CN103589694A - Tulip flavonoid-3'-hydroxylase TfF3' H protein and its coding gene - Google Patents
Tulip flavonoid-3'-hydroxylase TfF3' H protein and its coding gene Download PDFInfo
- Publication number
- CN103589694A CN103589694A CN201310533249.4A CN201310533249A CN103589694A CN 103589694 A CN103589694 A CN 103589694A CN 201310533249 A CN201310533249 A CN 201310533249A CN 103589694 A CN103589694 A CN 103589694A
- Authority
- CN
- China
- Prior art keywords
- tff3
- sequence
- protein
- seq
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
- C12Y114/13021—Flavonoid 3'-monooxygenase (1.14.13.21)
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a tulip flavonoid-3'-hydroxylase TfF3' H protein and its coding gene. The protein is the protein composed of the amino acid sequence shown as SEQ ID NO.4 or is the protein that is formed by subjecting the amino acid sequence shown as SEQ ID NO.4 to substitution, deletion or adding of one or several amino acids and has the activity of tulip flavonoid-3'-hydroxylase. The invention also provides a nucleic acid sequence shown as SEQ ID NO.3 coded with the protein. The tulip flavonoid-3'-hydroxylase involved in the invention is converted into tobaccos by constructing an overexpression vector, then the pink color of over 50% transgenic tobacco flowers deepens, and accordingly, the content of cyanidin in the transgenic tobacco petals increases by 20-30%. The results show that the tulip flavonoid-3'-hydroxylase can act on anthocyanin synthesis routes, influence cyanidin synthesis and change plant flower color.
Description
Technical field
The present invention relates to crux enzyme and encoding gene thereof in turmeric pattern glycosides route of synthesis, be specifically related to a kind of turmeric flavonoid-3 '-hydroxylase TfF3 ' H albumen and encoding gene thereof, belong to molecular engineering field.
Background technology
The color of flower is the important factor that affects plant pollination, also determines commodity value and the ornamental value of a kind of flowers simultaneously.Change pattern, create new pattern, can increase commodity value and the ornamental value of flowers.The color of flower is the result that accumulates cyanidin(e) in petal cell, and cyanidin(e) mainly comprises flavonoid, carotenoid and betaines.Flavonoid is a large class of cyanidin(e), and it makes flower produce the whole colors from Huang to purple.
Flavonoid-3 '-hydroxylase (flavonoid3 '-hydroxylase, F3 ' H, in document, be once called in the past flavanonol-3 '-hydroxylase, existing flavonoid-3 '-hydroxylases that are called) belong to cytochrome P 450 monooxygenases more, it has the function of the substrate oxidizing reaction of catalysis multiple dependence NADPH or NADH, in the synthetic and secondary metabolism of plant flavonoids, plays an important role.It can be at cofactor NADPH and O
2effect under, catalysis list Oxygenation, respectively by 3 ' position hydroxylation of naringenin (naringenin) and dihydrokaempferol (dihydrokaempferol) B-ring, catalysis forms eriodictyol and dihydroquercetin.And eriodictyol and dihydroquercetin are the important precursor of synthetic cyanidin(e), be the important colour former of flower, for the biosynthesizing of anthocyanidin, play an important role, thereby form different anthocyanins, make plant show different patterns.The expression activity of F3 ' H gene can directly affect the final color of flower, for example, in petunia, express F3 ' the H gene himself obtaining and make the pale pink petunia that lacks F3 ' H gene and F3 ' 5 ' H gene become deep pink; Rough gentian F3 ' H gene transformation is entered in tobacco, find in transgene tobacco body, to occur the increase of anthocyanidin and quercetin content, thereby affected the pattern of tobacco; During the disappearance of F3 ' H gene, pharbitis nilChoisy (Ipomoea nil), tricolo(u)r morning glory (Ipomoea tricolor) and Pharbitis purpurea (Ipomoea purpurea) can produce the ruddy flower that contains pelargonidin derivative.Therefore, to F3 ' H genetic expression to regulate and control be one of approach changing pattern.
F3 ' H gene is cloned in various plants, but in turmeric (Tulipa fosteriana), the clone of F3 ' H gene, expression pattern and F3 ' H albumen coded sequence it be not immediately clear.At present, there is not any bibliographical information relevant to turmeric F3 ' H albumen and coding gene sequence thereof.
Summary of the invention
The present invention fills up the blank of turmeric F3 ' H gene family member's clone, expression pattern analysis and turmeric F3 ' H albumen and encoding gene thereof, a kind of turmeric TfF3 ' H protein sequence is provided, and the present invention also provides a kind of coding above-mentioned nucleic acid sequences to proteins.The invention discloses the expression pattern that turmeric TfF3 ' H albumen and nucleotides sequence thereof are listed in turmeric Different Organs, different developmental phases; Build TfF3 ' H gene overexpression carrier transformation of tobacco, analyzed the impact of the synthetic and pattern of TfF3 ' H gene pairs transgene tobacco anthocyanogen.Utilize turmeric flavonoid provided by the invention 3 '-hydroxylase TfF3 ' H, can change pattern for genetically engineered, obtain innovation pattern a kind of effective technique means is provided.In addition, in addition, utilize turmeric TfF3 ' H gene of the present invention, by various conventional screening methods, can filter out and material, acceptor, inhibitor or the antagonist etc. of mutual use occur F3 ' H.
The present invention is achieved by the following technical solutions,
The object of the invention is to provide a kind of protein with turmeric flavonoid-3 '-hydroxylase activity, the protein that described protein is comprised of the aminoacid sequence as shown in SEQ ID NO.4; Or by aminoacid sequence shown in SEQ ID NO.4 through replacement, lack or add one or several amino acid and have turmeric flavonoid-3 '-hydroxylase activity by (a) derivative protein.This protein having that it's too late active size exist larger difference in painted stage of difference of flower petal, Different Organs.
Preferably, described protein be aminoacid sequence shown in SEQ ID NO.4 through 1～50 amino acid whose disappearance, insertion and/or replacement, or add 1～20 sequence obtaining with interior amino acid at C-terminal and/or N-terminal.
Further preferred, described protein be shown in SEQ ID NO.4 in aminoacid sequence 1～10 amino acid by the similar or close amino acid of character, replaced the sequence forming.
On the other hand, the invention provides the above-mentioned nucleic acid sequences to proteins of a kind of coding.
Preferably, described nucleotide sequence is specially:
(a) base sequence is as shown in 1st～1533 of SEQ ID NO.3;
Or (b) and the nucleic acid shown in 1st～1533 of SEQ ID NO.3 have the sequence of at least 70% homology;
Or the sequence that (c) can hybridize with the nucleic acid shown in 1st～1533 of SEQ ID NO.3.
Preferably, described nucleotide sequence is specially disappearance, insertion and/or the replacement of 1～90 Nucleotide in the nucleotide sequence shown in 1st～1533 of SEQ ID NO.3, and adds 60 sequences that form with inner nucleotide at 5 ' and/or 3 ' end.
In addition, the present invention also provides a kind of probe that detects above-mentioned nucleotide sequence, described probe is the nucleic acid molecule with 8～100 continuous nucleotides of above-mentioned nucleotide sequence, and this probe can be used for detecting in sample, whether have the relevant nucleic acid molecule of coding turmeric F3 ' H.
Isolated DNA molecular provided by the invention, this molecule comprises: the DNA molecular with nucleotide sequence shown in SEQ ID NO.3; Or coding has the nucleotide sequence of the polypeptide of turmeric TfF3 ' H protein active, and has at least 70% homology with sequence shown in SEQ ID NO.3; Or can with the nucleotide sequence hybridization of sequence shown in SEQ ID NO.3.
In the present invention, " separated DNA ", " DNA of purifying " refer to, this DNA or fragment have been arranged in the sequence of its both sides and have separated from native state, also refer to that this DNA or fragment with under native state follow the component of nucleic acid to separate, and separate with the protein accompanying in cell.
In the present invention, term " turmeric flavonoid-3 '-hydroxylase protein coded sequence " refers to that coding has the nucleotide sequence of the polypeptide of turmeric TfF3 ' H protein-active, nucleotide sequence and degenerate sequence thereof as shown in SEQ ID NO.3.This degenerate sequence refers to, is arranged in sequence shown in SEQ ID NO.3, the sequence that has one or more codons to be encoded to produce after the degenerate codon of same amino acid replaces.Due to the degeneracy of codon, so be low to moderate approximately 70% the degenerate sequence aminoacid sequence shown in SEQ ID NO.4 of also encoding out with the homology of sequence shown in SEQ ID NO.3.This term also comprise with sequence shown in SEQ ID NO.3 in from the nucleotide sequence of the homology at least 70% of the nucleotide sequence of 1st～1533, Nucleotide.
This term also comprises encoding to have the variant form of sequence shown in the SEQ ID NO.3 with the albumen of natural turmeric TfF3 ' H identical function.These variant forms comprise (but being not limited to): be generally disappearance, insertion and/or the replacement of 1～90 Nucleotide, and be added to 60 with inner nucleotide at 5 ' and/or 3 ' end.
In the present invention, term " turmeric flavonoid-3 '-hydroxylase TfF3 ' H albumen " refers to the polypeptide of sequence shown in the SEQ ID NO.4 with turmeric TfF3 ' H protein-active.This term also comprises having and the variant form relevant identical function of natural turmeric TfF3 ' H, sequence shown in SEQ ID NO.4.These variant forms comprise (but being not limited to): be generally 1～50 amino acid whose disappearance, insertion and/or replacement, and C-terminal and/or N-terminal add one or be 20 with interior amino acid.For example, in the art, while replacing with the close or similar amino acid of performance, conventionally can not change the function of protein.Again such as, at C-terminal and/or N-terminal, add one or several amino acid and conventionally also can not change the function of protein.This term also comprises active fragments and the reactive derivative of turmeric TfF3 ' H albumen.
The variant form of turmeric TfF3 ' H polypeptide of the present invention comprises: the albumen that homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, DNA that can DNA hybridization relevant to turmeric TfF3 ' H under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of turmeric TfF3 ' H polypeptide to obtain.
In the present invention, " turmeric TfF3 ' H conservative property variation polypeptide " refers to compare with the aminoacid sequence of sequence shown in SEQ ID NO.4, has at the most 10 amino acid be replaced by the similar or close amino acid of character and forms polypeptide.These conservative property variation polypeptide are preferably replaced and are produced according to table 1.
Table 1
| Initial residue | Representational replacement | Preferred replacement |
| Ala(A) | Val;Leu;Ile | Val |
| Arg(R) | Lys;Gln;Asn | Lys |
| Asn(N) | Gln;His;Lys;Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro;Ala | Ala |
| His(H) | Asn;Gln;Lys;Arg | Arg |
| Ile(I) | Leu;Val;Met;Ala;Phe | Leu |
| Leu(L) | Ile;Val;Met;Ala;Phe | Ile |
| Lys(K) | Arg;Gln;Asn | Arg |
| Met(M) | Leu;Phe;Ile | Leu |
| Phe(F) | Leu;Val;Ile;Ala;Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr;Phe | Tyr |
| Tyr(Y) | Trp;Phe;Thr;Ser | Phe |
| Val(V) | Ile;Leu;Met;Phe;Ala | Leu |
Invention also comprises the analogue of turmeric TfF3 ' H albumen or polypeptide.The difference of these analogues and turmeric TfF3 ' H related polypeptide can be the difference on aminoacid sequence, can be also the difference not affecting on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant natural or induction.Induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known molecular.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and the analogue with non-natural amino acid (as β, gamma-amino acid) that exist or synthetic.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide of enumerating.
(conventionally the not changing primary structure) form of modification comprises: the chemically derived form of the polypeptide that body is interior or external is as acetylize or carboxylated.Modify and also comprise glycosylation, as those carry out polypeptide glycosylation modified and that produce in procedure of processing in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and completes by polypeptide is exposed to.Modified forms also comprises the have phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, the expression pattern of methods analyst turmeric TfF3 ' the H gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of the mRNA transcript of analyzing TfF3 ' H gene in cell.
The present invention detects the detection method that whether has turmeric TfF3 ' H related nucleotide sequences in sample, comprises with above-mentioned probe and sample and hybridizing, and then whether detection probes combination has occurred.This sample is the product after pcr amplification, and wherein pcr amplification primer is corresponding to turmeric TfF3 ' H associated nucleotide encoding sequence, and can be positioned at both sides or the centre of this encoding sequence.Primer length is generally 15～50 Nucleotide.
In addition, according to turmeric TfF3 ' H nucleotide sequence of the present invention and aminoacid sequence, can be on the homology basis of nucleic acid homology or marking protein, the relevant homologous gene of screening turmeric TfF3 ' H or homologous protein.
In order to obtain the dot matrix with turmeric TfF3 ' H genes involved, can screen turmeric cDNA library with DNA probe, these probes are under low rigorous condition, use
32p relevant all or part of of turmeric TfF3 ' H cooked to radioactivity mark and.The cDNA library that is suitable for screening is the library from turmeric.Structure is that biology field is well-known from the method for the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example, purchased from Clontech, and Stratagene, Palo Alto, Cal..This screening method can be identified the nucleotide sequence of the gene family relevant to turmeric TfF3 ' H.
Turmeric TfF3 ' H associated nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually need to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplified is stitched together by proper order.
After having obtained relevant sequence, can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, then by ordinary method separation from the host cell propagation, obtains relevant sequence.
In addition, also can will suddenly change and introduce in protein sequence of the present invention by chemosynthesis.
Except producing with recombination method, the fragment of albumen of the present invention is available solid phase technique also, by direct peptide synthesis, produced (people such as Stewart, (1969) solid-phase polypeptide is synthetic, WH Freeman Co., San Francisco; Merrifield J.(1963) J.Am Chem.Soc85:2149-2154).Synthetic protein can carry out by hand or automatically in vitro.For example, can be with the 431A type peptide synthesizer (Foster City, CA) of Applied Biosystems from moving synthetic peptide.Can distinguish each fragment of chemosynthesis albumen of the present invention, then by chemical process, be connected to produce the molecule of total length.
Utilize turmeric TfF3 ' H albumen of the present invention, by various conventional screening methods, can filter out with turmeric TfF3 ' H interactional material occurs, or acceptor, inhibitor or antagonist etc.
Turmeric flavonoid-3 '-'-hydroxylase gene of the present invention or its homologous sequence can import plant tissue, cell or organ by plant expression vector; For the carrier that sets out that builds described plant expression vector, can be the carrier etc. that any one can be used for the binary vector of agrobacterium tumefaciens conversion of plant or can be used for plant micropellet bombardment.As pBin serial carrier, pBI serial carrier, Gateway
tMserial carrier, pCAMBIA serial carrier or other derivative plant expression vector, the described carrier that sets out can also be the carrier copying in prokaryotic organism, as pENTER-TOPO, pUC serial carrier etc.
While using in the present invention turmeric flavonoid-3 '-'-hydroxylase gene or its homologous sequence construction of expression vector, before transcribing super beginning Nucleotide, it can add any enhancement type, composing type, organizing specific type or inducible promoter.Constitutive promoter can be cauliflower mosaic virus (CAMV) 35S promoter etc.; Tissue-specific promoter can be the specific expressing promoters such as root, blade, petal, seed; Inducible promoter can be the promotor of inductions such as being subject to ethene, book alcohol.Above-mentioned promotor can be used separately or be combined with other plant promoter.In addition, while utilizing gene constructed plant expression vector of the present invention, also enhanser be can use, translational enhancer or transcriptional enhancer comprised.
For the ease of transgenic plant cells or plant are identified and are screened, can process plant expression vector used, the coding that can express in plant as added can produce the antibiotic marker thing gene of the enzyme of colour-change or luminophor gene, tool resistance or anti-chemical reagent marker gene etc.Described host plant cell, tissue or organ containing neomycin phosphotransferase (NPTII) gene can be screened by kantlex or its substituted derivatives etc., and the host plant cell, tissue or the organ that contain Totomycin transferase gene can be screened by Totomycin.After screening, aforesaid method also can adopt Southern, PCR or dot blot equimolecular detection means to detect transfer-gen plant, to determine whether it has transformed goal gene.
A specific embodiment of the present invention is to take the transformation carrier of pHB(pCambia1300 excision gus gene) be plant expression vector, build the mistake expression plant vector pHB-TfF3 ' H that contains turmeric flavonoid-3 '-'-hydroxylase gene of the present invention.Can pass through the plant expression vector that carries turmeric flavonoid-3 '-'-hydroxylase gene of the present invention or its homologous sequence protoplastis-chemical mediated method, Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, pollen tube importing, microinjection, electricity swash, combination transformed plant cells, tissue or the organ of any one or more method in particle gun, the conventional biological method such as agriculture bacillus mediated, cultivate synthetic plant cell, tissue or the organ changing of anthocyanogen, and further cultivate into plant; Described tissue and organ can comprise fruit, callus, root, stem apex, blade and the seed etc. of host plant.
Turmeric is one of the world's ten large cut-flowers, and ornamental value is high, is widely used, and people are also increasing to the demand of new pattern.Clone first the encoding sequence of key enzyme flavonoid-3 '-hydroxylase TfF3 ' H albumen at the synthetic forehearth limb's point of tulip petals flavonoid place, and turmeric flavonoid-3 '-'-hydroxylase gene in the present invention is transformed in tobacco by building over-express vector, found that under identical culture condition, transgene tobacco with contrast (unconverted) tobacco and compare, the pink colour of 50% above transgene tobacco pattern is deepened, correspondingly, Cyanidin content rising 20-30% in transgene tobacco petal.This shows that turmeric flavonoid-3 '-hydroxylase can act on anthocyanogen route of synthesis, affects Cyanidin synthetic, changes plant pattern.Flavonoid-3 '-hydroxylase is the synthetic key enzyme of Cyanidin in cyanidin(e) route of synthesis, turmeric flavonoid-3 '-'-hydroxylase gene TfF3 ' H provided by the invention, for utilizing genetically engineered to change pattern, obtains innovation pattern a kind of effective technique means is provided.In addition, utilize turmeric TfF3 ' H albumen of the present invention, by various conventional screening methods, can filter out with turmeric TfF3 ' H interactional material occurs, or acceptor, inhibitor or antagonist etc.
Accompanying drawing explanation
By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:
Fig. 1 is homology comparison (GAP) result of the nucleotide sequence of turmeric TfF3 ' H gene of the present invention and lily F3 ' H gene mRNA;
Fig. 2 is homology comparison (GAP) result of the nucleotide sequence of turmeric TfF3 ' H gene of the present invention and lily F3 ' H gene mRNA;
Fig. 3 is homology comparison (GAP) result of the nucleotide sequence of turmeric TfF3 ' H gene of the present invention and lily F3 ' H gene mRNA;
Fig. 4 is homology comparison (FASTA) result of the aminoacid sequence of turmeric TfF3 ' H of the present invention and lily F3 ' H, and wherein, identical amino acid marks with amino acid monocase between two sequences.
Fig. 5 is that plant over-express vector pHB-TfF3 ' H builds schematic diagram.
Fig. 6 is unconverted tobacco (a) and the pattern comparison that turns TfF3 ' H genetic tobacco (b, c).
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, for example Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
1. the acquisition of vegetable material
By health, tulip of the same size, (Tulipa fosteriana ' Shangnongzaoxia ', by Shanghai City crop varietal approval committee.Numbering: Shanghai agriculture product are recognized flowers 2011 No. 004) plant routinely and carry out field management, treating that flower is completely open, petal is complete gathers Petal when painted, for extracting RNA.
The extracting of 2.RNA
Utilize " RNA prep pure plant total RNA extraction reagent box " extracted total RNA (RNA prep pure Plant Kit: TIANGEN Biotech (Beijing) Co., Ltd.).With denaturing formaldehyde gel electrophoresis, identify the integrity of RNA, then in upper purity and the concentration of measuring RNA of spectrophotometer (Thermo Scientific NANODROP1000Spectrophotometer).
3. the full-length clone of gene
According to the amino acid conserved sequence of F3 ' H gene in other species, utilize homologous genes clone principle, adopt RACE method (3 '-Full RACE Core Set Ver.2.0: precious biotechnology (Dalian) company limited, SMARTer
tMrACEcDNA Amplification Kit:Clontech Laboratories, Inc.) carry out cDNA full-length clone, a minute three phases carries out:
(1) RT-PCR obtains gene intermediate segment
The RNA of extraction is carried out to reverse transcription (Prime Script II 1st Strand cDNA Synthesis Kit: precious biotechnology (Dalian) company limited), the first chain cDNA of take is template, utilize primers F 1(SEQ ID NO.1) and R1(SEQ ID NO.2) carry out PCR, amplification obtains about 1000bp fragment, reclaim and be connected on pMD18-T Simplevector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, at ABI377 sequenator (Perkin-Elmer, USA) on, check order.Sequencing result is by carrying out BLAST(in NCBI website
http:// blast.ncbi.nlm.nih.gov/) compare existing database (GenBank), know that the homology of its nucleotide sequence and proteins encoded and known lilium flavonoid-3 '-'-hydroxylase gene is very high, therefore tentatively think that it is flavonoid-3 '-'-hydroxylase gene.
（2）3’RACE
The sequence of 3 ' end is by being used 3 '-Full RACE Core Set Ver.2.0: precious biotechnology (Dalian) company limited obtains, and two take turns the amplification that nest-type PRC completes 3 ' end sequence:
The first round: Outerprimer+F3 ' H3-1(5 '-CTTCCACATCCCCAAGCACGCCACT-3 ')
Second takes turns: Innerprimer+F3 ' H3-2(5 '-GTGGAGTTCAAGCCTTCCCGGTTCA-3 ')
Outerprimer and Innerprimer provide for test kit.3 ' RACE obtains the 3 ' end sequence (476bp) of TfF3 ' H, reclaim, be connected on pMD18-T Simple vector carrier, with RV-M and M13-47, as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, on ABI377 sequenator (Perkin-Elmer, USA), check order, sequencing result is by carrying out BLAST(in NCBI website
http:// blast.ncbi.nlm.nih.gov/) compare existing database (GenBank), know that the homology of its nucleotide sequence and proteins encoded and known lilium flavonoid-3 '-'-hydroxylase gene is high.
（3）5’RACE
The sequence of 5 ' end is by being used SMARTer
tMrACE cDNA Amplification Kit obtains, and 5 ' the RACEready cDNA of take is template, takes turns nest-type PRC obtain by two:
The first round: UPM+F3 ' H5-1(5 '-GACTGCGGGGTCTCGAGCAATGG-3 ')
Second takes turns: NUP+F3 ' H5-2(5 '-ACCAGCACACTCAGCAAATCCCTCCCA-3 ')
UPM and NUP provide for test kit.5 ' RACE amplification obtains the 5 ' end sequence (836bp) of TfF3 ' H, reclaims after connecting by the method with above and checks order.
The sequencing result of the sequence obtaining by above-mentioned 3 kinds of methods is spliced, will splice sequence and submit to BLAST to analyze, TfF3 ' the H gene that result proof newly obtains from turmeric is really a gene relevant to flavonoid-3 '-hydroxylase.ORF Finding(by sequencing result in conjunction with NCBI
http:// www.ncbi.nlm.nih.gov/gorf) prediction, initiator codon and the terminator codon of turmeric TfF3 ' H gene have been found, according to the sequence obtaining, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F(5 '-ATGGAAGCTCAACCTCTCCTCCT-3 '), ORF-R(5 '-TCACTCCTTCCCATACGCCCTCGCT-3 '), the turmeric cDNA of take carries out PCR as template, and amplification obtains the total length coding gene sequence (SEQ ID NO.3) of 1533bp turmeric TfF3 ' H albumen.
embodiment 2, turmeric TfF3 ' H gene sequence information and homology analysis
Turmeric TfF3 ' H total length CDS opening code-reading frame sequence is 1533bp, and detailed sequence is shown in sequence shown in SEQ ID NO.3; According to CDS opening code-reading frame sequence, derive the aminoacid sequence of turmeric TfF3 ' H, totally 510 amino-acid residues, molecular weight is 55482.2 dalton, and iso-electric point (pI) is 6.98, and detailed sequence is shown in sequence shown in SEQ ID NO.4.
The aminoacid sequence of the CDS opening code-reading frame sequence of turmeric TfF3 ' H and proteins encoded thereof is carried out to Nucleotide and protein homology search with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that it and lily F3 ' H gene (the GenBank number of logging in AB699162.1) have 82% homogeny on nucleotide level, as shown in Figure 1, Figure 2 and Figure 3; On amino acid levels, it and lily F3 ' H gene (the GenBank number of logging in BAM28972.1) also have 88% consistence and 94% similarity, as shown in Figure 4.As can be seen here, all there is higher homology in turmeric TfF3 ' H gene and lily F3 ' H gene from nucleic acid or protein level.
embodiment 3, turmeric F3 ' H gene is at flower different developmental phases and the different expression in turmeric different tissues
1. the acquisition of material
4 different developmental phases of turmeric flower (bud, petal is not painted; Bud, petal starts painted; Flower is partly open, and petal is completely not painted; Flower is completely open, petal is completely painted), in field, take its petal, take its blade, terrestrial stem, flower portion organ stamen, gynoecium, petal (compound sample of each painted stage petal) simultaneously, after sample is wrapped with aluminium platinum paper respectively, drop at once in liquid nitrogen, then proceed to stored for future use in-80 ℃ of Ultralow Temperature Freezers.
The extraction of 2.RNA
Utilize RNA prep pure plant total RNA extraction reagent box (RNA prep pure Plant Kit: extract the petal of turmeric different developmental phases flower and the RNA in different tissues TIANGEN Biotech (Beijing) Co., Ltd.).
Determining of the integrity of 3.RNA, purity, concentration
With plain agar sugar gel electrophoresis (gum concentration 1.2%; 0.5 * TBE electrophoretic buffer; 150v, 15min) detection integrity; In electrophoretic band, maximum rRNA brightness should be 1.5-2.0 times of second rRNA brightness, otherwise represents the degraded of rRNA sample.The good RNA of purity, A
260/ A
280and A
260/ A
230be about 2.0 left and right; By spectrophotometric determination OD value and calculate rna content.
The acquisition of 4.cDNA
The total RNA of 500ng of take is template, according to the precious TaKaRa PrimeScript of biotech firm
tMit is standby that RT reagent Kit Perfect Real Time test kit operation instructions is carried out reverse transcription acquisition cDNA.
5. real-time fluorescence quantitative PCR is analyzed the expression amount of TfF3 ' H gene in each organ and tissue
According to the sequence of turmeric Tf3 ' the H gene having obtained, utilize primer-design software primer premier5.0 to be designed for the Auele Specific Primer that turmeric TfF3 ' H gene quantification in Real-time PCR is analyzed: F3 ' H-F(5 '-CCTTCCTCCAAGCCATCA-3 ') and F3 ' H-R(5 '-GTCGCTACCTTTCACATCCA-3 '), reference gene is the Actin(GenBank number of logging in AB456684), its primer is Actin-F(5 '-AGTCAGTCATACAGTGCCAATC-3 '), Actin-R(5 '-TCATAAGAGAGTCGGTCAAATCC-3 ').
6. make the typical curve of goal gene and reference gene
With EASY Dilution(test kit, provide) standard substance cDNA solution is carried out to gradient dilution, the cDNA solution of then take respectively after dilution is template, Auele Specific Primer with goal gene and reference gene carries out Real-time pcr amplification, and reaction finishes rear drafting solubility curve and typical curve; Analyze solubility curve, judge whether the solubility curve of goal gene and reference gene obtains simple spike, to judge, use this primer can obtain single pcr amplification product; By typical curve, determine the suitable extension rate of template cDNA.
7. the Real time PCR of goal gene in testing sample
The cDNA article one chain synthesizing of take is template, by the primer amplified of goal gene and internal reference gene, carry out quantitative fluorescence analysis respectively, Real-time PCR reaction is carried out on BIO-RAD Chromo4 real-time quantitative instrument, reaction system is 20 μ L, reaction adopts three-step approach, 94 ℃ of sex change 20s, then 41 circulations: 94 ℃ of 15s; 56 ℃ of 15s; 72 ℃ of 25s; After each amplification, all do solubility curve, take and check whether amplified production is special generation;
Adopt 2
-△ △ Ctmethod is made relative quantitative assay, and the expression level that result shows TfF3 ' H gene is along with the growth of flower is risen gradually and slowly risen.Petal complete when painted the expression amount of TfF3 ' H be 2.6 times of the firm painted stage expression amount of petal, what the expression of this gene and petal be described paintedly has a dependency; TfF3 ' H gene all has expression in stem, leaf, stamen, gynoecium, petal.Wherein, TfF3 ' H gene expression amount in stamen is the highest, and in blade, expression amount is minimum, and expression level is followed successively by stamen, gynoecium, petal, stem, leaf from high to low, and the expression of this explanation TfF3 ' H gene has obvious Spatial Difference.
embodiment 4, the functional verification of turmeric TfF3 ' H transgenosis
1. the structure of plant over-express vector
(1) structure of cloning vector
Upstream and downstream primer two ends at amplification TfF3 ' H gene ORF add respectively Hind III and Xbal I restriction enzyme site.Upstream primer sequence is 5 '-CCAAGCTTATGGAAGCTCAACCTCTCCT-3 ', and downstream primer is 5 '-GCTCTAGATCACTCCTTCCCATACGCC-3 '.By the ORF segment of pcr amplification TfF3 ' H cDNA, after electrophoresis, under ultraviolet lamp, extract object band, with Sanprep pillar DNA glue, reclaim test kit and reclaim segment.
Recovery segment is connected to pMD18-T vector, builds pMD18-TfF3 ' H cloning vector, linked system is shown in specification sheets.Freeze-thaw method transforms bacillus coli DH 5 alpha competence, is containing 100mgl
-137 ℃ of overnight incubation on the LB solid plate substratum of ammonia benzyl.The formula of LB substratum is: Tryptones 10gl
-1, yeast extract 5gl
-1, sodium-chlor 10gl
-1.Regulate pH to 7.0, sterilizing.LB solid culture based formulas is in LB liquid nutrient medium, adds 15gl
-1agar powder, sterilizing.Picking list bacterium colony PCR identifies, send positive bacterium colony order-checking to determine the exactness of order-checking.By the correct DH5 α bacterium colony containing pMD18-TfF3 ' H carrier of order-checking, add 2ml to contain 100mgl
-1the LB liquid nutrient medium incubated overnight of ammonia benzyl is to OD
600value is about 1.0.Use plasmid extraction kit (Tian Gen biochemical technology company limited) to extract pMD18-TfF3 ' H carrier, concrete operations reference reagent box specification sheets.
Use
restriction enzyme Hind III and Xbal I pMD18-TfF3 ' H carrier and plant expression vector pHB are carried out to double digestion at 37 ℃, time 15min simultaneously.Enzyme is cut system reference enzyme and is cut specification sheets.Enzyme is cut to product and carry out gel electrophoresis, after electrophoresis, with Sanprep pillar DNA glue recovery test kit, reclaim respectively enzyme and cut product.
The object segment of using DNA Ligation Kit test kit (TaKaRa, China) enzyme to be cut to rear acquisition is connected with pHB carrier, and method of attachment is referring to test kit specification sheets.To connect product freeze-thaw method transformed competence colibacillus DH5 α bacterial strain, contain 100mgl
-137 ℃ of overnight incubation on the LB solid plate substratum of kantlex, picking list bacterium colony, PCR evaluation and screening is containing the DH5 α bacterium colony of pHB-TfF3 ' H plant over-express vector.Picking, containing the DH5 α bacterium colony of pHB-TfF3 ' H plant over-express vector, adds 2ml to contain 100mgl
-1the LB liquid nutrient medium incubated overnight of kantlex is to OD
600value is about 1.0.Use plasmid extraction kit (Tian Gen biochemical technology company limited) to extract pHB-TfF3 ' H recombinant plasmid, concrete operations reference reagent box specification sheets.The formula of LB substratum is: Tryptones 10gl
-1, yeast extract 5gl
-1, sodium-chlor 10gl
-1, regulate pH to 7.0, sterilizing.LB solid culture based formulas is in LB liquid nutrient medium, adds 15gl
-1agar powder, sterilizing.
2. plant over-express vector transforms agrobacterium tumefaciens
By pHB-TfF3 ' H plant over-express vector frozen-thawed method transformed competence colibacillus agrobacterium tumefaciens bacterial strain GV3101, concrete grammar: add at least 6 μ l to connect product in 100 μ l GV3101 competent cells, mix gently, ice bath 30min, liquid nitrogen flash freezer 1min, 37 ℃ of thermal shock 3min, are placed in rapidly and upper 1-2min; Add 800 μ l containing 50mgl
-1rifampin and 50mgl
-1the YEB substratum of gentamicin, 28 ℃, 200rpm recovery 3h; The centrifugal 5min of 4000rpm, sops up 800 μ l LB substratum; Mix residue bacterium liquid, be applied in the gl containing 100m
-1kantlex, 50mgl
-1rifampin, and 50m gl
-1on the YEB flat board of gentamicin; Be inverted for 28 ℃ and cultivate 30-48h; Then take ORF-F and ORF-R detects as the PCR that primer carries out positive colony.YEB liquid culture based formulas: beef extract 5gl
-1, Tryptones 5gl
-1, yeast extract 1gl
-1, sucrose 5.0gl
-1, MgSO
47H
2o0.5gl
-1, regulate pH to 7.0, sterilizing.YEB solid culture based formulas for to add 15gl in YEB liquid nutrient medium
-1agar powder.
3. agriculture bacillus mediated tobacco genetic transformation
In this experiment, use material for tobacco (Nicotiana tabacum).Culture medium prescription used:
MS
o: MS powder+sucrose 30gl
-1, agar powder 15gl
-1, pH5.8;
MS
1(culture medium altogether): MS
o+ 6-BA2mgl
-1+ NAA O.2mgl
-1, pH5.8;
MS
2(screening culture medium): MS
1+ Totomycin 50mgl
-1+ cephamycin 300mgl
-1, pH5.8;
MS
3(root media): 1/2MS
o+ Totomycin 50mgl
-1+ cephamycin 300mgl
-1, pH5.8;
(1) activation of Agrobacterium
The positive list bacterium colony of Agrobacterium from picking YEB culture plate after PCR detects, is inoculated in 5ml YEB liquid nutrient medium (containing 100m gl
-1kantlex, 50mgl
-1rifampin, and 50mgl
-1gentamicin).28 ℃, 200rpm shaking culture 24h; Therefrom draw 800 μ l bacterium liquid again and transfer in 40ml containing in corresponding antibiotic YEB liquid nutrient medium, continue at 28 ℃, 200rpm shaking culture is to OD
600value 0.6.4 ℃, the centrifugal 10min of 5000rpm collects thalline, with the MS of certain volume
0liquid nutrient medium suspension thalline is to OD
600value is that 0.4 left and right is for contaminating.
(2) genetic transformation of tobacco
To plant at MS
0aseptic tobacco on minimum medium, cuts blade edge and main vein, is cut into 0.4 * 0.6cm size.By the explant cutting at above-mentioned OD
600be to soak 5min in 0.4 Agrobacterium bacterium liquid, with aseptic filter paper, blot the bacterium liquid on vegetable material surface, vanelets is placed in to the bud division culture medium MS that is covered with one deck filter paper
1on carry out common cultivation (leaf back upwards), 25 ℃ of dark cultivations 3 days.To transfer to and contain antibiotic resistant buds screening culture medium MS through the tobacco explant of cultivating altogether
2in cultivate, periodicity of illumination is that 16h illumination/8h is dark, 2-3 can be sprouted after week.Until resistant buds, grow to 1-2cm when high, cut budlet and proceed to root media MS
3middle root induction, just has afterwards adventive root 1-2 week and forms.After well developed root system, tobacco plant is taken out, with sterilized water, clean solid medium, then move in soil, just started several days by film cover several days, after plant to be planted stalwartness, take down again film, be placed in the cultivation of 25 ℃, greenhouse.
4. the mensuration of the evaluation of transgene tobacco and Minor centaury content
(1) evaluation of positive strain
Adopt CTAB method to extract with transgene tobacco genomic dna, take this DNA as template, with primer ORF-F and the ORF-R of amplification TfF3 ' H gene ORF segment, carry out pcr amplification with screening transgenic positive plant.PCR response procedures is: 94 ℃, and 5min; 94 ℃, 30s; 58 ℃, 1min; 72 ℃ 2min(35 circulation); 72 ℃ are extended 10min.
(2) mensuration of transfer-gen plant Cyanidin content
Mensuration transgenic positive plant and adjoining tree cyanidin content.The extracting method of total pattern aglycon is as follows: take the about 0.2g of fresh petal sample, liquid nitrogen is cooling rear direct grind into powder rapidly, with 2ml extracting solution (V
ethanol: V
acetic acid: V
water=10:1:9) 4 ℃ of lixiviates 24 hours; The centrifugal 10min of 4000rpm, gets the HCl that supernatant adds 4ml3M, at 100 ℃ of water-bath 90min in order to be hydrolyzed the glucosides of anthocyanogen; The primary isoamyl alcohol extraction pattern aglycon that adds 2ml after hydrolysis finishes.With Medium speed filter paper, filter afterwards, then it is to be measured to use strainer (0.2 μ m) to filter, is directly used in afterwards UPLC and analyzes.The separation of pattern aglycon and the mensuration of content adopt superelevation effect liquid phase chromatogram – diode array device (the Ultra performance liquid chromatography with a photodiode array detector of U.S. Waters company, UPLC – PAD) detect, this system equipment has high pressure binary gradient pump, constant temperature automatic sampler, column oven, diode-array detector, chromatographic working station (Masslynx V4.1 data processing software).Chromatographic column is the C18 post (2.1mm * 100mm, 1.7 μ m) of ACQUITYUPLC@BEH.UPLC analysis condition: 45 ℃ of column temperatures, flow velocity 0.5mlmin
-1, sampling volume 2 μ l.Mobile phase A liquid is 0.1% formic acid solution (V
formic acid: V
water=0.1:99.9); B liquid is the acetonitrile (V containing 0.1% formic acid
formic acid: V
acetonitrile=0.1:99.9).Gradient elution program: 0min, 93%A, 7%B; 1min, 93%A, 7%B; 11min, 82%A, 18%B; 11.5min, 10%A, 90%B; 13min, 10%A, 90%B; 13.1min, 93%A, 7%B; 15min, 93%A, 7%B.Under characteristic absorption wavelength 520nm, by the retention time comparison with standard substance cyanidin (Cyanidin chloride, Sigma), determine the component of cyanidin in extract.In Anthocyanin-rich Extract, the content of cyanidin converts by the ratio of the peak area with concentration known standard substance.
Result shows, plants in the transgene tobacco in greenhouse and compares with contrasting (unconverted) tobacco, and under identical culture condition, 50% above transgene tobacco pattern becomes deep pink from pink colour.As shown in Figure 6, a is unconverted tobacco flower, and b, c, for turning the flower of TfF3 ' H genetic tobacco, can see that the more unconverted tobacco color of flower pattern of transgene tobacco is darker.Correspondingly, Cyanidin content rising 20-30% in transgene tobacco petal.This shows that TfF3 ' H can act on anthocyanogen route of synthesis, affects Cyanidin synthetic, changes plant pattern.
embodiment 5, the functional verification of turmeric TfF3 ' H enzyme
1. the structure of prokaryotic expression carrier
Upstream and downstream primer two ends in amplification TfF3 ' H gene ORF segment add respectively BamH I and Xho I restriction enzyme site.Upstream primer sequence is 5 '-CGCGGATCCATGGAAGCTCAACCTCTCCT-3 ', and downstream primer is 5 '-ATTCTCGAGTCACTCCTTCCCATACGCC-3 '.By the ORF segment of pcr amplification TfF3 ' H cDNA, after electrophoresis, under ultraviolet lamp, extract object band, with Sanprep pillar DNA glue, reclaim test kit and reclaim segment.
Use
restriction enzyme BamH I and Xho I TfF3 ' H segment and prokaryotic expression carrier pMAL-c2x (New England BioLabs) are carried out to double digestion at 37 ℃, time 15min simultaneously.Enzyme is cut system reference enzyme and is cut specification sheets.Enzyme is cut to product and carry out gel electrophoresis, reclaim.
Use TfF3 ' the H segment after DNA Ligation Kit test kit (TaKaRa, China) is cut enzyme to be connected with pMAL-c2x, method of attachment is referring to test kit specification sheets.To connect product and transform e. coli bl21 competence.Containing 50mgl
-137 ℃ of overnight incubation on the LB solid plate substratum of ammonia Bian.Picking list bacterium colony, PCR send order-checking to confirm that TfF3 ' H segment is successfully connected with pMAL-c2x after identifying the positive.The formula of LB liquid and solid medium is the same.
The fusion rotein induction of 2.TfF3 ' H
Select the BL21 bacterial strain mono-clonal that growth conditions is good, be forwarded to 10ml containing 50mgl
-1in the LB liquid nutrient medium of ammonia Bian, 37 ℃, 200rpm incubated overnight.In the ratio of 1:50, be forwarded to 300ml containing 50mgl
-1in the LB liquid culture of ammonia Bian 37 ℃, 200rpm is cultured to OD
600it is 0.8 left and right.Culture is proceeded to 200rpm in the shaking table of 20 ℃, shaking culture 1 hour.Add 1ml1M IPTG (sec.-propyl-β-D-sulfo-galactopyranoside) (final concentration is 1mM), continue to cultivate 6h induction expressing fusion protein.
By 4 ℃ of overnight culture, the centrifugal 10min of 12000rpm, collects thalline, abandons supernatant.By the resuspended precipitation of 50ml cracked solution, then place 10min on ice.Under condition of ice bath, use ultrasonic wave (200-300w) smudge cells, ultrasonic/interval=10s/10s, 6 times.Subsequently by broken cell at 4 ℃, centrifugal 30min under 12000rpm condition, collects supernatant liquor for purifying.In supernatant, add 1ml to clean the also amylose resin (Amylose resin) (New England Biolabs) of balance by cracked solution, to solution, add another 50ml cracked solution again, 4 ℃ of overnight incubation simultaneously.Hatch latter 4 ℃, after the centrifugal 1min of 1000rpm, abandon supernatant.With after 50ml cleaning solution cleaning resin three times, with 1ml elute soln eluted protein.The formula of cellular lysate solution: 20mMTris-HCl, pH8.0,200mM sodium-chlor, 1mM PMSF, the proteinase inhibitor of EDTA-free (Promega), 10% glycerine; The formula of cleaning solution: 20mM Tris-HCl, pH8.0,200mM sodium-chlor, 10% glycerine; The formula of elute soln: 20mM Tris-HCl, pH8.0,200mM sodium-chlor, 50mM maltose, 10% glycerine.
TfF3 ' H fusion rotein and the substrate collected are hatched.The substrate of hatching is naringenin and dihydrokaempferol.Hatching system volume is 500 μ l, comprises 2-40 μ M substrate, 200mg TfF3 ' H fusion rotein extract, 1mM NADPH, 0.1M potassium primary phosphate (pH7.4).Reaction conditions: 25 ℃ of reaction 30min.Reaction finishes the ethyl acetate extracting twice of rear use 500 μ l, and extraction liquid is dissolved in 200 μ l methanol solutions after liquid nitrogen dries up again.Then carry out UPLC analysis.Standard substance flavanone [naringenin and eriodictyol (eriodictyol)] is purchased from Extrasynthese SA company, standard substance flavanonol [dihydrokaempferol and dihydroquercetin (hidydroquercetin)] Gou Ziyu PLANTECH company.Under 280nm wavelength, the elution time of corresponding standard substance is determined the kind of hatching product; According to the content of corresponding resultant in the concentration of respective standard product and peak area conversion product.
Result demonstration, naringenin and dihydrokaempferol have generated eriodictyol and dihydroquercetin under the effect of TfF3 ' H fusion rotein, and TfF3 ' H will be higher than the activity to naringenin to the activity of dihydrokaempferol.This result shows that TfF3 ' H can make the B ring 3 ' site of naringenin and dihydrokaempferol that hydroxylation occurs, and has the activity of flavonoid-3 '-hydroxylase, also has certain substrate selective simultaneously.
Above specific embodiments of the invention are described.It will be appreciated that, the present invention is not limited to above-mentioned specific implementations, and those skilled in the art can make various distortion or modification within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (6)
1. following (a) or protein (b):
(a) protein being formed by the aminoacid sequence as shown in SEQ ID NO.4;
(b) aminoacid sequence shown in SEQ ID NO.4 through replacement, lack or add one or several amino acid and have turmeric flavonoid-3 '-hydroxylase activity by (a) derivative protein.
2. protein as claimed in claim 1, it is characterized in that, described protein be aminoacid sequence shown in SEQ ID NO.4 through 1～50 amino acid whose disappearance, insertion and/or replacement, or add 1～20 sequence obtaining with interior amino acid at C-terminal and/or N-terminal.
3. protein as claimed in claim 2, is characterized in that, described protein be shown in SEQ ID NO.4 in aminoacid sequence 1～10 amino acid by the similar or close amino acid of character, replaced the sequence forming.
One kind coding claim 1 described in nucleic acid sequences to proteins.
5. nucleotide sequence as claimed in claim 4, is characterized in that, described nucleotide sequence is specially:
(a) base sequence is as shown in 1st～1533 of SEQ ID NO.3;
Or (b) and the nucleic acid shown in 1st～1533 of SEQ ID NO.3 have the sequence of at least 70% homology;
Or the sequence that (c) can hybridize with the nucleic acid shown in 1st～1533 of SEQ ID NO.3.
6. nucleotide sequence as claimed in claim 4, it is characterized in that, described nucleotide sequence is specially disappearance, insertion and/or the replacement of 1～90 Nucleotide in the nucleotide sequence shown in 1st～1533 of SEQ ID NO.3, or adds 60 sequences that form with inner nucleotide at 5 ' and/or 3 ' end.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310533249.4A CN103589694B (en) | 2012-10-31 | 2013-10-30 | Turmeric flavonoid-3 '-hydroxylase TfF3 ' H protein and encoding gene thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210429398.1 | 2012-10-31 | ||
| CN2012104293981A CN102965349A (en) | 2012-10-31 | 2012-10-31 | Tulip flavanonol-3'- hydroxylase TfF3' H protein, and coding gene and probe thereof |
| CN201310533249.4A CN103589694B (en) | 2012-10-31 | 2013-10-30 | Turmeric flavonoid-3 '-hydroxylase TfF3 ' H protein and encoding gene thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103589694A true CN103589694A (en) | 2014-02-19 |
| CN103589694B CN103589694B (en) | 2015-10-28 |
Family
ID=47795785
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012104293981A Pending CN102965349A (en) | 2012-10-31 | 2012-10-31 | Tulip flavanonol-3'- hydroxylase TfF3' H protein, and coding gene and probe thereof |
| CN201310533249.4A Expired - Fee Related CN103589694B (en) | 2012-10-31 | 2013-10-30 | Turmeric flavonoid-3 '-hydroxylase TfF3 ' H protein and encoding gene thereof |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012104293981A Pending CN102965349A (en) | 2012-10-31 | 2012-10-31 | Tulip flavanonol-3'- hydroxylase TfF3' H protein, and coding gene and probe thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN102965349A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105409972A (en) * | 2015-11-16 | 2016-03-23 | 湖南广播电视大学 | Compositions for promoting accumulation of anthocyanin in roselle calyx |
| CN112391300A (en) * | 2020-11-04 | 2021-02-23 | 江南大学 | Silybum marianum-derived flavone 3 beta-hydroxylase and application of coenzyme thereof |
| CN113322288A (en) * | 2020-02-28 | 2021-08-31 | 中国科学院分子植物科学卓越创新中心 | Novel flavone hydroxylase, microorganism for synthesizing flavone C-glycosides and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103756982B (en) * | 2013-12-16 | 2015-10-28 | 上海交通大学 | Tulipa fosteriana flavonol synthase TfFLS protein and encoding gene thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101870922A (en) * | 2009-04-27 | 2010-10-27 | 巧立永 | Method for preparing essence from tulip and Chinese sacred lily |
-
2012
- 2012-10-31 CN CN2012104293981A patent/CN102965349A/en active Pending
-
2013
- 2013-10-30 CN CN201310533249.4A patent/CN103589694B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101870922A (en) * | 2009-04-27 | 2010-10-27 | 巧立永 | Method for preparing essence from tulip and Chinese sacred lily |
Non-Patent Citations (2)
| Title |
|---|
| 刘鲜艳登: "百合ACC氧化酶基因全长cDAN的克隆及序列分析", 《西北植物学报》 * |
| 周小理登: "植物中查尔酮酶合成黄酮类化合物的研究进展", 《食品工业》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105409972A (en) * | 2015-11-16 | 2016-03-23 | 湖南广播电视大学 | Compositions for promoting accumulation of anthocyanin in roselle calyx |
| CN113322288A (en) * | 2020-02-28 | 2021-08-31 | 中国科学院分子植物科学卓越创新中心 | Novel flavone hydroxylase, microorganism for synthesizing flavone C-glycosides and application thereof |
| CN113322288B (en) * | 2020-02-28 | 2023-12-26 | 中国科学院分子植物科学卓越创新中心 | Novel flavone hydroxylase, microorganism for synthesizing flavone C-glycoside compounds and application thereof |
| CN112391300A (en) * | 2020-11-04 | 2021-02-23 | 江南大学 | Silybum marianum-derived flavone 3 beta-hydroxylase and application of coenzyme thereof |
| CN112391300B (en) * | 2020-11-04 | 2022-08-23 | 江南大学 | Application of flavone 3 beta-hydroxylase derived from silybum marianum and coenzyme thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103589694B (en) | 2015-10-28 |
| CN102965349A (en) | 2013-03-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2713208C (en) | Drought and heat tolerance in plants | |
| CN103614348B (en) | Turmeric flavanone-3-hydroxylase TfF3H albumen and encoding gene thereof | |
| CN111690662B (en) | Application of soybean bHLH transcription factor GmPIF1 gene in promotion of isoflavone synthesis | |
| WO2009125826A1 (en) | Gene capable of being expressed specifically in endosperm of plant, promoter for the gene, and use of the gene and the promoter | |
| CN103602686A (en) | Sweet wormwood MYC2 transcription factor protein coding sequence and applications thereof | |
| CN103695382A (en) | Tulip flavonoid 3-O-glucosyltransferase Tf3GT protein and coding gene thereof | |
| CN103589694B (en) | Turmeric flavonoid-3 '-hydroxylase TfF3 ' H protein and encoding gene thereof | |
| CN105524152B (en) | Rice high-temperature resistant gene and application thereof in crop high-temperature resistant breeding | |
| CN113025626A (en) | Application of tumorous stem mustard BjuEAR1 gene in regulation of plant stress resistance | |
| CN103614358B (en) | Tulip chalcone isomerase TfCHI protein and encoding gene thereof | |
| CN107475264B (en) | Application of DGM1 protein in improving plant root hair generation capability | |
| CN101210247B (en) | Endosperm specific expression promoter, albuminous cell specific gene and application thereof | |
| CN104745560A (en) | Eggplant chalcone synthase SmCHS1 protein and coding gene thereof | |
| CN102242111B (en) | Application of vector containing CYP704B2 gene | |
| CN103589698B (en) | Tulip chalcone synthase TfCHS albumen and encoding gene thereof | |
| CN114395023B (en) | Jatropha curcas early flowering gene JcRR1B and application thereof | |
| CN114560919B (en) | Plant drought tolerance related transcription factor VcMYB and coding gene and application thereof | |
| CN103074307B (en) | Tulipa gesneriana TfbHLH1 protein, encoding gene thereof and probe | |
| US6756524B2 (en) | Gene controlling fruit size and cell division in plants | |
| CN107326030B (en) | WRKY transcription factor for regulating low potassium tolerance and application thereof | |
| CN110343154B (en) | Clone of key gene SEM1 for controlling rice sink source flow and application thereof | |
| CN104017781B (en) | Agipanthus Plant hormones regulators,gibberellins synthesis dioxygenase APGA20ox albumen and encoding gene and probe | |
| CN106350525B (en) | A kind of rice grain shape gene DSS and its coding protein and application | |
| CN103695406A (en) | Tulip phenylalanine ammonialyase TfPAL protein and coding gene thereof | |
| CN102453704B (en) | Plant photooxidative stress-resistant gene carotenoid isomerase (CRTISO) and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20151028 Termination date: 20191030 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |