CN103756982A - Tulipa fosteriana flavonol synthase TfFLS protein and coding gene thereof - Google Patents
Tulipa fosteriana flavonol synthase TfFLS protein and coding gene thereof Download PDFInfo
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Abstract
The invention discloses a Tulipa fosteriana flavonol synthase TfFLS protein and a coding gene thereof. The protein is a protein composed by an amino acid sequence shown in SEQ ID No. 2 or a protein composed of an amino acid sequence obtained through replacement, deletion or addition of one or more amino acids of the amino acid sequence shown in the SEQ ID No. 2 and having the activity of Tulipa fosteriana flavonol synthase. The invention further provides a nucleic acid sequence used for coding the protein, and the nucleic acid sequence is shown in SEQ ID No. 1. The Tulipa fosteriana flavonol synthase TfFLS gene provided by the invention expresses a recombinant flavonol synthase protein in Escherichia coli cells and enables dihydrokaempferol to produce kaempferol, which proves that the gene has the functions of flavonol synthase. With the TfFLS gene, substances, receptors, inhibitors, antagonists or the like interacting with TfFLS can be screened by using a variety of conventional screening methods.
Description
Technical field
The invention belongs to molecular biology of plants field, relate to crux enzyme and encoding gene thereof in turmeric flavonoid route of synthesis, be specifically related to a kind of turmeric flavonol synthetic enzyme TfFLS albumen and encoding gene thereof.
Background technology
Flavonoids is a class polyphenolic compound, extensively be present in nature, according to the different modifying to center C ring, can be divided into the main large class such as flavones, isoflavones, flavanone, flavonol, Flavonol and anthocyanidin, wherein common with flavonols compound, account for 1/3 left and right of flavonoid compound sum.Flavonols compound is distributed in various plants widely, they are several kinase whose inhibitor relevant to Cellular Signaling Transduction Mediated and cell transformation, there is multiple biological activity, because they can block Cellular Signaling Transduction Mediated path selectively, so flavonoid compound is considered to treat the potential compound of tumour.Flavonol synthetic enzyme (flavonol synthase, FLS) is the synthetic direct regulation and control enzyme of flavonol in flavonoid route of synthesis, the C3 position hydroxylation in its catalysis flavones structure, thus form various flavonols, determining the synthetic of flavonol.Therefore FLS gene is one of key gene in the regulation and control of research Flavone metabolism.
Flavonoid is a large class of cyanidin(e) simultaneously, and it makes flower produce the whole colors from Huang to purple.Flavonol is generally yellow or colourless, can affect pattern as accessory pigment.The expression of FLS gene can directly affect the pattern of plant.In petunia, flavonol synthetic enzyme can be total to pigment synthesis or substrate competition mode by impact, changes the ratio of flavonol and anthocyanogen, thereby changes pattern (Mol J etc., 1998).Chinese Primrose (Primulasinensis Sabine ex L.) flavonol produces and controlled by gene B, and the pattern that contains common pigment is purple, and the pattern that lacks flavonol is dark reddish purple look (Forkman G, 1991).In petunia, the expression that increases flavonol synthase gene can make to spend aobvious blueness conventionally, and antisense expression FLS eDNA makes pattern obviously thin out (Holton TA etc., 1993).
At present, people have cloned FLS gene in the plants such as petunia (Petunia hybrida), potato (Solanum tuberosum), apple (Malus pumila), mandarin orange (citrus), grape (Vitis vinifera), ginkgo (Ginkgo biloba), but in turmeric (Tulipa fosteriana), clone and the expression pattern thereof of flavonol synthase gene it be unclear that.At present, there is not any bibliographical information relevant to turmeric FLS albumen and coding gene sequence thereof.
Summary of the invention
The object of the present invention is to provide a kind of turmeric flavonol synthetic enzyme TfFLS albumen and encoding gene thereof.The invention discloses turmeric TfFLS encoding gene at the expression pattern of turmeric Different Organs, different developmental phases; Turmeric TfFLS gene of the present invention is expressed the flavonol synthetase albumen of restructuring in Bacillus coli cells, can make dihydrokaempferol generate kaempferol.Utilize turmeric TfFLS gene of the present invention, by various conventional screening methods, can filter out material, acceptor, inhibitor or antagonist etc. that mutual use occurs with FLS.
The object of the invention is to be achieved through the following technical solutions,
First aspect, the present invention relates to a kind of protein with turmeric flavonol synthase activity, the protein that described protein is comprised of the aminoacid sequence as shown in SEQ ID NO.2; Or by aminoacid sequence shown in SEQ ID NO.2 through replacement, lack or add one or several amino acid and have turmeric flavonol synthase activity by (a) derivative protein.This protein having that it's too late active size exist larger difference in painted stage of difference of flower petal, Different Organs.
Preferably, described protein be aminoacid sequence shown in SEQ ID NO.2 through 1～50 amino acid whose disappearance, insertion and/or replacement, or add 1～20 sequence obtaining with interior amino acid at C-terminal and/or N-terminal.
Further preferred, described protein be shown in SEQ ID NO.2 in aminoacid sequence 1～10 amino acid by the similar or close amino acid of character, replaced the sequence forming.
Second aspect, the present invention relates to 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～999 of SEQ ID NO.1;
Or (b) and the nucleic acid shown in 1st～999 of SEQ ID NO.1 have the sequence of at least 70% homology;
Or the sequence that (c) can hybridize with the nucleic acid shown in 1st～999 of SEQ ID NO.1.
Preferably, described nucleotide sequence is specially disappearance, insertion and/or the replacement of 1～90 Nucleotide in the nucleotide sequence shown in 1st～999 of SEQ ID NO.1, and adds 60 sequences that form with inner nucleotide at 5 ' and/or 3 ' end.
The third aspect, the present invention relates to the purposes of a kind of above-mentioned nucleotide sequence in preparation restructuring flavonol synthetic enzyme.
Preferably, described preparation comprises the steps: to build the prokaryotic expression carrier containing described nucleotide sequence, described prokaryotic expression carrier is transformed in intestinal bacteria, and inducing culture, flavonol synthetic enzyme obtains recombinating
Fourth aspect, the present invention relates to a kind of restructuring flavonol synthetic enzyme, described restructuring flavonol synthetic enzyme be prepare by the following method and: build the prokaryotic expression carrier containing above-mentioned nucleotide sequence, described prokaryotic expression carrier is transformed in intestinal bacteria, inducing culture, obtains described restructuring flavonol synthetic enzyme.
Isolated DNA molecular provided by the invention, this molecule comprises: the DNA molecular with nucleotide sequence shown in SEQ ID NO.1; Or coding has the nucleotide sequence of the polypeptide of turmeric TfFLS protein active, and has at least 70% homology with sequence shown in SEQ ID NO.3; Or can with the nucleotide sequence hybridization of sequence shown in SEQ ID NO.1.
In the present invention, " separated DNA ", " 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 flavonol synthetic enzyme TfFLS protein coding gene " refers to that coding has the nucleotide sequence of the polypeptide of turmeric TfFLS protein-active, nucleotide sequence and degenerate sequence thereof as shown in SEQ ID NO.1.This degenerate sequence refers to, is arranged in sequence shown in SEQ ID NO.1, 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 aminoacid sequence shown in SEQ ID NO.2 of also encoding out with the homology of sequence shown in SEQ ID NO.1.This term also comprise with sequence shown in SEQ ID NO.1 in from the nucleotide sequence of the homology at least 70% of the nucleotide sequence of 1st～999, Nucleotide.
This term also comprises encoding to have the variant form of sequence shown in the SEQ ID NO.1 with the albumen of natural turmeric TfFLS identical function.These variant forms comprise (but being not limited to): be generally disappearance, insertion and/or the replacement of 1～90 Nucleotide, and be added to 60 with inner nucleotide at 5 ' and/or 3 ' end.
In the present invention, term " turmeric flavonol synthetic enzyme TfFLS albumen " refers to the polypeptide of sequence shown in the SEQ ID NO.2 with turmeric TfFLS protein-active.This term also comprises having and the variant form relevant identical function of natural turmeric TfFLS, sequence shown in SEQ ID NO.2.These variant forms comprise (but being not limited to): be generally 1～50 amino acid whose disappearance, insertion and/or replacement, and C-terminal and/or N-terminal add one or be 20 with interior amino acid.For example, in the art, while replacing with the close or similar amino acid of performance, conventionally can not change the function of protein.Again such as, at C-terminal and/or N-terminal, add one or several amino acid and conventionally also can not change the function of protein.This term also comprises active fragments and the reactive derivative of turmeric TfFLS albumen.
The variant form of turmeric TfFLS polypeptide 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 TfFLS under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of turmeric TfFLS polypeptide to obtain.
In the present invention, " turmeric TfFLS conservative property variation polypeptide " refers to compare with the aminoacid sequence of sequence shown in SEQ ID NO.2, has at the most 10 amino acid be replaced by the similar or close amino acid of character and forms 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 |
[0028]?
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；ms；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；Phc | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phc | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phc；Ile | Leu |
| Phc(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phc | Tyr |
| Tyr(Y) | Trp；Phc；Thr；Ser | Phc |
| Val(V) | Ile；Leu；Met；Phc；Ala | Leu |
Invention also comprises the analogue of turmeric TfFLS albumen or polypeptide.The difference of these analogues and turmeric TfFLS 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 molecular.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and the analogue with 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: the chemically derived form of the polypeptide that body is interior or external is 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 TfFLS gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of the mRNA transcript of analyzing TfFLS gene in cell.
In addition, according to turmeric TfFLS 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 TfFLS or homologous protein.
In order to obtain the dot matrix with turmeric TfFLS 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 TfFLS 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 TfFLS.
Turmeric TfFLS 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 need to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplified is stitched together by proper order.
After having obtained relevant sequence, can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, then by ordinary method separation from the host cell propagation, 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.
The bacillus coli DH 5 alpha the present invention relates to, BL21 bacterial strain is at < < Pehanorm Brooker J, Russell D W. molecular cloning experiment guide [M]. Huang Peitang, Wang Jiaxi, the thick plinth of Zhu, Deng translating. the 3rd edition. Beijing: Science Press, open in 2002 > >; Bacillus coli DH 5 alpha, pET-28a (+), BL21 (DE3) etc. can obtain by disclosing commercially available commercial channel.
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 pattern.Beneficial effect of the present invention is: clone first flavonol synthetic enzyme TfFLS albumen and encoding gene thereof in tulip petals flavonoid route of synthesis, and TfFLS is transformed in intestinal bacteria by building prokaryotic expression carrier, find that the flavonol synthetic enzyme of restructuring can make dihydrokaempferol generate kaempferol.TfFLS provided by the invention is for utilizing genetically engineered to regulate pattern that a kind of approach is provided; Utilize turmeric TfFLS albumen of the present invention, by various conventional screening methods, can filter out with turmeric TfFLS interactional material occurs, or acceptor, inhibitor or antagonist etc.
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 the building process schema of prokaryotic expression carrier pET-28a (+)-TfFLS;
Fig. 2 is the high performance liquid chromatography detected result that turmeric TfFLS proteoplast exoenzyme of the present invention is lived and reacted; Wherein, a is the liquid chromatogram of dihydrokaempferol (dihydrokaempferol) standard substance; The vitro enzyme reaction product alive that b carries out for using TfFLS albumen; The negative control reaction of c; D is kaempferol (Kaempferol) standard substance liquid chromatograms.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in detail.Following examples will contribute to those skilled in the art further to understand the present invention, but not limit in any form the present invention.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, can also make some distortion and improvement.These all belong to protection scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, for example Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
the clone of embodiment 1, turmeric TfFLS gene
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 are recognized flowers 2011 No. 004) plant routinely and carry out field management, treating that flower is completely open, petal is complete gathers Petal when painted, for extracting RNA.
The extracting of 2.RNA
Utilize " 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 FLS gene in other species, utilize homologous genes clone principle, adopt RACE method (3 '-Full RACE Core Set Vet.2.0: precious biotechnology (Dalian) company limited, SMARTer
tMrACE cDNA Amplification KIt:Clontech Laboratories, Inc.) carry out cDNA full-length clone, a minute three phases carries out:
(1) RT-PCR obtains gene intermediate segment
The RNA of extraction is carried out to reverse transcription (Prime Script II lst Strand cDNA SynthesisKit, treasured biotechnology (Dalian) company limited), the first chain cDNA of take is template, utilize degenerate primer TfFLS-F (5 '-ATYGAAGGNTAYGGMCANAARHTNC-3 ') (sequence is as shown in SEQ ID NO.3) and TfFLS-R (5 '-WRAAHACNGGCCAYGACATNCK-3 ') (sequence is as shown in SEQ ID NO.4) to carry out PCR, intermediate segment increases to obtain, reclaim and be connected on pMDl8-T 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 compared existing database (GenBank) by carry out BLAST (http://blast.ncbi.nlm.nih.gov/) in NCBI website, the homology of knowing its nucleotide sequence and proteins encoded and known onion (Allium capa) flavonol synthase gene is very high, therefore tentatively think that it is a flavonol synthase gene.
(2)3’RACE
The sequence of 3 ' end is by being used test kit 3 '-Full RACE Core Set Vet.2.0 (precious biotechnology (Dalian) company limited) to carry out pcr amplification acquisition., upstream primer is TfFLS31 (5 '-TGTGAGTGATGAGAGTCCTGCGAAA-3 ') (sequence is as shown in SEQ ID NO.5), the Outer primer that downstream primer provides for test kit;
3 ' the end sequence that 3 ' RACE is obtained to TfFLS reclaims, and is connected on pMDl8-T vector carrier, take RV-M and M13-47 as primer, serves extra large Invitrogen company and checks order.Sequencing result is compared existing database (GenBank) by carry out BLAST (http://blast.ncbi.nlm.nih.gov/) in NCBI website, knows that the homology of its nucleotide sequence and proteins encoded and known onion (Allium capa) flavonol synthase gene is high.
(3)5’RACE
The sequence of 5 ' end is by being used SMARTer
tMrACE cDNA Amplification Kit obtains, and 5 ' the RACE ready cDNA of take is template, obtains the 5 ' terminal sequence of TfFLS by PCR.The UPM of upstream primer for providing for test kit, downstream primer is TFLS51 (5 '-AAGTGAAACAAGTAATCCACCCACGCCT-3 ') (sequence is as shown in SEQ ID NO.6).5 ' the end sequence that 5 ' RACE amplification is obtained to TfFLS reclaims, connects, checks order.Sequencing result is compared existing database (GenBank) by carry out BLAST (http://blast.ncbi.nlm.nih.gov/) in NCBI website, knows that the homology of its nucleotide sequence and proteins encoded and known onion (Allium capa) flavonol synthase gene is high.
The sequencing result of the sequence obtaining by above-mentioned 3 kinds of methods is spliced, will splice sequence and submit to BLAST to analyze, the TfFLS gene that result proof newly obtains from turmeric is really a gene relevant to flavonol synthetic enzyme.ORF Finding (http://www.ncbi.nlm.nih.gov/gorf) prediction by sequencing result in conjunction with NCBI, initiator codon and the terminator codon of turmeric TfFLS gene have been found, according to the sequence obtaining, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F (5 '-ATGGAGGTGGAAAGAGTGCAG-3 ') (sequence is as shown in SEQ ID NO.7), ORF-R (5 '-TTATTGTGGAAGCTTGTTAATCTTG-3 ') (sequence is as shown in SEQ ID NO.8), the turmeric cDNA of take carries out PCR as template, amplification obtains the coding gene sequence (SEQ ID NO.1) of 999bp turmeric TfFLS albumen.
sequence information and the homology analysis of embodiment 2, turmeric TfFLS gene
Turmeric TfFLS total length CDS opening code-reading frame sequence is 999bp, and detailed sequence is shown in sequence shown in SEQ ID NO.1; According to CDS opening code-reading frame sequence, derive the aminoacid sequence of turmeric TfFLS, totally 332 amino-acid residues, molecular weight is 37672 dalton, and iso-electric point (pI) is 5.39, and detailed sequence is shown in sequence shown in SEQ ID NO.2.
The aminoacid sequence of the CDS opening code-reading frame sequence of turmeric TfFLS and proteins encoded thereof is carried out to Nucleotide and protein homology search with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database.Table 2 is homology comparison (GAP) result of the nucleotide sequence of turmeric TfFLS gene of the present invention and onion (Alliumcepa) FLS gene mRNA; As shown in Table 2, turmeric TfFLS gene and onion (Allium cepa) FLS gene (the GenBank number of logging in AY221247.1) has 71% homogeny on nucleotide level; Table 3 is homology comparison (FASTA) result of the aminoacid sequence of turmeric TfFLS gene of the present invention and onion (Allium cepa) FLS gene mRNA, and wherein, identical amino acid marks with amino acid monocase between two sequences; As shown in Table 3, on amino acid levels, it and onion (Allium cepa) FLS gene (the GenBank number of logging in AA063023.1) also have 85% consistence and 74% similarity, from table 2 and table 3, all there is higher homology in turmeric TfFLS gene and onion FLS gene from nucleic acid or protein level.
Table 2
Table 3
embodiment 3, turmeric TfFLS gene are at flower different developmental phases and the different expression in turmeric different tissues
1. the acquisition of material
4 different developmental phases of turmeric flower (bud, petal is not painted; Bud, petal starts painted; Flower is partly open, and petal is completely not painted; Flower is completely open, petal is completely painted), in field, take its bulb, terrestrial stem, blade and petal (compound sample of each painted stage petal), 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: extract the petal of turmeric different developmental phases flower and the RNA in different tissues TIANGEN Biotech (Beijing) Co., Ltd.).
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) detection integrity; In electrophoretic band, maximum rRNA brightness should be 1.5-2.0 times of second 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 0D value and calculate rna content.
The acquisition of 4.cDNA
The total RNA of 500ng of take is 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. real-time fluorescence quantitative PCR is analyzed the expression amount of TfFLS gene in each organ and tissue
According to the sequence of the turmeric TFLS gene having obtained, utilize primer-design software primer premier5.0 to be designed for the Auele Specific Primer that turmeric TfFLS gene quantification in Real-time PCR is analyzed: TfFLS-qF (5 '-AGGAGGAGATT6C66CT6T6-3 ') (sequence is as shown in SEQ ID NO.9) and TfFLS-qR (5 '-TAGTCGGTAGGGTTCTT666-3 ') (sequence is as shown in SEQ ID NO.10), reference gene is Actin (the GenBank number of logging in AB456684), its primer is Actin-F (5 '-AGTCAGTCATACAGTGCCAATC-3 ') (sequence is as shown in SEQ ID NO.11), Actin-R (5 '-TCATAAGAGAGTCGGTCAAATCC-3 ') (sequence is as shown in SEQ ID NO.12).
6. make the typical curve of goal gene and reference gene
With EASY Dilution (test kit provides), standard substance cDNA solution is carried out to gradient dilution, the cDNA solution of then take respectively after dilution is 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
The cDNA article one chain synthesizing of take is 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 25s; 72 ℃ of 20s; After each amplification, all do solubility curve, take and check whether amplified production is special generation.
Adopt 2
-△ △ Ctmethod is made relative quantitative assay.The expression level that result shows TfFLS gene reduces gradually along with the growth of flower, and in the end the expression amount of an etap is 0.72 times of first stage expression amount.Research before us shows in flowering process in petal that flavonol total content declines gradually, and therefore, the expression variation of TfFLS is consistent with the variation tendency of flavonols content, shows the synthetic positive correlation of TfFLS and flavonol.TfFLS gene all has expression in bulb, terrestrial stem, leaf, petal, and wherein, TfFLS gene expression amount in petal and terrestrial stem is higher, and in bulb, expression amount is minimum.Expression amount in petal is respectively 1.4,2.6,5.5 times of expression amount in stem, blade, bulb on the ground, be consistent with the difference of flavonol content in these organs, a nearly step confirms that TfFLS gene and flavonol are synthetic and is proportionate, and the while also shows that the expression of TfFLS has certain Spatial Difference.
embodiment 4, the functional verification of turmeric TfFLS enzyme
The structure of 1.pET-28a (+)-TfFLS prokaryotic expression carrier
The building process of pET-28a (+)-TfFLS prokaryotic expression carrier as shown in Figure 1.
Upstream and downstream primer two ends in amplification TfFLS gene ORF segment add respectively Sac I and Xho I restriction enzyme site.Upstream primer sequence is 5 '-C
gAGCTCaTGGAGGTGGAAAGAGTGCA6-3 ' (sequence is as shown in SEQ ID NO.13), downstream primer is 5 ' CC
cTCGAGtTATTGTGGAAGCTTGTTAATCTT6-3 ' (sequence is as shown in SEQ ID NO.14).Underscore represents respectively Sac I and Xho I restriction endonuclease recognition sequence.
By increase the respectively ORF segment of TfFLS cDNA of PCR, after electrophoresis, under ultraviolet lamp, extract object band, with Sanprep pillar DNA glue, reclaiming test kit (Shanghai Sheng Gong biotechnology company limited) reclaims, be connected to pMD18-Tvector, build pMD18-TfFLS cloning vector, linked system is shown in specification sheets.Freeze-thaw method transforms bacillus coli DH 5 alpha competence, is containing 100mgl
-137 ℃ of overnight incubation on the LB solid plate substratum of ammonia benzyl.The formula of LB substratum is: Tryptones 10gl, 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 order-checking to determine the exactness of order-checking.By the correct DH5 α bacterium colony containing pMD18-TfFLS carrier of order-checking, add 2ml to contain 100mg1
-1the LB liquid nutrient medium incubated overnight of ammonia benzyl is to OD
600value is about 1.0.Use plasmid extraction kit (Tian Gen biochemical technology company limited) to extract pMD18-TfFLS carrier, concrete operations reference reagent box specification sheets.
Use
restriction enzyme Sac I and Xho I respectively simultaneously to cloning vector pMD18-TfFLS and and prokaryotic expression carrier pET-28a (+) (U.S. Novagen company) at 37 ℃, carry out double digestion, time 15min.Enzyme is cut system reference enzyme and is cut specification sheets.Enzyme is cut to product and carry out gel electrophoresis, reclaim.
Use the TfFLS segment after DNA Ligation Kit test kit (TaKaRa, China) is cut enzyme to be connected with pET-28a (+), method of attachment is referring to test kit specification sheets.To connect product and transform e. coli bl21 (DE3) competence.Containing 50mgl
-137 ℃ of overnight incubation on the LB solid plate substratum of ammonia Bian.Picking list bacterium colony, PCR send order-checking to confirm that TfFLS segment is successfully connected with pET-28a (+) after identifying the positive.
The fusion rotein induction of 2.TfFLS
Select the BL21 that growth conditions is good (DE3) bacterial strain mono-clonal, be forwarded to 10ml containing 50mgl
-1in the LB liquid nutrient medium of ammonia Bian, 37 ℃, 200rpm incubated overnight.In the ratio of 1:50, be forwarded to 300ml containing 50mgl
-1in the LB liquid culture of ammonia Bian 37 ℃, 200rpm is cultured to 0D
600it is 0.6 left and right.Culture is proceeded to 200rpm in the shaking table of 20 ℃, shaking culture 1 hour.Add 1ml1M IPTG (sec.-propyl-β-D-sulfo-galactopyranoside) (final concentration is 1mM), continue to cultivate 4-6h induction expressing fusion protein.
By 4 ℃ of overnight culture, the centrifugal 10min of 12000rpm, collects thalline, abandons supernatant.In the PBS for ratio of 5:1 (140mM NaCl, 2.7mM KCl, 10mM Na
2hPO
4, 1.8mM KH
2pO
4) dissolve centrifugal collection thalline, the broken thalline of ultrasonic wave (200w-300w), ultrasonic/interval=10sec/sec, 6 times.With buffer B (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, all the other are distilled water, pH8.0) resuspended bacterial sediment (20-200ml cell culture).Buffer B consumption is 5mlg
-1weight in wet base, stirring at room to the solution shape that is translucent.4 ℃, the centrifugal 30min of 10000rpm, discards precipitation, collects supernatant for upper column purification.
Suspension 50%Ni-NTA solution, dress post, avoids producing bubble.Ni-NTA consumption is every milliliter of resin 5-10mg albumen.Deng resin natural subsidence, with 5 times of column volume ddH2O, cross post and clean chromatography column, then add 1 * Ni-NTA buffer B balance chromatography column of 5-10 times of column volume.Sample upper prop, washes post, 1ml.min with 1 * Ni-NTA buffer B of 5-10 times of volume
-1flow velocity, collects effluent liquid.With 1 * Ni-NTA buffer C of 5-10 times of column volume (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, all the other are distilled water, pH6.3) wash post, collect effluent liquid.With 1 * Ni-NTA buffer E of 5 times of volumes (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, all the other are distilled water, pH4.5) wash post, and target protein is carried out to wash-out.
TfFLS fusion rotein and the substrate collected are hatched.The substrate of hatching is dihydrokaempferol (Dihydrokaempferol).Hatching system volume is 360 μ l, comprises 4.5-22 μ g TfFLS fusion rotein extract, 100 μ M substrates, 83 μ M2-ketoisocaproics (2-oxoglutarate), 42 μ M ammonium sulfate iron (II), 2.5mM sodium ascorbate (Sodium ascorbate), 2mg.ml
-1catalase (ox) (bovine catalase).Incubation condition: 37 ℃, pH5.0, slightly shakes 5-10min.Use the full EDTA aqueous solution closing of 15 μ l to finish reaction.Reaction finishes the ethyl acetate extracting twice of rear use 75 μ l, then carries out UPLC (Waters, USA) and analyzes.UPLC analyzes and uses two kinds of moving phases, and mobile phase A is 20% methanol solution, and the methanol solution that B is 100% carries out linear elution.Standard substance dihydrokaempferol standard substance are purchased from SIGMA (USA), and standard substance kaempferol (kaempferol) is purchased from national standard material net.At 350nm place, detect product.
The TfFLS albumen that purifying is obtained, after renaturation, for the vitro enzyme experiment of living, is usingd and is not added albumen that the recombinant plasmid bacterial classification of IPTG induction produces as negative control.High performance liquid chromatography detected result demonstration as shown in Figure 2, TfFLS fusion rotein makes dihydrokaempferol generate kaempferol, and negative control reaction does not have corresponding product to generate.This result shows that TfFLS can make flavanonol generate flavonol, has the activity of flavonol synthetic enzyme.
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 (9)
1. following (a) or protein (b):
(a) protein being formed by the aminoacid sequence as shown in SEQ ID NO.2;
(b) aminoacid sequence shown in SEQ ID NO.2 through replacement, lack or add one or several amino acid and have turmeric flavonol synthase activity by (a) derivative protein.
2. protein as claimed in claim 1, it is characterized in that, described protein be aminoacid sequence shown in SEQ ID NO.2 through 1～50 amino acid whose disappearance, insertion and/or replacement, or add 1～20 sequence obtaining with interior amino acid at C-terminal and/or N-terminal.
3. protein as claimed in claim 2, is characterized in that, described protein be shown in SEQ ID NO.2 in aminoacid sequence 1～10 amino acid by the similar or close amino acid of character, replaced the sequence forming.
One kind coding claim 1 described in nucleic acid sequences to proteins.
5. nucleotide sequence as claimed in claim 4, is characterized in that, described nucleotide sequence is specially:
(a) base sequence is as shown in 1st～999 of SEQ ID NO.1;
Or (b) and the nucleic acid shown in 1st～999 of SEQ ID NO.1 have the sequence of at least 70% homology;
Or the sequence that (c) can hybridize with the nucleic acid shown in 1st～999 of SEQ ID NO.1.
6. nucleotide sequence as claimed in claim 4, it is characterized in that, described nucleotide sequence is specially disappearance, insertion and/or the replacement of 1～90 Nucleotide in the nucleotide sequence shown in 1st～999 of SEQ ID NO.1, or adds 60 sequences that form with inner nucleotide at 5 ' and/or 3 ' end.
7. a nucleotide sequence as claimed in claim 4 is being prepared the purposes of recombinating in flavonol synthetic enzyme.
8. purposes as claimed in claim 7, is characterized in that, described preparation comprises the steps: to build the prokaryotic expression carrier containing described nucleotide sequence, described prokaryotic expression carrier is transformed in intestinal bacteria, and inducing culture, flavonol synthetic enzyme obtains recombinating.
9. a restructuring flavonol synthetic enzyme, it is characterized in that, described restructuring flavonol synthetic enzyme be prepare by the following method and: build containing the prokaryotic expression carrier of nucleotide sequence as claimed in claim 4, described prokaryotic expression carrier is transformed in intestinal bacteria, inducing culture, obtains described restructuring flavonol synthetic enzyme.
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| CN108048415A (en) * | 2018-02-01 | 2018-05-18 | 浙江大学 | The application of two myricetin alcohol synzyme MrFLSs albumen and its encoding gene |
| CN112662685A (en) * | 2021-01-11 | 2021-04-16 | 浙江万里学院 | Rhododendron Belgium FLS gene, primer group for cloning same, cloning method thereof and fluorescent quantitative primer group |
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| CN112662685A (en) * | 2021-01-11 | 2021-04-16 | 浙江万里学院 | Rhododendron Belgium FLS gene, primer group for cloning same, cloning method thereof and fluorescent quantitative primer group |
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