CN103074307A

CN103074307A - Tulipa gesneriana TfbHLH1 protein, encoding gene thereof and probe

Info

Publication number: CN103074307A
Application number: CN2013100026539A
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: 2013-01-05
Filing date: 2013-01-05
Publication date: 2013-05-01
Anticipated expiration: 2033-01-05
Also published as: CN103074307B

Abstract

The invention provides a tulipa gesneriana TfbHLH1 protein, encoding gene thereof and a probe. The protein is a protein (a) or (b): (a), the protein is composed of an amino acid sequence shown in SEQ ID NO. 4; and (b), the protein derived from (a) with the activity of the protein of tulipa gesneriana bHLH, and obtained through performing replacement and/or deletion and/or addition of one or more amino acids for the amino acid sequence shown in SEQ ID NO. 4. The invention further provides a nucleotide sequence for encoding the protein and a probe for detecting the nucleotide sequence. According to the invention, the genetic engineering technology is utilized, the flavonoid content is effectively increased or reduced and a foundation is provided for color change through adjusting the transcriptional level of a plurality of structural genes in the expression regulation flavonoid biosynthetic pathway of the TfbHLH1 gene, and important theoretical and practical application value can be obtained.

Description

Turmeric TfbHLH1 albumen and encoding gene thereof and probe

Technical field

The present invention relates to key enzyme and encoding gene and probe in the turmeric pattern glycosides route of synthesis, particularly, relate to a kind of turmeric TfbHLH1 albumen and encoding gene and probe.

Background technology

Flower is the important reproductive organ of plant, and pattern is the prerequisite that plant normally breeds the offspring, also is that the ornamental plant Important Economic is worth the place.The molecule mechanism of research pattern substance metabolism is the basis for the strange pattern flower variety of seed selection.The color of flower is the result of cyanidin(e) accumulation.Cyanidin(e) mainly comprises flavonoid, carotenoid and betaines.The biosynthetic pathway of flavonoid is one of the most deep pathways metabolism of at present research.

The flavonoid biosynthetic pathway is mainly controlled by two genoids: structure gene and regulatory gene.Wherein, the various enzymes that the structure gene direct coding is relevant with the biosynthesizing of flavonoid secondary metabolism, regulatory gene then is a genoid of control texture genetic expression intensity and phraseology.The transcription factor of regulatory gene coding can be combined by the energy that contains in structure gene promotor cis-acting elements identified by it, thereby activates the expression of a plurality of genes in the flavonoid biosynthetic pathway, effectively starts the flavonoid biosynthetic pathway.In recent years, utilize genetic engineering technique, activate the coordinate expression of a plurality of structure genes in the flavonoid biosynthetic pathway by the expression of regulating transcription factor gene, the transcription factor gene that maybe will separate from specified plant transforms in different in addition plants, effectively improve or reduce the content of flavonoid in the transgenic plant, reached the purpose that changes flavonoid substances content or pattern.In some plants, found at present the regulatory gene of regulation and control flavonoid biosynthetic pathway, and cloned the gene of the part encoding transcription factor with technology such as transposon taggings, wherein bHLH type transcription factor is one of transcription factor of main research, and it can regulate and control with MYB type transcription factor synergy the transcriptional level of a plurality of structure genes in the flavonoid route of synthesis.

Up to the present, from Common Snapdragon, petunia, corn, Arabidopis thaliana, rough gentian and African chrysanthemum, clone the transcription factor of more than ten bHLH families, all participated in the regulation and control of anthocyanin biosynthetic pathway.The fundamental research of turmeric genetic molecule is seldom only registered 245 nucleotide sequences of Tulipa (Tulipa) on the NCBI at present, and wherein the gene relevant with pattern has 5.BHLH protein coding gene sequence and expression pattern it be unclear that in turmeric, and any bibliographical information relevant with turmeric bHLH albumen and coding gene sequence thereof do not arranged yet.

Summary of the invention

The object of the invention is to fill up the blank of turmeric bHLH gene family member's clone, expression pattern analysis and turmeric bHLH albumen, a kind of turmeric bHLH albumen TfbHLH1 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 TfbHLH1 albumen and nucleotide sequence thereof at the expression pattern of turmeric Different Organs, different developmental phases, by analyzing the expression pattern of structure gene in TfbHLH1 gene and the flavonoid route of synthesis, infer that the transcription factor of TfbHLH1 genes encoding is relevant with the transcriptional level of structure gene.The present invention is for utilizing genetic engineering technique, transcriptional level by a plurality of structure genes in the expression regulation flavonoid biosynthetic pathway of regulating the TfbHLH1 gene, thereby the content, the change pattern that effectively improve or reduce flavonoid provide the foundation, and have important using value.

On the one hand, the invention provides a kind of protein with turmeric bHLH protein-active, 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 replacing, lack or adding one or several amino acid and have the protein of turmeric bHLH protein-active.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～2079 of SEQ ID NO.3; Or (b) and the nucleic acid shown in the 1st～2079 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～2079 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～2079 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 bHLH albumen.

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 TfbHLH1 albumen coded sequence " refer to the encode nucleotide sequence of polypeptide with turmeric bHLH protein-active, the 1st～2079 nucleotide sequence shown in SEQ ID NO.3 and degenerate sequence thereof.This degenerate sequence refers to, is arranged in the 1st～2079 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 approximately 70% the degenerate sequence sequence shown in the SEQ ID NO.4 of also encoding out with the 1st～2079 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 bHLH 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 TfbHLH1 albumen " refers to have the polypeptide of sequence shown in the SEQ IDNO.4 of turmeric TfbHLH1 protein-active.This term also comprises having and variant form natural turmeric TfbHLH1 albumen identical function, SEQ IDNO.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 TfbHLH1 albumen.

The variant form of turmeric TfbHLH1 albumen of the present invention comprises: the albumen that homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, DNA that can DNA hybridization relevant with turmeric TfbHLH1 under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of turmeric TfbHLH1 albumen to obtain.

In the present invention, " turmeric TfbHLH1 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 TfbHLH1 albumen or polypeptide.The difference of these analogues and turmeric TfbHLH1 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 TfbHLH1 gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of mRNA transcript in cell of namely analyzing turmeric TfbbHLH1 gene.

The detection method that whether has turmeric TfbHLH1 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 TfbHLH1 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 TfbHLH1 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 TfbHLH1 or homologous protein.

In order to obtain the dot matrix with turmeric TfbbHLH1 oncogene related gene, can screen the turmeric cDNA library with dna probe, these probes are under low rigorous condition, use ³²P turmeric TfbHLH1 gene-correlation all or part of 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 bHLH albumen.

Turmeric TfbHLH1 gene-correlation 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.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 TfbHLH1 albumen of the present invention, by various conventional screening methods, can filter out the interactional material of generation relevant with turmeric TfbHLH1 albumen, perhaps acceptor, inhibitor or antagonist etc.

Compared with prior art, the present invention has following beneficial effect: 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 TfbHLH1 albumen in the flavonoid biosynthetic pathway in the turmeric plant materials first, and adopt fluorescence real-time quantitative PCR methods analyst the TfbHLH1 gene expression pattern and with the flavonoid biosynthetic pathway in the relation of expression of structural gene pattern.For utilizing from now on genetic engineering technique, activate the coordinate expression of a plurality of structure genes in the flavonoid biosynthetic pathway by the expression of regulating the TfbHLH1 gene, or the TfbHLH1 gene transformed in addition different plants, effectively improve or reduce the content of flavonoid in the transgenic plant, reach the purpose that changes pattern and provide the foundation, have important 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 TfbHLH1 gene of the present invention and lily LhbHLH6 gene mRNA compares (GAP) as a result figure;

Fig. 2 is that the homology of the nucleotide sequence of turmeric TfbHLH1 gene of the present invention and lily LhbHLH6 gene mRNA compares (GAP) as a result figure;

Fig. 3 is that the homology of the nucleotide sequence of turmeric TfbHLH1 gene of the present invention and lily LhbHLH6 gene mRNA compares (GAP) as a result figure;

Fig. 4 is that the homology of the nucleotide sequence of turmeric TfbHLH1 gene of the present invention and lily LhbHLH6 gene mRNA compares (GAP) as a result figure;

Fig. 5 is that the homology of the amino acid conserved domain sequence of turmeric TfbHLH1 albumen of the present invention and lily bHLH transcription factor compares (FASTA) as a result figure, 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 HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1, turmeric TfbHLH1 gene cloning

1. the acquisition of vegetable material

(Tulipa fosteriana ' Shangnong zaoxia ' 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 extracting 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.), identify the integrity of RNA with the denaturing formaldehyde gel electrophoresis, 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 bHLH albumen 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 ^TMRACEcDNA 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(shown in SEQ ID NO.1) and primer R1(shown in SEQ ID NO.2) carry out PCR, amplification obtains the 1528bp 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), the homology of knowing its nucleotide sequence and proteins encoded and known lilium bHLH protein coding gene is very high, thinks that tentatively it is a bHLH protein coding gene;

（2）3′RACE

Two take turns the amplification that nest-type PRC is finished 3 ' end sequence, and Outerprimer and Innerprimer provide for test kit.

The first round: Outerprimer+TfbHLH1 3-1(5 '-GACACTAGACTTGGGAACAACACCT-3 ')

Second takes turns: Innerprimer+TfbHLH1 3-2(5 '-GGTTCCCTCTATTACCAAGGCTGA-3 ')

3 ' RACE obtains the 3 ' end sequence (791bp) of TfbHLH1 gene, reclaim, be connected on the pMD18-T Simplevector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (BigDye, Perkin-Elmer, USA) method, at ABI377 sequenator (PerkinElmer, 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 that the homology of its nucleotide sequence and proteins encoded and known lilium bHLH protein coding gene is very high;

（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, wherein UPM and NUP provide for test kit;

The first round: UPM+TfbHLH1 5-1(5 '-CTATCCGCAAAGGAAGCGTTCGTCAC-3 ')

Second takes turns: NUP+TfbHLH1 5-1(5 '-ATCCATCCCTCCATGCCAACACCCCT-3 ')

5 ' RACE obtains the 5 ' end sequence (408bp) of turmeric TfbHLH1 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 TfbHLH1 gene that result's proof newly obtains from turmeric is a gene relevant with coding bHLH albumen 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 TfbHLH1 gene have been found, according to the sequence that obtains, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F(5 '-ATGGAGGGGCTTGATATTCAGCAGG-3 ') and ORF-R(5 '-TTATTCAAGAGCTCTGACATGTACA-3 '), carry out PCR take turmeric cDNA as template, amplification obtains the complete encoding sequence (shown in SEQ ID NO.3) of 2079bp turmeric TfbHLH1 albumen.

Embodiment 2, turmeric TfbHLH1 gene sequence information and homology analysis

The new turmeric TfbHLH1 full length gene opening code-reading frame sequence of the present invention is 2079bp, detailed sequence is shown in SEQ IDNO.3, derive the aminoacid sequence of turmeric TfbHLH1 albumen according to the opening code-reading frame sequence, totally 692 amino-acid residues, molecular weight is 77655.3 dalton, iso-electric point (pI) is 4.87, and detailed sequence is seen sequence shown in the SEQ ID NO.4;

The opening code-reading frame sequence of turmeric TfbHLH1 gene and the aminoacid sequence of proteins encoded thereof are carried out Nucleotide and protein homology search with blast program in GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that it and lily LhbHLH1 gene (the GenBank accession number is AB222075.1) have 76% homogeny at nucleotide level, such as Fig. 1, Fig. 2, Fig. 3 and (Query: the coding gene sequence of turmeric TfbHLH1 albumen shown in Figure 4; Sbjct: the mRNA sequence of lily LhbHLH1); On amino acid levels, it and lily bHLH transcription factor (GenBank accession number BAE20057.1) also have 63% consistence and 75% similarity, as shown in Figure 5 (Query: the aminoacid sequence of turmeric TfbHLH1 albumen; Sbjct: the aminoacid sequence of lily bHLH transcription factor), this shows, all there are higher homology in turmeric TfbHLH1 gene and lily bHLH protein coding gene on nucleic acid or protein level.

Embodiment 3, turmeric TfbHLH1 gene is at the different expression of different tissues, flower different developmental phases

1. the acquisition of material: (bud, petal are not painted in 4 different developmental phases of turmeric (Tulipa fosteriana ' Shangnong zaoxia ') 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 terrestrial stem, blade, stamen, gynoecium and petal (compound sample of each etap 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 PlantKit: 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 TfbHLH1 gene order that has obtained, utilize primer-design software to be designed for the Auele Specific Primer that the TfbHLH1 gene quantification is analyzed among the Real-time PCR, primer B-QF(5 '-AAACGACTCAAGCAACGG-3 '), primer B-QR(5 '-TGAAGGACATGCAAACCAC-3 '), reference gene is that the Actin(GenBank accession number is AB456684), primer is Actin-F(5 '-AGTCAGTCATACAGTGCCAATC-3 ') and 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 Chromo4 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 20s; 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 turmeric TfbHLH1 gene does not all detect transcript in gynoecium, in stem, leaf, stamen and petal, all can detect transcript, wherein, expression amount in stem is the highest, be respectively 1.2,7.2,3.4 times of in leaf, stamen, petal expression amount, show that the expression of TfbHLH1 gene has tissue specificity; The expression level of TfbHLH1 gene increases in the growth course of flower gradually, and front 3 etap increase slowly, and the last stage significantly improves; TfbHLH1 gene expression amount when flower the last stage (flower is fully open, and petal is fully painted) is the highest, is respectively 3.5,3.2,1.9 times of front 3 stage expression amounts; In the expression pattern of TfbHLH1 gene in the flower growth course and the tulip petals in the anthocyanogen route of synthesis structure gene TfCHS, TfF3HTfDFR and TfANS gene and the expression pattern of regulatory gene TfMYB2 gene in this process corresponding, infer that there are mutually work in TfbHLH1 and TfMYB2, regulate and control the expression level of structure gene in the anthocyanogen route of synthesis jointly.

Above specific embodiments of the invention are described.It will be appreciated that, the present invention is not limited to above-mentioned specific implementations, and those skilled in the art can make various distortion or modification within the scope of the claims, and this does not affect flesh and blood of the present invention.

Claims

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 TfbHLH1 protein-active by (a) derivative protein.

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～2079 of SEQ ID NO.3;

Or (b) and the nucleic acid shown in the 1st～2079 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～2079 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～2079 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.