CN103087168B

CN103087168B - Tulip TfMYB2 protein and coding gene thereof as well as probe

Info

Publication number: CN103087168B
Application number: CN201310002614.9A
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: 2014-04-16
Anticipated expiration: 2033-01-05
Also published as: CN103087168A

Abstract

The invention provides a tulip TfMYB2 protein and a coding gene thereof as well as a probe. The protein is the protein in (a) or (b): (a) the protein comprising an amino acid sequence shown by SEQ ID NO.4; and (b) the protein which is derived from the protein in (a) through replacing, missing or adding one or more amino acids to the amino acid sequence shown by SEQ ID NO.4, and has the tulip MYB protein activity. The invention also provides a nucleotide sequence coding the protein, and the probe for detecting the nucleotide sequence. Expressions of a plurality of structural genes in biosynthetic pathways of flavonoid can be regulated and controlled through regulating the expression of TfMYB2 gene by utilizing the genetic engineering technology, or the TfMYB2 gene is transformed in other different plants, so that the content of flavonoid in transgenic plants can be effectively improved or lowered, the basis for achieving the purpose of flower color changing is provided, and the tulip TfMYB2 protein has important application value.

Description

Turmeric TfMYB2 albumen and encoding gene thereof and probe

Technical field

The present invention relates to key enzyme and encoding gene and probe in turmeric pattern glycosides route of synthesis, particularly, relate to a kind of turmeric TfMYB2 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 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 biosynthetic pathway is mainly by two genoid controls: 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 a genoid of control texture genetic expression intensity and phraseology.The transcription factor of regulatory gene coding can be identified by it by the energy containing in structure gene promotor cis-acting elements be combined, thereby activate the expression of multiple genes in flavonoid biosynthetic pathway, effectively start flavonoid biosynthetic pathway.Therefore, regulatory gene can regulate the formation of specific secondary metabolite to a certain extent.Although utilize transgenic technology the key gene of control flavonoid secondary metabolism or its antisense sequences importing plant can be promoted or be suppressed the expression of this gene, changed flavonoid secondary metabolism approach, change pattern, but plant flavonoids biosynthetic pathway complexity, relating to enzyme of a great variety, is not that indivedual key enzyme is controlled.By transcription factor effect, likely activate the expression of multiple structure genes in flavonoid secondary metabolism approach, the effect reaching is by more obvious than the effect that imports certain structure gene.

In some plants, found at present the regulatory gene of regulation and control flavonoid biosynthetic pathway, and by technology such as transposon taggings, cloned the gene of the part encoding transcription factor, wherein MYB class transcription factor family be research the most widely, it can regulate and control the transcriptional level of multiple structure genes in flavonoid route of synthesis.Myb transcription factor family member's common trait is to contain MYB structural domain, and MYB structural domain is high conservative between different plant species.The fundamental research of turmeric genetic molecule is little, only registers 245 nucleotide sequences of Tulipa (Tulipa) at present on NCBI, and wherein the gene relevant to pattern has 5.In turmeric, MYB protein coding gene sequence and expression pattern it be unclear that, and also do not have any bibliographical information relevant to turmeric MYB albumen and coding gene sequence thereof.

Summary of the invention

The object of the invention is to fill up the blank of turmeric myb gene family member's clone, expression pattern analysis and turmeric MYB albumen, a kind of turmeric MYB albumen TfMYB2 is provided, and the present invention also provides a kind of above-mentioned nucleic acid sequences to proteins and detect the probe of described nucleotide sequence of encoding; The invention discloses turmeric TfMYB2 albumen and nucleotide sequence thereof the expression pattern in turmeric Different Organs, different developmental phases.By analyzing the expression pattern of structure gene in TfMYB2 gene and flavonoid route of synthesis, infer that the transcription factor of TfMYB2 genes encoding is relevant to the transcriptional level of structure gene.The present invention is for utilizing genetic engineering technique, by regulating the expression of TfMYB2 gene, thereby the expression level of multiple structure genes in regulation and control flavonoid biosynthetic pathway, or TfMYB2 gene is transformed in different in addition plants, effectively improve or reduce the content of flavonoid in transgenic plant, the object that reaches change pattern provides the foundation, and has important using value.

On the one hand, the invention provides a kind of protein with turmeric MYB protein-active, the protein that described protein is comprised of the aminoacid sequence as shown in SEQ ID NO.4; Or by aminoacid sequence shown in SEQ ID NO.4 through replacing, lack or adding one or several amino acid and there is the protein of turmeric MYB protein-active.This protein having that it's too late active size exist larger difference in the different developmental phases of flower, Different Organs.

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

Further preferred, described protein is replaced by the similar or close amino acid of character the sequence forming for 1～10 amino acid in aminoacid sequence shown in SEQ ID NO.4.

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 1st～657 of SEQ ID NO.3; Or (b) and the nucleic acid shown in 1st～657 of SEQ ID NO.3 have the sequence of at least 70% homology; Or (c) sequence that can hybridize with the nucleic acid shown in 1st～657 of 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 1st～657 of 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 detecting in sample, whether have the relevant nucleic acid molecule of coding turmeric MYB 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 from native state, also refer to that this DNA or fragment with under native state follow the component of nucleic acid to separate, and separate with the protein accompanying in cell.

In the present invention, term " turmeric TfMYB2 albumen coded sequence " refers to that coding has the nucleotide sequence of polypeptide of turmeric MYB protein-active, 1st～657 nucleotide sequences as shown in SEQ ID NO.3 and degenerate sequence thereof.This degenerate sequence refers to, is arranged in 1st～657 Nucleotide shown in 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.Due to the degeneracy of codon, so be low to moderate approximately 70% the degenerate sequence sequence shown in SEQ ID NO.4 of also encoding out with 1st～657 nucleotide sequence homologies shown in SEQ ID NO.3.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, SEQ ID NO.3 of the natural turmeric MYB 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 TfMYB2 albumen " refers to have the polypeptide of sequence shown in the SEQ IDNO.4 of turmeric TfMYB2 protein-active.This term also comprises having and variant form natural turmeric TfMYB2 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 add one or be amino acid in 20 at C-terminal and/or N-terminal.For example, in the art, while replacing with the close or similar amino acid of performance, conventionally can not change the function of protein.Again such as, the function of adding or several amino acid and conventionally also can not change protein at C-terminal and/or N-terminal.This term also comprises active fragments and the reactive derivative of turmeric TfMYB2 albumen.

The variant form of turmeric TfMYB2 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 to turmeric TfMYB2 under high or low rigorous condition are coded and polypeptide or the albumen that utilizes the antiserum(antisera) of turmeric TfMYB2 albumen to obtain.

In the present invention, " turmeric TfMYB2 conservative property variation polypeptide " refer to compared with the aminoacid sequence shown in SEQ ID NO.4, has at the most 10 amino acid be replaced by the similar or close amino acid of character and form polypeptide.These conservative property variation polypeptide are preferably replaced and are produced according to table 1.

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 TfMYB2 albumen or polypeptide.The difference of these analogues and turmeric TfMYB2 related polypeptide can be the difference on aminoacid sequence, can be also the difference not affecting on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant natural or induction.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-(as D-amino acid), and has the analogue of non-natural amino acid (as β, gamma-amino acid) that exist or synthetic.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide of enumerating.

(conventionally the not changing primary structure) form of modification comprises: in body or the chemically derived form of external polypeptide as acetylize or carboxylated.Modify and also comprise glycosylation, as those carry out polypeptide glycosylation modified and that produce in procedure of processing in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and completes by polypeptide is exposed to.Modified forms also comprises the have phosphorylated amino acid residue sequence of (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 TfMYB2 gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of the mRNA transcript of analyzing turmeric TfMYB2 gene in cell.

The present invention detects the detection method that whether has turmeric TfMYB2 related nucleotide sequences in sample, comprises with above-mentioned probe and sample and hybridizing, and then whether detection probes combination has occurred.This sample is the product after pcr amplification, and wherein pcr amplification primer is corresponding to turmeric TfMYB2 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 TfMYB2 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 TfMYB2 or homologous protein.

In order to obtain and the dot matrix of turmeric TfMYB2 genes involved, can screen turmeric cDNA library with DNA probe, these probes are under low rigorous condition, use ³²p relevant all or part of of turmeric TfMYB2 cooked to 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, purchased from Clontech, and Stratagene, Palo Alto, Cal..This screening method can be identified the nucleotide sequence of the gene family relevant to turmeric MYB albumen.

Turmeric TfMYB2 associated nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic conventionally.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 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 amplifying for each time is stitched together by proper order.

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

In addition, also can will suddenly change and introduce in protein sequence of the present invention by chemosynthesis.

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

Utilize turmeric TfMYB2 albumen of the present invention, by various conventional screening methods, can filter out the interactional material of generation relevant to turmeric TfMYB2 albumen, or 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 in turmeric plant materials the encoding sequence of the transcription factor TfMYB2 albumen of adjusted and controlled activity of gene expression in flavonoid biosynthetic pathway first, and adopt fluorescence real-time quantitative PCR methods analyst TfMYB2 gene expression pattern and with flavonoid biosynthetic pathway in the relation of expression of structural gene pattern.For utilizing from now on genetic engineering technique, by regulating the expression of TfMYB2 gene, thereby the expression of multiple structure genes in regulation and control flavonoid biosynthetic pathway, or TfMYB2 gene is transformed in different in addition plants, effectively improve or reduce the content of flavonoid in transgenic plant, the object that reaches change pattern provides the foundation, and has important using value.

Accompanying drawing explanation

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 homology comparison (GAP) the result figure of the nucleotide sequence of turmeric TfMYB2 gene of the present invention and lily LhMYB6 gene mRNA;

Fig. 2 is homology comparison (FASTA) the result figure of the amino acid conserved domain sequence of turmeric TfMYB2 albumen of the present invention and lily R2R3-MYB transcription factor, and wherein, identical amino acid marks with amino acid monocase between two sequences.

Embodiment

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

embodiment 1, turmeric TfMYB2 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 are recognized flowers 2011 No. 004) plant routinely and carry out field management, treat that flower is completely open, petal is complete gathers Petal when painted, 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.), with denaturing formaldehyde gel electrophoresis, identify the integrity of RNA, then in upper purity and the concentration of measuring RNA of spectrophotometer (Thermo Scientific NANODROP1000Spectrophotometer);

3. the full-length clone of gene

According to the amino acid conserved sequence of MYB albumen in other species, utilize homologous genes clone principle, adopt RACE method (3 '-Full RACE Core Set Ver.2.0: precious biotechnology (Dalian) company limited, SMARTer ^tMrACEcDNA 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 to reverse transcription (Prime Script II 1st Strand cDNA Synthesis Kit: precious biotechnology (Dalian) company limited), take the first chain cDNA as template, utilize primers F 1(SEQ ID NO.1) and R1(SEQ ID NO.2) carry out PCR, amplification obtains 188bp fragment, reclaim and be connected on pMD18-T Simplevector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, at ABI377 sequenator (Perkin-Elmer, USA) on, check order, sequencing result is by carrying out BLAST(http in NCBI website: //blast.ncbi.nlm.nih.gov/) compare existing database (GenBank), the homology of knowing its nucleotide sequence and proteins encoded and known lilium MYB protein coding gene is very high, tentatively think that it is a MYB protein coding gene,

（2）3′RACE

Two take turns nest-type PRC completes the amplification of 3 ' end sequence.

The first round: Outerprimer+TfMYB23-1(5 '-TCGAAGAGACGAGGATGACCT-3 ')

Second takes turns: Innerprimer+TfMYB23-2(5 '-TCAGGCTTCATAATCTCTTGGGC-3 ')

Outerprimer and Innerprimer provide for test kit.3 ' RACE obtains the 3 ' end sequence (650bp) of TfMYB2 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) on, check order, sequencing result is by carrying out BLAST(http in NCBI website: //blast.ncbi.nlm.nih.gov/) compare existing database (GenBank), the homology of knowing its nucleotide sequence and proteins encoded and known lilium MYB protein coding gene is very high,

（3）5′RACE

Take 5 ' RACE ready cDNA as template, take turns nest-type PRC and complete the amplification of 5 ' end sequence by two, wherein UPM and NUP provide for test kit.

The first round: UPM+TfMYB25-1(5 '-TGGTTCGGAGTCCTCACGCATCATAAG-3 ')

Second takes turns: NUP+TfMYB25-1(5 '-ACGAGCCTTCCTGCTTTCATTCAACAA-3 ')

5 ' RACE obtains the 5 ' end sequence (487bp) of turmeric TfMYB2 gene, reclaim connect after with checking order with the method above, the sequencing result of the sequence obtaining by above-mentioned 3 kinds of methods is spliced, to splice sequence submits to BLAST to analyze, result proves that the TfMYB2 gene newly obtaining from turmeric is a gene relevant to coding MYB albumen really, ORF Finding(http by sequencing result in conjunction with NCBI: //www.ncbi.nlm.nih.gov/gorf) prediction, initiator codon and the terminator codon of turmeric TfMYB2 gene have been found, according to the sequence obtaining, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F(5 '-ATGACGAGCAACCCTTCCTCCTACC-3 '), ORF-R(5 '-CTAGTTCTGTCCAAAAGTCTCCTCC-3 '), take turmeric cDNA as template, carry out PCR, amplification obtains the complete encoding sequence (as shown in SEQ ID NO.3) of 657bp turmeric TfMYB2 albumen.

embodiment 2, turmeric TfMYB2 gene sequence information and homology analysis

The new turmeric TfMYB2 full length gene opening code-reading frame sequence of the present invention is 657bp, and its detailed sequence is as shown in SEQ IDNO.3; According to opening code-reading frame sequence, derive the aminoacid sequence of turmeric TfMYB2 albumen, totally 218 amino-acid residues, molecular weight is 24851.8 dalton, and iso-electric point (pI) is 8.25, and its detailed sequence is as shown in SEQ ID NO.4;

The aminoacid sequence of the opening code-reading frame sequence of turmeric TfMYB2 gene and proteins encoded thereof is carried out to 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 LhMYB6 gene (GenBank accession number is AB534587.1) have 77% homogeny, (Query: the coding gene sequence of turmeric TfMYB2 albumen as shown in Figure 1 on nucleotide level; Sbjct: the mRNA sequence of lily LhMYB6); On amino acid levels, it and lily R2R3-MYB transcription factor (GenBank accession number BAJ22983.1) have 58% consistence and 74% similarity, as shown in Figure 2 (Query: the aminoacid sequence of turmeric TfMYB2 albumen in total length; Sbjct: the aminoacid sequence of lily R2R3-MYB transcription factor).TfMYB2 contains the conservative R2 of MYB albumen and R3 region (sequence in the DNA region of specific binding) on aminoacid sequence, wherein in R3 region, contain a conservative bHLH in conjunction with territory ([D/E] Lx2[R/K] x3Lx6Lx3R), except R2 and R3 region, TfMYB2 also contains a motif6 region, and this region is the conservative region of the general synthetic relevant MYB family member of all and anthocyanidin in Arabidopis thaliana.As can be seen here, from nucleic acid or protein level, all there is higher homology in turmeric TfMYB2 gene and lily MYB protein coding gene, and be under the jurisdiction of the R2R3MYB gene group relevant to Kuromanine biosynthesizing, in sequence, there is the conserved domain of this monoid, structural domain sequence has high conservative, therefore infers that the Kuromanine of TfMYB2 regulation and control turmeric is synthetic.

embodiment 3, turmeric TfMYB2 gene different expression

1. the acquisition of material: 4 different developmental phases of 3 different assortment of turmeric (Tulipa fosteriana) (it is pink, pale red, bright red that color is respectively) flower (bud, petal is not painted; Bud, petal starts painted; Flower part is open, and petal is completely not painted; Flower is completely open, petal is completely painted), in field, take its petal, take subterraneous root, terrestrial stem, blade and petal (compound sample of each etap flower petal) simultaneously, after sample is wrapped with aluminium platinum paper respectively, drop at once in liquid nitrogen, then proceed to stored for future use in-80 ℃ 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 the RNA in petal and the different tissues of turmeric different developmental phases flower;

Determining 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; The good RNA of purity, A ₂₆₀/ A ₂₈₀and A ₂₆₀/ A ₂₃₀be about 2.0 left and right, by spectrophotometric determination OD value and calculate rna content;

The acquisition of 4.cDNA: take total RNA of 500ng as template, according to the precious TaKaRa PrimeScript of biotech firm ^tMit is standby that RT reagent Kit Perfect Real Time test kit operation instructions is carried out reverse transcription acquisition cDNA;

5. 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 TfMYB2 gene order having obtained, utilize primer-design software to be designed for the Auele Specific Primer that in Real-time PCR, TfMYB2 gene quantification is analyzed, primer M-QF(5 '-CAAGTGGACCCGTGTTCCC-3 '), primer M-QR(5 '-ATCCGGCCTGCTATTAGCG-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 to gradient dilution, then respectively take dilution after cDNA solution as template, Auele Specific Primer with goal gene and reference gene carries out Real-time pcr amplification, and reaction finishes 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, use this primer can obtain single pcr amplification product; By typical curve, determine the suitable extension rate of template cDNA;

7. the Real time PCR of goal gene in testing sample: take the cDNA Article 1 chain that synthesizes as template, by the primer amplified of goal gene and internal reference gene, carry out quantitative fluorescence analysis respectively, 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 ℃ of sex change 20s, then 41 circulations: 94 ℃ of 15s; 56 ℃ of 20s; 72 ℃ of 25s; After each amplification, all do solubility curve, to check amplified production whether as special generation;

8. adopt 2 ^{-△ △ Ct}method is made relative quantitative assay, and result shows that turmeric TfMYB2 gene only can detect transcript in petal, at root, stem, Ye Zhongjun, transcript do not detected, shows that this transcription factor is closely related with the growth of flower; TfMYB2 gene in petal expression level along with the growth course of flower, raise gradually,, petal completely open at flower complete when painted expression amount the highest, be respectively 5.6,1.5 and 1.2 times of first three flower developmental stage expression amount; In the 3 kinds of same growth grade of the red tulipine kind of difference petals, anthocyanin content is that the pale red ﹥ of bright red ﹥ is pink, correspondingly be, it is pink that the expression level of TfMYB2 gene in these three kinds also shows as the pale red ﹥ of bright red ﹥, supposition TfMYB2 gene is synthetic relevant to anthocyanogen, the expression level of structure gene in regulation and control anthocyanogen route of synthesis.

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. the protein being formed by the aminoacid sequence 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, is characterized in that, described nucleotide sequence is specially:

Base sequence is as shown in 1st～657 of SEQ ID NO.3.

4. for detection of a probe for nucleotide sequence as claimed in claim 2, it is characterized in that, described probe is the nucleic acid molecule that includes 8～100 continuous nucleotides of described nucleotide sequence.