CN103589694B

CN103589694B - Turmeric flavonoid-3 '-hydroxylase TfF3 ' H protein and encoding gene thereof

Info

Publication number: CN103589694B
Application number: CN201310533249.4A
Authority: CN
Inventors: 袁媛; 史益敏; 唐东芹; 马晓红
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2012-10-31
Filing date: 2013-10-30
Publication date: 2015-10-28
Anticipated expiration: 2033-10-30
Also published as: CN102965349A; CN103589694A

Abstract

The present invention relates to a kind of turmeric flavonoid-3 '-hydroxylase TfF3 ' H protein and encoding gene thereof, does is described protein as SEQ? ID? shown in NO.4 aminoacid sequence composition protein or as SEQ? ID? aminoacid sequence shown in NO.4 is through replacing, lacking or add one or several amino acid and have turmeric flavonoid-3 ' protein of-hydroxylase activity.Present invention also offers a kind of above-mentioned protein of encoding as SEQ? ID? nucleotide sequence shown in NO.3.Turmeric flavonoid-3 in the present invention '-'-hydroxylase gene deepened by more than 50% transgene tobacco pattern pink colour after building over-express vector and being transformed in tobacco, correspondingly, Cyanidin content rising 20-30% in transgene tobacco petal.This shows turmeric flavonoid-3 '-hydroxylase can act on anthocyanogen route of synthesis, and affect Cyanidin synthesis, change flower color.

Description

Turmeric flavonoid-3 '-hydroxylase TfF3 ' H protein and encoding gene thereof

Technical field

The present invention relates to the crux enzyme in turmeric anthocyanogen route of synthesis and encoding gene thereof, be specifically related to a kind of turmeric flavonoid-3 '-hydroxylase TfF3 ' H protein and encoding gene thereof, belong to molecular engineering field.

Background technology

The color of flower is the important factor affecting plant pollination, also determines commodity value and the ornamental value of a kind of flowers simultaneously.Change pattern, create new pattern, commodity value and the ornamental value of flowers can be increased.The color of flower is the result accumulating cyanidin(e) in petal cell, and cyanidin(e) mainly comprises flavonoid, carotenoid and betaines.Flavonoid is a large class of cyanidin(e), and it makes the whole colors of flower generation from Huang to purple.

Flavonoid-3 ' and-hydroxylase (flavonoid3 '-hydroxylase, F3 ' H, once flavanonol-3 was called in the past in document '-hydroxylase, now be called flavonoid-3 more '-hydroxylase) belong to cytochrome P 450 monooxygenases, it has the function of the substrate oxidation reaction of the multiple dependence of catalysis NADPH or NADH, plays an important role in the synthesis and secondary metabolism of plant flavonoids.It can at cofactor NADPH and O ₂effect under, catalysis list Oxygenation, respectively by 3 ' position hydroxylation of naringenin (naringenin) and dihydrokaempferol (dihydrokaempferol) B-ring, catalysis forms eriodictyol and dihydroquercetin.And eriodictyol and dihydroquercetin are the important precursor of synthesis cyanidin(e), be the important colour former of flower, the biosynthesizing for anthocyanidin plays an important role, thus forms different anthocyanins, makes the pattern that plant performance is different.The expression activity of F3 ' H gene directly can affect the final color of flower, such as, in petunia, express the F3 ' H gene himself obtained make the pale pink petunia lacking F3 ' H gene and F3 ' 5 ' H gene become deep pink; Rough gentian F3 ' H gene is transformed in tobacco, finds the increase occurring anthocyanidin and quercetin content in transgene tobacco body, thus have impact on the pattern of tobacco; During the disappearance of F3 ' H gene, pharbitis nilChoisy (Ipomoea nil), tricolo(u)r morning glory (Ipomoea tricolor) and Pharbitis purpurea (Ipomoea purpurea) can produce the ruddy flower containing pelargonidin derivative.Therefore, regulating and controlling the expression of F3 ' H gene is one of approach changing pattern.

F3 ' H gene is cloned in various plants, but in turmeric (Tulipa fosteriana), and the clone of F3 ' H gene, expression pattern and F3 ' H protein encoding sequence it be not immediately clear.At present, any bibliographical information relevant to turmeric F3 ' H protein and coding gene sequence thereof is not had.

Summary of the invention

The present invention fills up the blank of the clone of turmeric F3 ' H gene family member, expression pattern analysis and turmeric F3 ' H protein and encoding gene thereof, provide a kind of turmeric TfF3 ' H protein sequence, present invention also offers the above-mentioned nucleic acid sequences to proteins of a kind of coding.The invention discloses the expression pattern that turmeric TfF3 ' H protein and nucleotides sequence thereof are listed in turmeric Different Organs, different developmental phases; Construct TfF3 ' H gene over-express vector and transformation of tobacco, analyze TfF3 ' H gene to the synthesis of transgene tobacco anthocyanogen and the impact of pattern.Utilize turmeric flavonoid provided by the invention 3 '-hydroxylase TfF3 ' H, pattern can be changed for genetically engineered, obtain innovation pattern and a kind of effective technique means is provided.In addition, in addition, utilize turmeric TfF3 ' H gene of the present invention, by various conventional screening assays, can to filter out and mutual material, acceptor, inhibitor or antagonist etc. occur F3 ' H.

The present invention is achieved by the following technical solutions,

The object of the invention is to provide one to have turmeric flavonoid-3 ' protein of-hydroxylase activity, 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 turmeric flavonoid-3 ' protein derivative by (a) of-hydroxylase activity.This protein having that it's too late active size exist larger difference in the different tinting stage, Different Organs of flower petal.

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 ~ 1533;

Or the nucleic acid shown in (b) and SEQ ID NO.3 1st ~ 1533 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 ~ 1533.

Preferably, described nucleotide sequence is specially the disappearance of 1 ~ 90 Nucleotide in the nucleotide sequence shown in SEQ ID NO.3 1st ~ 1533, 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 F3 ' H.

Isolated DNA molecular provided by the invention, this molecule comprises: the DNA molecular with nucleotide sequence shown in SEQ ID NO.3; Or coding has the nucleotide sequence of the polypeptide of turmeric TfF3 ' H protein matter activity, and has the homology of at least 70% with sequence shown in SEQ IDNO.3; Or can with the nucleotide sequence hybridization of sequence shown in SEQ ID NO.3.

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 flavonoid-3 '-hydroxylase protein coded sequence " refers to that coding has the nucleotide sequence of the polypeptide of turmeric TfF3 ' H protein activity, nucleotide sequence and degenerate sequence thereof as shown in SEQ ID NO.3.This degenerate sequence refers to, is arranged in sequence 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 aminoacid sequence shown in SEQ ID NO.4 so the degenerate sequence being low to moderate about 70% with the homology of sequence shown in SEQ ID NO.3 also can be encoded out.This term also comprise with sequence shown in SEQ ID NO.3 in from the nucleotide sequence of the homology at least 70% of the nucleotide sequence of 1st ~ 1533, Nucleotide.

This term also comprises encoding and has the variant form with sequence shown in the SEQ ID NO.3 of the albumen of natural turmeric TfF3 ' H identical function.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 flavonoid-3 '-hydroxylase TfF3 ' H protein " refer to there is turmeric TfF3 ' H protein activity SEQ ID NO.4 shown in the polypeptide of sequence.This term also comprise have to the relevant identical function of natural turmeric TfF3 ' H, the variant form of sequence shown in SEQ ID NO.4.These variant forms comprise (but being not limited to): be generally 1 ~ 50 amino acid whose disappearance, insertion and/or replacement, and 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 TfF3 ' H protein.

The variant form of turmeric TfF3 ' H polypeptide of the present invention comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutants, under high or low high stringency conditions can to the albumen coded by the DNA of the relevant DNA hybridization of turmeric TfF3 ' H and the polypeptide utilizing the antiserum(antisera) of turmeric TfF3 ' H polypeptide to obtain or albumen.

In the present invention, " turmeric TfF3 ' H conservative variation polypeptide " refers to compared with the aminoacid sequence of 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 TfF3 ' H protein or polypeptide.The difference of these analogues and turmeric TfF3 ' H related polypeptide can be the difference on aminoacid sequence, also can be 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 molecular.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 methods analyst turmeric TfF3 ' the H gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of the mRNA transcript namely analyzing TfF3 ' H gene in cell.

The present invention detects in sample the detection method that whether there is turmeric TfF3 ' H related nucleotide sequences, 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 TfF3 ' H 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 TfF3 ' H nucleotide sequence of the present invention and aminoacid sequence, can on the homology basis of nucleic acid homology or marking protein, screening turmeric TfF3 ' H associated homologous gene or homologous protein.

In order to obtain the dot matrix with turmeric TfF3 ' H genes involved, can screen turmeric cDNA library with DNA probe, these probes are under low high stringency conditions, use ³²what P was correlated with to turmeric TfF3 ' H all or part ofly does radioactivity mark 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 TfF3 ' H.

Turmeric TfF3 ' H 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 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, relevant sequence can be obtained 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 TfF3 ' H protein of the present invention, by various conventional screening assays, can filter out, with turmeric TfF3 ' H, interactional material occur, or acceptor, inhibitor or antagonist etc.

Turmeric flavonoid-3 of the present invention '-'-hydroxylase gene or its homologous sequence import plant tissue, cell or organ by plant expression vector; The carrier that sets out for building described plant expression vector can be any one binary vector that can be used for Agrobacterium tumefaciens transformation plant or can be used for the carrier etc. of plant micropellet bombardment.As pBin serial carrier, pBI serial carrier, Gateway ^tMserial carrier, pCAMBIA serial carrier or other derivative plant expression vector, the described carrier that sets out can also be the carrier copied in prokaryotic organism, as pENTER-TOPO, pUC serial carrier etc.

Use turmeric flavonoid-3 in the present invention '-'-hydroxylase gene or its homologous sequence construction of expression vector time, any one enhancement type, composing type, organizing specific type or inducible promoter can be added before it transcribes super beginning Nucleotide.Constitutive promoter can be cauliflower mosaic virus (CAMV) 35S promoter etc.; Tissue-specific promoter can be the specific expressing promoters such as root, blade, petal, seed; Inducible promoter can be the promotor by the induction such as ethene, book alcohol.Above-mentioned promotor can be used alone or is combined with other plant promoter.In addition, when utilizing gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer.

For the ease of identifying transgenic plant cells or plant and screening, can process plant expression vector used, the coding can expressed in plant as added can produce the antibiotic marker thing gene or chemical resistance reagent marker gene etc. of the enzyme of colour-change or luminophor gene, tool resistance.Described host plant cell, tissue or organ containing neomycin phosphotransferase (NPTII) gene can be screened by kantlex or its substituted derivatives etc., can be screened containing the host plant cell of hygromycin phosphotransferase gene, tissue or organ by Totomycin.After aforesaid method screens, Southern, PCR or dot blot equimolecular detection means also can be adopted to detect transfer-gen plant, to determine whether it has transformed goal gene.

A specific embodiment of the present invention excises the transformation carrier of gus gene with pHB(pCambia1300), for plant expression vector, build containing turmeric flavonoid-3 of the present invention ' the process LAN plant vector pHB-TfF3 ' H of-'-hydroxylase gene.Carrying turmeric flavonoid-3 of the present invention ' plant expression vector of-'-hydroxylase gene or its homologous sequence is by the combination transformed plant cells of any one or more method in protoplastis-chemical mediated method, Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, pollen tube importing, microinjection, electric shock, particle gun, the conventional biology methods such as agriculture bacillus mediated, tissue or organ, cultivate the plant cell of anthocyanogen synthesis change, tissue or organ, and cultivate into plant further; Described tissue and organ can comprise the fruit of host plant, callus, root, stem apex, blade and seed etc.

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 pattern.Clone the key enzyme flavonoid-3 at some place of tulip petals flavonoid synthesis forehearth limb first ' encoding sequence of-hydroxylase TfF3 ' H protein, and by turmeric flavonoid-3 in the present invention '-'-hydroxylase gene by build over-express vector be transformed in tobacco, found that under identical culture condition, transgene tobacco is compared with contrast (unconverted) tobacco, the pink colour of more than 50% transgene tobacco pattern is deepened, correspondingly, Cyanidin content rising 20-30% in transgene tobacco petal.This shows turmeric flavonoid-3 '-hydroxylase can act on anthocyanogen route of synthesis, and affect Cyanidin synthesis, change flower color.Flavonoid-3 '-hydroxylase be in cyanidin(e) route of synthesis Cyanidin synthesis key enzyme, turmeric flavonoid-3 provided by the invention '-'-hydroxylase gene TfF3 ' H is for utilizing genetically engineered change pattern, and acquisition innovation pattern provides a kind of effective technique means.In addition, utilize turmeric TfF3 ' H protein of the present invention, by various conventional screening assays, can filter out, with turmeric TfF3 ' H, interactional material occur, or acceptor, inhibitor or antagonist etc.

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) result of the nucleotide sequence of turmeric TfF3 ' H gene of the present invention and lily F3 ' H gene mRNA;

Fig. 2 is Homology search (GAP) result of the nucleotide sequence of turmeric TfF3 ' H gene of the present invention and lily F3 ' H gene mRNA;

Fig. 3 is Homology search (GAP) result of the nucleotide sequence of turmeric TfF3 ' H gene of the present invention and lily F3 ' H gene mRNA;

Fig. 4 is Homology search (FASTA) result of the aminoacid sequence of turmeric TfF3 ' H of the present invention and lily F3 ' H, and wherein, identical amino acid marks with amino acid monocase between two sequences.

Fig. 5 is that plant over-express vector pHB-TfF3 ' H builds schematic diagram.

Fig. 6 is that unconverted tobacco (a) and the pattern turning TfF3 ' H gene tobacco (b, c) compare.

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 HarborLaboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.

embodiment 1, turmeric TfF3 ' H gene clone

1. the acquisition of vegetable material

By health, tulip of the same size, (Tulipa fosteriana ' Shangnongzaoxia ', 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

Utilize " RNA prep pure plant total RNA extraction reagent box " extracted total RNA (RNA prep pure PlantKit: 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 F3 ' H gene 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 ^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 reverse transcription (Prime Script II 1st Strand cDNA Synthesis Kit: precious biotechnology (Dalian) company limited), with the first chain cDNA for template, utilize primers F 1(SEQ ID NO.1) and R1(SEQ ID NO.2) carry out PCR, amplification obtains about 1000bp 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) check order on.Sequencing result is by carrying out BLAST(in NCBI website http:// blast.ncbi.nlm.nih.gov/) the existing database of comparison (GenBank), ' homology of-'-hydroxylase gene very high, therefore tentatively think that it is a flavonoid-3 '-'-hydroxylase gene of knowing its nucleotide sequence and proteins encoded and known lilium flavonoid-3.

（2）3’RACE

3 ' the sequence of holding is by use 3 '-Full RACE Core Set Ver.2.0: precious biotechnology (Dalian) company limited obtains, and two take turns the amplification that nest-type PRC completes 3 ' end sequence:

The first round: Outerprimer+F3 ' H3-1(5 '-CTTCCACATCCCCAAGCACGCCACT-3 ')

Second takes turns: Innerprimer+F3 ' H3-2(5 '-GTGGAGTTCAAGCCTTCCCGGTTCA-3 ')

Outerprimer and Innerprimer provides for test kit.3 ' RACE obtains the 3 ' end sequence (476bp) of TfF3 ' H, 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, ABI377 sequenator (Perkin-Elmer, USA) checks order, and sequencing result is by carrying out BLAST(in NCBI website http:// blast.ncbi.nlm.nih.gov/) the existing database of comparison (GenBank), know its nucleotide sequence and proteins encoded and known lilium flavonoid-3 ' homology of-'-hydroxylase gene is high.

（3）5’RACE

5 ' the sequence of holding is by using SMARTer ^tMrACE cDNA Amplification Kit obtains, and with 5 ' RACEready cDNA for template, takes turns nest-type PRC obtain by two:

The first round: UPM+F3 ' H5-1(5 '-GACTGCGGGGTCTCGAGCAATGG-3 ')

Second takes turns: NUP+F3 ' H5-2(5 '-ACCAGCACACTCAGCAAATCCCTCCCA-3 ')

UPM and NUP provides for test kit.5 ' RACE amplification obtains the 5 ' end sequence (836bp) of TfF3 ' H, reclaims after connecting and checks order with the method above.

The sequencing result of the sequence obtained by above-mentioned 3 kinds of methods is spliced, will splice sequence submission BLAST analysis, result prove the TfF3 ' H gene newly obtained from turmeric be really one to flavonoid-3 ' gene that-hydroxylase is relevant.By the ORF Finding(of sequencing result in conjunction with NCBI http:// www.ncbi.nlm.nih.gov/gorf) prediction, initiator codon and the terminator codon of turmeric TfF3 ' H gene are found, according to the sequence obtained, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F(5 '-ATGGAAGCTCAACCTCTCCTCCT-3 '), ORF-R(5 '-TCACTCCTTCCCATACGCCCTCGCT-3 '), with turmeric cDNA for template carries out PCR, amplification obtains the total length coding gene sequence (SEQ ID NO.3) of 1533bp turmeric TfF3 ' H protein.

embodiment 2, the sequence information of turmeric TfF3 ' H gene and homology analysis

Turmeric TfF3 ' H total length CDS opening code-reading frame sequence is 1533bp, and detailed sequence is shown in sequence shown in SEQ ID NO.3; Derive the aminoacid sequence of turmeric TfF3 ' H according to CDS opening code-reading frame sequence, totally 510 amino-acid residues, molecular weight is 55482.2 dalton, and iso-electric point (pI) is 6.98, and detailed sequence is shown in sequence shown in SEQ ID NO.4.

The CDS opening code-reading frame sequence of turmeric TfF3 ' H and the aminoacid sequence blast program of proteins encoded thereof are carried out Nucleotide and protein homology search in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database, found that it and lily F3 ' H gene (the GenBank number of logging in AB699162.1) have the homogeny of 82% on nucleotide level, as shown in Figure 1, Figure 2 and Figure 3; On amino acid levels, it and lily F3 ' H gene (the GenBank number of logging in BAM28972.1) also have the consistence of 88% and the similarity of 94%, as shown in Figure 4.As can be seen here, all there is higher homology in turmeric TfF3 ' H gene and lily F3 ' H gene from nucleic acid or protein level.

embodiment 3, turmeric F3 ' H gene is at flower different developmental phases and the different expression in turmeric different tissues

1. the acquisition of material

Turmeric 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 its blade, terrestrial stem, flower portion organ stamen, gynoecium, petal (compound sample of each tinting stage 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

RNA prep pure plant total RNA extraction reagent box (RNA prep pure Plant Kit: TIANGEN Biotech (Beijing) Co., Ltd.) is utilized 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 the 1.5-2.0 of Article 2 rRNA brightness doubly, 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 KitPerfect Real Time test kit operation instructions carries out reverse transcription acquisition cDNA.

5. real-time fluorescence quantitative PCR analyzes TfF3 ' H gene at each organ and the expression amount in tissue

According to the sequence of the turmeric Tf3 ' H gene obtained, utilize primer-design software primer premier5.0 to be designed for the Auele Specific Primer of turmeric TfF3 ' H gene quantitative analysis in Real-time PCR: F3 ' H-F(5 '-CCTTCCTCCAAGCCATCA-3 ') and F3 ' H-R(5 '-GTCGCTACCTTTCACATCCA-3 '), reference gene is the Actin(GenBank number of logging in AB456684), its primer is Actin-F(5 '-AGTCAGTCATACAGTGCCAATC-3 '), Actin-R(5 '-TCATAAGAGAGTCGGTCAAATCC-3 ').

6. make the typical curve of goal gene and reference gene

There is provided with EASY Dilution(test kit) 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 of synthesis 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 15s; 72 DEG C of 25s; Whether, after each amplification completes, all do solubility curve, be special generation to check amplified production;

Adopt 2 ^{-△ △ Ct}method makes relative quantitative assay, and result shows that the expression level of TfF3 ' H gene slowly rises along with the growth of flower is risen gradually.Petal complete painted time TfF3 ' H expression amount be 2.6 times of the firm tinting stage expression amount of petal, illustrate that the expression of this gene and the painted of petal have dependency; TfF3 ' H gene all has expression in stem, leaf, stamen, gynoecium, petal.Wherein, TfF3 ' H gene expression amount in stamen is the highest, and in blade, expression amount is minimum, and expression level is followed successively by stamen, gynoecium, petal, stem, leaf from high to low, and this illustrates that the expression of TfF3 ' H gene has obvious Spatial Difference.

embodiment 4, the functional verification of turmeric TfF3 ' H transgenosis

1. the structure of plant over-express vector

(1) structure of cloning vector

Hind III and Xbal I restriction enzyme site is added respectively at the upstream and downstream primer two ends of amplification TfF3 ' H gene ORF.Upstream primer sequence is 5 '-CCAAGCTTATGGAAGCTCAACCTCTCCT-3 ', and downstream primer is 5 '-GCTCTAGATCACTCCTTCCCATACGCC-3 '.By the ORF segment of pcr amplification TfF3 ' H cDNA, after electrophoresis, under ultraviolet lamp, extract object band, reclaim test kit with Sanprep pillar DNA glue and reclaim segment.

Recovery segment is connected to pMD18-T vector, and build pMD18-TfF3 ' H cloning vector, linked system is shown in specification sheets.Freeze-thaw method transformation of E. coli DH5 α competence, is containing 100mgl ^-137 DEG C of overnight incubation on the LB solid plate substratum of ammonia benzyl.The formula of LB substratum is: Tryptones 10gl ^-1, yeast extract 5gl ^-1, sodium-chlor 10gl ^-1.Regulate pH to 7.0, sterilizing.LB solid culture based formulas is in LB liquid nutrient medium, adds 15gl ^-1agar powder, sterilizing.Picking list bacterium colony PCR identifies, send positive bacterium colony to check order and determines the exactness of order-checking.By the DH5 α bacterium colony containing pMD18-TfF3 ' H carrier correct for order-checking, add 2ml containing 100mgl ^-1the LB liquid nutrient medium incubated overnight of ammonia benzyl is to OD ₆₀₀value is about 1.0.Plasmid extraction kit (Tian Gen biochemical technology company limited) is used to extract pMD18-TfF3 ' H carrier, concrete operations reference reagent box specification sheets.

Use restriction enzyme Hind III and Xbal I at 37 DEG C, double digestion is carried out to pMD18-TfF3 ' H carrier and plant expression vector pHB, time 15min simultaneously.Enzyme is cut system reference enzyme and is cut specification sheets.Gel electrophoresis is carried out to digestion products, reclaims test kit with Sanprep pillar DNA glue after electrophoresis and reclaim digestion products respectively.

Use DNA Ligation Kit test kit (TaKaRa, China) to be connected with pHB carrier the object segment that enzyme cuts rear acquisition, method of attachment is see test kit specification sheets.Product freeze-thaw method transformed competence colibacillus DH5 α bacterial strain will be connected, containing 100mgl ^-137 DEG C of overnight incubation on the LB solid plate substratum of kantlex, picking list bacterium colony, PCR evaluation and screening is containing the DH5 α bacterium colony of pHB-TfF3 ' H plant over-express vector.Picking, containing the DH5 α bacterium colony of pHB-TfF3 ' H plant over-express vector, adds 2ml containing 100mgl ^-1the LB liquid nutrient medium incubated overnight of kantlex is to OD ₆₀₀value is about 1.0.Plasmid extraction kit (Tian Gen biochemical technology company limited) is used to extract pHB-TfF3 ' H recombinant plasmid, concrete operations reference reagent box specification sheets.The formula of LB substratum is: Tryptones 10gl ^-1, yeast extract 5gl ^-1, sodium-chlor 10gl ^-1, regulate pH to 7.0, sterilizing.LB solid culture based formulas is in LB liquid nutrient medium, adds 15gl ^-1agar powder, sterilizing.

2. plant over-express vector transform Agrobacterium tumefaciens

By pHB-TfF3 ' H plant over-express vector frozen-thawed method transformed competence colibacillus Agrobacterium tumefaciens strain GV3101, concrete grammar: add at least 6 μ l and connect product in 100 μ l GV3101 competent cells, mix gently, ice bath 30min, liquid nitrogen flash freezer 1min, 37 DEG C of thermal shock 3min, are placed in rapidly and upper 1-2min; Add 800 μ l containing 50mgl ^-1rifampin and 50mgl ^-1the YEB substratum of gentamicin, 28 DEG C, 200rpm recovery 3h; The centrifugal 5min of 4000rpm, sops up 800 μ l LB substratum; Mixing residue bacterium liquid, is applied in containing 100m gl ^-1kantlex, 50mgl ^-1rifampin, and 50m gl ^-1on the YEB flat board of gentamicin; Be inverted for 28 DEG C and cultivate 30-48h; Then the PCR carrying out positive colony with ORF-F and ORF-R for primer detects.YEB liquid culture based formulas: beef extract 5gl ^-1, Tryptones 5gl ^-1, yeast extract 1gl ^-1, sucrose 5.0gl ^-1, MgSO ₄7H ₂o0.5gl ^-1, regulate pH to 7.0, sterilizing.YEB solid culture based formulas for add 15gl in YEB liquid nutrient medium ^-1agar powder.

3. agriculture bacillus mediated tobacco genetic transformation

Use material for tobacco (Nicotiana tabacum) in this experiment.Culture medium prescription used:

MS _o: MS powder+sucrose 30gl ^-1, agar powder 15gl ^-1, pH5.8;

MS ₁(Dual culture substratum): MS _o+ 6-BA2mgl ^-1+ NAA O.2mgl ^-1, pH5.8;

MS ₂(screening culture medium): MS ₁+ Totomycin 50mgl ^-1+ cephamycin 300mgl ^-1, pH5.8;

MS ₃(root media): 1/2MS _o+ Totomycin 50mgl ^-1+ cephamycin 300mgl ^-1, pH5.8;

(1) activation of Agrobacterium

From the positive list bacterium colony of Agrobacterium of picking YEB culture plate after PCR detects, be inoculated in 5ml YEB liquid nutrient medium (containing 100m gl ^-1kantlex, 50mgl ^-1rifampin, and 50mgl ^-1gentamicin).28 DEG C, 200rpm shaking culture 24h; Therefrom draw 800 μ l bacterium liquid again to transfer in 40ml containing in corresponding antibiotic YEB liquid nutrient medium, continue at 28 DEG C, 200rpm shaking culture is to OD ₆₀₀value 0.6.4 DEG C, the centrifugal 10min of 5000rpm collects thalline, with the MS of certain volume ₀liquid nutrient medium suspension thalline is to OD ₆₀₀value be about 0.4 for contaminate.

(2) genetic transformation of tobacco

To plant at MS ₀aseptic tobacco on minimum medium, cuts blade edge and main vein, is cut into 0.4 × 0.6cm size.By the explant that cuts at above-mentioned OD ₆₀₀be soak 5min in the Agrobacterium bacterium liquid of 0.4, blot the bacterium liquid of plant material surface with aseptic filter paper, vanelets is placed in the Bud polarization substratum MS being covered with one deck filter paper ₁on carry out Dual culture (leaf back upwards), 25 DEG C of light culture 3 days.Tobacco explants through Dual culture is transferred to containing antibiotic resistant buds screening culture medium MS ₂in cultivate, periodicity of illumination is that 16h illumination/8h is dark, can sprout after 2-3 week.When resistant buds grows to 1-2cm height, cut budlet and proceed to root media MS ₃middle root induction, 1-2 week just has Adventitious root initiation afterwards.After well developed root system, tobacco plant is taken out, cleans solid medium with sterilized water, then move in soil, just started several days by film cover several days, taken down film again after plant to be planted stalwartness, be placed in the cultivation of 25 DEG C, greenhouse.

4. the qualification of transgene tobacco and the mensuration of Minor centaury content

(1) qualification of positive strain

Adopt CTAB method to extract with transgene tobacco genomic dna, with this DNA for template, carry out pcr amplification with screening transgenic positive plant with primer ORF-F and ORF-R of amplification TfF3 ' H gene ORF segment.PCR response procedures is: 94 DEG C, 5min; 94 DEG C, 30s; 58 DEG C, 1min; 72 DEG C 2min(35 circulation); 72 DEG C extend 10min.

(2) mensuration of transfer-gen plant Cyanidin content

To measure transgenic positive plant and adjoining tree cyanidin content.The extracting method of total pattern aglycon is as follows: take fresh petal samples and be about 0.2g, and liquid nitrogen cools rear direct grind into powder rapidly, with 2ml extracting solution (V _ethanol: V _{acetic acid}: V _water=10:1:9) 4 DEG C of lixiviates 24 hours; The centrifugal 10min of 4000rpm, gets the HCl that supernatant adds 4ml3M, at 100 DEG C of water-bath 90min in order to be hydrolyzed the glucosides of anthocyanogen; The primary isoamyl alcohol extraction pattern aglycon of 2ml is added after hydrolysis terminates.Filter with Medium speed filter paper afterwards, then use strainer (0.2 μm) to filter to be measured, be directly used in UPLC afterwards and analyze.The separation of pattern aglycon and the mensuration of content adopt superelevation effect liquid phase chromatogram – diode array device (the Ultra performance liquid chromatography with a photodiode arraydetector of Waters, US, UPLC – PAD) detect, this system equipment has high pressure binary gradient pump, thermostated autosampler, column oven, diode-array detector, chromatographic working station (Masslynx V4.1 data processing software).Chromatographic column is the C18 post (2.1mm × 100mm, 1.7 μm) of ACQUITYUPLC@BEH.UPLC analysis condition: column temperature 45 DEG C, flow velocity 0.5mlmin ^-1, sampling volume 2 μ l.Mobile phase A liquid is the formic acid solution (V of 0.1% _{formic acid}: V _water=0.1:99.9); B liquid is the acetonitrile (V containing 0.1% formic acid _{formic acid}: V _acetonitrile=0.1:99.9).Gradient elution program: 0min, 93%A, 7%B; 1min, 93%A, 7%B; 11min, 82%A, 18%B; 11.5min, 10%A, 90%B; 13min, 10%A, 90%B; 13.1min, 93%A, 7%B; 15min, 93%A, 7%B.Under characteristic absorption wavelength 520nm, by the retention time comparison with standard substance cyanidin (Cyanidin chloride, Sigma), determine the component of cyanidin in extract.In Anthocyanin-rich Extract, the content of cyanidin is converted by the ratio of the peak area with concentration known standard substance.

Result shows, plant transgene tobacco in greenhouse compared with contrast (unconverted) tobacco, under identical culture condition, more than 50% transgene tobacco pattern becomes deep pink from pink colour.As shown in Figure 6, a is unconverted tobacco flower, and b, c, for turning the flower of TfF3 ' H gene tobacco, can see that the flower pattern more unconverted tobacco color of transgene tobacco is darker.Correspondingly, Cyanidin content rising 20-30% in transgene tobacco petal.This shows that TfF3 ' H can act on anthocyanogen route of synthesis, affects Cyanidin synthesis, changes flower color.

embodiment 5, the functional verification of turmeric TfF3 ' H enzyme

1. the structure of prokaryotic expression carrier

BamH I and Xho I restriction enzyme site is added respectively at the upstream and downstream primer two ends of amplification TfF3 ' H gene ORF segment.Upstream primer sequence is 5 '-CGCGGATCCATGGAAGCTCAACCTCTCCT-3 ', and downstream primer is 5 '-ATTCTCGAGTCACTCCTTCCCATACGCC-3 '.By the ORF segment of pcr amplification TfF3 ' H cDNA, after electrophoresis, under ultraviolet lamp, extract object band, reclaim test kit with Sanprep pillar DNA glue and reclaim segment.

Use restriction enzyme BamH I and Xho I at 37 DEG C, double digestion is carried out, time 15min to TfF3 ' H segment and prokaryotic expression carrier pMAL-c2x (New England BioLabs) simultaneously.Enzyme is cut system reference enzyme and is cut specification sheets.Gel electrophoresis is carried out to digestion products, reclaims.

TfF3 ' H segment after using DNA Ligation Kit test kit (TaKaRa, China) to cut enzyme is connected with pMAL-c2x, and method of attachment is see test kit specification sheets.Product conversion e. coli bl21 competence will be connected.Containing 50mgl ^-137 DEG C of overnight incubation on the LB solid plate substratum of ammonia Bian.Picking list bacterium colony, PCR send order-checking confirmation TfF3 ' H segment to be successfully connected with pMAL-c2x after identifying the positive.The formula of LB liquid and solid medium is the same.

The fusion rotein induction of 2.TfF3 ' H

Select the BL21 bacterial strain mono-clonal that growth conditions is good, be forwarded to 10ml containing 50mgl ^-1in the LB liquid nutrient medium of ammonia Bian, 37 DEG C, 200rpm incubated overnight.300ml is forwarded to containing 50mgl in the ratio of 1:50 ^-1in the LB liquid culture of ammonia Bian 37 DEG C, 200rpm is cultured to OD ₆₀₀be about 0.8.Culture is proceeded to 200rpm in the shaking table of 20 DEG C, shaking culture 1 hour.Add 1ml1M IPTG (isopropyl-beta D-thio galactopyranoside) (final concentration is 1mM), continue to cultivate 6h induced fusion protein expression.

By overnight culture 4 DEG C, the centrifugal 10min of 12000rpm, collects thalline, abandons supernatant.By the resuspended precipitation of 50ml cracked solution, then place 10min on ice.Under condition of ice bath, use ultrasonic wave (200-300w) smudge cells, ultrasonic/interval=10s/10s, 6 times.Subsequently by the cell of fragmentation at 4 DEG C, centrifugal 30min under 12000rpm condition, collect supernatant liquor be used for purifying.In supernatant, add 1ml cracked solution clean and the amylose resin balanced (Amylose resin) (New England Biolabs), add another 50ml cracked solution to solution again, 4 DEG C of overnight incubation simultaneously.Hatch latter 4 DEG C, after the centrifugal 1min of 1000rpm, abandon supernatant.After 50ml cleaning solution cleaning resin three times, with 1ml elute soln eluted protein.The formula of cellular lysate solution: 20mMTris-HCl, pH8.0,200mM sodium-chlor, the proteinase inhibitor (Promega) of 1mM PMSF, EDTA-free, 10% glycerine; The formula of cleaning solution: 20mM Tris-HCl, pH8.0,200mM sodium-chlor, 10% glycerine; The formula of elute soln: 20mM Tris-HCl, pH8.0,200mM sodium-chlor, 50mM maltose, 10% glycerine.

TfF3 ' the H fusion rotein collected and substrate are hatched.The substrate of hatching is naringenin and dihydrokaempferol.Hatching system volume is 500 μ l, comprises 2-40 μM of substrate, 200mg TfF3 ' H fusion rotein extract, 1mMNADPH, 0.1M potassium primary phosphate (pH7.4).Reaction conditions: 25 DEG C of reaction 30min.Reaction terminates the extraction into ethyl acetate twice of rear use 500 μ l, and extraction liquid is dissolved in 200 μ l methanol solutions after liquid nitrogen dries up again.Then UPLC analysis is carried out.Standard substance flavanone [naringenin and eriodictyol (eriodictyol)] is purchased from ExtrasyntheseSA company, and standard substance flavanonol [dihydrokaempferol and dihydroquercetin (hidydroquercetin)] structure is from PLANTECH company.The elution time of corresponding standard substance determines the kind of hatching product at a wavelength of 280 nm; According to the content of corresponding resultant in the concentration of respective standard product and peak area conversion product.

Result shows, and naringenin and dihydrokaempferol generate eriodictyol and dihydroquercetin under the effect of TfF3 ' H fusion rotein, and the activity of TfF3 ' H to dihydrokaempferol is higher than the activity to naringenin.This result shows that TfF3 ' H can make the B ring 3 ' site of naringenin and dihydrokaempferol that hydroxylation occurs, and has flavonoid-3 ' activity of-hydroxylase, also there is certain substrate selective simultaneously.

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 one kind following (a):

A protein that () is made up of the such as aminoacid sequence 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:

A () base sequence is as shown in SEQ ID NO.3 1st ~ 1533.