CN103333233B

CN103333233B - Agapanthus praecox auxin receptor protein TIR1 and coding gene and probe thereof

Info

Publication number: CN103333233B
Application number: CN201310270238.1A
Authority: CN
Inventors: 申晓辉; 张荻; 岳建华; 任丽
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2013-06-28
Filing date: 2013-06-28
Publication date: 2014-12-10
Anticipated expiration: 2033-06-28
Also published as: CN103333233A

Abstract

The invention relates to an agapanthus praecox auxin receptor protein TIR1 and a coding gene and probe thereof. The protein is: (a) a protein consisting of the amino acid sequence shown in SEQ ID No.4; or (b) a protein obtained by substituting, losing or adding one or multiple amino acids of the amino acid sequence shown in SEQ ID No.4, having agapanthus praecox auxin receptor protein activity and derived from (a). The invention also provides a nucleotide sequence for coding the protein and a probe for detecting the nucleotide sequence. According to the agapanthus praecox auxin receptor protein TIR1 and the coding gene and probe thereof provided by the invention, the agapanthus praecox auxin signal transduction pathway is regulated by the genetic engineering technology to reach an aim of controlling the growth development and organ morphogenesis, thereby providing a theoretical basis for molecular breeding and creating great application value.

Description

Agipanthus growth hormone receptor albumen TIR1 and encoding gene and probe

Technical field

Key protein TIR1 and encoding gene and probe in Agipanthus Auxin Signal Tranducation approach of the present invention, be specifically related to a kind of Agipanthus growth hormone receptor albumen TIR1 and encoding gene and probe.

Background technology

Endogenous hormones is grown Growth of Ornamental Plants and fancy points regulation and control have important effect, and growth hormone is unique a kind of endogenous hormones with polarity Transport Characteristics in plant materials.The content of growth hormone and distribution relation are to the growth of plant and polar structure and the spatial shape of development rate and each organ-tissue.It is comparatively clear that the signal transduction path of growth hormone is studied at present, and wherein growth hormone must be combined and could regulate and control downstream gene network with its receptor protein, cause physiological responses, and TIR1 albumen is topmost growth hormone receptor albumen.

(body embryo) occurs somatic embryo is plant molecular breeding and the most effective technical system of rapid propagation in vitro, there are some researches show that plant materials embryonal induction mainly depends on exogenous auxin regulation and control, and exogenous growth hormone material picloram is to unifacial leaf flower bulbs body embryonal induction tool wholesomeness.Agipanthus is tropical perennial flowers, and flower amount is large, the florescence is long, ornamental value is high, has underground stem tuber tissue.The Agipanthus body idiosome system that we set up in earlier stage shows that growth hormone signal has decisive role to Agipanthus body embryonal induction, body embryo form, body embryo quantity and body embryo seedling.In addition, application external source regulation and control substance interrupts Polar Transport of Auxin all has obvious regulating and controlling effect to Agipanthus florescence, plant dwarfing, morphology of terminal inflorescence.Therefore, growth hormone signal is produced with breeding improvement work and is had vital effect Flower Industrialization.

The encoding gene of TIR1 is cloned out from various plants, comprising: Arabidopis thaliana, paddy rice, corn, grape, castor-oil plant etc.But for ornamental plant, especially in flower bulbs, TIR1 clone, expression pattern and protein sequence it be unclear that.At present, there is not any bibliographical information relevant to Agipanthus TIR1 albumen and coding gene sequence thereof.

Summary of the invention

The object of the invention is to fill up the blank of clone, expression pattern analysis and the Agipanthus TIR1 albumen of Agipanthus TIR1 gene, a kind of Agipanthus TIR1 albumen 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 Agipanthus TIR1 albumen and nucleotide sequence thereof the expression pattern in Agipanthus Different Organs and body embryonic development process, for utilizing from now on genetic engineering technique to regulate and control the space-time characterisation of TIR1 genetic expression, thereby for body embryo occurs, regulation of plant form provides theoretical foundation, there is very large using value.

On the one hand, the invention provides and there is Agipanthus growth hormone receptor albumen, the protein that described protein is comprised of the aminoacid sequence as shown in SEQ ID NO.4; Or by aminoacid sequence shown in SEQ ID NO.4 through replacing, lack or adding one or several amino acid and there is the protein of Agipanthus growth hormone receptor protein-active.There is larger difference in the expression amount of this protein in not consubstantiality Embryogenesis, Different Organs.

Preferably, described protein be aminoacid sequence shown in SEQ ID NO.4 through 1～50 amino acid whose disappearance, insertion and/or replacement, or 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.4 in aminoacid sequence 1～10 amino acid by the similar or close amino acid of character, replaced the sequence forming.

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～1767 of SEQ ID NO.3; Or (b) and the nucleic acid shown in 1st～1767 of SEQ ID NO.3 have the sequence of at least 70% homology; Or the sequence that (c) can hybridize with the nucleic acid shown in 1st～1767 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～1767 of SEQ ID NO.3, and 5 ' and/or 3 ' end add 60 sequences that form with inner nucleotide.

In addition, the present invention also provides a kind of probe that detects above-mentioned nucleotide sequence, described probe is the nucleic acid molecule with 8～100 continuous nucleotides of above-mentioned nucleotide sequence, and this probe can be used for detecting in sample, whether have the relevant nucleic acid molecule of coding Agipanthus TIR1.

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 " Agipanthus TIR1 albumen coded sequence " refers to that coding has the nucleotide sequence of polypeptide of Agipanthus TIR1 protein-active, 1st～1767 nucleotide sequences as shown in SEQ ID NO.3 and degenerate sequence thereof.This degenerate sequence refers to, is arranged in 1st～1767 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～1767 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 TIR1 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 TIR1 albumen " refers to have the polypeptide of sequence shown in the SEQ ID NO.4 of Agipanthus TIR1 protein-active.This term also comprises having and variant form natural Agipanthus TIR1 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, 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 Agipanthus TIR1 albumen.

The variant form of Agipanthus TIR1 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 TIR1 under high or low rigorous condition are coded and the polypeptide or the albumen that utilize the antiserum(antisera) of Agipanthus TIR1 albumen to obtain.

In the present invention, " Agipanthus TIR1 conservative property variation polypeptide " refers to compare with the aminoacid sequence shown in SEQ ID NO.4, has at the most 10 amino acid be replaced by the similar or close amino acid of character and 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
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 TIR1 albumen or polypeptide.The difference of these analogues and Agipanthus TIR1 related polypeptide can be the difference on aminoacid sequence, can be also the difference not affecting on the modified forms of sequence, or have both at the same time.These polypeptide comprise genetic variant natural or induction.Induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and 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 Agipanthus TIR1 gene product of available real-time fluorescence quantitative PCR, whether and quantity the existence of the mRNA transcript of analyzing Agipanthus TIR1 gene in cell.

The present invention detects the detection method that whether has Agipanthus TIR1 related nucleotide sequences in sample, comprises with above-mentioned probe and sample and hybridizing, and then whether detection probes combination has occurred.This sample is the product after pcr amplification, and wherein pcr amplification primer is corresponding to Agipanthus TIR1 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 TIR1 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 TIR1 or homologous protein.

In order to obtain the dot matrix with Agipanthus TIR1 genes involved, can screen Agipanthus cDNA library with DNA probe, these probes are under low rigorous condition, use ³²p relevant all or part of of Agipanthus TIR1 cooked to 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 TIR1.

Agipanthus TIR1 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, just can obtain in large quantity relevant sequence with recombination method.This is normally cloned into carrier, then proceeds to cell, then by ordinary method 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.

Utilize Agipanthus TIR1 albumen of the present invention, by various conventional screening methods, can filter out the interactional material of generation relevant to Agipanthus TIR1 albumen, or inhibitor and antagonist etc.

Agipanthus ornamental value is high, is widely used, and its scape is tall and straight is good fresh cutting flower kind, is also the agapanthus that can express love except rose, and its market requirement is also increasing.The present invention clones the encoding sequence of the key receptor albumen TIR1 in Auxin Signal Tranducation approach in Agipanthus plant materials first, and adopt the expression pattern of the methods analyst TIR1 gene of fluorescence real-time quantitative PCR, for controlling from now on plant tip growth advantage, regulation of plant form and reducing somatocyte Embryos generation ratio aspect, there is important application prospect, also for breeding of new variety aspect provides theoretical foundation, there is very large using value.

Accompanying drawing explanation

By reading the detailed description of non-limiting example being done with reference to the following drawings, it is more obvious that other features, objects and advantages of the present invention will become:

Fig. 1 is homology comparison (GAP) result of the nucleotide sequence of Agipanthus TIR1 gene of the present invention and castor-oil plant TIR1 gene mRNA;

Fig. 2 is homology comparison (FASTA) result of the aminoacid sequence of Agipanthus TIR1 albumen of the present invention and grape TIR1 albumen, and wherein, identical amino acid marks with amino acid monocase between two sequences;

Fig. 3 is the expression amount variation diagram of Agipanthus TIR1 gene under external source IAA and NPA regulation and control;

Fig. 4 is the gene expression amount variation diagram of Agipanthus TIR1 gene in somatic embryo development process.

Embodiment

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, for example Sambrook equimolecular 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 TIR1 gene clone

1. the acquisition of vegetable material

Get the adult seedling leaf tissue of Agipanthus, for extracting RNA; Agipanthus was introduced China in 2000 by South Africa, and in Shanghai, carried out introduction and Experiment.Sun Bo is in research > > mono-literary composition of the master thesis < < Agipanthus pot culturing and dwarfing of delivering for 2011, Basic Biological Character to Agipanthus, introduces a fine variety historical grade and is described in more detail.The Agipanthus material that the present embodiment relates to can obtain by disclosed channel.

The extracting of 2.RNA

By " RNA prep pure plant total RNA extraction reagent box " extracted total RNA (Trizol:Invitrogen), 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

The protein function annotation result of transcribing group order-checking (RNA-seq) according to Agipanthus, obtains Agipanthus TIR1 gene core fragment.Adopt RACE method (SMARTer ^tMrACE cDNA Amplification Kit:Clonetech) carry out cDNA full-length clone, a minute three phases carries out:

(1) RT-PCR obtains gene intermediate segment

By the RNA of extraction carry out reverse transcription (Prime Script II 1st Strand cDNA Synthesis Kit: precious biotechnology (Dalian) company limited),

The first chain cDNA of take is template, utilizes primer to carry out PCR:

TIR1F5′-ATGGGCAATCCCTAATCT-3′（SEQ?ID?NO.1）

And TIR1R5 '-CTCGTGCTTTCCCTGATG-3 ' (SEQ ID NO.2)

Amplification obtains 1476bp 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, check order, sequencing result is by carrying out BLAST(http in NCBI website: //blast.ncbi.nlm.nih.gov/) compare existing database (GenBank), know its nucleotide sequence and proteins encoded and known grape, comospore poplar, the homology of the TIR1 gene of castor-oil plant is very high, tentatively think that it is a TIR1 gene,

（2）3′RACE

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

The first round: UPM+3 '-GSP1(5 '-TGTCCTCTTCTTCGCCGCCTTTGGGT-3 ') (SEQ ID NO.5)

Second takes turns: NUP+3 '-GSP2(5 '-CAACGCCTTGCTGTTTCGGGTCTATT-3 ') (SEQ ID NO.6)

UPM and NUP provide for test kit.3 ' RACE obtains 3 ' end sequence (1887bp) of Agipanthus TIR1, reclaim, be connected on pMD18-T Simple vector carrier, with RV-M and M13-47 as universal primer, adopt and stop thing fluorescent mark (Big-Dye, Perkin-Elmer, USA) method, at ABI377 sequenator (Perkin-Elmer, USA) on, check order, sequencing result is by carrying out BLAST(http in NCBI website: //blast.ncbi.nlm.nih.gov/) compare existing database (GenBank), know its nucleotide sequence and proteins encoded and known grape, comospore poplar, the homology of the TIR1 gene of castor-oil plant is very high,

（3）5′RACE

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

The first round: UPM+5 '-GSP1(5 '-ATCCATTGGCTCGTTGGTGAGGTAGTCG-3 ') (SEQ ID NO.7)

Second takes turns: NUP+5 '-GSP2(5 '-CCTTGCATACCAAAGACACGGCGCTAC-3 ') (SEQ ID NO.8)

UPM and NUP provide for test kit.5 ' RACE obtains the 5 ' end sequence (1607bp) of Agipanthus TIR1 gene, after reclaim connecting, by the method with above, check order, the sequencing result of the sequence obtaining by above-mentioned 3 kinds of methods is spliced, to splice sequence submits to BLAST to analyze, the TIR1 gene that result proof newly obtains from Agipanthus is a gene that growth hormone receptor albumen is relevant really, ORF Finding(http by sequencing result in conjunction with NCBI: //www.ncbi.nlm.nih.gov/gorf) prediction, initiator codon and the terminator codon of Agipanthus TIR1 gene have been found, according to the sequence obtaining, respectively from initiator codon and terminator codon design Auele Specific Primer,

ORF-F（5′-ATGGACGCGAAGAGGAAGAAAGA-3′），（SEQ?ID?NO.9）

ORF-R（5′-TCAGAGTGTGACAACAAAAGGAGGC-3′），（SEQ?ID?NO.10）

The Agipanthus cDNA of take carries out PCR as template, and amplification obtains the complete encoding sequence (SEQ ID NO.3) of 1767bp Agipanthus TIR1 albumen.

embodiment 2, Agipanthus TIR1 gene sequence information and homology analysis

The new Agipanthus TIR1 full length gene opening code-reading frame sequence of the present invention is 1767bp, and detailed sequence is shown in sequence shown in SEQ ID NO.3.According to opening code-reading frame sequence, derive the aminoacid sequence of Agipanthus TIR1 albumen, totally 588 amino-acid residues, molecular weight is 65.8kDa, and iso-electric point (pI) is 5.83, 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 TIR1 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 castor-oil plant TIR1 gene (accession number: XM_002524640.1) there is 72% homogeny on nucleotide level, as shown in Figure 1 (Query: the coding gene sequence of Agipanthus TIR1; Sbjct: the mRNA sequence of castor-oil plant TIR1); On amino acid levels, it and grape TIR1 gene (accession number: XP_002271412.2) also have 78% consistence and 87% similarity, as shown in Figure 2 (Query: the aminoacid sequence of Agipanthus TIR1 albumen; Sbjct: the aminoacid sequence of grape TIR1 albumen).As can be seen here, all there is higher homology in the TIR1 gene of Agipanthus TIR1 gene and other known species from nucleic acid or protein level.

embodiment 3, Agipanthus TIR1 gene is at body embryo different developmental phases and the different expression under external source regulating and controlling effect

1. the acquisition of material: at 4 different developmental phases (callus of Agipanthus somatic embryo; Embryo callus; Somatic embryo; Body embryo seedling) material samples; In field, utilize in addition external source regulation and control substance to spray 3 years raw plant regulation and control growth hormone signals of Agipanthus, external source regulation and control substance is respectively IAA (100ppm) and NPA (polar auxin transport inhibitor: 100ppm), process with the blade of NPA processing plant and sample respectively after processing with control sample (non-processor), IAA.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 the total RNA of RNA prep pure plant to extract (Trizol:Invitrogen); Extract the total RNA in the different sample tissue of Agipanthus;

Determining of the integrity of 3.RNA, purity, concentration: with plain agar sugar gel electrophoresis (gum concentration 1.2%; 0.5 * TBE electrophoretic buffer; 150v, 15min) detect integrity, in electrophoretic band, maximum rRNA brightness should be 1.5～2.0 times of 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 OD 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. design Auele Specific Primer is to carry out the expression amount of real-time fluorescence quantitative PCR analyzing gene in each organ and tissue, according to the Agipanthus TIR1 gene order having obtained, utilize primer-design software to be designed for the Auele Specific Primer primer that in Real-time PCR, TIR1 gene quantification is analyzed

qT-F（5′-GATTGTGGCGAGGTGTAG-3′），（SEQ?ID?NO.11）

qT-R（5′-AGCGTGTTCAGGTTCTTG-3′）。（SEQ?ID?NO.12）

Reference gene is Agipanthus Actin gene, and primer is:

Actin-F（5′-CAGTGTCTGGATTGGAGG-3′），（SEQ?ID?NO.13）

Actin-R（5′-TAGAAGCACTTCCTGTG-3′）。（SEQ?ID?NO.14）

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 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 40 circulations: 94 ℃ of 15s; 57 ℃ of 15s; 72 ℃ of 25s; After each amplification, all do solubility curve, take and check whether amplified production is special generation;

8. adopt 2 ^{-△ △ Ct}method is made relative quantitative assay, the expression level that result shows Agipanthus TIR1 gene is processed TIR1 gene expression amount in lower blade decrease (declining 8%) at external source IAA, and process TIR1 gene expression amount in lower blade at NPA, risen 7.7 times (Fig. 3), between two processing, TIR1 gene expression amount variation tendency is completely contradicted.Above result shows to have reverse feedback regulation mechanism between growth hormone content and its receptor protein, and growth hormone content weakens Auxin Signal Tranducation level higher than normal level plant self by reducing the expression amount of its receptor protein, and vice versa.In addition, in Agipanthus somatic embryogenesis, TIR1 gene expression amount content in callus is minimum, in embryo callus, content rises 2.27 times, in somatic embryo, TIR1 gene expression amount rises 1.84 times, and TIR1 expression amount is the highest in body embryo seedling, be 7.32 times (Fig. 4) of callus.Illustrate that TIR1 gene has obvious space and time difference in Agipanthus body embryonic development process.Therefore, the acquisition of Agipanthus TIR1 albumen coded sequence, for having important application prospect and using value aspect regulation and control growth hormone signal control plant tip growth advantage, regulation of plant form and reduction somatocyte Embryos ratio from now on.

Above specific embodiments of the invention are described.It will be appreciated that, the present invention is not limited to above-mentioned specific implementations, and those skilled in the art can make various distortion or modification within the scope of the claims, and this does not affect flesh and blood of the present invention.

Claims

1. the protein that aminoacid sequence as shown in SEQ ID NO.4 forms.

One kind coding claim 1 described in nucleic acid sequences to proteins.

3. nucleotide sequence as claimed in claim 2, is characterized in that, described nucleotide sequence is specifically as shown in 1st～1767 of SEQ ID NO.3.