CN103074307B

CN103074307B - Tulipa gesneriana TfbHLH1 protein, encoding gene thereof and probe

Info

Publication number: CN103074307B
Application number: CN201310002653.9A
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: 2015-01-14
Anticipated expiration: 2033-01-05
Also published as: CN103074307A

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 in turmeric anthocyanogen route of synthesis and encoding gene thereof and probe, particularly, relate to a kind of turmeric TfbHLH1 albumen and encoding gene thereof and probe.

Background technology

Flower is the important reproductive organ of plant, and pattern is the prerequisite that plant normally breeds offspring, is also that ornamental plant Important Economic is worth 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 research at present.

Flavonoid biosynthesis pathway controls primarily of two genoids: structure gene and regulatory gene.Wherein, the various enzymes that structure gene direct coding is relevant with the biosynthesizing of flavonoid secondary metabolism, regulatory gene is then a genoid of control texture Gene expression intensities and phraseology.The transcription factor of regulatory gene coding can be identified by it by the energy contained in structure gene promotor cis-acting elements is combined, thus the expression of multiple gene in activation flavonoid biosynthesis pathway, starts flavonoid biosynthesis pathway effectively.In recent years, utilize genetic engineering technique, by the coordinate expression regulating the expression of transcription factor gene to activate multiple structure gene in flavonoid biosynthesis pathway, or the transcription factor gene be separated from specified plant is transformed in plants different in addition, effectively improve or reduce the content of flavonoid in transgenic plant, reach the object changing flavonoid substances content or pattern.In some plants, found the regulatory gene regulating and controlling flavonoid biosynthesis pathway at present, and the gene of code segment transcription factor has been cloned by technology such as transposon taggings, wherein bHLH type transcription factor is one of transcription factor of main research, and it can regulate and control the transcriptional level of multiple structure gene in flavonoid route of synthesis with MYB type transcription factor synergy.

Up to the present, from Common Snapdragon, petunia, corn, Arabidopis thaliana, rough gentian and African chrysanthemum, cloned the transcription factor of ten Yu Ge bHLH families, all take part in the regulation and control of anthocyanin biosynthetic pathway.The fundamental research of turmeric genetic molecule is little, and current NCBI only registers Tulipa (Tulipa) 245 nucleotide sequences, wherein relevant to pattern gene has 5.In turmeric, bHLH protein coding gene sequence and expression pattern it be unclear that, and also do not have any bibliographical information relevant to turmeric bHLH albumen and coding gene sequence thereof.

Summary of the invention

The object of the invention is to fill up the blank of the clone of turmeric bHLH gene family member, expression pattern analysis and turmeric bHLH albumen, provide a kind of turmeric bHLH albumen TfbHLH1, present invention also offers a kind of above-mentioned nucleic acid sequences to proteins and detect the probe of described nucleotide sequence of encoding; The invention discloses turmeric TfbHLH1 albumen and nucleotide sequence thereof the expression pattern in turmeric Different Organs, different developmental phases, by analyzing the expression pattern of structure gene in TfbHLH1 gene and flavonoid route of synthesis, infer that the transcription factor of TfbHLH1 genes encoding is relevant to the transcriptional level of structure gene.The present invention is for utilizing genetic engineering technique, by the transcriptional level of multiple structure gene in the expression regulation flavonoid biosynthesis pathway of adjustment TfbHLH1 gene, thus effectively improve or reduce flavonoid content, change pattern provide the foundation, there is 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 made up of the such as aminoacid sequence shown in SEQ ID NO.4; Or by the aminoacid sequence shown in SEQ ID NO.4 through replacing, lacking or add one or several amino acid and there is the protein of turmeric bHLH protein-active.This protein having that it's too late active size exist larger difference in the different developmental phases, Different Organs of flower.

Preferably, described protein for aminoacid sequence shown in SEQ ID NO.4 is through 1 ~ 50 amino acid whose disappearance, insertion and/or replacement, or within C-terminal and/or N-terminal add 1 ~ 20 amino acid and the sequence that obtains.

Further preferred, described protein for 1 ~ 10 amino acid in aminoacid sequence shown in SEQ ID NO.4 replace by the similar or close amino acid of character and the sequence that formed.

On the other hand, the invention provides the above-mentioned nucleic acid sequences to proteins of a kind of coding.

Preferably, described nucleotide sequence is specially: (a) base sequence is as shown in SEQ ID NO.3 1st ~ 2079; Or the nucleic acid shown in (b) and SEQ ID NO.3 1st ~ 2079 has the sequence of the homology of at least 70%; Or (c) can carry out the sequence of hybridizing with the nucleic acid shown in SEQ ID NO.3 1st ~ 2079.

Preferably, described nucleotide sequence is specially the disappearance of 1 ~ 90 Nucleotide in the nucleotide sequence shown in SEQ ID NO.3 1st ~ 2079, insertion and/or replacement, and 5 ' and/or 3 ' sequence of being formed with inner nucleotide of end interpolation 60.

In addition, present invention also offers a kind of probe detecting above-mentioned nucleotide sequence, described probe is the nucleic acid molecule with above-mentioned nucleotide sequence 8 ~ 100 continuous nucleotides, and this probe can be used for detecting in sample whether there is the relevant nucleic acid molecule of coding turmeric bHLH albumen.

In the present invention, " DNA of separation ", " DNA of purifying " refer to, the sequence that this DNA or fragment have been arranged in its both sides from native state is separated, also refer to that this DNA or fragment with under native state are separated with the component of nucleic acid, and separate with the protein accompanied in cell.

In the present invention, term " turmeric TfbHLH1 albumen coded sequence " refers to encode the nucleotide sequence of the polypeptide with turmeric bHLH protein-active, 1st ~ 2079 nucleotide sequences as shown in SEQ ID NO.3 and degenerate sequence thereof.This degenerate sequence refers to, is arranged in 1st ~ 2079 Nucleotide shown in SEQ ID NO.3, have one or more codon replace by the degenerate codon of same amino acid of encoding after produce sequence.Due to the degeneracy of codon, the sequence shown in SEQ ID NO.4 so the degenerate sequence being low to moderate about 70% with 1st ~ 2079 nucleotide sequence homologies shown in SEQ ID NO.3 also can be encoded out.This term also comprises the nucleotide sequence with the homology at least 70% of the nucleotide sequence shown in SEQ ID NO.3.

This term also comprises the variant form of sequence shown in the identical function of natural turmeric bHLH albumen of encoding, SEQ ID NO.3.These variant forms comprise (but being not limited to): be generally the disappearance of 1 ~ 90 Nucleotide, insertion and/or replacement, and 5 ' and/or 3 ' end be added to 60 with inner nucleotide.

In the present invention, term " turmeric TfbHLH1 albumen " refer to have turmeric TfbHLH1 protein-active SEQ ID NO.4 shown in the polypeptide of sequence.This term also comprise have with natural turmeric TfbHLH1 albumen identical function, the variant form of 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 add one or amino acid within being 20 at C-terminal and/or N-terminal.Such as, in the art, when replacing with similar nature or similar amino acid, the function of protein can not usually be changed.Again such as, add at C-terminal and/or N-terminal the function that or several amino acid also can not change protein usually.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: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutants, can the albumen coded by DNA of DNA hybridization relevant to turmeric TfbHLH1 and the polypeptide utilizing the antiserum(antisera) of turmeric TfbHLH1 albumen to obtain or albumen under high or low high stringency conditions.

In the present invention, " turmeric TfbHLH1 conservative variation polypeptide " refers to compared with the aminoacid sequence shown in SEQ ID NO.4, have 10 amino acid at the most replace by the similar or close amino acid of character and form polypeptide.These conservative variation's polypeptide preferably carry out replacing according to table 1 and produce.

Table 1

Initial residue	Representational replacement	Preferred replacement
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile

Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

Invention also comprises the analogue of turmeric TfbHLH1 albumen or polypeptide.The difference of these analogues and turmeric TfbHLH1 related polypeptide can be the difference on aminoacid sequence, can be also the difference do not affected on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant that is natural or induction.Induce variation body can be obtained by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also by site-directed mutagenesis or the biological technology of other known moleculars.Analogue also comprises the analogue with the residue (as D-amino acid) being different from natural L-amino acids, and has the analogue of amino acid (as β, gamma-amino acid) that is that non-natural exists or synthesis.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide enumerated.

(usually the not changing primary structure) form of modification comprises: the chemically derived form of the polypeptide that body is interior or external is as acetylize or carboxylated.Modify and also comprise glycosylation, as carried out glycosylation modified and polypeptide that is that produce in those in the synthesis of polypeptide and processing or further procedure of processing.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) by being exposed to by polypeptide and completing.Modified forms also comprises the sequence with phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Also comprise and modified thus improve its anti-proteolysis performance or optimize the polypeptide of solubility property.

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 the mRNA transcript namely analyzing turmeric TfbHLH1 gene in cell.

The present invention detects the detection method that whether there is turmeric TfbHLH1 related nucleotide sequences in sample, and comprise and hybridizing with above-mentioned probe and sample, then whether detection probes there occurs combination.This sample is the product after pcr amplification, and wherein pcr amplification primer corresponds to turmeric TfbHLH1 related nucleosides coding sequences, 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 on the homology basis of nucleic acid homology or marking protein, screening turmeric TfbHLH1 associated homologous gene or homologous protein.

In order to obtain the dot matrix with turmeric TfbHLH1 oncogene related gene, can screen turmeric cDNA library with DNA probe, these probes are under low high stringency conditions, use ³²p does radioactivity mark to turmeric TfbHLH1 gene-correlation all or part of and obtains.The cDNA library being suitable for screening is the library from turmeric.The method built from the cDNA library of interested cell or tissue is that biology field is well-known.In addition, many such cDNA libraries also can buy, such as, purchased from Clontech, Stratagene, Palo Alto, Cal..This screening method can identify the nucleotide sequence of the gene family relevant to turmeric bHLH albumen.

Turmeric TfbHLH1 gene-correlation Nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the cDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.When sequence is longer, usually needs to carry out twice or repeatedly pcr amplification, and then the fragment that each time amplifies is stitched together by proper order.

After obtaining relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.

In addition, also by chemosynthesis, sudden change is introduced in protein sequence of the present invention.

Except producing with recombination method, the fragment also available solid phase technique of albumen of the present invention, is produced (people such as Stewart, (1969) Solid phase peptide synthssis, WH Freeman Co., San Francisco by direct peptide synthesis; Merrifield J. (1963) J.Am Chem.Soc85:2149-2154).Synthetic protein can carry out by hand or automatically in vitro.Such as, can with the 431A type peptide synthesizer (Foster City, CA) of Applied Biosystems from dynamic synthetic peptide.Each fragment of chemosynthesis albumen of the present invention can be distinguished, then chemically connected the molecule producing total length.

Utilize turmeric TfbHLH1 albumen of the present invention, by various conventional screening assays, can filter out and relevant to turmeric TfbHLH1 albumen interactional material occur, or acceptor, inhibitor or antagonist etc.

Compared with prior art, the present invention has following beneficial effect: turmeric is one of large cut-flower in the world ten, 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 turmeric plant materials in flavonoid biosynthesis pathway first, and adopts the expression pattern of the methods analyst of fluorescence real-time quantitative PCR TfbHLH1 gene and the relation with expression of structural gene pattern in flavonoid biosynthesis pathway thereof.For utilizing genetic engineering technique from now on, by the coordinate expression regulating the expression of TfbHLH1 gene to activate multiple structure gene in flavonoid biosynthesis pathway, or TfbHLH1 gene is transformed in plants different in addition, effectively improve or reduce the content of flavonoid in transgenic plant, reach the object changing pattern to provide the foundation, there is important using value.

Accompanying drawing explanation

By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:

Fig. 1 is Homology search (GAP) the result figure of the nucleotide sequence of turmeric TfbHLH1 gene of the present invention and lily LhbHLH6 gene mRNA;

Fig. 2 is Homology search (GAP) the result figure of the nucleotide sequence of turmeric TfbHLH1 gene of the present invention and lily LhbHLH6 gene mRNA;

Fig. 3 is Homology search (GAP) the result figure of the nucleotide sequence of turmeric TfbHLH1 gene of the present invention and lily LhbHLH6 gene mRNA;

Fig. 4 is Homology search (GAP) the result figure of the nucleotide sequence of turmeric TfbHLH1 gene of the present invention and lily LhbHLH6 gene mRNA;

Fig. 5 is Homology search (FASTA) the result figure of the conservative domain sequence of turmeric TfbHLH1 albumen of the present invention and lily bHLH transcription factor, and wherein, identical amino acid marks with amino acid monocase between two sequences.

Embodiment

Below in conjunction with specific embodiment, set forth the present invention further.These embodiments are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, such as Sambrook equimolecular clone: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.

embodiment 1, turmeric TfbHLH1 gene clone

1. the acquisition of vegetable material

By health, tulip of the same size, (Tulipa fosteriana ' Shangnong zaoxia ', by Shanghai City crop varietal approval committee.Numbering: Shanghai agriculture product recognize flowers 2011 No. 004) plant routinely and carry out field management, treat flower opening completely, petal complete painted time gather Petal, for extracting RNA;

The extracting of 2.RNA

By " RNA prep pure plant total RNA extraction reagent box " extracted total RNA (RNA prep pure Plant Kit: TIANGEN Biotech (Beijing) Co., Ltd.), by the integrity of denaturing formaldehyde gel electrophoresis qualification RNA, then in upper purity and the concentration measuring RNA of spectrophotometer (Thermo Scientific NANODROP1000Spectrophotometer);

3. the full-length clone of gene

According to the amino acid consensus sequence of bHLH albumen in other species, utilize homologous genes cloning mechanisms, adopt 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 cDNA full-length clone, a point three phases carries out:

(1) RT-PCR obtains gene intermediate segment

The RNA of extraction is carried out reverse transcription (Prime Script II 1st Strand cDNA Synthesis Kit: precious biotechnology (Dalian) company limited), with the first chain cDNA for template, primers F 1 (as shown in SEQ ID NO.1) and primer R1 (as shown in SEQ ID NO.2) is utilized to carry out PCR, amplification obtains 1528bp fragment, reclaim and be connected on pMD18-T Simple vector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, at ABI377 sequenator (Perkin-Elmer, USA) check order on, sequencing result is by carrying out the BLAST existing database of (http://blast.ncbi.nlm.nih.gov/) comparison (GenBank) in NCBI website, know that the homology of its nucleotide sequence and proteins encoded and known lilium bHLH protein coding gene is very high, tentatively think that it is a bHLH protein coding gene,

(2)3′RACE

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

The first round: Outerprimer+TfbHLH13-1 (5 '-GACACTAGACTTGGGAACAACACCT-3 ')

Second takes turns: Innerprimer+TfbHLH13-2 (5 '-GGTTCCCTCTATTACCAAGGCTGA-3 ')

3 ' RACE obtains the 3 ' end sequence (791bp) of TfbHLH1 gene, reclaim, be connected on pMD18-T Simple vector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, at ABI377 sequenator (Perkin-Elmer, USA) check order on, sequencing result is by carrying out the BLAST existing database of (http://blast.ncbi.nlm.nih.gov/) comparison (GenBank) in NCBI website, know that the homology of its nucleotide sequence and proteins encoded and known lilium bHLH protein coding gene is very high,

(3)5′RACE

With 5 ' RACE ready cDNA for template, take turns by two the amplification that nest-type PRC completes 5 ' end sequence, wherein UPM and NUP provides for test kit;

The first round: UPM+TfbHLH15-1 (5 '-CTATCCGCAAAGGAAGCGTTCGTCAC-3 ')

Second takes turns: NUP+TfbHLH15-1 (5 '-ATCCATCCCTCCATGCCAACACCCCT-3 ')

5 ' RACE obtains the 5 ' end sequence (408bp) of turmeric TfbHLH1 gene, reclaim after connecting and check order with the method above, the sequencing result of the sequence obtained by above-mentioned 3 kinds of methods is spliced, to splice sequence submits to BLAST to analyze, result proves that the TfbHLH1 gene newly obtained from turmeric is a gene relevant to coding bHLH albumen really, the ORF Finding (http://www.ncbi.nlm.nih.gov/gorf) of sequencing result in conjunction with NCBI is predicted, initiator codon and the terminator codon of turmeric TfbHLH1 gene are found, according to the sequence obtained, Auele Specific Primer ORF-F (5 '-ATGGAGGGGCTTGATATTCAGCAGG-3 ') and ORF-R (5 '-TTATTCAAGAGCTCTGACAT GTACA-3 ') is designed respectively from initiator codon and terminator codon, with turmeric cDNA for template carries out PCR, amplification obtains the complete encoding sequence (as shown in SEQ ID NO.3) of 2079bp turmeric TfbHLH1 albumen.

embodiment 2, the sequence information of turmeric TfbHLH1 gene and homology analysis

The new turmeric TfbHLH1 full length gene opening code-reading frame sequence of the present invention is 2079bp, detailed sequence is as shown in SEQ ID NO.3, the aminoacid sequence of turmeric TfbHLH1 albumen is derived according to 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 shown in sequence shown in SEQ ID NO.4;

The opening code-reading frame sequence of turmeric TfbHLH1 gene and the aminoacid sequence blast program of proteins encoded thereof are carried out Nucleotide and protein homology search in GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that it and lily LhbHLH1 gene (GenBank accession number is AB222075.1) have the homogeny of 76% on nucleotide level, as Fig. 1, Fig. 2, (the coding gene sequence of Query: turmeric TfbHLH1 albumen shown in Fig. 3 and Fig. 4, the mRNA sequence of Sbjct: lily LhbHLH1), on amino acid levels, it and lily bHLH transcription factor (GenBank accession number BAE20057.1) also have the consistence of 63% and the similarity of 75%, as shown in Figure 5 (the aminoacid sequence of Query: turmeric TfbHLH1 albumen, the aminoacid sequence of Sbjct: lily bHLH transcription factor), as can be seen here, all there is higher homology in turmeric TfbHLH1 gene and lily bHLH protein coding gene from 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: turmeric (Tulipa fosteriana ' Shangnong zaoxia ') flower 4 different developmental phases (bud, petal is not painted; Bud, petal starts painted; Floral parts is open, and petal is completely not painted; Flower is opening completely, petal is completely painted), its petal is taked in field, take terrestrial stem, blade, stamen, gynoecium and petal (compound sample of each etap petal) simultaneously, drop at once in liquid nitrogen after sample is wrapped with aluminium platinum paper respectively, then proceed to stored for future use in-80 DEG C of Ultralow Temperature Freezers;

The extraction of 2.RNA: utilize RNA prep pure plant total RNA extraction reagent box (RNA prep pure Plant Kit: TIANGEN Biotech (Beijing) Co., Ltd.) to extract RNA in the petal of turmeric different developmental phases flower and different tissues;

The determination of the integrity of 3.RNA, purity, concentration: with plain agar sugar gel electrophoresis (gum concentration 1.2%; 0.5 × TBE electrophoretic buffer; 150v, 15min) detect integrity, in electrophoretic band, maximum rRNA brightness should be 1.5 ~ 2.0 times of Article 2 rRNA brightness, otherwise represents the degraded of rRNA sample; Purity good RNA, A ₂₆₀/ A ₂₈₀and A ₂₆₀/ A ₂₃₀be about about 2.0, calculate rna content by spectrophotometric determination OD value;

The acquisition of 4.cDNA: with the total serum IgE of 500ng for template, according to precious biotech firm TaKaRa PrimeScript ^tMit is for subsequent use that RT reagent Kit Perfect Real Time test kit operation instructions carries out reverse transcription acquisition cDNA;

5. design Auele Specific Primer to carry out real-time fluorescence quantitative PCR analyzing gene at each organ and the expression amount in tissue, according to the turmeric TfbHLH1 gene order obtained, primer-design software is utilized to be designed for the Auele Specific Primer that in Real-time PCR, TfbHLH1 gene quantification is analyzed, primer B-QF (5 '-AAACGACTCAAGCAACGG-3 '), primer B-QR (5 '-TGAAGGACATGCAAACCAC-3 '), reference gene is 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: with EASY Dilution (test kit provides), standard substance cDNA solution is carried out gradient dilution, then respectively with dilution after cDNA solution for template, carry out Real-time pcr amplification with the Auele Specific Primer of goal gene and reference gene, reaction terminates rear drafting solubility curve and typical curve; Analyze solubility curve, judge whether the solubility curve of goal gene and reference gene obtains simple spike, to judge to use this primer can obtain single pcr amplification product; By the appropriate dilutions multiple of typical curve determination template cDNA;

7. the Real time PCR of goal gene in testing sample: with the cDNA Article 1 chain synthesized for template, quantitative fluorescence analysis is carried out respectively by the primer amplified of goal gene and internal reference gene, Real-time PCR reaction is carried out on BIO-RAD Chromo4 real-time quantitative instrument, reaction system is 20 μ L, reaction adopts three-step approach, 94 DEG C of sex change 20s, then 41 circulations: 94 DEG C of 15s; 56 DEG C of 20s; 72 DEG C of 25s; Whether, after each amplification completes, all do solubility curve, be special generation to check amplified production;

8. adopt 2 ^{-△ △ Ct}method makes relative quantitative assay, result shows that turmeric TfbHLH1 gene does not all detect transcript in gynoecium, all transcript can be detected in stem, leaf, stamen and petal, wherein, expression amount in stem is the highest, be respectively 1.2,7.2,3.4 times of expression amount in leaf, stamen, petal, show that the expression of TfbHLH1 gene has tissue specificity; The expression level of TfbHLH1 gene increases gradually in the growth course of flower, and front 3 etap increase slowly, and the last stage significantly improves; TfbHLH1 gene expression amount when flower the last stage (flower is opening completely, and petal is completely painted) is the highest, is respectively 3.5,3.2,1.9 times of front 3 stage expression amounts; The expression pattern of TfbHLH1 gene in flower growth process is corresponding with structure gene TfCHS, TfF3H, TfDFR and TfANS gene and regulatory gene TfMYB2 gene expression pattern in this process in anthocyanogen route of synthesis in tulip petals, infer that TfbHLH1 and TfMYB2 exists to do mutually, the expression level of structure gene in common regulation and control anthocyanogen route of synthesis.

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

Claims

1. the protein of the aminoacid sequence composition as shown in SEQ ID NO.4.

2. nucleic acid sequences to proteins described in a coding claim 1.

3. nucleotide sequence as claimed in claim 2, it is characterized in that, described nucleotide sequence is specially base sequence as shown in SEQ ID NO.3 1st ~ 2079.