CN101165186A - Flavonoid-3,5'-hydroxylase gene cloned from moth orchid, its coded sequence and application - Google Patents
Flavonoid-3,5'-hydroxylase gene cloned from moth orchid, its coded sequence and application Download PDFInfo
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Abstract
The present invention belongs to the field of molecular biology and gene technology, and is especially the nucleotide coding sequence of flavoid-3', 5'-hydroxylase gene expressed in butterfly orchid, and its application in changing flower color. The present invention relates to the recombinant expression vector including the said gene, the transgenic plant, nucleotide primer sequence for obtaining the gene, the oligonucleotide sequence probe for detecting endogenous expression mode and method of analyzing and identifying the expression mode of the endogenous gene in butterfly orchid. Transforming the gene into petunia obtains transgenic plant with differentially expressed flavoid-3', 5'-hydroxylase gene and altered flower color.
Description
Technical field
The invention belongs to molecular biology, gene engineering technology field, be specifically related to the nucleotide coding sequence of a kind of flavonoid-3 ' 5 ' of in butterfly orchid, expressing-'-hydroxylase gene, the recombinant expression vector that comprises said gene, transfer-gen plant, and the method for the expression pattern of this native gene of butterfly orchid being carried out Analysis and Identification.
Background technology
The color of occurring in nature flower is ever-changing, and this and the pathways metabolism of its complexity and various meta-bolites are undivided.As far back as 1664, Robert Boyle just studied the influence of plant color the acidity-basicity ph value.Pattern is mainly by flavonoid, carotenoid and the decision of alkaloid three class materials.Wherein the flavonoid class pigment is a class vacuole Bao Su, mainly exists in the vacuole, and the accumulation of different pigments in vacuole of flavonoid produced various patterns, and the different of the composition of pigment and concentration also are the reasons that various patterns occur simultaneously.And the generation of various coloring matters is to begin to be formed by plurality of enzymes catalysis successively along the pathways metabolism of complexity from phenylalanine.Wherein cyanidin(e) and anthocyanin are important color decision materials, also are the important detection indexs of pattern physiological level.And flavonoid-3 ' 5 '-hydroxylase (F3 ' 5 ' H) be synthetic 3 '-, the key enzyme and the rate-limiting enzyme of 5 '-hydroxyl cyanidin(e) (for example delphisine), this class pigment presents bluish voilet, is the reason of blue flower and pale reddish brown formation.Therefore the research about it just becomes important in a pattern research field integral part.
F3 ', 5 ' H gene belong to cytopigment 450 monooxygenases (P450) superfamily.Cytopigment 450 monooxygenases (P450) are the oxydase that protoheme relies on, and extensively in distribution and the various biologies, have very complicated function, can the thousands of reaction of catalysis.Vegitabilia distribute also very extensively, can catalytic reaction surpass 50 kinds, also relate to the synthetic of flavonoid.It makes plant show distinct colors at the adding hydroxyl of 3 ' and 5 ' regioselectivity of flavanonol thereby produce different anthocyanins.
At present cloned F3 ' 5 ' the H gene that has obtained several plant, as Nielsen﹠amp; Podiwinsky is (Nielsen KM, 1997) from prairie gentian Eustoma grandiflorum, and Okinaka (Okinaka Y, 2003) from bellflower Campan μ la medium clones and obtained F3 ' 5 ' H gene.
S.T.Jeong etc. are by the analysis to pigment, find leaf and the shade of fruit and the expression positive correlation (S.T.Jeong of F3 ' 5 ' H in the grape, 2005), Effendi Leonard etc. has expressed F3 ' 5 ' H gene (Effendi Leonard, 2005) from intestinal bacteria (Escherichia coli).Yuko Fukui etc. changes F3 ' 5 ' H gene in the carnation over to, finds that pattern has produced obvious variation, has become purple (Yuko Fukuia, 2002) from pink colour.YukihisaShimada etc. have also expressed F3 ' 5 ' H gene in tobacco and petunia, pattern all has significantly and deepens (YukihisaShimada, 1999).Nakatsuka T etc. inserts transposon among the F3 ' 5 ' H, thereby causes the inactivation of gene, and pattern becomes pink colour (Nakatsuka T, 2006) from purple.S.Mori etc. will clone F3 ' 5 ' the H gene that obtains and change in the petunia from Vinca, variation has taken place the pattern of some flower, has wherein detected the expression (S.Mori, 2004) of F3 ' 5 ' H.Christian Seitz etc. thinks that the specifically expressing of F3 ' 5 ' H gene in some feverfew is because the evolution (Christian Seitz, 2006) that hepatic petal can attract insect to produce effectively.
The present invention has studied a kind of flavonoid-3 ' 5 ' that clones-'-hydroxylase gene from butterfly orchid, changed the pattern phenotype after changing petunia over to.And before Invention Announce, any butterfly orchid flavonoid-3 ' 5 '-'-hydroxylase gene sequence of mentioning in the present patent application of openly reporting is not arranged as yet, its protein sequence or the application in transfer-gen plant.
Summary of the invention
The objective of the invention is to propose a kind of flavonoid-3 ' 5 ' that from butterfly orchid, clones-'-hydroxylase gene, its encoding sequence and application.
An aspect of of the present present invention provides a kind of new butterfly orchid gene, is designated as phf3 ' 5 ' h, and this gene is flavonoid-3 ' 5 '-'-hydroxylase gene, belongs to Cytochrome P450 family.Registered in GenBank/EMBL/DDBJ, the number of landing is: DQ148458, its nucleotides sequence classify as shown in the SEQ ID NO.1.
The present invention also provides the said gene protein sequence that coding translates, and its aminoacid sequence is shown in the SEQ ID NO.2.
The present invention also provides a kind of to have butterfly orchid flavonoid-3 ' 5 '-'-hydroxylase gene with above-mentioned coding and changes petunia over to change the method for pattern, and concrete steps are as follows:
(1) will the encode exercisable connection plant expression vector of nucleotide sequence of butterfly orchid flavonoid-3 ' 5 '-'-hydroxylase gene forms the carrier that contains nucleotide sequence shown in the SEQ ID NO.1; (see figure 4)
(2) change the expression vector in the step (1) over to Agrobacterium, and with bacterium liquid and petunia blade mixed culture.(see figure 5)
(3) by antibiotic-screening, PCR identifies, obtains transformant and regeneration of transgenic plant.
The present invention also provides a kind of method that is used for test sample butterfly orchid flavonoid-3 ' 5 '-'-hydroxylase gene copy number, it uses foregoing nucleotide sequence SEQ ID NO.1 that the butterfly orchid genome DNA sample is carried out Southern hybridization, and testing goal is segmental then has or not.Sample is the genomic dna of butterfly orchid, the nucleotide fragments behind digestion with restriction enzyme.
The present invention also provides and has been used for transferring acquisition butterfly orchid sample flavonoid-3 ' 5 '-'-hydroxylase gene nucleotide primer series.At first use F, the R primer obtains the part fragment of goal gene, uses AP then, AUAP, 3GSP1,3GSP2,36SP3,3 ' the end sequence that the acquisition of 3GSP4 primer links to each other with fragment re-uses 5-1a, 5-2,5-3,5-4,5-5,5-6,5-7,5-8,3-1b, 3-2,3-3 primer series obtains 5 ' end sequence.Each primer sequence is concrete as shown in SEQ ID NO.3.
At last, content of the present invention has also comprised transgenic petunia petal pigment glycosides content detecting method:
1. the extraction of anthocyanin:
(1) prepares liquid nitrogen, mortar and tweezers.The centrifuge tube of the number of finishing is weighed.
(2) petal is put into liquid nitrogen and ground fully, move in the centrifuge tube, weigh.
(3) every pipe adds the acidic methanol that 1ml prepares (methyl alcohol: HCl=24: 1), fully vibration disperses the petal fragment on vibrator.
(4) 4 ℃, 100rpm, shaking table jolting 2 days is vibrated on vibrator twice therebetween.
2. the measurement of anthocyanin:
(1) gets jolting liquid 800 μ l and be diluted to 2ml.
(2) measure the absorbancy of A530 and A657 respectively with spectrophotometer.
(3) calculate anthocyanin content.
Description of drawings:
The Southern hybridization of Fig. 1 butterfly orchid phf35h.Each 15 μ g of genomic dna use Bgl II (B) respectively, EcoR V (E) and Hind III (H) digestion.The right digitized representation molecular weight size (bp).The result shows that phf35h is single copy.
Fig. 2 RT-PCR detects the expression pattern of phf35h in the butterfly orchid.A, five open phases of flower; B, phf35h is at the expression of these five open phases; C, phf35h is spending (W) in vain, the expression in chrysanthemum (Y) and pale reddish brown (P) petal; D, phf35h be at butterfly orchid petal (Pe), leaf (L), the expression in the root (R).Figure left side band is the DL2000 molecular weight marker.
Fig. 3 F3 ' 5 ' H protein structure.Form by 17 alpha-helixs of 8 beta sheets.It is divided into two structural domains, α-Jie Gou territory and β-Jie Gou territory.The α-Jie Gou territory has comprised most alpha-helix and 3 little beta sheets, and the β-Jie Gou territory has comprised most beta sheet alpha-helix and 3 little alpha-helixs.
Fig. 4 vector plasmid transformed into escherichia coli PCR result, 1-5 are five bacterium colony PCR results of any picking.Transformation efficiency is 80%.
Fig. 5 shuttle plasmid transforms Agrobacterium PCR result, and 1-8 is eight bacterium colony PCR results of any picking.Transformation efficiency is 50%.
The group training upgrowth situation of Fig. 6 transgenic petunia.
The PCR result of Fig. 7 transgenic petunia genome Km gene.The petunia Km resistance of wild-type is very poor, amplifies specific band explanation plant by successful transgenosis.1-8 is transgenic petunia with Km primer (this primer serves as to obtain according to general bibliographic reference, non-this patent protection content) PCR result among the figure, and specific band is all arranged.The transgenosis success is described.
Fig. 8 transfer-gen plant changes comparison diagram than the wild-type pattern.
Embodiment:
Further explain the present invention below in conjunction with concrete embodiment.Should be understood that these examples only are not used in to be used to the present invention is described limits the scope of the invention.Unreceipted concrete experimental technique in the following example all can carry out according to ordinary method.Clone as the Sambrook equimolecular: condition described in the laboratory manual (New York:Cold Spring Harbor Labortary Press, 1989), or according to the operation instruction of making production firm.
The clone of butterfly orchid flavonoid-3 ' 5 '-'-hydroxylase gene
1 butterfly orchid (Phalaenopsis hybrida) material picks up from vegetable gardening institute of Shanghai Academy of Agricultural Sciences, and kind is Formosa rosa, and pattern is a purple, white, yellow.Vegetable material under the greenhouse normal condition, grow (L/D, 16h/8h; 25-28 ℃).
2 RNA extract.Get 100 milligrams of materials that the left and right sides is fresh, liquid nitrogen fully grinds.Add 1mL TriBlue reagent, firmly shake 15s, room temperature is placed 5min.Add the 0.2mL chloroform, Deproteinization, supernatant are transferred to new centrifuge tube, add the equal-volume Virahol, abundant mixing, and room temperature is placed 10min.75% ethanol 1mL washing precipitation with the DEPC preparation repeats once.Drying at room temperature 5～10min is dissolved in the 20 μ LDEPC water, surveys the OD value, electrophoresis detection.
The clone of 3 genes.By F3 ' 5 ' the H gene order of having delivered being carried out homology relatively,, carry out RT-PCR according to conserved regions design degenerate primer F and R.Primer sequence is seen SEQ ID NO.3.
Because gene fragment is excessive, abovementioned steps can't obtain complete gene order, obtains 3 ' end of F3 ' 5 ' H gene with 3 ' RACE.Extract total RNA, reverse transcription is cDNA.Use many wheel primers to comprise AP successively, AUAP, 3GSP1,3GSP2,3GSP3,3GSP4 carry out the nest-type PRC amplification.Primer sequence is seen SEQ ID NO.3 RACE primer series of tables.
5 ' end and upstream sequence thereof obtain with the IPCR method.Genomic dna is carried out enzyme with commercially available XbaI, XhoI and three kinds of enzymes of SphI to be cut.Enzyme is cut product from connecting, with trans pcr amplification again.Carry out the three-wheel amplified reaction, each take turns all use previous round product as masterplate.The special product of the specific PCR that obtains at last is connected to the pGEM-T carrier, clone, order-checking.The primer that uses comprises 5-1a, 5-2, and 5-3,5-4,5-5,5-6,5-7,5-8,3-1b, 3-2,3-3, concrete primer sequence is seen the trans PCR primer of SEQ ID NO.3 table.
The copy number analysis of butterfly orchid flavonoid-3 ' 5 '-'-hydroxylase gene
Extract the butterfly orchid genome with the CTAB method, get 10 μ gDNA and use Bgl II respectively, three kinds of commercially available enzymes of EcoR V and Hind III digest, and 37 ℃ of enzymes are cut and spent the night.With 0.8% sepharose enzyme is cut product and carry out electrophoretic separation.Transfer DNA also is fixed on the nylon membrane.Probe hybridization determines that it is but copies.(see figure 1)
The eukaryotic expression of butterfly orchid flavonoid-3 ' 5 '-'-hydroxylase gene in the petunia plant and the phenotypic evaluation of transfer-gen plant.
The structure of 1 expression vector.At first amplify complete coding and read frame, and on forward and reverse primer, introduce restriction enzyme enzyme recognition site (deciding) respectively, so that construction of expression vector on the carrier of selecting for use.Amplified production with acquisition among the embodiment 1 is a template, behind pcr amplification, butterfly orchid flavonoid-3 ' 5 '-'-hydroxylase gene forward is cloned among the carrier p2301.
2 change Agrobacterium over to.Preparation Agrobacterium competence utilizes the heat shock conversion method that plasmid is imported Agrobacterium.
3 Agrobacterium leaf dish methods transform petunia.
(1) the petunia blade is cut into 0.8 * 0.8cm (1cm) square, puts in the MSO liquid that contains Agrobacterium 190rpm shaking table, 10min into.
(2) blot blade and be placed on the MS1 that adds lid layer filter paper, seal film and seal, 22 ℃ of dark cultivations.
(3) wash bacterium, blot excessive moisture, be transferred to MS2 (vacuum side of blade down).
(4) illumination cultivation, callus forms, and 20d breaks up bud.
(5) reincarnation root substratum, 2～7d is taken root.
(6) tissue cultured seedling is transplanted and is buried hot-house culture.(see figure 6)
The mensuration of 4 transgenic petunia petal anthocyanins:
The extraction of anthocyanin:
(1) prepares liquid nitrogen, mortar and tweezers.The centrifuge tube of the number of finishing is weighed.
(2) petal is put into liquid nitrogen and ground fully, move in the centrifuge tube, weigh.
(3) every pipe adds the acidic methanol (methyl alcohol: HCl=24: 1), fully vibrate the dispersion of petal fragment on vibrator that 1ml prepares.
(4) 4 ℃, 100rpm, shaking table jolting 2 days is vibrated on vibrator twice therebetween.
The measurement of anthocyanin:
(1) gets jolting liquid 800 μ l and be diluted to 2ml.
(2) measure the absorbancy of A530 and A657 respectively with spectrophotometer.
(3) calculate anthocyanin content (U/g).
The concrete outcome of different sample number into spectrum see Table 1 and table 2 shown in (transfer-gen plant cyanidin(e) content and the comparison of pattern phenotypic evaluation)
The extraction average value measured of table 1 transgenic petunia petal anthocyanin
| Sample number into spectrum | A530 is average | A637 is average | A proofreaies and correct average | Average U/ | SD | |
| 1 | 0.063333 | 0.008667 | 0.06225 | 4.98 | 0.23388 |
| 16 | 0.190667 | 0.026333 | 0.187375 | 5.730122 | 0.169709 |
| 25-2 | 0.06 | 0.008667 | 0.058917 | 12.02381 | 0.204612 |
| 25-4 | 0.060667 | 0.006333 | 0.059875 | 4.714567 | 0.125661 |
| WT5 | 0.324333 | 0.120667 | 0.30925 | 6.063725 | 0.125288 |
| WT1 | 0.157 | 0.026 | 0.15375 | 8.401639 | 0.094648 |
WT is a wild-type, and its anthocyanin content (U/g) is between the 6-8 through survey; Be lower than 5 on the low side, as sample 1,25-4; Be higher than 10 for higher, as sample 25-2.
Table 2 transgenic petunia cyanidin(e) content and pattern depth relation statistics
| Sample number into spectrum | Cyanidin(e) value (average U/g) | Pattern |
| 1 | (4.98 on the low side) | Shoal deformity |
| 25-4 | (4.714567 on the low side) | Shoal |
| 16 | (5.730122 normally) | Consistent with wild-type |
| 25-2 | (12.02381 higher) | Deepen |
Sequence table
SEQ ID NO.1：
1 GCATGAACATCCTTCCTTCTGCTTATTCAAACTGCTGCAACTGCGCCAAAA
52 ATACTTAGTTGTTAAATATAAAAAATTTAAAGAAAATGATTTTTGATCGAA
103 AATATAAGAAAATGTAATTCTATTTTAAAATATCCAATTTTTAAAATGCCC
154 ATCACTCGACCTATATAAAACGCAGAACGAAATACTCCCGATCATCACAGA
205 ACCATGTCCATCTTCCTCATCGCAACCCTCTTCCTCTCTCTTTCCCTCCAC
256 CTCCTACTCCGCCGCTTCCGCCGCCGCCGCCGCATCCTCCCCCTCCCTCCC
307 GGACCCCTCAACTTCCCCATCGTCGGCGCACTCCCCTTCATCGGCTCCATG
358 CCCCACTCAGGCCTCGCCCTCCTCTCCCGCCGCTACGGCCCCATTATGTTC
409 CTCAAAATGGGCATCCGCCAAGTGGTCGTCGCCTCCTCCTCCTCCGCCGCA
460 CGCTCCTTTCTCAAAACTCACGACTCCCGCTTCTCCGACCGCCCTCTCGAC
511 ATCATCTCGAAGCAAGTCAGCTACAACGGCCAGAACATGGTCTTCGCCGAC
562 TACGGTCCCAAGTGGAAGCTCCTCCGCAAAGTCTCCAATCTCCATCTCTTT
613 GGCCCCAAGGCCATGTCCCGTTGGGCCGACGTGCGGCGGGACGAAGCTTTC
664 TCCATGTCCCACTTCCTGAAGAAACAGAGCGATTCCAAAAACCCTGTTTTG
715 CTTTCCAACTTGCTGGTTTGTTCCATGGCGAATGTGATTGGGAGAATTTCG
766 ATGAGCAAGAGGGTGTTCGATGAGGAGGGGAAGGAGGCGAAGGAGTTTAAG
817 GAGATAATTAAGGAGCTTTTGGTCGGGCAGGGGGCTTCGAATATTGGGGAT
868 TTGGTGCCGGCGATGAGGTGGCTGGATCCGCAGGGGGCGAGGAAGAAGCTG
919 CTGGGATTGAATCAGAGGTTTGTTAGGATGATAAGTAAGTTCTTGGCGGAG
970 CACGGTGAGAGTAGAGGGGAGCGGGAGGGGAATCCTGATCTGCTTGATCTT
1021 ATTGTGGCTGATAAGATCGCCGGCGACGACGGGGAAGGGCTTTCTGAGGAG
1072 AACATCAAGGGCTTCATATCTGATCTATTCGTGGCGGGCACAGACACATCC
1123 GCTATGGTCATCGAGTGGGCGATGGCTGAGATGCTAAAAAATCCGGCGATC
1174 CTCCGCCGAGTACAGGAAGAAACCGACCGCATCGTCGGCCGCGATCGCCTT
1225 CTCGAAGAATCCGACATACCAAATCTCCCCTACCTCCAAGCCATATGCAAG
1276 GAAGCTCTGCGCAAGCATCCCCCAACTCCTCTCAGCATACCTCACTACGCT
1327 AGCGAGCCCTGCGAGGTAGAAGGTTACCACATTCCCGGCAAGACCTGGCTT
1378 TTGGTCAACATATGGGCCATCGGGCGAGACCCAGAGGTGTGGGAGAAGCCA
1429 TTGGAGTTCGATCCGGAGAGGTTTATGGAAGGGAAGATGGCGAGGATTGAT
1480 CCGATGGGGAACGATTTTGAGCTGATACCGTTCGGCGCTGGGAGGAGGATT
1531 TGCGCGGGGAAGTTGATGGGGATGGTTATGGTGCAGTACTTTTTGGGAGTG
1582 TTGGTGCAGGGTTTTGACTGGAGTTTGCCGGAAGGGGTGGTGGAGCTGGAC
1633 ATGGAGGAGGGGCCGGGATTGGTGTTGCCGAAGGCGGTGCCGCTGTTGGTG
1684 ACGGCGAGGCCGCGGCTGCCGGCGGCGGCGTATGGGGTTGTTTGATAAAGG
1735 AGAGTTTTTTCTAGTTTATGTACTGCGTGTTAAGAGTTTTGAATAATGAAT
1786 AATTAATCTATTTCGATGTAAAAAAAAAAAAAA
SEQ ID NO. 2：
MSIFLIATLFLSLSLHLLLRRFRRRRRILPLPPGPLNFPIVGALPFIGSMPHSGLALLSRRYGPIMFLKMG
IRQVVVASSSSAARSFLKTHDSRFSDRPLDIISKQVSYNGQNMVFADYGPKWKLLRKVSNLHLFGPKAMSRWAD
VRRDEAFSMSHFLKKQSDSKNPVLLSNLLVCSMANVIGRISMSKRVFDEEGKEAKEFKEIIKELLVGQGASNIG
DLVPAMRWLDPQGARKKLLGLNQRFVRMISKFLAEHGESRGEREGNPDLLDLIVADKIAGDDGEGLSEENIKGF
ISDLFVAGTDTSAMVIEWAMAEMLKNPAILRRVQEETDRIVGRDRLLEESDIPNLPYLQAICKEALRKHPPTPL
SIPHYASEPCEVEGYHIPGKTWLLVNIWAIGRDPEVWEKPLEFDPERFMEGKMARIDPMGNDFELIPFGAGRRI
CAGKLMGMVMVQYFLGVLVQGFDWSLPEGVVELDMEEGPGLVLPKAVPLLVTARPRLPAAAYGVV
SEQ ID NO.3：
Degenerate primer:
F：5’-GCC ATG GC(A/G/C)AA(C/T)ATG(A/T)T(A/C)GG-3’
R：5’-CTC T(T/C)C C(A/G)A T(T/G/A)G CCC ATA TG-3’。
RACE primer series of tables:
| The primer title | Sequence (5 ' → 3 ') |
| AP | GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT |
| AUAP | GGCCACGCGTCGACTAGTAC |
| 3GSP1 | GGTCGGGCAGGGGGCTTCGA |
| 3GSP2 | GCGGGCACAGACACATCCGCT |
| 3GSP3 | TCGAGTGGGCGATGGCTGAGAT |
| 3GSP4 | CCTACCTCCAAGCCATATGC |
Trans PCR primer table:
| The primer numbering | Primer sequence (5 ' → 3 ') | The primer numbering | Primer sequence (5 ' → 3 ') |
| 5-1a | CTCACCGTGCTCCGCCAAGA | 3-1b | CCTACCTCCAAGCCATATGC |
| 5-2 | GGCACCAAATCCCCAATA | 3-2 | CATCCCCCAACTCCTCTTAG |
| 5-3 | ACTCCTTCGCCTCCTTCC | 3-3 | CGGTGCCGCTATTGGTGACG |
| 5-4 | TCCCAATCACATTCGCCA | ||
| 5-5 | GCTTCGTCCCGCCGCACA | ||
| 5-6 | GAGGGCGGTCAGAGAAGC | ||
| 5-7 | AGCGGCGGGAGAGGAGGG | ||
| 5-8 | TGGGGAAGTTGAGGGGTC |
Claims (5)
1. isolated flavonoid-3 ' 5 '-'-hydroxylase gene, it is characterized in that for having the dna molecular of particular sequence, the exon that comprises two long 885bp of being respectively and 636bp, and longly between two exons be the intron of 213bp, its nucleotides sequence is classified SEQ ID NO.1 as.
2. one kind by flavonoid-3 ' 5 ' as claimed in claim 1-'-hydroxylase gene encoded protein matter molecule, it is characterized in that long 507 amino-acid residues, and its aminoacid sequence is SEQ ID NO.2.
3. method that is used for test sample butterfly orchid flavonoid-3 ' 5 '-'-hydroxylase gene copy number, it is characterized in that using according to claim 1, nucleotide sequence SEQ ID NO.1 carries out southern hybridization to the butterfly orchid genome DNA sample, testing goal is segmental then has or not, sample is the genomic dna of butterfly orchid, the nucleotide fragments behind digestion with restriction enzyme.
4. be used for transferring acquisition butterfly orchid sample flavonoid-3 ' 5 '-'-hydroxylase gene nucleotide primer sequence, comprise degenerate primer according to the described flavonoid-3 ' 5 ' of claim 1-'-hydroxylase gene family conservative region design, a series of RACE primers and trans PCR primer, these primer sequences are shown in SEQ ID NO.3.
5. the nucleotide sequence that coding as claimed in claim 1 is had butterfly orchid flavonoid-3 ' 5 '-'-hydroxylase gene changes petunia over to change the method for pattern, and concrete steps are as follows:
(1) will the encode exercisable plant expression vector that is connected in of nucleotide sequence SEQ ID NO.1 of butterfly orchid flavonoid-3 ' 5 '-'-hydroxylase gene forms the carrier that contains nucleotide sequence shown in the SEQ ID NO.1;
(2) change the expression vector in the step (1) over to Agrobacterium, and with bacterium liquid and petunia blade mixed culture;
(3) by antibiotic-screening, PCR identifies, obtains transformant and regeneration of transgenic plant.
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