caagcatccg 960

cgggtaatga aaaagctcca agacgaggtc aaaactgtca tcggaatgaa taggatggtc 1020

gaggaggccg acttgcctaa gctaccatac ctcgacatgg tagtgaaaga gaccatgagg 1080

ttacaccctc ctggaccatt gctcgtgccc cgagagtcca tggaagacat cacaatcaac 1140

ggatactaca tacctaagaa atcgcgaatc attgtcaacg cctgggcaat tgggcgtgat 1200

acaaacgcct ggtctaataa cgcgcacgag ttcttcccag agaggtttat gagtagcaat 1260

gtggacttac agggacaaga tttccaactt atcccattcg ggtccggtcg gagagggtgc 1320

cccgggatgc gcctaggcct cacaaccgtt cgattagtgt tagcgcagct cattcattgt 1380

ttcgacttgg agcttcctaa gggaaccgtg gcgaccgact tggacatgag tgagaaattc 1440

gggttggcaa tgcccagagc ccagcacttg cttgcatttc caacctatcg cttggagtcc 1500

taa 1503

<210> 3

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer SaSSY-5-BamH1

<400> 3

ccggatccat ggattcttcc accgccaccg ccatgac 37

<210> 4

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer SaSSY-3-3ns-spe1

<400> 4

ccactagtct actcctcgcc gagaggaata gggtcg 36

<210> 5

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer SaCYP167F-BamH1

<400> 5

ccggatccat gtctccggca acagccgtta tcc 33

<210> 6

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer SaCYP167-3ns-spe1

<400> 6

ccactagtgg actccaagcg ataggtt 27

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Forward primer 35SsyIE

<400> 7

tatgaccatg attacgaatt c 21

<210> 8

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> reverse primer RBCsyIE

<400> 8

taccgagctc gaattcatcg attgatgcat gttgtcaa 38

<210> 9

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Forward primer 35s167ISal

<400> 9

atcctctaga gtcgactatg accatgatta cgaattc 37

<210> 10

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> reverse primer RBC167ISal

<400> 10

atgcctgcag gtcgacatcg attgatgcat gttgtcaa 38

<210> 11

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer SaSSY-5

<400> 11

atggattctt ccaccgccac cgccatgac 29

<210> 12

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> primer SaSSY-3

<400> 12

ctactcctcg ccgagaggaa tagggtcg 28

<210> 13

<211> 12789

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> pHB vector

<400> 13

catggatggc taaaatgaga atatcaccgg aattgaaaaa actgatcgaa aaataccgct 60

gcgtaaaaga tacggaagga atgtctcctg ctaaggtata taagctggtg ggagaaaatg 120

aaaacctata tttaaaaatg acggacagcc ggtataaagg gaccacctat gatgtggaac 180

gggaaaagga catgatgcta tggctggaag gaaagctgcc tgttccaaag gtcctgcact 240

ttgaacggca tgatggctgg agcaatctgc tcatgagtga ggccgatggc gtcctttgct 300

cggaagagta tgaagatgaa caaagccctg aaaagattat cgagctgtat gcggagtgca 360

tcaggctctt tcactccatc gacatatcgg attgtcccta tacgaatagc ttagacagcc 420

gcttagccga attggattac ttactgaata acgatctggc cgatgtggat tgcgaaaact 480

gggaagaaga cactccattt aaagatccgc gcgagctgta tgatttttta aagacggaaa 540

agcccgaaga ggaacttgtc ttttcccacg gcgacctggg agacagcaac atctttgtga 600

aagatggcaa agtaagtggc tttattgatc ttgggagaag cggcagggcg gacaagtggt 660

atgacattgc cttctgcgtc cggtcgatca gggaggatat cggggaagaa cagtatgtcg 720

agctattttt tgacttactg gggatcaagc ctgattggga gaaaataaaa tattatattt 780

tactggatga attgtttatg gctaaaatga gaatatcacc ggaattgaaa aaactgatcg 840

aaaaataccg ctgcgtaaaa gatacggaag gaatgtctcc tgctaaggta tataagctgg 900

tgggagaaaa tgaaaaccta tatttaaaaa tgacggacag ccggtataaa gggaccacct 960

atgatgtgga acgggaaaag gacatgatgc tatggctgga aggaaagctg cctgttccaa 1020

aggtcctgca ctttgaacgg catgatggct ggagcaatct gctcatgagt gaggccgatg 1080

gcgtcctttg ctcggaagag tatgaagatg aacaaagccc tgaaaagatt atcgagctgt 1140

atgcggagtg catcaggctc tttcactcca tcgacatatc ggattgtccc tatacgaata 1200

gcttagacag ccgcttagcc gaattggatt acttactgaa taacgatctg gccgatgtgg 1260

attgcgaaaa ctgggaagaa gacactccat ttaaagatcc gcgcgagctg tatgattttt 1320

taaagacgga aaagcccgaa gaggaacttg tcttttccca cggcgacctg ggagacagca 1380

acatctttgt gaaagatggc aaagtaagtg gctttattga tcttgggaga agcggcaggg 1440

cggacaagtg gtatgacatt gccttctgcg tccggtcgat cagggaggat atcggggaag 1500

aacagtatgt cgagctattt tttgacttac tggggatcaa gcctgattgg gagaaaataa 1560

aatattatat tttactggat gaattgtttg gtgaccagct cgaatttccc cgatcgttca 1620

aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 1680

atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 1740

tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 1800

aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 1860

gatcgggaat taaactatca gtgtttgaca ggatatattg gcgggtaaac ctaagagaaa 1920

agagcgttta ttagaataac ggatatttaa aagggcgtga aaaggtttat ccgttcgtcc 1980

atttgtatgt gcatgccaac cacagggttc ccctcgggat caaagtactt tgatccaacc 2040

cctccgctgc tatagtgcag tcggcttctg acgttcagtg cagccgtctt ctgaaaacga 2100

catgtcgcac aagtcctaag ttacgcgaca ggctgccgcc ctgccctttt cctggcgttt 2160

tcttgtcgcg tgttttagtc gcataaagta gaatacttgc gactagaacc ggagacatta 2220

cgccatgaac aagagcgccg ccgctggcct gctgggctat gcccgcgtca gcaccgacga 2280

ccaggacttg accaaccaac gggccgaact gcacgcggcc ggctgcacca agctgttttc 2340

cgagaagatc accggcacca ggcgcgaccg cccggagctg gccaggatgc ttgaccacct 2400

acgccctggc gacgttgtga cagtgaccag gctagaccgc ctggcccgca gcacccgcga 2460

cctactggac attgccgagc gcatccagga ggccggcgcg ggcctgcgta gcctggcaga 2520

gccgtgggcc gacaccacca cgccggccgg ccgcatggtg ttgaccgtgt tcgccggcat 2580

tgccgagttc gagcgttccc taatcatcga ccgcacccgg agcgggcgcg aggccgccaa 2640

ggcccgaggc gtgaagtttg gcccccgccc taccctcacc ccggcacaga tcgcgcacgc 2700

ccgcgagctg atcgaccagg aaggccgcac cgtgaaagag gcggctgcac tgcttggcgt 2760

gcatcgctcg accctgtacc gcgcacttga gcgcagcgag gaagtgacgc ccaccgaggc 2820

caggcggcgc ggtgccttcc gtgaggacgc attgaccgag gccgacgccc tggcggccgc 2880

cgagaatgaa cgccaagagg aacaagcatg aaaccgcacc aggacggcca ggacgaaccg 2940

tttttcatta ccgaagagat cgaggcggag atgatcgcgg ccgggtacgt gttcgagccg 3000

cccgcgcacg tctcaaccgt gcggctgcat gaaatcctgg ccggtttgtc tgatgccaag 3060

ctggcggcct ggccggccag cttggccgct gaagaaaccg agcgccgccg tctaaaaagg 3120

tgatgtgtat ttgagtaaaa cagcttgcgt catgcggtcg ctgcgtatat gatgcgatga 3180

gtaaataaac aaatacgcaa ggggaacgca tgaaggttat cgctgtactt aaccagaaag 3240

gcgggtcagg caagacgacc atcgcaaccc atctagcccg cgccctgcaa ctcgccgggg 3300

ccgatgttct gttagtcgat tccgatcccc agggcagtgc ccgcgattgg gcggccgtgc 3360

gggaagatca accgctaacc gttgtcggca tcgaccgccc gacgattgac cgcgacgtga 3420

aggccatcgg ccggcgcgac ttcgtagtga tcgacggagc gccccaggcg gcggacttgg 3480

ctgtgtccgc gatcaaggca gccgacttcg tgctgattcc ggtgcagcca agcccttacg 3540

acatatgggc caccgccgac ctggtggagc tggttaagca gcgcattgag gtcacggatg 3600

gaaggctaca agcggccttt gtcgtgtcgc gggcgatcaa aggcacgcgc atcggcggtg 3660

aggttgccga ggcgctggcc gggtacgagc tgcccattct tgagtcccgt atcacgcagc 3720

gcgtgagcta cccaggcact gccgccgccg gcacaaccgt tcttgaatca gaacccgagg 3780

gcgacgctgc ccgcgaggtc caggcgctgg ccgctgaaat taaatcaaaa ctcatttgag 3840

ttaatgaggt aaagagaaaa tgagcaaaag cacaaacacg ctaagtgccg gccgtccgag 3900

cgcacgcagc agcaaggctg caacgttggc cagcctggca gacacgccag ccatgaagcg 3960

ggtcaacttt cagttgccgg cggaggatca caccaagctg aagatgtacg cggtacgcca 4020

aggcaagacc attaccgagc tgctatctga atacatcgcg cagctaccag agtaaatgag 4080

caaatgaata aatgagtaga tgaattttag cggctaaagg aggcggcatg gaaaatcaag 4140

aacaaccagg caccgacgcc gtggaatgcc ccatgtgtgg aggaacgggc ggttggccag 4200

gcgtaagcgg ctgggttgtc tgccggccct gcaatggcac tggaaccccc aagcccgagg 4260

aatcggcgtg acggtcgcaa accatccggc ccggtacaaa tcggcgcggc gctgggtgat 4320

gacctggtgg agaagttgaa ggccgcgcag gccgcccagc ggcaacgcat cgaggcagaa 4380

gcacgccccg gtgaatcgtg gcaagcggcc gctgatcgaa tccgcaaaga atcccggcaa 4440

ccgccggcag ccggtgcgcc gtcgattagg aagccgccca agggcgacga gcaaccagat 4500

tttttcgttc cgatgctcta tgacgtgggc acccgcgata gtcgcagcat catggacgtg 4560

gccgttttcc gtctgtcgaa gcgtgaccga cgagctggcg aggtgatccg ctacgagctt 4620

ccagacgggc acgtagaggt ttccgcaggg ccggccggca tggccagtgt gtgggattac 4680

gacctggtac tgatggcggt ttcccatcta accgaatcca tgaaccgata ccgggaaggg 4740

aagggagaca agcccggccg cgtgttccgt ccacacgttg cggacgtact caagttctgc 4800

cggcgagccg atggcggaaa gcagaaagac gacctggtag aaacctgcat tcggttaaac 4860

accacgcacg ttgccatgca gcgtacgaag aaggccaaga acggccgcct ggtgacggta 4920

tccgagggtg aagccttgat tagccgctac aagatcgtaa agagcgaaac cgggcggccg 4980

gagtacatcg agatcgagct agctgattgg atgtaccgcg agatcacaga aggcaagaac 5040

ccggacgtgc tgacggttca ccccgattac tttttgatcg atcccggcat cggccgtttt 5100

ctctaccgcc tggcacgccg cgccgcaggc aaggcagaag ccagatggtt gttcaagacg 5160

atctacgaac gcagtggcag cgccggagag ttcaagaagt tctgtttcac cgtgcgcaag 5220

ctgatcgggt caaatgacct gccggagtac gatttgaagg aggaggcggg gcaggctggc 5280

ccgatcctag tcatgcgcta ccgcaacctg atcgagggcg aagcatccgc cggttcctaa 5340

tgtacggagc agatgctagg gcaaattgcc ctagcagggg aaaaaggtcg aaaaggtctc 5400

tttcctgtgg atagcacgta cattgggaac ccaaagccgt acattgggaa ccggaacccg 5460

tacattggga acccaaagcc gtacattggg aaccggtcac acatgtaagt gactgatata 5520

aaagagaaaa aaggcgattt ttccgcctaa aactctttaa aacttattaa aactcttaaa 5580

acccgcctgg cctgtgcata actgtctggc cagcgcacag ccgaagagct gcaaaaagcg 5640

cctacccttc ggtcgctgcg ctccctacgc cccgccgctt cgcgtcggcc tatcgcggcc 5700

gctggccgct caaaaatggc tggcctacgg ccaggcaatc taccagggcg cggacaagcc 5760

gcgccgtcgc cactcgaccg ccggcgccca catcaaggca ccctgcctcg cgcgtttcgg 5820

tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta 5880

agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg 5940

gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg 6000

gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc 6060

gtaaggagaa aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc 6120

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 6180

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 6240

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 6300

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 6360

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 6420

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 6480

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 6540

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 6600

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 6660

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 6720

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 6780

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 6840

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 6900

aacgaaaact cacgttaagg gattttggtc atgcattcta ggtactaaaa caattcatcc 6960

agtaaaatat aatattttat tttctcccaa tcaggcttga tccccagtaa gtcaaaaaat 7020

agctcgacat actgttcttc cccgatatcc tccctgatcg accggacgca gaaggcaatg 7080

tcataccact tgtccgccct gccgcttctc ccaagatcaa taaagccact tactttgcca 7140

tctttcacaa agatgttgct gtctcccagg tcgccgtggg aaaagacaag ttcctcttcg 7200

ggcttttccg tctttaaaaa atcatacagc tcgcgcggat ctttaaatgg agtgtcttct 7260

tcccagtttt cgcaatccac atcggccaga tcgttattca gtaagtaatc caattcggct 7320

aagcggctgt ctaagctatt cgtataggga caatccgata tgtcgatgga gtgaaagagc 7380

ctgatgcact ccgcatacag ctcgataatc ttttcagggc tttgttcatc ttcatactct 7440

tccgagcaaa ggacgccatc ggcctcactc atgagcagat tgctccagcc atcatgccgt 7500

tcaaagtgca ggacctttgg aacaggcagc tttccttcca gccatagcat catgtccttt 7560

tcccgttcca catcataggt ggtcccttta taccggctgt ccgtcatttt taaatatagg 7620

ttttcatttt ctcccaccag cttatatacc ttagcaggag acattccttc cgtatctttt 7680

acgcagcggt atttttcgat cagttttttc aattccggtg atattctcat tttagccatt 7740

tattatttcc ttcctctttt ctacagtatt taaagatacc ccaagaagct aattataaca 7800

agacgaactc caattcactg ttccttgcat tctaaaacct taaataccag aaaacagctt 7860

tttcaaagtt gttttcaaag ttggcgtata acatagtatc gacggagccg attttgaaac 7920

cgcggtgatc acaggcagca acgctctgtc atcgttacaa tcaacatgct accctccgcg 7980

agatcatccg tgtttcaaac ccggcagctt agttgccgtt cttccgaata gcatcggtaa 8040

catgagcaaa gtctgccgcc ttacaacggc tctcccgctg acgccgtccc ggactgatgg 8100

gctgcctgta tcgagtggtg attttgtgcc gagctgccgg tcggggagct gttggctggc 8160

tggtggcagg atatattgtg gtgtaaacaa attgacgctt agacaactta ataacacatt 8220

gcggacgttt ttaatgtact gaattaacgc cgaattaatt cgggggatct ggattttagt 8280

actggatttt ggttttagga attagaaatt ttattgatag aagtatttta caaatacaaa 8340

tacatactaa gggtttctta tatgctcaac acatgagcga aaccctatag gaaccctaat 8400

tcccttatct gggaactact cacacattat tatggagaaa ctcgagtcaa atctcggtga 8460

cgggcaggac cggacggggc ggtaccggca ggctgaagtc cagctgccag aaacccacgt 8520

catgccagtt cccgtgcttg aagccggccg cccgcagcat gccgcggggg gcatatccga 8580

gcgcctcgtg catgcgcacg ctcgggtcgt tgggcagccc gatgacagcg accacgctct 8640

tgaagccctg tgcctccagg gacttcagca ggtgggtgta gagcgtggag cccagtcccg 8700

tccgctggtg gcggggggag acgtacacgg tcgactcggc cgtccagtcg taggcgttgc 8760

gtgccttcca ggggcccgcg taggcgatgc cggcgacctc gccgtccacc tcggcgacga 8820

gccagggata gcgctcccgc agacggacga ggtcgtccgt ccactcctgc ggttcctgcg 8880

gctcggtacg gaagttgacc gtgcttgtct cgatgtagtg gttgacgatg gtgcagaccg 8940

ccggcatgtc cgcctcggtg gcacggcgga tgtcggccgg gcgtcgttct gggctcatgg 9000

tagactcgag agagatagat ttgtagagag agactggtga tttcagcgtg tcctctccaa 9060

atgaaatgaa cttccttata tagaggaagg tcttgcgaag gatagtggga ttgtgcgtca 9120

tcccttacgt cagtggagat atcacatcaa tccacttgct ttgaagacgt ggttggaacg 9180

tcttcttttt ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca 9240

gaggcatctt gaacgatagc ctttccttta tcgcaatgat ggcatttgta ggtgccacct 9300

tccttttcta ctgtcctttt gatgaagtga cagatagctg ggcaatggaa tccgaggagg 9360

tttcccgata ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc tgagactgta 9420

tctttgatat tcttggagta gacgagagtg tcgtgctcca ccatgttatc acatcaatcc 9480

acttgctttg aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg 9540

ggtccatctt tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg 9600

caatgatggc atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag 9660

atagctgggc aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca 9720

atagcccttt ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg 9780

tgctccacca tgttggcaag ctgctctagc caatacgcaa accgcctctc cccgcgcgtt 9840

ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc 9900

gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc 9960

ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct 10020

atgaccatga ttacgaattc gcccggggat ctcctttgcc ccagagatca caatggacga 10080

cttcctatat ctctacgatc tagtcaggaa gttcgacgga gaaggtgacg ataccatgtt 10140

caccactgat aatgagaaga ttagcctttt caatttcaga aagaatccta acccacagat 10200

ggttagagac gcttacgcag caggtctcat caagacgatc tacccgagca ataatctcca 10260

ggagatcaaa taccttccca agaaggttaa agatgcagtc aaaagattca ggactaactg 10320

catcaagaac acagagaaag atatatttct caagatcaga agtactattc cagtatggac 10380

gattcaaggc ttgcttcaca aaccaaggca agtaatagag attggagtct ctaaaaaggt 10440

agttcccact gaatcaaagg ccatggagtc aaagattcaa atagaggacc taacagaact 10500

cgccgtaaag actggcgaac agttcataca gagtctctta cgactcaatg acaagaagaa 10560

aatcttcgtc aacatggtgg agcacgacac gcttgtctac ctccaaaaat atcaaagata 10620

cagtctcaga agaccaaagg gaattgagac ttttcaacaa agggtaatat ccggaaacct 10680

cctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg aaaaggaagg 10740

tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag atgcctctgc 10800

cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt 10860

tccaaccacg tcttcaaagc aagtggattg atgtgataac atggtggagc acgacacgct 10920

tgtctacctc caaaaatatc aaagatacag tctcagaaga ccaaagggaa ttgagacttt 10980

tcaacaaagg gtaatatccg gaaacctcct cggattccat tgcccagcta tctgtcactt 11040

tattgtgaag atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg 11100

aaaggccatc gttgaagatg cctctgccga cagtggtccc aaagatggac ccccacccac 11160

gaggagcatc gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg 11220

tgatatctcc actgacgtaa gggatgacgc acaatcccac tatccttcgc aagacccttc 11280

ctctatataa ggaagttcat ttcatttgga gaggacacgc tgaaatcacc agtctctctc 11340

taagcttgga tcctcgagct gcaggagctc gaattgatcc tctagagctt tcgttcgtat 11400

catcggtttc gacaacgttc gtcaagttca atgcatcagt ttcattgcgc acacaccaga 11460

atcctactga gttcgagtat tatggcattg ggaaaactgt ttttcttgta ccatttgttg 11520

tgcttgtaat ttactgtgtt ttttattcgg ttttcgctat cgaactgtga aatggaaatg 11580

gatggagaag agttaatgaa tgatatggtc cttttgttca ttctcaaatt aatattattt 11640

gttttttctc ttatttgttg tgtgttgaat ttgaaattat aagagatatg caaacatttt 11700

gttttgagta aaaatgtgtc aaatcgtggc ctctaatgac cgaagttaat atgaggagta 11760

aaacacttgt agttgtacca ttatgcttat tcactaggca acaaatatat tttcagacct 11820

agaaaagctg caaatgttac tgaatacaag tatgtcctct tgtgttttag acatttatga 11880

actttccttt atgtaatttt ccagaatcct tgtcagattc taatcattgc tttataatta 11940

tagttatact catggatttg tagttgagta tgaaaatatt ttttaatgca ttttatgact 12000

tgccaattga ttgacaacat gcatcaatcg atggcactgg ccgtcgtttt acaacgtcgt 12060

gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 12120

agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 12180

aatggcgaat gctagagcag cttgagcttg gatcagattg tcgtttcccg ccttcagttt 12240

agcttcatgg agtcaaagat tcaaatagag gacctaacag aactcgccgt aaagactggc 12300

gaacagttca tacagagtct cttacgactc aatgacaaga agaaaatctt cgtcaacatg 12360

gtggagcacg acacacttgt ctactccaaa aatatcaaag atacagtctc agaagaccaa 12420

agggcaattg agacttttca acaaagggta atatccggaa acctcctcgg attccattgc 12480

ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc 12540

catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa 12600

gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca 12660

aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat 12720

ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag aacacggggg 12780

actcttgac 12789

<210> 14

<211> 3688

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 35S-SaSSY-RBCterminator

<400> 14

tatgaccatg attacgaatt cgcccgggga tctcctttgc cccagagatc acaatggacg 60

acttcctata tctctacgat ctagtcagga agttcgacgg agaaggtgac gataccatgt 120

tcaccactga taatgagaag attagccttt tcaatttcag aaagaatcct aacccacaga 180

tggttagaga cgcttacgca gcaggtctca tcaagacgat ctacccgagc aataatctcc 240

aggagatcaa ataccttccc aagaaggtta aagatgcagt caaaagattc aggactaact 300

gcatcaagaa cacagagaaa gatatatttc tcaagatcag aagtactatt ccagtatgga 360

cgattcaagg cttgcttcac aaaccaaggc aagtaataga gattggagtc tctaaaaagg 420

tagttcccac tgaatcaaag gccatggagt caaagattca aatagaggac ctaacagaac 480

tcgccgtaaa gactggcgaa cagttcatac agagtctctt acgactcaat gacaagaaga 540

aaatcttcgt caacatggtg gagcacgaca cgcttgtcta cctccaaaaa tatcaaagat 600

acagtctcag aagaccaaag ggaattgaga cttttcaaca aagggtaata tccggaaacc 660

tcctcggatt ccattgccca gctatctgtc actttattgt gaagatagtg gaaaaggaag 720

gtggctccta caaatgccat cattgcgata aaggaaaggc catcgttgaa gatgcctctg 780

ccgacagtgg tcccaaagat ggacccccac ccacgaggag catcgtggaa aaagaagacg 840

ttccaaccac gtcttcaaag caagtggatt gatgtgataa catggtggag cacgacacgc 900

ttgtctacct ccaaaaatat caaagataca gtctcagaag accaaaggga attgagactt 960

ttcaacaaag ggtaatatcc ggaaacctcc tcggattcca ttgcccagct atctgtcact 1020

ttattgtgaa gatagtggaa aaggaaggtg gctcctacaa atgccatcat tgcgataaag 1080

gaaaggccat cgttgaagat gcctctgccg acagtggtcc caaagatgga cccccaccca 1140

cgaggagcat cgtggaaaaa gaagacgttc caaccacgtc ttcaaagcaa gtggattgat 1200

gtgatatctc cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt 1260

cctctatata aggaagttca tttcatttgg agaggacacg ctgaaatcac cagtctctct 1320

ctaagcttgg atccatgaca gctccattca ttgatcctac tgatcatgtg aatctcaaaa 1380

ctgatacgga tgcctcagag aatcgaagga tgggaaatta taaacccagc atttggaatt 1440

atgatttttt acaatcactt gcaactcatc acaatattgt ggaagagagg catctaaagc 1500

tagctgagaa gctgaagggc caagtgaagt ttatgtttgg ggcaccaatg gagccgttag 1560

caaagctgga gcttgtggat gtggttcaaa ggcttgggct aaaccaccta tttgagacag 1620

agatcaagga agcgctgttt agtatttaca aggatgggag caatggatgg tggtttggcc 1680

accttcatgc gacatctctc cgatttaggc tgctacgaca gtgtgggctt tttattcccc 1740

aagatgtgtt taaaacgttc caaaacaaga ctggggaatt tgatatgaaa ctttgtgaca 1800

acgtaaaagg gctgctgagc ttatatgaag cttcatactt gggatggaag ggtgaaaaca 1860

tcctagatga agccaaggcc ttcaccacca agtgcttgaa aagtgcatgg gaaaatatat 1920

ccgaaaagtg gttagccaaa agagtgaagc atgcattggc tttgcctttg cattggagag 1980

tccctcgaat cgaagctaga tggttcattg aggcatatga gcaagaagcg aatatgaacc 2040

caacactact caaactcgca aaattagact ttaatatggt gcaatcaatt catcagaaag 2100

agattgggga attagcaagg tggtgggtga ctactggctt ggataagtta gcctttgcca 2160

ggaataattt actgcagagc tatatgtgga gctgcgcgat tgcttccgac ccgaagttca 2220

aacttgctag agaaactatt gtcgaaatcg gaagtgtact cacagttgtt gacgatggat 2280

atgacgtcta tggttcaatc gacgaacttg atctctacac aagctccgtt gaaaggtgga 2340

gctgtgtgga aattgacaag ttgccaaaca cgttaaaatt aatttttatg tctatgttca 2400

acaagaccaa tgaggttggc cttcgagtcc agcatgagcg aggctacaat agcatcccta 2460

cttttatcaa agcgtgggtt gaacagtgta aatcatacca gaaagaagca agatggttcc 2520

acgggggaca cacgcctcca ttggaagaat atagcttgaa tggacttgtt tccataggat 2580

tccctctctt gttaatcacg ggctacgtgg caatcgctga gaacgaggct gcactggata 2640

aagtgcaccc ccttcctgat cttctgcact actcctccct ccttagtcgc ctcatcaatg 2700

atataggaac gtctccggat gagatggcaa gaggcgataa tctgaagtca atccattgtt 2760

acatgaacga aactggggct tccgaggaag ttgctcgtga gcacataaag ggagtaatcg 2820

aggagaattg gaaaatactg aatcagtgct gctttgatca atctcagttt caggagcctt 2880

ttataacctt caatttgaac tctgttcgag ggtctcattt cttctatgaa tttggggatg 2940

gctttggggt gacggatagc tggacaaagg ttgatatgaa gtccgttttg atcgacccta 3000

ttcctctcgg cgaggagtag tctagagctt tcgttcgtat catcggtttc gacaacgttc 3060

gtcaagttca atgcatcagt ttcattgcgc acacaccaga atcctactga gttcgagtat 3120

tatggcattg ggaaaactgt ttttcttgta ccatttgttg tgcttgtaat ttactgtgtt 3180

ttttattcgg ttttcgctat cgaactgtga aatggaaatg gatggagaag agttaatgaa 3240

tgatatggtc cttttgttca ttctcaaatt aatattattt gttttttctc ttatttgttg 3300

tgtgttgaat ttgaaattat aagagatatg caaacatttt gttttgagta aaaatgtgtc 3360

aaatcgtggc ctctaatgac cgaagttaat atgaggagta aaacacttgt agttgtacca 3420

ttatgcttat tcactaggca acaaatatat tttcagacct agaaaagctg caaatgttac 3480

tgaatacaag tatgtcctct tgtgttttag acatttatga actttccttt atgtaatttt 3540

ccagaatcct tgtcagattc taatcattgc tttataatta tagttatact catggatttg 3600

tagttgagta tgaaaatatt ttttaatgca ttttatgact tgccaattga ttgacaacat 3660

gcatcaatcg atgaattcga gctcggta 3688

<210> 15

<211> 3521

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 35S-SaCYP736A 167-RBCterminator expression cassette

<400> 15

atcctctaga gtcgactatg accatgatta cgaattcgcc cggggatctc ctttgcccca 60

gagatcacaa tggacgactt cctatatctc tacgatctag tcaggaagtt cgacggagaa 120

ggtgacgata ccatgttcac cactgataat gagaagatta gccttttcaa tttcagaaag 180

aatcctaacc cacagatggt tagagacgct tacgcagcag gtctcatcaa gacgatctac 240

ccgagcaata atctccagga gatcaaatac cttcccaaga aggttaaaga tgcagtcaaa 300

agattcagga ctaactgcat caagaacaca gagaaagata tatttctcaa gatcagaagt 360

actattccag tatggacgat tcaaggcttg cttcacaaac caaggcaagt aatagagatt 420

ggagtctcta aaaaggtagt tcccactgaa tcaaaggcca tggagtcaaa gattcaaata 480

gaggacctaa cagaactcgc cgtaaagact ggcgaacagt tcatacagag tctcttacga 540

ctcaatgaca agaagaaaat cttcgtcaac atggtggagc acgacacgct tgtctacctc 600

caaaaatatc aaagatacag tctcagaaga ccaaagggaa ttgagacttt tcaacaaagg 660

gtaatatccg gaaacctcct cggattccat tgcccagcta tctgtcactt tattgtgaag 720

atagtggaaa aggaaggtgg ctcctacaaa tgccatcatt gcgataaagg aaaggccatc 780

gttgaagatg cctctgccga cagtggtccc aaagatggac ccccacccac gaggagcatc 840

gtggaaaaag aagacgttcc aaccacgtct tcaaagcaag tggattgatg tgataacatg 900

gtggagcacg acacgcttgt ctacctccaa aaatatcaaa gatacagtct cagaagacca 960

aagggaattg agacttttca acaaagggta atatccggaa acctcctcgg attccattgc 1020

ccagctatct gtcactttat tgtgaagata gtggaaaagg aaggtggctc ctacaaatgc 1080

catcattgcg ataaaggaaa ggccatcgtt gaagatgcct ctgccgacag tggtcccaaa 1140

gatggacccc cacccacgag gagcatcgtg gaaaaagaag acgttccaac cacgtcttca 1200

aagcaagtgg attgatgtga tatctccact gacgtaaggg atgacgcaca atcccactat 1260

ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag gacacgctga 1320

aatcaccagt ctctctctaa gcttggatcc atgtctccgg caacagccgt tatcctcact 1380

ctcctcgtgg ccctagggct atccatcctt ttgcggcggc gccaaaaaag aaataatcta 1440

cctcccggtc cacccgcttt accgatcatc ggaaacatcc acatattggg gacccttcct 1500

caccagagcc tctacaactt ggccaagaag tatggtccca tcatgtcaat gaggctgggg 1560

ctcgtgccgg ctgttgtgat atcctctccg gaggccgccg agctcgtcct caagacccac 1620

gatatcgttt tcgccagccg gcccagactc caagttgcgg actacttcca ttacgggaca 1680

aagggcgtca tcctgacgga gtatggtaca tattggcgca acatgcgaag gctgtgcacc 1740

gtgaagcttc tcaacacggt gaaaatcgat tctttcgcag ggacaaggaa gaaggaggtg 1800

gcatcgttcg tgcagtccct taaggaggct tcggtggcac acaaaatggt gaatttgagc 1860

gcgagggtgg cgaacgtcat tgaaaacatg gtgtgcctta tggtgatcgg gcgaagtagc 1920

gatgagaggt ttaagctaaa ggaggtcatc caggaggcag cgcagttggc gggagctttc 1980

aatatagggg attatgttcc attccttatg ccccttgacc tacagggatt aactcggcgc 2040

ataaagtcag gaagtaaagc tttcgacgac atcttggaag tcataatcga cgagcacgtg 2100

caagacatta aggaccatga tgatgaacaa catggagact tcattgatgt gttgctggca 2160

atgatgaaca agcccatgga ttcgcgggag ggtcttagta tcattgaccg aacaaacatc 2220

aaagcgatcc tagtggacat gattggagct gcaatggaca cttcaacaag tggcgtcgag 2280

tgggcgattt cagagctcat caagcatccg cgggtaatga aaaagctcca agacgaggtc 2340

aaaactgtca tcggaatgaa taggatggtc gaggaggccg acttgcctaa gctaccatac 2400

ctcgacatgg tagtgaaaga gaccatgagg ttacaccctc ctggaccatt gctcgtgccc 2460

cgagagtcca tggaagacat cacaatcaac ggatactaca tacctaagaa atcgcgaatc 2520

attgtcaacg cctgggcaat tgggcgtgat acaaacgcct ggtctaataa cgcgcacgag 2580

ttcttcccag agaggtttat gagtagcaat gtggacttac agggacaaga tttccaactt 2640

atcccattcg ggtccggtcg gagagggtgc cccgggatgc gcctaggcct cacaaccgtt 2700

cgattagtgt tagcgcagct cattcattgt ttcgacttgg agcttcctaa gggaaccgtg 2760

gcgaccgact tggacatgag tgagaaattc gggttggcaa tgcccagagc ccagcacttg 2820

cttgcatttc caacctatcg cttggagtcc taatctagag ctttcgttcg tatcatcggt 2880

ttcgacaacg ttcgtcaagt tcaatgcatc agtttcattg cgcacacacc agaatcctac 2940

tgagttcgag tattatggca ttgggaaaac tgtttttctt gtaccatttg ttgtgcttgt 3000

aatttactgt gttttttatt cggttttcgc tatcgaactg tgaaatggaa atggatggag 3060

aagagttaat gaatgatatg gtccttttgt tcattctcaa attaatatta tttgtttttt 3120

ctcttatttg ttgtgtgttg aatttgaaat tataagagat atgcaaacat tttgttttga 3180

gtaaaaatgt gtcaaatcgt ggcctctaat gaccgaagtt aatatgagga gtaaaacact 3240

tgtagttgta ccattatgct tattcactag gcaacaaata tattttcaga cctagaaaag 3300

ctgcaaatgt tactgaatac aagtatgtcc tcttgtgttt tagacattta tgaactttcc 3360

tttatgtaat tttccagaat ccttgtcaga ttctaatcat tgctttataa ttatagttat 3420

actcatggat ttgtagttga gtatgaaaat attttttaat gcattttatg acttgccaat 3480

tgattgacaa catgcatcaa tcgatgtcga cctgcaggca t 3521

Claims (10)

1. A method for preparing santalol from artemisia annua, which comprises introducing SaSSY and SaCYP736a167 into the artemisia annua.

2. The method of claim 1, wherein the SaSSY comprises the nucleic acid sequence set forth in SEQ ID No. 1.

3. The method of claim 1, wherein the SaCYP736a167 comprises the nucleic acid sequence set forth in SEQ ID No. 2.

4. The method of claim 1, wherein said introducing comprises transforming said Artemisia annua with an Agrobacterium tumefaciens-mediated expression vector comprising SaSSY and SaCYP736A 167.

5. The method of claim 4, wherein the Agrobacterium tumefaciens is Agrobacterium tumefaciens EHA 105.

6. The method of claim 4, wherein the expression vector is plasmid pCAMBIA 1300.

7. The method of claim 6, wherein said expression vector is transferred into said Agrobacterium tumefaciens by freeze-thaw.

8. The method according to claim 6, characterized in that the nucleic acid sequence comprising SaSSY and SaCYP736A167, obtained by amplification using the primers shown in SEQ ID No.7 and 8, is ligated into the plasmid pCAMBIA 1300.

9. The method of claim 1, wherein the santalol comprises α -santalol and/or β -santalol.

Use of SaSSY including a nucleic acid sequence shown as SEQ ID No.1 and SaCYP736a167 including a nucleic acid sequence shown as SEQ ID No.2 in the preparation of santalol using artemisia annua.

Images