CN103288942A - African agapanthus flowering gene ApFT protein and coding gene and probe thereof - Google Patents
African agapanthus flowering gene ApFT protein and coding gene and probe thereof Download PDFInfo
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Abstract
本发明涉及百子莲开花基因ApFT蛋白及其编码基因和探针，所述蛋白质为如下（a）或（b）的蛋白质：（a）由如SEQ ID NO.4所示的氨基酸序列组成的蛋白质；（b）SEQ ID NO.4所示的氨基酸序列经过取代、缺失或者添加一个或几个氨基酸且具有百子莲ApFT活性的由（a）衍生的蛋白质。本发明还提供了一种编码上述蛋白质的核酸序列，以及检测上述核酸序列的探针；本发明为利用基因工程技术调控百子莲开花相关基因ApFT的时空表达特性，从而改变花期调控技术提供了理论依据，具有很大的应用价值。
The present invention relates to Agapanthus flowering gene ApFT protein and its coding gene and probe. The protein is the following (a) or (b): (a) protein composed of the amino acid sequence shown in SEQ ID NO.4 (b) The amino acid sequence shown in SEQ ID NO.4 is substituted, deleted or added with one or several amino acids, and the protein derived from (a) has the activity of Agapanthus japonicus ApFT. The present invention also provides a nucleic acid sequence encoding the above-mentioned protein, and a probe for detecting the above-mentioned nucleic acid sequence; the present invention provides a theory for regulating the temporal and spatial expression characteristics of Agapanthus flowering-related gene ApFT by using genetic engineering technology, thereby changing the flowering regulation technology It has great application value.
Description
Technical field
The present invention relates to the key gene FLOWERING LOCUS T(FT in the Agipanthus flower development approach) and encoding gene and probe, be specifically related to a kind of Agipanthus bloom genes involved ApFT albumen and encoding gene and probe.
Background technology
Bloom is that plant is from nourishing and growing to the transition process of reproductive growth.FLOWERING LOCUS T(FT) gene is the Rendezvous Point of flower development approach, and it can integrate the signal from different flower development approach such as photoperiod approach, vernalization approach and autonomous approach, plays an important role in the plant flowers growth course.The transcriptional level of FT shows diel rhythm under the long day condition.Up-to-date studies show that, FT albumen be exactly can long-distance transportation the plain signaling molecule of blooming, it moves to the stem end by phloem from blade.In stem end meristematic tissue, FT albumen and bZIP transcription factor FLOWERING LOCUS D(FD) do mutually, the FT/FD mixture can activate floral meristem Gene A PETALA1(AP1) express, thereby promote into the flower conversion and start the flower development process.The mutual work of FT and FD albumen is all verified on different plant species such as wheat, Arabidopis thaliana, corn.
Along with molecular biological continuous development, in plants such as Arabidopis thaliana, paddy rice, wheat, tomato, oranges and tangerines, cloned the homologous gene of FT, and the part plant carried out genetic transformation, the overexpression of FT genoid has all caused the early flowering of transfer-gen plant to some extent in the transgenic plant.But for flower bulbs Agipanthus (Agapanthus praecox ssp.orientalis), the clone of FT gene, expression pattern and FT albumen coded sequence it be not immediately clear.Before the present invention comes forth, the relevant patent report of Agipanthus FT protein sequence mentioned in any and the present patent application is not arranged.
Summary of the invention
Purpose of the present invention, be to fill up the blank of Agipanthus FT gene family member's clone, expression pattern analysis and Agipanthus FT albumen coded sequence, a kind of Agipanthus FT albumin A pFT is provided, and the present invention also provides a kind of probe of encoding above-mentioned nucleic acid sequences to proteins and detecting described nucleotide sequence; The invention discloses and the closely-related protein gene of Agipanthus ApFT and Agipanthus ApFT protein sequence and nucleotide sequence thereof, and at the expression pattern of Agipanthus Different Organs, different development stage, for utilizing genetic engineering technique from now on the space-time of ApFT genetic expression is regulated and control, thereby the change florescence provides theoretical foundation, has very big using value.
On the one hand, the invention provides and have the Agipanthus genes involved ApFT protein of blooming, the protein that described protein is made up of the aminoacid sequence shown in SEQ ID NO.4; Or by the aminoacid sequence shown in the SEQ ID NO.4 through replacing, lack or adding one or several amino acid and have the Agipanthus genes involved ApFT protein of blooming.This protein having that it's too late there is larger difference in active size in Agipanthus different developmental phases, Different Organs.
Preferably, described protein is that aminoacid sequence shown in the SEQ ID NO.4 is through 1～50 amino acid whose disappearance, insertion and/or replacement, perhaps in C-terminal and/or 1～20 sequence that obtains with interior amino acid of N-terminal interpolation.
Further preferred, described protein be shown in the SEQ ID NO.4 in the aminoacid sequence 1～10 amino acid replaced the sequence that forms by similar performance or close amino acid.
On the other hand, the invention provides the above-mentioned nucleic acid sequences to proteins of a kind of coding.
Preferably, described nucleotide sequence is specially: (a) base sequence is shown in the 1st～534 of SEQ ID NO.3; Or (b) and the nucleic acid shown in the 1st～534 of the SEQ ID NO.3 sequence of at least 70% homology is arranged; Or the sequence that (c) can hybridize with the nucleic acid shown in the 1st～534 of the SEQ ID NO.3.
Preferably, described nucleotide sequence is specially disappearance, insertion and/or the replacement of 1～90 Nucleotide in the nucleotide sequence shown in the 1st～534 of the SEQ ID NO.3, and 5 ' and/or 3 ' end add 60 sequences that form with inner nucleotide.
In addition, the present invention also provides a kind of probe that detects above-mentioned nucleotide sequence, described probe is the nucleic acid molecule with 8～100 continuous nucleotides of above-mentioned nucleotide sequence, and this probe can be used for whether existing in the test sample the relevant nucleic acid molecule of coding Agipanthus ApFT.
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 under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein that in cell, accompanies.
In the present invention, term " Agipanthus bloom genes involved ApFT albumen coded sequence " refer to the encode nucleotide sequence of polypeptide with Agipanthus ApFT protein-active, the 1st～534 nucleotide sequence shown in SEQ ID NO.3 and degenerate sequence thereof.This degenerate sequence refers to, is arranged in the 1st～534 Nucleotide shown in the SEQ ID NO.3, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, so be low to moderate about 70% the degenerate sequence sequence shown in the SEQ ID NO.4 of also encoding out with the 1st～534 nucleotide sequence homology shown in the SEQ ID NO.3.This term also comprises the nucleotide sequence with the homology of nucleotide sequence at least 70% shown in the SEQ ID NO.3.
This term also comprises the variant form of sequence shown in the identical function, SEQ ID NO.3 of the natural Agipanthus ApFT 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 " Agipanthus ApFT albumen " refers to have polypeptide of sequence shown in the SEQ ID NO.4 of Agipanthus ApFT protein-active.This term also comprises having and variant form natural Agipanthus ApFT albumen identical function, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): be generally 1～50 amino acid whose disappearance, insertion and/or replacement, and C-terminal and/or N-terminal add one or be 20 with interior amino acid.For example, in the art, when replacing with the close or similar amino acid of performance, can not change the function of protein usually.Again such as, add the function that or several amino acid also can not change protein usually at C-terminal and/or N-terminal.This term also comprises active fragments and the reactive derivative of Agipanthus ApFT albumen.
The variant form of Agipanthus ApFT albumen of the present invention comprises: the albumen that homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, DNA that can relevant DNA hybridization with Agipanthus ApFT under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of Agipanthus ApFT albumen to obtain.
In the present invention, " Agipanthus ApFT conservative property variation polypeptide " refers to compare with the aminoacid sequence shown in the SEQ ID NO.4, has at the most 10 amino acid be replaced by similar performance or close amino acid and forms polypeptide.These conservative property variation polypeptide are preferably replaced according to table 1 and are produced.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val;Leu;Ile | Val |
| Arg(R) | Lys;Gln;Asn | Lys |
| Asn(N) | Gln;His;Lys;Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro;Ala | Ala |
| His(H) | Asn;Gln;Lys;Arg | Arg |
| Ile(I) | Leu;Val;Met;Ala;Phe | Leu |
| Leu(L) | Ile;Val;Met;Ala;Phe | Ile |
| Lys(K) | Arg;Gln;Asn | Arg |
| Met(M) | Leu;Phe;Ile | Leu |
| Phe(F) | Leu;Val;Ile;Ala;Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr;Phe | Tyr |
| Tyr(Y) | Trp;Phe;Thr;Ser | Phe |
| Val(V) | Ile;Leu;Met;Phe;Ala | Leu |
Invention also comprises the analogue of Agipanthus ApFT albumen or polypeptide.The difference of these analogues and Agipanthus ApFT related polypeptide can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise genetic variant natural or that induce.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and the analogue with that non-natural exists or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide of enumerating.
(the not changing primary structure usually) form of modification comprises: chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (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 Agipanthus ApFT gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of mRNA transcript in cell of namely analyzing Agipanthus ApFT gene.
The detection method that whether has Agipanthus ApFT related nucleotide sequences in the test sample of the present invention comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.This sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to Agipanthus ApFT 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 Agipanthus ApFT 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 Agipanthus ApFT or homologous protein.
In order to obtain the dot matrix with Agipanthus ApFT genes involved, can screen Agipanthus cDNA library with dna probe, these probes are under low rigorous condition, use
32Relevant all or part of of the Agipanthus ApFT of P do the radioactivity mark and.The cDNA library that is suitable for screening is the library from Agipanthus.Structure is that biology field is well-known from the method in the cDNA library of interested cell or tissue.In addition, many such cDNA libraries also can buy, for example available from Clontech, and Stratagene, Palo Alto, Cal..This screening method can be identified the nucleotide sequence of the gene family relevant with Agipanthus ApFT.
Agipanthus ApFT associated nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
After having obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also can will suddenly change by chemosynthesis and introduce in the protein sequence of the present invention.
Except producing with recombination method, the fragment of albumen of the present invention is available solid phase technique also, produced by direct peptide synthesis (people such as Stewart, (1969) solid-phase polypeptide is synthetic, WH Freeman Co., San Francisco; Merrifield J.(1963) J.Am Chem.Soc85:2149-2154).Can carry out by hand or automatically at external synthetic protein.For example, can use 431A type peptide synthesizer (Foster City, CA) the next automatic synthetic peptide of Applied Biosystems.Can distinguish each fragment of chemosynthesis albumen of the present invention, be connected to produce the molecule of total length then with chemical process.
Utilize Agipanthus ApFT albumen of the present invention, by various conventional screening methods, can filter out the interactional material of relevant generation with Agipanthus ApFT albumen, perhaps acceptor, inhibitor or antagonist etc.
The Agipanthus sight is strong, and the florescence is long, flower is big and intensive, pattern is unique, is the high-grade fresh cutting flower of western countries.In Europe, Agipanthus is the agapanthus that can express love except Rose.From 2003, Holland successfully held nine " agapanthus Agipanthus joints ".In addition, Agipanthus also is plant resources commonly used in the external plant landscaping, its strong resistance, and impoverishment tolerant, maintenance management is extensive, has very broad range of application.Often be applied to presbyopic glasses, garland, flower arrangement, fresh cutting flower, potted plant, littoral by with plant landscaping in.The present invention has substantive distinguishing features and marked improvement, cloned the methods analyst ApFT expression of gene pattern that Agipanthus becomes to spend the encoding sequence of first key gene ApFT albumen that changes and adopts fluorescence real-time quantitative PCR of controlling first, for utilizing genetically engineered that the FT gene is regulated and control from now on, and florescence control technology etc. provides scientific basic.Therefore, the present invention has very big using value.
Description of drawings
By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:
Fig. 1 is that the homology of the nucleotide sequence of Agipanthus ApFT gene of the present invention and satsuma orange CuMFT1 gene mRNA compares (GAP) result;
Fig. 2 is that the homology of the aminoacid sequence of Agipanthus ApFT albumen of the present invention and satsuma orange CuMFT1 gene compares (FASTA) result, and wherein, identical amino acid marks with the amino acid monocase between two sequences.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
1. the acquisition of vegetable material
Test materials is 4 years living potted plant seedlings of Agipanthus (Agapanthus praecox ssp.orientalis), gathers stem apex and the blade of vegetative growth phase, bud inductive phase, flower bud differentiation period, is used for extracting RNA and real time fluorescent quantitative qRT-PCR analyzes.
2.RNA extracting
With " plant RNA extracts test kit in a small amount " extracted total RNA, test kit is purchased in Shanghai Lay maple bio tech ltd.1% agarose gel electrophoresis is identified the integrity of RNA, measures purity and the concentration of RNA then at spectrophotometer (Thermo Scientific NANODROP1000Spectrophotometer).
3. the full-length clone of gene
According to transcribing the group sequencing result, obtain and the Agipanthus higher core fragment of genes involved FT homology of blooming, adopt RACE method (3 '-Full RACE Core Set Ver.2.0: precious biotechnology (Dalian) company limited, SMARTer
TMRACE cDNA Amplification Kit:Clontech Laboratories Inc.) carries out the cDNA full-length clone, divides three phases to carry out:
(1) RT-PCR obtains the gene intermediate segment
To extract RNA and carry out reverse transcription (Trabs Script
TMOne-Step gDNA Removal and cDNA Synthesis Super Mix: the Beijing Quanshijin Biotechnology Co., Ltd), be template with the first chain cDNA, utilize software design primers F TS1(SEQ ID NO.1 according to core fragment) and FTA1(SEQ ID NO.2), pcr amplification obtains the 373bp core fragment, reclaim and be connected on the pMD18-T Simple vector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, (Perkin-Elmer checks order on USA) at the ABI377 sequenator.Sequencing result is by carrying out BLAST(http in the NCBI website: //blast.ncbi.nlm.nih.gov/) compare existing database (Genebank), the homology of knowing its nucleotide sequence and proteins encoded and known satsuma orange (BAF93494.1), paddy rice (ABA92722.1), grape (XP_003634198), Arabidopis thaliana plant FT genes such as (NP_173250.1) is very high, so think that tentatively it is the FT gene that a control is bloomed.
（2）3’RACE
Be template with 3 ' RACE ready cDNA, two take turns the amplification that nest-type PRC is finished 3 ' end sequence.
The first round: Outer primer+FTS1 (5 '-TGTCGGTGAGCTACGGTTCCAAGCA-3 ')
Second takes turns: Inner primer+FTS2 (5 '-TTGACCCTGATGCACCAAGCCCAAGT-3 ')
Obtain ApFT3 ' end sequence (268bp), reclaim by glue, be connected on the pMD18-T Simple vector carrier, with RV-M and M13-47 as universal primer, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer checks order on USA) at the ABI377 sequenator.Sequencing result is by carrying out BLAST(http in the NCBI website: //blast.ncbi.nlm.nih.gov/) compare existing database (Genebank), know that the homology of its nucleotide sequence and proteins encoded and known satsuma orange CuMFT1 gene is very high.
（3）5′RACE
Be template with 5 ' RACE ready cDNA, take turns the amplification that nest-type PRC is finished 5 ' end sequence by two.
The first round: UPM+FTA1(5 '-CATAACGGGCCGCAAAGTACCGAGTG-3 ')
Second takes turns: NUP+FTA2(5 '-CCTTGGGAAGGATCTGTTCCTCCTGGTA-3 ')
UPM and NUP provide for test kit.5 ' RACE obtains Agipanthus ApFT5 ' end sequence (174bp), uses with top the same method after recovery connects and checks order.
The sequencing result of the sequence that will obtain by above-mentioned 3 kinds of methods splices, and will splice sequence and submit to BLAST to analyze, and the ApFT gene that result's proof newly obtains from Agipanthus really is the FT family homologous gene of blooming relevant.With the ORF Fingding(http of sequencing result in conjunction with NCBI: //www.ncbi.nlm.nih.gov/gorf) webpage prediction, found initiator codon and the terminator codon of Agipanthus ApFT gene.According to the sequence that obtains, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F(5 '-ATGCATATGGCAAGCTGTGTGGATC-3 '), ORF-R(5 '-TCATATATGCCGTCTCCG GCGACTC-3 '), be that template is carried out PCR with the Agipanthus leaf cDNA, amplification obtains the complete encoding sequence (SEQ ID NO.3) of 534bp Agipanthus ApFT albumen.
Embodiment 2, Agipanthus ApFT gene sequence information and homology analysis
The new Agipanthus ApFT full length gene opening code-reading frame sequence of the present invention is 534bp, and detailed sequence is seen sequence shown in the SEQ ID NO.3.Derive the aminoacid sequence of Agipanthus ApFT according to the opening code-reading frame sequence, totally 177 amino-acid residues, molecular weight is 19490 dalton, and iso-electric point (pI) is 8.67, and detailed sequence is seen sequence shown in the SEQ ID NO.4;
The opening code-reading frame sequence of Agipanthus ApFT and the aminoacid sequence of proteins encoded thereof are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that it and satsuma orange CuMFT1 gene (the GenBank accession number is AB304142.1) have 65.51% similarity at nucleotide level, as shown in Figure 1 (Query: the coding gene sequence of Agipanthus ApFT; Sbjct: the mRNA sequence of satsuma orange CuMFT1 gene); On amino acid levels, it and satsuma orange CuMFT1 gene (GenBank accession number BAF93494.1) also have 72% consistence and 85% similarity, (Query: the aminoacid sequence of Agipanthus ApFT as shown in Figure 2; Sbjct: the aminoacid sequence of satsuma orange CuMFT1).This shows that all there are higher homology in Agipanthus ApFT gene and satsuma orange CuMFT1 gene on nucleic acid still is protein level.
Embodiment 3, the differential expression of ApFT gene in Agipanthus different developmental phases different tissues
1. the acquisition of material: gather blade and the stem apex of Agipanthus vegetative growth phase, bud inductive phase, flower bud differentiation period respectively, drop at once in the liquid nitrogen after sample is wrapped with aluminium platinum paper respectively, put into-80 ° of C Ultralow Temperature Freezer stored for future use subsequently.
2.RNA extraction: utilize " plant RNA extracts test kit in a small amount " (Shanghai Lay maple bio tech ltd), extract the blade of Agipanthus different developmental phases and the RNA in the stem-tip tissue.
3.RNA the determining of integrity, purity, concentration: with plain agar sugar gel electrophoresis (gum concentration 1%; 1 * TAE electrophoretic buffer; 120v 30min) detects integrity.Maximum rRNA brightness should be 1.5-2.0 times of second rRNA brightness in the electrophoretic band, otherwise represents the degraded of rRNA sample.Purity is RNA preferably, A
260/ A
280And A
260/ A
230Be about about 2.0.Measure the OD value and calculate rna content with Thermo NanoDrop1000 trace ultraviolet spectrophotometer.
4.cDNA acquisition: the total RNA with 500ng is template, according to Trabs Script
TMIt is standby that One-Step gDNARemoval and cDNA Synthesis Super Mix test kit operation instructions is carried out reverse transcription acquisition cDNA.
5. the design Auele Specific Primer is used for the real-time fluorescence quantitative PCR analyzing gene at the expression amount of each organ with tissue.According to the Agipanthus ApFT gene order that has obtained, utilize the Auele Specific Primer that the ApFT gene quantification is analyzed among the Beacon Designer7 software design Real-time PCR, ApFT-F(5 '-GCGACTGTCTACTTCAATG-3 '), ApFT-R(5 '-TTACATCAACCTTCACTAATCC-3 ').Internal control gene is Actin, and its primer sequence is Actin-F (5 '-CAGTGTCTGGATTGGAGG-3 '), Actin-R(5 '-TAGAAGCACTTCCTGTG-3 ').
6. make the typical curve of goal gene and internal control gene.Provide with EASY Dilution(test kit) standard substance cDNA solution is carried out gradient dilution, be template with the cDNA solution after the dilution respectively then, Auele Specific Primer with goal gene and internal control gene carries out the Real-time pcr amplification, and reaction finishes the back and draws solubility curve and typical curve.Analyze solubility curve, judge whether the solubility curve of goal gene and internal control gene obtains simple spike, use this primer can obtain single pcr amplification product to judge.Determine the suitable extension rate of template cDNA by typical curve.
7. the real time fluorescent quantitative analysis of goal gene in the testing sample.Be template with synthetic cDNA article one chain, carry out quantitative fluorescence analysis with goal gene and interior primer amplified with reference to gene respectively, Real-time PCR is reflected on the BIO-RAD Chromo4 real-time quantitative instrument and carries out the quantitative fluorescent PCR reaction system: SYBR Premix Ex Taq II(2X) 10 μ L, primer(10 μ M) each 0.5 μ L, cDNA2.0 μ L, RNase-free H
2O up to20 μ L.Response procedures is 94 ℃ of sex change 60s; 94 ℃ of sex change 10s, 53 ℃ of annealing 15s, 72 ℃ are extended 25s, totally 40 circulations.Whether after each amplification is finished, all do solubility curve, be special generation with the check amplified production.
8. adopt 2
-△ △ CtMethod is made relative quantitative assay.The result shows ApFT expression of gene level in the bud differentiation process, and the content in the blade is higher than content in the stem apex.Along with the bud differentiation process, expression amount decline trend in blade, expression amount is the highest in the vegetative growth phase blade, and expression amount is minimum in the flower bud differentiation period blade, infers that this gene may play a significant role in regulating the bud differentiation process.Expression amount is 2.33 times of expression amount in the flower bud differentiation period blade in the vegetative phase blade; In the stem apex ApFT gene inductive phase expression amount the highest, the flower bud differentiation period expression amount is minimum, high expression level amount is 2.02 times of minimum expression amount.The ApFT expression amount is in the stem apex 22.21 times in the vegetative growth phase blade; Bud leaf expression inductive phase amount is 11.40 times in the stem apex; Inflorescence bud formation phase leaf expression amount is 12.20 times in the stem apex.Illustrate that this gene mainly expresses in blade, express and have the tangible space parallax opposite sex.
Claims (7)
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