CN102965349A - Tulip flavanonol-3'- hydroxylase TfF3' H protein, and coding gene and probe thereof - Google Patents
Tulip flavanonol-3'- hydroxylase TfF3' H protein, and coding gene and probe thereof Download PDFInfo
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- 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
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- 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)
Abstract
The invention relates to a tulip flavonoid 3'- hydroxylase TfF3' H protein, and a coding gene and a probe thereof. The protein is (a) or (b) as follows: (a) a protein composed of an amino acid sequence shown as a SEQ ID NO.4; and (b) a protein obtained by substitution, deletion or addition of one or several amino acids on the amino acid sequence shown as the SEQ ID NO.4, having tulip hydrogen flavonoid 3'-hydroxylase activity and derived from (a). The invention also provides a nucleic acid sequence encoding the protein, and the probe to detect the above nucleic acid sequence. The invention employs genetic engineering technology to regulate spatio-temporal expression characteristic of the TfF3' H gene of tulip, so as to provide theoretical basis for color change and color innovation, and has important application value.
Description
Technical field
The present invention relates to crux enzyme and encoding gene and probe in the turmeric pattern glycosides route of synthesis, be specifically related to a kind of turmeric flavanonol-3 '-hydroxylase TfF3 ' H albumen and encoding gene and probe.
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 of accumulation cyanidin(e) in the petal cell, and cyanidin(e) mainly comprises flavonoid, carotenoid and betaines.Flavonoid is a large class of cyanidin(e), and it makes the whole colors of flower generation from the Huang to the purple.In the route of synthesis of flavonoid, flavanone forms flavanonol through flavanone-3-hydroxylase (F3H) catalysis hydroxylation, flavanonol can flavanonol 3 '-hydroxylase (flavonoid3 ' hydroxylase, F3 ' H) and flavanonol 3 ', 5 '-form respectively dihydro quercetin and dihydromyricetin flavones under the katalysis of hydroxylase.The former arrives purplish red pigment at the final brick red that produces based on Cyanidin; The final pigment from the purple to the blueness that produces based on delphinidin of the latter.If flavanonol 3 ' with 5 ' all not by hydroxylation then change bolarious pelargonidin into.When F3 ' H inactivation, can not accumulate cyanin in spending, and the more pelargonidins of accumulation, it is orange red that flower is presented more.Therefore, the expression activity of F3 ' H gene can directly affect the final color of flower, to regulate and control be one of approach of change pattern in genetic expression to F3 ' H.
F3 ' H gene has obtained the clone in various plants, but in turmeric (Tulipa fosteriana), F3 ' H gene cloning, expression pattern and F3 ' H albumen coded sequence it be not immediately clear.At present, any bibliographical information relevant with turmeric F3 ' H albumen and coding gene sequence thereof do not arranged.
Summary of the invention
Purpose of the present invention, be to fill 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 albumen is provided, and the present invention also provides a kind of probe of encoding above-mentioned nucleic acid sequences to proteins and detecting described nucleotide sequence.The invention discloses the expression pattern that turmeric TfF3 ' H albumen and nucleotides sequence thereof are listed in turmeric Different Organs, different developmental phases, for utilizing from now on the expression pattern of genetic engineering technique regulation and control TfF3 ' H gene, thereby change pattern, innovation pattern provide theoretical foundation, have great using value.
The present invention is achieved by the following technical solutions,
On the one hand, the invention provides a kind of have turmeric flavanonol-3 '-protein of hydroxylase activity, the protein that described protein is comprised of the aminoacid sequence shown in SEQ ID NO.4; Or by the aminoacid sequence shown in the SEQ ID NO.4 through replacement, lack or add one or several amino acid and have turmeric flavanonol-3 '-protein of being derived by (a) of hydroxylase activity.This protein having that it's too late there is larger difference in active size in different painted stage of flower petal, Different Organs.
Preferably, described protein is that aminoacid sequence shown in the SEQ ID NO.4 is through 1～50 amino acid whose disappearance, insertion and/or replacement, perhaps in C-terminal and/or 1～20 sequence that obtains with interior amino acid of N-terminal interpolation.
Further preferred, described protein be shown in the SEQ ID NO.4 in the aminoacid sequence 1～10 amino acid replaced the sequence that forms by similar performance or close amino acid.
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 shown in the 1st～1533 of SEQ ID NO.3;
Or (b) and the nucleic acid shown in the 1st～1533 of the SEQ ID NO.3 sequence of at least 70% homology is arranged;
Or the sequence that (c) can hybridize with the nucleic acid shown in the 1st～1533 of the 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 the 1st～1533 of the SEQ ID NO.3, and 5 ' and/or 3 ' end add 60 sequences that form with inner nucleotide.
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 whether existing in the test sample 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 the SEQ ID NO.3; Perhaps coding has the nucleotide sequence of the polypeptide of turmeric TfF3 ' H protein active, and with sequence shown in the SEQ ID NO.3 at least 70% homology is arranged; Perhaps can with the nucleotide sequence hybridization of sequence shown in the SEQ ID NO.3.
In the present invention, " DNA of separation ", " DNA of purifying " refer to, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein that in cell, accompanies.
In the present invention, term " turmeric flavanonol-3 '-hydroxylase protein coded sequence " refer to the encode nucleotide sequence of polypeptide with turmeric TfF3 ' H protein-active, nucleotide sequence and degenerate sequence thereof shown in SEQ ID NO.3.This degenerate sequence refers to, is arranged in sequence shown in the 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.Because the degeneracy of codon, so be low to moderate about 70% the degenerate sequence aminoacid sequence shown in the SEQ ID NO.4 of also encoding out with the homology of sequence shown in the SEQ ID NO.3.This term also comprise with sequence shown in the SEQ ID NO.3 in from the nucleotide sequence of the homology at least 70% of the nucleotide sequence of the 1st～1533 in 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 5 ' and/or 3 ' end be added to 60 with inner nucleotide.
In the present invention, term " turmeric flavanonol-3 '-hydroxylase TfF3 ' H albumen " refers to have the polypeptide of sequence shown in the SEQ ID NO.4 of 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 the 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, when replacing with the close or similar amino acid of performance, usually can not change the function of protein.Again such as, add the function that or several amino acid also can not change protein usually at C-terminal and/or N-terminal.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 relevant DNA hybridization with 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 the SEQ ID NO.4, has at the most 10 amino acid be replaced by similar performance or close amino acid and forms polypeptide.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.
Table 1
| Initial residue | Representational replacement | The 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 the aminoacid sequence, also can be the difference that does not affect on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise genetic variant natural or that induce.The 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 moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(such as D-amino acid), and the analogue with that non-natural exists or synthetic amino acid (such as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide of enumerating.
(usually the not changing primary structure) form of modification comprises: chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing such as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (such as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (such 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 the methods analyst turmeric TfF3 ' H gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of mRNA transcript in cell of namely analyzing TfF3 ' H gene.
Whether there is the detection method of turmeric TfF3 ' H related nucleotide sequences in the test sample of the present invention, comprises with above-mentioned probe and sample and hybridizing then whether detection probes combination has occured.This sample is the product behind the pcr amplification, and wherein the 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 the 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 the 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 available from Clontech, and Stratagene, Palo Alto, Cal..This screening method can be identified the nucleotide sequence of the gene family relevant with turmeric TfF3 ' H.
Turmeric TfF3 ' H associated nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the 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 needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
After having obtained relevant sequence, can obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell over to again, then separates obtaining relevant sequence from the host cell after the propagation by ordinary method.
In addition, also can will suddenly change by chemosynthesis and introduce in the protein sequence of the present invention.
Except producing with recombination method, the fragment of albumen of the present invention is available solid phase technique also, produced by direct peptide synthesis (people such as Stewart, (1969) solid-phase polypeptide is synthetic, WH Freeman Co., San Francisco; Merrifield J.(1963) J.Am Chem.Soc85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can come automatic pressing to become peptide with the 431A type peptide synthesizer (Foster City, CA) of Applied Biosystems.Can distinguish each fragment of chemosynthesis albumen of the present invention, then be connected to produce the molecule of total length with chemical process.
Utilize turmeric TfF3 ' H albumen of the present invention, by various conventional screening methods, can filter out the interactional material of relevant generation with turmeric TfF3 ' H, perhaps acceptor, inhibitor or antagonist etc.
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 key enzyme flavanonol-3 at the synthetic forehearth limb of tulip petals flavonoid point place '-encoding sequence of hydroxylase TfF3 ' H albumen, and the spatial and temporal expression characteristics of the methods analyst TfF3 ' H gene of employing fluorescence real-time quantitative PCR, for utilizing genetic engineering technique thereby the expression pattern of TfF3 ' H gene is regulated and control to change pattern, cultivate new fancy variety theoretical foundation is provided, have great using value.
Description of drawings
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 be turmeric flavanonol-3 of the present invention '-homology of the nucleotide sequence of hydroxylase TfF3 ' H gene and lily flavanonol-3 ' '-hydroxylase gene mRNA (GAP) result relatively;
Fig. 2 be turmeric flavanonol-3 of the present invention '-homology of the nucleotide sequence of hydroxylase TfF3 ' H gene and lily flavanonol-3 ' '-hydroxylase gene mRNA (GAP) result relatively;
Fig. 3 be turmeric flavanonol-3 of the present invention '-hydroxylase TfF3 ' H gene and lily flavanonol-3 '-homology of the nucleotide sequence of '-hydroxylase gene mRNA (GAP) result relatively;
Fig. 4 be turmeric flavanonol-3 of the present invention '-hydroxylase TfF3 ' H and lily flavanonol-3 '-homology of the aminoacid sequence of hydroxylase (FASTA) result relatively, wherein, identical amino acid marks with the amino acid monocase between two sequences.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the 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
(Tulipa fosteriana ' Shangnongzaoxia ' is by the Shanghai City crop varietal approval committee with health, tulip of the same size.Numbering: farming product in Shanghai are recognized flowers 2011 No. 004) plant routinely and carry out field management, treat that flower is fully open, petal is complete to gather Petal when painted, is used for extraction RNA;
2.RNA extracting
Utilize " RNA prep pure plant total RNA extraction reagent box " extracted total RNA (RNA prep pure Plant Kit: TIANGEN Biotech (Beijing) Co., Ltd.).With the integrity of denaturing formaldehyde gel electrophoresis evaluation RNA, then measure purity and the concentration of RNA at 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, employing RACE method (3 '-Full RACE Core Set Ver.2.0: precious biotechnology (Dalian) company limited, SMARTer
TMRACE cDNA Amplification Kit:Clontech Laboratories, Inc.) carry out the cDNA full-length clone, a minute three phases carries out:
(1) RT-PCR obtains the gene intermediate segment
The RNA that extracts is carried out reverse transcription (Prime Script II 1st Strand cDNA Synthesis Kit: precious biotechnology (Dalian) company limited), take the first chain cDNA as 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 the 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, check order at ABI377 sequenator (Perkin-Elmer, USA).Sequencing result is by carrying out BLAST(http in the NCBI website: //blast.ncbi.nlm.nih.gov/) compare existing database (GenBank), know its nucleotide sequence and proteins encoded and known lilium flavanonol-3 '-homology of '-hydroxylase gene is very high, so tentatively think it be a flavanonol-3 '-'-hydroxylase gene;
（2）3′RACE
The sequence of 3 ' end by use 3 '-Full RACE Core Set Ver.2.0: precious biotechnology (Dalian) company limited obtains, and two take turns the amplification that nest-type PRC is finished 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 3 ' end sequence (476bp) of TfF3 ' H, reclaim, be connected on the 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, at ABI377 sequenator (Perkin-Elmer, USA) check order on, sequencing result is by carrying out BLAST(http in the NCBI website: //blast.ncbi.nlm.nih.gov/) compare existing database (GenBank), know its nucleotide sequence and proteins encoded and known lilium flavanonol-3 '-homology of '-hydroxylase gene is high;
（3）5′RACE
The sequence of 5 ' end is by using SMARTer
TMRACE cDNA Amplification Kit obtains, and take 5 ' RACE ready cDNA as template, takes turns nest-type PRC by two and obtains:
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 5 ' end sequence (836bp) of TfF3 ' H, reclaims the method for using after connecting with top and checks order;
The sequencing result of the sequence that will obtain by above-mentioned 3 kinds of methods splices, will splice sequence and submit to BLAST to analyze, the result prove the TfF3 ' H gene that from turmeric, newly obtains really be one with flavanonol-3 '-gene that hydroxylase is relevant.With the ORF Finding(http of sequencing result in conjunction with NCBI: //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 that obtains, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F(5 ' ATGGAAGCTCAACCTCTCCTCCT-3 '), ORF-R(5 '-TCACTCCTTCCCATACGCCCTCGCT-3 '), carry out PCR take turmeric cDNA as template, 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
The new turmeric TfF3 ' H total length CDS opening code-reading frame sequence of the present invention is 1533bp, and detailed sequence is seen sequence shown in the SEQ ID NO.3; Derive the aminoacid sequence of turmeric TfF3 ' H according to CDS opening code-reading frame sequence, totally 510 amino-acid residues, molecular weight is 55482.2 dalton, and iso-electric point (pI) is 6.98, and detailed sequence is seen sequence shown in the SEQ ID NO.4;
The CDS opening code-reading frame sequence of turmeric TfF3 ' H and the aminoacid sequence of proteins encoded thereof are carried out 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 at nucleotide level, such as Fig. 1, Fig. 2 and shown in Figure 3 (Query: turmeric flavanonol-3 '-coding gene sequence of hydroxylase TfF3 ' H; Sbjct: lily flavanonol-3 '-the mRNA sequence of hydroxylase); 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 (Query: turmeric flavanonol-3 '-aminoacid sequence of hydroxylase TfF3 ' H; Sbjct: lily flavanonol-3 '-aminoacid sequence of hydroxylase).This shows that all there are higher homology in turmeric TfF3 ' H gene and lily F3 ' H gene on nucleic acid or protein level.
Embodiment 3, turmeric F3 ' H gene is at flower different developmental phases and different expression in the turmeric different tissues
1. the acquisition of material: (bud, petal are not painted in 4 different developmental phases of turmeric flower; Bud, petal begins painted; Flower is partly open, and petal is fully not painted; Flower is fully open, petal is fully painted), take its petal in the field, take simultaneously its blade, terrestrial stem, flower section organ stamen, gynoecium, petal (compound sample of each painted stage petal), drop at once in the liquid nitrogen after sample wrapped with aluminium platinum paper respectively, then change stored for future use in-80 ℃ of Ultralow Temperature Freezers over to;
2.RNA extraction: 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 the different tissues TIANGEN Biotech (Beijing) Co., Ltd.);
3.RNA the determining of integrity, purity, concentration: with plain agar sugar gel electrophoresis (gum concentration 1.2%; 0.5 * TBE electrophoretic buffer; 150v, 15min) the detection integrity; Maximum rRNA brightness should be 1.5-2.0 times of second rRNA brightness in the electrophoretic band, otherwise represents the degraded of rRNA sample.Purity is RNA preferably, A
260/ A
280And A
260/ A
230Be about about 2.0; With spectrophotometric determination OD value and calculating rna content;
4.cDNA acquisition: take total RNA of 500ng as template, according to the precious TaKaRa PrimeScript of biotech firm
TMIt is for subsequent use that RT reagent Kit Perfect Real Time test kit operation instructions is carried out reverse transcription acquisition cDNA;
5. the design Auele Specific Primer is to carry out the expression amount of real-time fluorescence quantitative PCR analyzing gene in each organ and tissue: according to the sequence of the turmeric Tf3 ' H gene that has obtained, utilize primer-design software primer premier5.0 to be designed for the Auele Specific Primer that turmeric TfF3 ' H gene quantification among the 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 '), Ac t i n-R(5 '-TCATAAGAGAGTCGGTCAAATCC-3 ');
6. make the typical curve of goal gene and reference gene: provide with EASY Dilution(test kit) standard substance cDNA solution is carried out gradient dilution, then respectively take the dilution after cDNA solution as template, Auele Specific Primer with goal gene and reference gene carries out the Real-time pcr amplification, draws solubility curve and typical curve after reaction finishes; Analyze solubility curve, judge whether the solubility curve of goal gene and reference gene obtains simple spike, use this primer can obtain single pcr amplification product to judge; Determine the suitable extension rate of template cDNA by typical curve;
7. the Real time PCR of goal gene in the testing sample: take synthetic cDNA article one chain as template, carry out quantitative fluorescence analysis with the primer amplified of goal gene and internal reference gene respectively, Real-time PCR reaction is carried out at BIO-RAD Chromo4 real-time quantitative instrument, reaction system is that three-step approach is adopted in 20 μ L reaction, 94 ℃ of sex change 20s, then 41 circulations: 94 ℃ of 15s; 56 ℃ of 15s; 72 ℃ of 25s; After each amplification is finished, all do solubility curve, to check amplified production whether as special generation;
8. adopt 2
-△ △ CtMethod is made relative quantitative assay, and the expression level that the result shows TfF3 ' H gene rises gradually and slowly rises along with the growth of flower.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 expression amount is minimum in blade, 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.
Claims (7)
1. one kind following (a) or protein (b):
(a) protein that is formed by the aminoacid sequence shown in SEQ ID NO.4;
(b) aminoacid sequence shown in the SEQ ID NO.4 through replacement, lack or add one or several amino acid and have turmeric flavanonol-3 '-protein of being derived by (a) of hydroxylase activity.
2. protein as claimed in claim 1, it is characterized in that, described protein is that aminoacid sequence shown in the SEQ ID NO.4 is through 1～50 amino acid whose disappearance, insertion and/or replacement, perhaps in C-terminal and/or 1～20 sequence that obtains with interior amino acid of N-terminal interpolation.
3. protein as claimed in claim 2 is characterized in that, described protein be shown in the SEQ ID NO.4 in the aminoacid sequence 1～10 amino acid replaced the sequence that forms by similar performance or close amino acid.
4. coding claim 1 described 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 shown in the 1st～1533 of SEQ ID NO.3;
Or (b) and the nucleic acid shown in the 1st～1533 of the SEQ ID NO.3 sequence of at least 70% homology is arranged;
Or the sequence that (c) can hybridize with the nucleic acid shown in the 1st～1533 of the 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 the 1st～1533 of the SEQ ID NO.3, perhaps 5 ' and/or 3 ' end add 60 sequences that form with inner nucleotide.
7. one kind for detection of the probe of nucleotide sequence as claimed in claim 4, it is characterized in that described probe is the nucleic acid molecule that includes 8～100 continuous nucleotides of described nucleotide sequence.
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| CN103756982A (en) * | 2013-12-16 | 2014-04-30 | 上海交通大学 | Tulipa fosteriana flavonol synthase TfFLS protein and coding gene thereof |
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| 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 |
| 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 |
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| CN103756982A (en) * | 2013-12-16 | 2014-04-30 | 上海交通大学 | Tulipa fosteriana flavonol synthase TfFLS protein and coding gene thereof |
| CN103756982B (en) * | 2013-12-16 | 2015-10-28 | 上海交通大学 | Tulipa fosteriana flavonol synthase TfFLS protein and encoding gene thereof |
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