CN103483437A

CN103483437A - Protein of key gene ApCO for photoperiod and flowering pathway of agapanthus praecox ssp.orientalis as well as coding gene and probe of gene

Info

Publication number: CN103483437A
Application number: CN201310431725.1A
Authority: CN
Inventors: 王玲; 石玉波; 卓丽环
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2013-09-22
Filing date: 2013-09-22
Publication date: 2014-01-01

Abstract

The invention relates to protein of a key gene ApCO for photoperiod and flowering pathway of agapanthus praecox ssp.orientalis as well as a coding gene and a probe thereof. The protein is shown in (a) or (b): (a) a protein formed by an amino-acid sequence shown in SEQ ID NO. 4; (b) a protein formed by an amino-acid sequence shown in SEQ ID NO. 4, which is replaced and deleted by or added with one or more amino-acids, has ApCO activity of the agapanthus praecox ssp.orientalis and is derived from the protein shown in the (a). The invention further provides a nucleic acid sequence for coding the protein as well as the probe for detecting the nucleic acid sequence. According to the invention, the temporal and spatial expression characteristics of agapanthus praecox ssp.orientalis ApCO are adjusted and controlled by utilizing the genetic engineering technique, as a result, a theory basis is provided for an adjusting and monitoring technique for changing florescence, and great application value is obtained.

Description

Bloom pathway key gene ApCO albumen and encoding gene and probe of Agipanthus photoperiod

Technical field

The present invention relates to the key gene CONSTANS(CO that Agipanthus (Agapanthus praecox ssp.orientalis) Photoperiod is bloomed in approach) and encoding gene and probe, be specifically related to a kind of key gene ApCO albumen and encoding gene and probe determined in Photoperiod pathway that Agipanthus blooms.

Background technology

Photoperiod is the important environmental factor that affects flowering of plant, CONSTANS(CO) gene is the key gene in Photoperiod pathway, and it is by inducing FLOWERING LOCUS T(FT), TWIN SISTER OF FT(TSF) and SUPPRESSOR OF OVEREREXPRESSION OF CONATANS1(SOC1) expression promote flowering of plant.In the flower development regulation process, CO is mainly at blade place activation FT, and the FT protein delivery activates the expression of floral meristem Gene A P1 to apical meristem place and FD protein binding subsequently, and then promotes flowering of plant.In many plants, found at present that coding has the CONSTANS-like(COL of N end class zinc fingers B-box and C end CCT conserved domain) gene, but their function is not quite similar.In Arabidopis thaliana, CO is the final exporter of photoperiodic signal under the long day condition, and its up-regulated expression promotes the expression of flowering hormone gene FT, but, under the short day condition, CO is also little to the promoter action of blooming.And CO-like gene Hd1 Accelerate bloom under the short day condition of paddy rice suppresses on the contrary to bloom under the long day condition.As can be seen here, CO and homologous gene thereof may be different in different plants and the effect under different condition.

Agipanthus (Agapanthus praecox ssp.orientalis), have another name called blue lily, is Agapanthaceae Agapanthus plant, originates in African southern subtropical zone, is the good class flowers of viewing and admiring.As the key gene in the Photoperiod approach, the clone of CO gene, expression pattern and CO albumen coded sequence have no report in the research of monocotyledons Agipanthus.Before the present invention comes forth, the patent report that any to mentioned in present patent application Agipanthus CO protein sequence is relevant is not arranged.

Summary of the invention

Purpose of the present invention, be to fill up the blank of Agipanthus CONSTANS gene family member's clone, expression pattern analysis and Agipanthus CONSTANS albumen coded sequence, a kind of Agipanthus CONSTANS albumin A pCO 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 ApCO and Agipanthus ApCO protein sequence and nucleotide sequence thereof, and the expression pattern in Agipanthus different developmental phases and different tissues, for utilizing from now on genetic engineering technique to be regulated and controled the space-time of ApCO genetic expression, thereby the change florescence provides theoretical foundation, has very large using value.

On the one hand, the invention provides and there is the bloom key gene ApCO protein of approach of Agipanthus photoperiod, the protein that described protein is comprised of the aminoacid sequence as shown in SEQ ID NO.4; Or by the aminoacid sequence shown in SEQ ID NO.4 through replacing, lack or adding one or several amino acid and there is the Agipanthus photoperiod pathway key gene ApCO protein of blooming.This protein having that it's too late there is larger difference in active size in the Agipanthus different tissues.

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

Further preferred, described protein be shown in SEQ ID NO.4 in aminoacid sequence 1～10 the amino acid amino acid similar or close by character replace the sequence 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 1st～1167 of SEQ ID NO.3; Or (b) and the nucleic acid shown in 1st～1167 of SEQ ID NO.3 the sequence of at least 70% homology is arranged; Or the sequence that (c) can be hybridized with the nucleic acid shown in 1st～1167 of SEQ ID NO.3.

Preferably, described nucleotide sequence is specially disappearance, insertion and/or the replacement of 1～90 Nucleotide in the nucleotide sequence shown in 1st～1167 of SEQ ID NO.3, and 5 ' and/or 3 ' end add 60 sequences that form with inner nucleotide.

In addition, the present invention also provides a kind of probe that detects above-mentioned nucleotide sequence, described probe is the nucleic acid molecule with 8～100 continuous nucleotides of above-mentioned nucleotide sequence, and this probe can be used for detecting in sample whether have a coding Agipanthus ApCO relevant nucleic acid molecule.

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

In the present invention, term " Agipanthus photoperiod bloom pathway key gene ApCO albumen coded sequence " refers to that coding has the nucleotide sequence of the polypeptide of Agipanthus ApCO protein-active, 1st～1167 nucleotide sequences as shown in SEQ ID NO.3 and degenerate sequence thereof.This degenerate sequence refers to, is arranged in 1st～1167 Nucleotide shown in SEQ ID NO.3, the sequence that has one or more codons to be encoded to produce after the degenerate codon of same amino acid replaces.Due to the degeneracy of codon, so be low to moderate approximately 70% the degenerate sequence sequence shown in SEQ ID NO.4 of also encoding out with 1st～1167 nucleotide sequence homologies shown in SEQ ID NO.3.This term also comprises the nucleotide sequence with the homology at least 70% of the nucleotide sequence shown in SEQ ID NO.3.

This term also comprises the variant form of sequence shown in the identical function, SEQ ID NO.3 of the natural Agipanthus ApCO 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 ApCO albumen " refers to have the polypeptide of sequence shown in the SEQ ID NO.4 of Agipanthus ApCO protein-active.This term also comprises having and natural Agipanthus ApCO albumen identical function, variant form 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 close or similar amino acid is replaced by performance, usually can not change the function of protein.Again such as, add one or several amino acid at C-terminal and/or N-terminal and usually also can not change the function of protein.This term also comprises active fragments and the reactive derivative of Agipanthus ApCO albumen.

The variant form of Agipanthus ApCO albumen of the present invention comprises: the albumen that homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, DNA that can DNA hybridization relevant to Agipanthus ApCO under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of Agipanthus ApCO albumen to obtain.

In the present invention, " Agipanthus ApCO conservative property variation polypeptide " refers to compare with the aminoacid sequence shown in SEQ ID NO.4, has at the most 10 amino acid amino acid similar or close by character replace and forms polypeptide.These conservative property variation polypeptide are preferably replaced and are produced according to table 1.

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

(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 to comprise glycosylation, in the synthetic and processing of polypeptide or further carry out glycosylation modified and polypeptide that produce as those in procedure of processing.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 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 ApCO gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of mRNA transcript in cell of analyzing Agipanthus ApCO gene.

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

In order to obtain the dot matrix with Agipanthus ApCO genes involved, can screen the Agipanthus cDNA library with DNA probe, these probes are under low rigorous condition, use ³²all or part of relevant to Agipanthus ApCO 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 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 Agipanthus ApCO.

Agipanthus ApCO 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 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 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, just can obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier by it, then proceeds to cell, then by ordinary method, from the host cell propagation, separates and obtains relevant sequence.

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

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

Utilize Agipanthus ApCO albumen of the present invention, by various conventional screening methods, can filter out the interactional material of generation relevant to Agipanthus ApCO albumen, or acceptor, inhibitor or antagonist etc.

The Agipanthus sight is strong, and the florescence is long, flower is large 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, is also plant resources commonly used in 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 first the encoding sequence of a key gene ApCO albumen in Agipanthus photoperiod flowering regulation pathways, and the expression pattern of the methods analyst ApCO gene of employing fluorescence real-time quantitative PCR, for utilizing from now on genetically engineered to be regulated and controled the ApCO gene, and florescence control technology etc. provides scientific basic.Therefore, the present invention has very large using value.

The 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 Agipanthus ApCO gene of the present invention and grape (Vitis vinifera) CONSTANS-LIKE(CO) homology of the nucleotide sequence of gene mRNA (GAP) result relatively;

The homology of the aminoacid sequence that Fig. 2 is Agipanthus ApCO albumen of the present invention and grape (Vitis vinifera) CO gene is (FASTA) result relatively, 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 and limit the scope of the invention for the present invention is described.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.

embodiment 1, Agipanthus ApCO gene the clone

1. the acquisition of vegetable material

Test materials is 4 years living potted plant seedlings of Agipanthus (Agapanthus praecox ssp.orientalis), chooses health, without the disease and pest plant, is put in unified management in greenhouse, gathers respectively the tissues such as blade, stem apex for extracting RNA.

2.RNA extracting

By " plant RNA extracts test kit in a small amount " extracted total RNA, test kit is purchased from Shanghai Lay maple bio tech ltd.1% agarose gel electrophoresis is identified 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 early stage Agipanthus transcribe the group sequencing analysis result, obtain the bloom higher core fragment of key gene CONSTANS homology of approach of 1 Photoperiod, adopt RACE method (3 '-Full RACE Core Set Ver.2.0: precious biotechnology (Dalian) company limited, SMARTer ^tMrACE cDNA Amplification Kit:Clontech Laboratories, Inc.) carry out the cDNA full-length clone, a minute three phases carries 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: Beijing Quanshijin Biotechnology Co., Ltd), the first chain cDNA of take is template, utilize software design primer CO-S1(SEQ ID NO.1 according to core fragment) and CO-A1(SEQ ID NO.2), pcr amplification obtains the 229bp core fragment, reclaim and be connected on pMD18-T Simple vector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, at ABI377 sequenator (Perkin-Elmer, USA) on, checked order.Sequencing result is by carrying out BLAST(http in the NCBI website: //blast.ncbi.nlm.nih.gov/) compare existing database (GeneBank), know its nucleotide sequence and proteins encoded and known paddy rice (AGG54062.1), cucumber (XP_004151929.1), Arabidopis thaliana (NP_188826.1), tomato (XP_004240291.1), grape (XP_002264506.2), the homology of the plant CONSTANS genes such as two fringe false bromegrasses (XP_003570474.1) is very high, therefore tentatively think that it is to control the CONSTANS gene that Agipanthus blooms in Photoperiod pathway.

（2）3’RACE

Agipanthus 3 ' the RACE ready cDNA of take is template, completes the amplification of 3 ' end sequence through the three-wheel nest-type PRC.

The first round: UPM+CO-S1 (5 '-ATGGATCCCGTGTGCGACTCGTGTG-3 ')

Second takes turns: NUP+CO-S2 (5 '-GCCTCCAGTGCCACCTCTCCGAC-3 ')

Third round: NUP+CO-S3 (5 '-TCACGAAAAGCCAGAGCAGATACGC-3 ')

By glue, reclaim, be connected on pMD18-T Simple vector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, at ABI377 sequenator (Perkin-Elmer, USA) on, checked order, sequence obtains ApCO3 ' end sequence 1522bp after splicing.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 CONSTANS-like gene gene of the plants such as its nucleotide sequence and proteins encoded and known cocoa (EOX99181.1), soybean (NP_001241023.1), grape (XP_002274384.2), strawberry (XP_004289156.1), clover (XP_003597033.1) is very high.

（3）5′RACE

5 ' the RACE ready cDNA of take is template, by two, takes turns the amplification that nest-type PRC completes 5 ' end sequence.

The first round: UPM+CO-A1(5 '-GAACGTCGGAGAGGTGGCACTGG-3 ')

Second takes turns: NUP+CO-A2(5 '-CAGAGGAGGGATCGTAGGTGGCG-3 ')

UPM and NUP provide for test kit.5 ' RACE obtains Agipanthus ApCO5 ' end encoding sequence (269bp), uses after reclaim connecting with the method top and is checked order.

The sequencing result of the sequence that will obtain by above-mentioned 3 kinds of methods is spliced, to splice sequence submits to BLAST to analyze, the ApCO gene that result proof newly obtains from Agipanthus is really the bloom key gene CONSTANS(CO of approach of Photoperiod) family's homologous gene.ORF Fingding(http by sequencing result in conjunction with NCBI: //www.ncbi.nlm.nih.gov/gorf) webpage prediction, found initiator codon and the terminator codon of Agipanthus ApCO gene.According to the sequence obtained, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F(5 '-ATGGATCCCGTGTGCGACTCGTGTG-3 '), ORF-R(5 '-TCAGCTATTTCTTGATAGAAGAGGT-3 '), the Agipanthus leaf cDNA of take is carried out PCR as template, and amplification obtains the complete encoding sequence (SEQ ID NO.3) of 1167bp Agipanthus ApCO albumen.

embodiment 2, Agipanthus ApCO gene sequence information and homology analysis

The new Agipanthus ApCO full length gene opening code-reading frame sequence of the present invention is 1167bp, and detailed sequence is shown in sequence shown in SEQ ID NO.3.Derive the aminoacid sequence of Agipanthus ApCO according to the opening code-reading frame sequence, totally 388 amino-acid residues, molecular weight is 42.5KD, and iso-electric point (pI) is 5.22, and detailed sequence is shown in sequence shown in SEQ ID NO.4.

The aminoacid sequence of the opening code-reading frame sequence of Agipanthus ApCO 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, found that it and grape (Vitis vinifera) CO gene (XP_002274384) have 59.97% similarity on nucleotide level, as shown in Figure 1 (Query: the coding gene sequence of Agipanthus ApCO; Sbjct: the mRNA sequence of grape CONSTANS gene); On amino acid levels, it and grape CONSTANS gene (GenBank accession number XP_002274384.2) also have 42% consistence and 55% similarity, (Query: the aminoacid sequence of Agipanthus ApCO as shown in Figure 2; Sbjct: the aminoacid sequence of grape CONSTANS).As can be seen here, all there are higher homology in Agipanthus ApCO gene and grape CONSTANS gene from nucleic acid or protein level.

embodiment 3, the different expression of ApCO gene in Agipanthus different developmental phases and different tissues

1. the acquisition of material: the 4 years living Agipanthus seedlings of take are the examination material, get respectively stem apex and the blade of vegetative growth phase, Flower induction phase, flower bud differentiation period, and the young fruit of the root of early flowering season, stem, leaf, scape, bennet, petal, ovary and productive phase, drop at once in liquid nitrogen after sample is wrapped with aluminium platinum paper respectively, put into subsequently-80 ℃ of Ultralow Temperature Freezer stored for future use.

2.RNA extraction: utilize " plant RNA extracts test kit in a small amount " (Shanghai Lay maple bio tech ltd), extract the RNA in the Agipanthus different tissues.

3.RNA the determining of integrity, purity, concentration: with plain agar sugar gel electrophoresis (gum concentration 1%; 1 * TAE electrophoretic buffer; 120v, 30min) the detection integrity.In electrophoretic band, maximum rRNA brightness should be 1.5-2.0 times of second rRNA brightness, otherwise means the degraded of rRNA sample.Purity is RNA preferably, A ₂₆₀/ A ₂₈₀and A ₂₆₀/ A ₂₃₀be about 2.0 left and right.Measure the OD value and calculate rna content with Thermo NanoDrop1000 trace ultraviolet spectrophotometer.

4.cDNA acquisition: the total RNA of 500ng of take is template, according to Trabs Script ^tMit is standby that One-Step gDNA Removal and cDNA Synthesis Super Mix test kit operation instructions is carried out reverse transcription acquisition cDNA.

5. design Auele Specific Primer, for the real-time fluorescence quantitative PCR analyzing gene at each organ the expression amount with tissue.According to the Agipanthus ApCO gene order obtained, utilize the Auele Specific Primer that in Beacon Designer7 software design Real-time PCR, the ApCO gene quantification is analyzed, ApCO-F(5 '-CTTCTTCTTCGGCACAAC-3 '), ApCO-R(5 '-TCCTCATAATCAGCAACACT-3 ').Reference 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 reference gene.With EASY Dilution(test kit, provide) standard substance cDNA solution is carried out to gradient dilution, the cDNA solution of then take respectively after diluting is template, Auele Specific Primer with goal gene and reference gene carries out the Real-time pcr amplification, after reaction finishes, draws solubility curve and typical curve.Analyze solubility curve, judge whether the solubility curve of goal gene and reference gene obtains simple spike, with judgement, use this primer can obtain single pcr amplification product.Determine the suitable extension rate of template cDNA by typical curve.

7. the Real time PCR of goal gene in testing sample.The cDNA article one chain synthesized of take is template, carry out quantitative fluorescence analysis by the primer amplified of goal gene and internal reference gene respectively, Real-time PCR reaction is carried out on BIO-RAD Chromo4 real-time quantitative instrument, 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, 56 ℃ of annealing 15s, 72 ℃ are extended 25s, totally 40 circulations.After each the amplification, all do solubility curve, whether the check amplified production of take is special generation.

8. adopt 2 ^{-△ △ Ct}method is made relative quantitative assay.Result shows that the expression level of ApCO gene all has expression in Agipanthus different developmental phases and different tissues, and has certain difference.In whole Process of Flower Bud Differentiation, in blade, the ApCO expression amount is all higher than the expression amount in stem apex.Three period Leaf expression amount be respectively 1.53,5.66 and 2.57 times of expression amount in stem apex.The ApCO trend that expression amount is fallen after rising in blade, Flower induction phase leaf expression amount is the highest, is respectively vegetative phase and Leaf during Flower Bud Differentiation 1.85 and 2.10 times.ApCO is in the expression of stem apex the trend that first descends and rise afterwards, and in the vegetative growth phase stem apex, expression amount is the highest, is respectively inductive phase and bud and divides 1.99 and 1.89 times of florescence.Infer that this gene may the certain effect of performance in regulating Process of Flower Bud Differentiation.Spatial and temporal expression studies show that, ApCO gene expression level in root, stem, blade, bennet, petal, ovary, fruit is followed successively by bennet, blade, root, fruit, ovary, petal, scape, stem from high to low, high expression level amount is minimum expression amount 114.3 times, illustrate that the expression of Agipanthus ApCO gene has obvious spatial diversity.

Claims

1. following (a) or protein (b):

(a) protein formed by the aminoacid sequence as shown in SEQ ID NO.4;

(b) aminoacid sequence shown in SEQ ID NO.4 is through replacing, lack or adding one or several amino acid and have the bloom derivative protein by (a) of pathway key gene ApCO activity of Agipanthus photoperiod.

2. protein as claimed in claim 1, it is characterized in that, described protein be aminoacid sequence shown in SEQ ID NO.4 through 1～50 amino acid whose disappearance, insertion and/or replacement, or add 1～20 sequence obtained 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.4 in aminoacid sequence 1～10 the amino acid amino acid similar or close by character replace the sequence formed.

4. coding claim 1 a described 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～1167 of SEQ ID NO.3;

Or (b) and the nucleic acid shown in 1st～1167 of SEQ ID NO.3 the sequence of at least 70% homology is arranged;

Or the sequence that (c) can be hybridized with the nucleic acid shown in 1st～1167 of SEQ ID NO.3.

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～1167 of SEQ ID NO.3, or 5 ' and/or 3 ' end add 60 sequences that form with inner nucleotide.

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