CN102978194A

CN102978194A - Tulip chalcone isomerase TfCHI protein and coding gene thereof and probe

Info

Publication number: CN102978194A
Application number: CN2012104284662A
Authority: CN
Inventors: 袁媛; 史益敏; 唐东芹; 马晓红
Original assignee: Shanghai Jiao Tong University
Current assignee: Shanghai Jiao Tong University
Priority date: 2012-10-31
Filing date: 2012-10-31
Publication date: 2013-03-20
Also published as: CN103614358B; CN103614358A

Abstract

The present invention relates to a tulip chalcone isomerase TfCHI protein and its coding gene and probe. The protein is the following (a) or (b) protein: (a) composed of the protein as shown in SEQ ID NO.4 A protein composed of an amino acid sequence; (b) a protein derived from (a) in which the amino acid sequence shown in SEQ ID NO.4 has been substituted, deleted or added with one or several amino acids and has tulip chalcone isomerase activity. The present invention also provides a nucleic acid sequence encoding the above-mentioned protein, and a probe for detecting the above-mentioned nucleic acid sequence; the present invention uses genetic engineering technology to regulate the temporal and spatial expression characteristics of the tulip chalcone isomerase TfCHI gene, thereby changing flower color and creating The new suit provides a theoretical basis and has great application value.

Description

Turmeric enzyme, namely chalcone isomerase TfCHI 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 enzyme, namely chalcone isomerase TfCHI albumen and encoding gene and probe.

Background technology

The color of flower is one of important factor that determines its commodity value and ornamental value.The color of flower is the result of cyanidin(e) accumulation, and cyanidin(e) mainly comprises flavonoid, carotenoid and betaines.Flavonoid makes the whole colors of flower generation from the Huang to the purple as one of three large cyanidin(e).The flavonoid biosynthetic pathway is present more clearly one of pathways metabolism of research, and wherein the biosynthetic pathway of anthocyanogen is studied the most extensive.Enzyme, namely chalcone isomerase (Chalcone isomerase CHI) is second enzyme that is right after chalcone synthase (CHS) in the anthocyanogen biosynthetic pathway, and it is catalytically conveted to colourless flavanone with the cinnamophenone of yellow.Nearly all flavonoid class compound all is to be derived by flavanone in the plant materials, so this enzyme plays an important role to the flavonoid class compou nd synthesis.Flower can become yellow when the CHI activity is suppressed.As by the CHI gene in the method sudden change carnation petal that inserts transposon, can make the flower yellowing of white; Yellow or jade-green pollen etc. when the promotor inactivation of CHI gene in the petunia flower pesticide, have been produced.These results show that the expression level of regulation and control CHI gene can affect the color of flower.

The encoding gene of CHI is a multigene family, this gene is separated from various plants, such as petunia, Kidney bean, corn etc., but for flower bulbs turmeric (Tulipa fosteriana), CHI gene cloning, expression pattern and CHI protein sequence it be not immediately clear.At present, any bibliographical information relevant with turmeric CHI albumen and coding gene sequence thereof do not arranged.

Summary of the invention

The object of the invention is to fill up the blank of turmeric CHI gene family member's clone, expression pattern analysis and turmeric CHI albumen, a kind of turmeric CHI albumen TfCHI 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 turmeric TfCHI albumen and nucleotide sequence thereof at the expression pattern of turmeric Different Organs, different developmental phases, for utilizing from now on genetic engineering technique the space-time characterisation of TfCHI genetic expression is regulated and control, thereby change pattern, the new pattern of creation provide theoretical foundation, have very large using value.

On the one hand, the invention provides the protein with turmeric enzyme, namely chalcone isomerase activity, the protein that described protein is comprised of the aminoacid sequence shown in SEQ ID NO.4; Or by the protein of the aminoacid sequence shown in the SEQ ID NO.4 through replacing, lacking or add one or several amino acid and have turmeric enzyme, namely chalcone isomerase 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 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～693 of SEQ ID NO.3; Or (b) and the nucleic acid shown in the 1st～693 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～693 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～693 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 TfCHI.

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 enzyme, namely chalcone isomerase TfCHI albumen coded sequence " refer to the encode nucleotide sequence of polypeptide with turmeric TfCHI protein-active, the 1st～693 nucleotide sequence shown in SEQ ID NO.3 and degenerate sequence thereof.This degenerate sequence refers to, is arranged in the 1st～693 Nucleotide 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 sequence shown in the SEQ ID NO.4 of also encoding out with the 1st～693 nucleotide sequence homology shown in the SEQ ID NO.3.This term also comprises the nucleotide sequence with the homology at least 70% of the nucleotide sequence shown in the SEQ ID NO.3.

This term also comprises the variant form of sequence shown in the identical function, SEQ ID NO.3 of the natural turmeric TfCHI albumen of encoding.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 enzyme, namely chalcone isomerase TfCHI albumen " refers to have the polypeptide of sequence shown in the SEQ ID NO.4 of turmeric TfCHI protein-active.This term also comprises having and variant form natural turmeric TfCHI albumen identical function, SEQ ID NO.4 sequence.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 TfCHI albumen.

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

In the present invention, " turmeric TfCHI 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

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

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

In order to obtain the dot matrix with turmeric TfCHI 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 TfCHI 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 CHI.

Turmeric TfCHI 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, just 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 TfCHI albumen of the present invention, by various conventional screening methods, can filter out the interactional material of relevant generation with turmeric TfCHI 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 people are also increasing to the demand of new fancy variety.The present invention clones the encoding sequence of the key enzyme enzyme, namely chalcone isomerase TfCHI albumen in the flavonoid biosynthetic pathway in the turmeric plant materials first, and the expression pattern of the methods analyst TfCHI gene of employing fluorescence real-time quantitative PCR, for utilizing from now on the spatial and temporal expression of genetic engineering technique regulation and control TfCHI gene, thereby change pattern, the new pattern of creation provide theoretical foundation, have very large 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 is that the homology of the nucleotide sequence of turmeric TfCHI gene of the present invention and America oil palm enzyme, namely chalcone isomerase gene mRNA compares (GAP) result;

Fig. 2 is that the homology of the aminoacid sequence of turmeric TfCHI albumen of the present invention and America oil palm enzyme, namely chalcone isomerase 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 TfCHI 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 CHI 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 SynthesisKit: 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 the 338bp 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, 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), it is very high to know that its nucleotide sequence and proteins encoded and known America oil palm belong to the enzyme, namely chalcone isomerase gene homology, thinks that tentatively it is an enzyme, namely chalcone isomerase gene;

（2）3′RACE

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

The first round: Outerprimer+CHI3-1(5 '-TTCACCCAGTCCCCTTCAGGTTCAC-3 ')

Second takes turns: Innerprimer+CHI3-2(5 '-TCTCTCAGAGGCAGTATTGGAGTCG-3 ')

Outerprimer and Innerprimer provide for test kit.3 ' RACE obtains 3 ' end sequence (372bp) of TfCHI, 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), it is very high to know that its nucleotide sequence and proteins encoded and known America oil palm belong to the enzyme, namely chalcone isomerase gene homology;

（3）5′RACE

Take 5 ' RACE ready cDNA as template, take turns the amplification that nest-type PRC is finished 5 ' end sequence by two,

The first round: UPM+CHI5-1(5 '-TCGTGAGTGAACCTGAAGGGGACTGGG-3 ')

Second takes turns: NUP+CHI 5-1(5 '-CTGCTCGGCTGCAACACCTTCAGCTTC-3 ')

UPM and NUP provide for test kit.5 ' RACE obtains the 5 ' end sequence (450bp) of turmeric TfCHI gene, use after reclaim connecting with the method top and check order, the sequencing result of the sequence that will obtain by above-mentioned 3 kinds of methods splices, to splice sequence submits to BLAST to analyze, the TfCHI gene that result's proof newly obtains from turmeric is a gene relevant with enzyme, namely chalcone isomerase really, 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 TfCHI gene have been found, according to the sequence that obtains, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F(5 '-ATGGCAGCAGTCGCGCCCAAGCTTG-3 '), ORF-R(5 '-TCACACAGAGACAGTGGCGGGTAGC-3 '), carry out PCR take turmeric cDNA as template, amplification obtains the complete encoding sequence (SEQ ID NO.3) of 693bp turmeric TfCHI albumen.

Embodiment 2, turmeric TfCHI gene sequence information and homology analysis

The new turmeric TfCHI full length gene opening code-reading frame sequence of the present invention is 693bp, and detailed sequence is seen sequence shown in the SEQ ID NO.3.Derive the aminoacid sequence of turmeric TfCHI according to the opening code-reading frame sequence, totally 230 amino-acid residues, molecular weight is 24374.9 dalton, and iso-electric point (pI) is 5.15, and detailed sequence is seen sequence shown in the SEQ ID NO.4;

The opening code-reading frame sequence of turmeric TfCHI 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 America oil palm CHI gene (the GenBank accession number is FJ940770.1) have 75% homogeny, as shown in Figure 1 (Query: the coding gene sequence of turmeric enzyme, namely chalcone isomerase TfCHI at nucleotide level; Sbjct: the mRNA sequence of America oil palm enzyme, namely chalcone isomerase); On amino acid levels, it and America oil palm CHI gene (GenBank accession number ACQ41836.1) also have 76% consistence and 86% similarity, as shown in Figure 2 (Query: the aminoacid sequence of turmeric enzyme, namely chalcone isomerase TfCHI; Sbjct: the aminoacid sequence of America oil palm enzyme, namely chalcone isomerase).This shows that all there are higher homology in turmeric TfCHI gene and America oil palm CHI gene on nucleic acid or protein level.

Embodiment 3, turmeric TfCHI gene is at flower different developmental phases and the 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) 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; 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 TfCHI gene order that has obtained, utilize primer-design software to be designed for the Auele Specific Primer that the TfCHI gene quantification is analyzed among the Real-time PCR, primer CI-F(5 '-CCGCTGACTGGACAACAA-3 '), primer CI-R(5 '-CCCGGATTCAGGAATAGAAC-3 '), reference gene is Actin(GenBank accession number 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, 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 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 expression level first sharply more slowly rising of decline in the growth course of flower of turmeric TfCHI gene, the expression amount in painted stage is the not highest at bud, the expression amount that begins when painted when bud is minimum, flower is fully open, petal complete when painted expression level go up to some extent again.Wherein high expression level amount is 8.73 times of minimum expression amount, illustrates that the expression of this gene and the growth course of flower have obvious dependency, has the time expression specificity; Turmeric TfCHI gene has expression in stem, leaf, gynoecium, petal, but can't detect transcript in stamen.TfCHI gene expression level in stem, leaf, gynoecium, petal is followed successively by stem, gynoecium, leaf, petal from high to low, illustrates that the expression of turmeric TfCHI gene 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 enzyme, namely chalcone isomerase 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～693 of SEQ ID NO.3;

Or (b) and the nucleic acid shown in the 1st～693 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～693 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～693 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.