CN102965352A

CN102965352A - Tulip chalcone synthase TfCHS protein, and coding gene and probe thereof

Info

Publication number: CN102965352A
Application number: CN201210429585XA
Authority: CN
Inventors: 袁媛; 史益敏; 唐东芹; 马晓红
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2012-10-31
Filing date: 2012-10-31
Publication date: 2013-03-13
Also published as: CN103589698B; CN103589698A

Abstract

The invention relates to a tulip chalcone synthase TfCHS 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 chalcone synthase 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 provides theoretical basis for regulation of spatio-temporal expression characteristic of the TfCHS gene of tulip for color change and color innovation by using a genetic engineering technology, and has important application value.

Description

Turmeric chalcone synthase TfCHS albumen and encoding gene and probe

Technical field

The present invention relates to key enzyme and encoding gene and probe in the turmeric pattern glycosides route of synthesis, be specifically related to a kind of turmeric chalcone synthase TfCHS albumen and encoding gene and probe.

Background technology

The color of flower is one of important factor that determines florist's product value and ornamental value.Change pattern, innovate pattern, can increase commodity value and the ornamental value of flowers.The color of flower is the result of cyanidin(e) accumulation in the petal cell, and cyanidin(e) mainly comprises flavonoid, carotenoid and betaines.Flavonoid makes the color of flower generation from the Huang to the purple as a large class of cyanidin(e).The biosynthetic pathway of flavonoid be for can be divided into three phases, the fs be by phenylalanine to coumaric acyl CoA, this is that many secondary metabolisms are total; To flavanonol, this is the committed step of flavonoid metabolism to subordinate phase by coumaric acyl CoA, its synthetic anthocyanidin and other flavonoid substance.The synthetic precursor of all flavonoids all is that 4-coumaric acyl CoA and malonyl-CoA produce cinnamophenone and begin under the effect of chalcone synthase (Chalcone synthase CHS).Phase III is the synthetic of various anthocyanins.

Because CHS is synthetic flavanone, flavones, flavonol and the necessary enzyme of anthocyanogen, therefore be considered to a key enzyme of flavonoid biosynthetic pathway, be the multigene family coding.Have notable difference on the different CHS gene functions, the spatial and temporal expression characteristics in different lines are also different.Utilizing genetic engineering technique that the expression of CHS gene is regulated and control is one of approach that changes the flower color.Disturb the expression of reticent CHS gene that cyanidin(e) is synthesized such as microRNA and interrupted, thereby make pattern thin out, even become white; The cDNA Opposite direction connection of CHS on 35S promoter, is connected binary vector again and transforms petunia and can make pattern become pink and come adularescent with red-purple, and some flower is white in color fully; Chrysanthemum CHS gene justice is imported the chrysanthemum Cultivar produced white flowers and the different color hemp nettle of pattern afterwards; The CHS gene promoter that the clone obtains from three look rough gentian can be specific expressed at the petal lip of petunia; The CHS inverted defined gene changed over to obtained flower that has at colored wave edge etc. in the blue pig ear.

At present, in a lot of plants, found the CHS multigene family, such as petunia, grape, sweet potato, Arabidopis thaliana, angle violet etc.But for flower bulbs turmeric (Tulipa fosteriana), CHS gene cloning, expression pattern and CHS albumen coded sequence it be not immediately clear.At present, any bibliographical information relevant with turmeric CHS albumen and encoding gene thereof do not arranged.

Summary of the invention

The object of the invention is to fill up the blank of turmeric CHS gene family member's clone, expression pattern analysis and turmeric CHS albumen and encoding gene thereof, a kind of turmeric CHS albumen TfCHS 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 provides the expression pattern that turmeric TfCHS albumen and nucleotides sequence thereof are listed in turmeric Different Organs, different developmental phases, for utilizing from now on genetic engineering technique the spatial and temporal expression characteristic of TfCHS gene is regulated and control, thereby change pattern, innovation pattern provide theoretical foundation, have very large using value.

The present invention is achieved by the following technical solutions,

On the one hand, the invention provides a kind of protein with turmeric chalcone synthase activity, the protein that described protein is comprised of the aminoacid sequence shown in SEQ ID NO.4; Or by the protein of by (a) being derived of the aminoacid sequence shown in the SEQ ID NO.4 through replacing, lack or adding one or several amino acid and have turmeric chalcone synthase activity.This protein having that it's too late there is larger difference in active size in the different developmental phases of flower, 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 present invention also provides a kind of coding above-mentioned nucleic acid sequences to proteins.

Preferably, described nucleotide sequence is specially:

(a) base sequence is shown in the 1st～1179 of SEQ ID NO.3;

Or (b) and the nucleic acid shown in the 1st～1179 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～1179 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～1179 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 TfCHS.

In the present invention, " separation ", " purifying " DNA 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 " TfCHS albumen coded sequence " refer to the encode nucleotide sequence of polypeptide with turmeric TfCHS protein-active, the 1st～1179 nucleotide sequence and degenerate sequence thereof in the sequence shown in SEQ ID NO.3.This degenerate sequence refers to: be arranged in the 1st～1179 Nucleotide of 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, thus with sequence shown in the SEQ ID NO.3 in the 1st～1179 nucleotide sequence homology be low to moderate about 70% the degenerate sequence aminoacid sequence shown in the SEQ ID NO.4 of also encoding out.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～1179 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 TfCHS 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 " TfCHS albumen " refers to have the polypeptide of sequence shown in the SEQ ID NO.4 of turmeric TfCHS protein-active.This term also comprises having and the variant form relevant identical function of natural turmeric TfCHS, 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 TfCHS albumen.

The variant form of turmeric TfCHS albumen 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 TfCHS under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of turmeric TfCHS albumen to obtain.

In the present invention, " turmeric TfCHS conservative property variation polypeptide " refers to compare with the aminoacid 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

The present invention also comprises the analogue of turmeric chalcone synthase TfCHS albumen or polypeptide.The difference of these analogues and TfCHS 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 chalcone synthase TfCHS gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of mRNA transcript in cell of namely analyzing the TfCHS gene.

The detection method that whether has turmeric TfCHS 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 TfCHS 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 TfCHS 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 TfCHS or homologous protein.

In order to obtain the dot matrix with turmeric TfCHS 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 TfCHS 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 TfCHS.

Turmeric TfCHS 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 obtaining 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.Soc 85:2149-2154).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 TfCHS albumen of the present invention, by various conventional screening methods, can filter out the interactional material of relevant generation with turmeric TfCHS albumen, 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 the market requirement of new fancy variety is also increasing.The present invention clones the encoding sequence of first key enzyme chalcone synthase of flavonoid biosynthetic pathway TfCHS albumen in the turmeric plant materials first, and the expression pattern of the methods analyst TfCHS gene of employing fluorescence real-time quantitative PCR, for utilizing from now on genetic engineering technique the spatial and temporal expression characteristic of TfCHS gene is regulated and control, thereby change pattern, creation new fancy variety provide theoretical foundation, have very large reference 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 is that the homology of the nucleotide sequence of TfCHS gene of the present invention and lilium chalcone synthase genes mRNA compares (GAP) result;

Fig. 2 is that the homology of the nucleotide sequence of TfCHS gene of the present invention and lilium chalcone synthase genes mRNA compares (GAP) result;

Fig. 3 is that the homology of the aminoacid sequence of TfCHS albumen of the present invention and lilium chalcone synthase compares (FASTA) result, and 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.

Embodiment 1, turmeric TfCHS gene cloning

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

With " 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 NANODROP 1000Spectrophotometer);

3. the full-length clone of gene

According to the amino acid conserved sequence of CHS gene, 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 840bp fragment, reclaim and 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, check order at ABI 377 sequenators (Perkin-Elmer, USA).Sequencing result is by carrying out BLAST(http in the NCBI website: //blast.ncbi.nlm.nih.gov/) compare existing database (GenBank), the homology of knowing its nucleotide sequence and proteins encoded and known lilium chalcone synthase genes is very high, so think that tentatively it is a chalcone synthase genes;

（2）3′RACE

Two take turns the amplification that nest-type PRC is finished 3 ' end sequence;

The first round: Outerprimer+CHS3-1 (5 '-TGTCGTCTGCTCTGAAATCACTGCC-3 ')

Second takes turns: Innerprimer+CHS3-2 (5 '-AGCCAGACCATCCTCCCAGATTCGG-3 ')

Outerprimer and Innerprimer provide for test kit.3 ' RACE obtains 3 ' end sequence (about 590bp) of TfCHS, reclaim, be connected on the pMD18-T Simple vector carrier, with RV-M and M13-47 as universal primer, adopt the method that stops thing fluorescent mark (Big-Dye, Perkin-Elmer, USA), check order at ABI 377 sequenators (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 that the homology of its nucleotide sequence and proteins encoded and known lilium chalcone synthase genes is high;

（3）5′RACE

The sequence of 5 ' end is by using SMARTer ^TMRACE cDNA Amplification Kit obtains, take 5 ' RACE ready cDNA as template, provide by primer UPM(test kit) with CHS 5-1(5 '-ATGAAGCGGTTGACGGAGGGGCGGA-3 ') amplification obtains 5 ' end sequence (about 510bp) of TfCHS, reclaim the method for using after the connection with top and check order;

The sequencing result of the sequence that will obtain by above-mentioned 3 kinds of methods splices, and will splice sequence and submit to BLAST to analyze, and the result proves that the TfCHS gene that newly obtains really is a gene relevant with chalcone synthase from turmeric; With the ORF Finding(http of sequencing result in conjunction with NCBI: //www.ncbi.nlm.nih.gov/gorf) prediction, found initiator codon and the terminator codon of TfCHS gene; According to the sequence that obtains, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F(5 ' ATGGCGAATGTCGATGAGATCCGGC-3 ') and ORF-R(5 '-TCAGTTGGAGGTGATTGGAAGGCTG-3 '), carry out PCR take turmeric cDNA as template, amplification obtains the total length coding gene sequence (shown in SEQ ID NO.3) of 1179bp turmeric TfCHS albumen.

Embodiment 2, turmeric TfCHS gene sequence information and homology analysis

The new turmeric TfCHS total length CDS open reading frame sequence of the present invention is 1179bp, detailed sequence is seen the sequence shown in the SEQ ID NO.3, derive the aminoacid sequence of turmeric TfCHS according to the CDS open reading frame sequence, totally 392 amino-acid residues, molecular weight is 42861.3 dalton, iso-electric point (pI) is 5.90, and detailed sequence is seen sequence shown in the SEQ ID NO.4;

The CDS open reading frame sequence of turmeric TfCHS 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 CHS gene (the GenBank number of logging in HQ161731.1) have 86% homogeny at nucleotide level, (Query: the encoding sequence of turmeric chalcone synthase TfCHS, Sbjct: the mRNA sequence of lilium chalcone synthase) as depicted in figs. 1 and 2; On amino acid levels, it and lily CHS gene (the GenBank number of logging in ACX31605.1) also have 94% consistence and 99% similarity, (Query: the encoding sequence of turmeric chalcone synthase TfCHS, Sbjct: the aminoacid sequence of lilium chalcone synthase) as shown in Figure 3.This shows that all there are higher homology in turmeric TfCHS gene and lily CHS gene on nucleic acid or protein level.

Embodiment 3, turmeric TfCHS gene is at flower different developmental phases and differential 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) detect 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, and 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 turmeric TfCHS gene order that has obtained, utilize primer-design software to be designed for the Auele Specific Primer that the TfCHS gene quantification is analyzed among the Real-time PCR, CS-F(5 ' AGCCAGACCATCCTCCCA-3 '), CS-R(5 '-GCCAGCTTCAACTCCACC-3 '); Reference gene is the Actin(GenBank number of logging in AB456684), primer is Actin-F(5 '-AGTCAGTCATACAGTGCCAATC-3 '), Actin-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, after finishing, reaction draws solubility curve and typical curve, analyze solubility curve, whether the solubility curve of judging 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 Chromo 4 real-time quantitative instrument, reaction system is 20 μ L, three-step approach is adopted in 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 ^{-Δ Δ Ct}Method is made relative quantitative assay, the result shows that the expression level of TfCHS gene constantly rises in the growth course of flower, flower is fully open, petal complete when painted the expression amount of TfCHS be significantly higher than other painted stage, be 4.8 times of the firm painted stage expression amount of petal, illustrate that the expression of this gene and the coloring process of petal have obvious dependency; The TfCHS gene all has expression in leaf, stem, stamen, gynoecium, petal, expression amount is respectively stem＞petal＞leaf＞stamen＞gynoecium from more to less.The expression level of TfCHS gene in stem with in petal, leaf without significant difference, but be significantly higher than expression level in stamen and gynoecium, be respectively its 5.8 times and 6.3 times, this expression that TfCHS gene is described has obvious Spatial Difference.

Claims

1. one kind following (a) or protein (b):

(a) protein that is formed by the aminoacid sequence shown in SEQ ID NO.4;

(b) protein of by (a) being derived of the aminoacid sequence shown in the SEQ ID NO.4 through replacing, lack or adding one or several amino acid and have turmeric chalcone synthase 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～1179 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 the 1st～1179 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.