CN104961814A - Agapanthus auxin signal transcriptional control protein Aux/IAA3 and encoding gene and probe thereof - Google Patents
Agapanthus auxin signal transcriptional control protein Aux/IAA3 and encoding gene and probe thereof Download PDFInfo
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- C07—ORGANIC CHEMISTRY
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- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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Abstract
The invention discloses agapanthus auxin signal transcriptional control protein Aux/IAA3 and an encoding gene and a probe thereof. The protein is protein shown in a, namely protein formed by an amino acid sequence shown in SEQ IN NO.4 or b, namely protein which is formed by replacement or deletion or addition of one or more amino acids on the amino acid sequence shown in SEQ ID NO.4, has agapanthus Aux/IAA3 protein activity and is derived from a. The invention further provides a nucleotide sequence for encoding the protein and a probe for detecting the nucleotide sequence. An agapanthus auxin signal transcriptional pathway is controlled through the gene engineering technology, so that the purposes of controlling growth and development of agapanthus and organ shape establishment are achieved, the theoretical basis is provided for molecular breeding, and high application value is achieved.
Description
Technical field
The present invention relates to Agipanthus growth hormone signal transcription modulin and encoding gene thereof and probe, be specifically related to a kind of Agipanthus growth hormone signal transcription modulin Aux/IAA3 and encoding gene thereof 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 polar translocation feature in plant materials.The concentration and distribution of growth hormone is related to the growth of plant and the polar structure of development rate and each organ-tissue and spatial shape.It is comparatively clear that the signal transduction path of growth hormone is studied at present, and wherein Aux/IAA is important growth hormone transcription inhibitory factor, when it with growth hormone in conjunction with time can the transcribing of Developing restraint element, reduce growth hormone content.
(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 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 flowering perennial, 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 has vital effect to Flower Industrialization production and breeding improvement work.
The encoding gene of Aux/IAA clones out from various plants, comprising: Arabidopis thaliana, paddy rice, corn, grape, castor-oil plant etc.But for ornamental plant, especially in flower bulbs, the clone of Aux/IAA, expression pattern and protein sequence it be unclear that.At present, any bibliographical information relevant to Agipanthus Aux/IAA3 albumen and coding gene sequence thereof is not had.
Summary of the invention
The object of the invention is to fill up the blank of the clone of Agipanthus Aux/IAA3 gene, expression pattern analysis and Agipanthus Aux/IAA3 albumen, provide a kind of Agipanthus growth hormone signal transcription modulin Aux/IAA3, present invention also offers a kind of above-mentioned nucleic acid sequences to proteins and detect the probe of described nucleotide sequence of encoding; The invention discloses the physiological effect after Agipanthus Aux/IAA3 gene transformation Arabidopis thaliana and expression pattern, regulate and control for utilizing the space-time characterisation of genetic engineering technique to Aux/IAA3 genetic expression from now on, thus for body embryo occur, regulation of plant form provides theoretical foundation, has very large using value.
First aspect, the invention provides Agipanthus growth hormone signal transcription modulin, the protein that described protein is made up of the aminoacid sequence such as shown in SEQIDNO.4; Or by the aminoacid sequence shown in SEQ IDNO.4 through replacing, lacking or add one or several amino acid and there is the protein of Agipanthus growth hormone signal transcription modulin feature.
Preferably, described protein for aminoacid sequence shown in SEQ ID NO.4 is through 1 ~ 50 amino acid whose disappearance, insertion and/or replacement, or within C-terminal and/or N-terminal add 1 ~ 20 amino acid and the sequence that obtains.
Further preferred, described protein for 1 ~ 10 amino acid in aminoacid sequence shown in SEQ ID NO.4 replace by the similar or close amino acid of character and the sequence that formed.
Second aspect, the invention provides the above-mentioned nucleic acid sequences to proteins of a kind of coding.
Preferably, described nucleotide sequence is specially: (a) base sequence is as shown in SEQ ID NO.3 1st ~ 549; Or the nucleic acid shown in (b) and SEQ ID NO.3 1st ~ 549 has the sequence of the homology of at least 70%; Or (c) can carry out the sequence of hybridizing with the nucleic acid shown in SEQ ID NO.3 1st ~ 549.
Preferably, described nucleotide sequence is specially the disappearance of 1 ~ 90 Nucleotide in the nucleotide sequence shown in SEQ ID NO.3 1st ~ 549, insertion and/or replacement, and 5 ' and/or 3 ' sequence of being formed with inner nucleotide of end interpolation 60.
The third aspect, present invention also offers a kind of probe detecting above-mentioned nucleotide sequence, described probe is the nucleic acid molecule with above-mentioned nucleotide sequence 8 ~ 100 continuous nucleotides, and this probe can be used for detecting in sample whether there is the relevant nucleic acid molecule of coding Agipanthus Aux/IAA3.
In the present invention, " DNA of separation ", " DNA of purifying " refer to, the sequence that this DNA or fragment have been arranged in its both sides from native state is separated, also refer to that this DNA or fragment with under native state are separated with the component of nucleic acid, and separate with the protein accompanied in cell.
In the present invention, the nucleotide sequence of term " the Agipanthus growth hormone signal transcription modulin Aux/IAA3 albumen coded sequence " polypeptide with Agipanthus Aux/IAA3 protein-active that refers to encode, 1st ~ 549 nucleotide sequences as shown in SEQ ID NO.3 and degenerate sequence thereof.This degenerate sequence refers to, is arranged in 1st ~ 549 Nucleotide shown in SEQ ID NO.3, have one or more codon replace by the degenerate codon of same amino acid of encoding after produce sequence.Due to the degeneracy of codon, the sequence shown in SEQ ID NO.4 so the degenerate sequence being low to moderate about 70% with 1st ~ 549 nucleotide sequence homologies shown in SEQ ID NO.3 also can be encoded out.This term also comprises the nucleotide sequence with the homology at least 70% of the nucleotide sequence shown in SEQ IDNO.3.
This term also comprises the variant form of sequence shown in the identical function of natural Agipanthus Aux/IAA3 albumen of encoding, SEQ ID NO.3.These variant forms comprise (but being not limited to): be generally the disappearance of 1 ~ 90 Nucleotide, insertion and/or replacement, and 5 ' and/or 3 ' end be added to 60 with inner nucleotide.
In the present invention, term " Agipanthus growth hormone signal transcription modulin Aux/IAA3 " refer to have Agipanthus Aux/IAA3 protein-active SEQ ID NO.4 shown in the polypeptide of sequence.This term also comprise have with natural Agipanthus Aux/IAA3 albumen identical function, the variant form of 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 add one or amino acid within being 20 at C-terminal and/or N-terminal.Such as, in the art, when replacing with similar nature or similar amino acid, the function of protein can not usually be changed.Again such as, add at C-terminal and/or N-terminal the function that or several amino acid also can not change protein usually.This term also comprises active fragments and the reactive derivative of Agipanthus Aux/IAA3 albumen.
The variant form of Agipanthus growth hormone signal transcription modulin Aux/IAA3 of the present invention comprises: homologous sequence, conservative variant, allelic variant, natural mutation, induced mutants, can the albumen coded by DNA of DNA hybridization relevant to Agipanthus Aux/IAA3 and the polypeptide utilizing the antiserum(antisera) of Agipanthus Aux/IAA3 albumen to obtain or albumen under high or low high stringency conditions.
In the present invention, " Agipanthus Aux/IAA3 conservative variation polypeptide " refers to compared with the aminoacid sequence shown in SEQ ID NO.4, have 10 amino acid at the most replace by the similar or close amino acid of character and form polypeptide.These conservative variation's polypeptide preferably carry out replacing according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | Preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Invention also comprises the analogue of Agipanthus Aux/IAA3 albumen or polypeptide.The difference of these analogues and Agipanthus Aux/IAA3 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 that is natural or induction.Induce variation body can be obtained by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also by site-directed mutagenesis or the biological technology of other known moleculars.Analogue also comprises the analogue with the residue (as D-amino acid) being different from natural L-amino acids, and has the analogue of amino acid (as β, gamma-amino acid) that is that non-natural exists or synthesis.Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide enumerated.
(usually the not changing primary structure) form of modification comprises: the chemically derived form of the polypeptide that body is interior or external is as acetylize or carboxylated.Modify and also comprise glycosylation, as carried out glycosylation modified and polypeptide that is that produce in those in the synthesis of polypeptide and processing or further procedure of processing.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) by being exposed to by polypeptide and completing.Modified forms also comprises the sequence with phosphorylated amino acid residue (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Also comprise and modified thus improve its anti-proteolysis performance or optimize the polypeptide of solubility property.
Utilize Agipanthus growth hormone signal transcription modulin Aux/IAA3 of the present invention, by various conventional screening assays, can filter out and relevant to Agipanthus Aux/IAA3 albumen interactional material occur, or inhibitor and antagonist etc.
The present invention detects the detection method that whether there is Agipanthus Aux/IAA3 related nucleotide sequences in sample, and comprise and hybridizing with above-mentioned probe and sample, then whether detection probes there occurs combination.This sample is the product after pcr amplification, and wherein pcr amplification primer corresponds to Agipanthus Aux/IAA3 related nucleosides coding sequences, and can be positioned at both sides or the centre of this encoding sequence.Primer length is generally 15 ~ 50 Nucleotide.
In addition, according to Agipanthus Aux/IAA3 nucleotide sequence of the present invention and aminoacid sequence, can on the homology basis of nucleic acid homology or marking protein, screening Agipanthus Aux/IAA3 associated homologous gene or homologous protein.
In order to obtain the dot matrix with Agipanthus Aux/IAA3 genes involved, can screen Agipanthus cDNA library with DNA probe, these probes are under low high stringency conditions, use
32what P was correlated with to Agipanthus Aux/IAA3 all or part ofly does radioactivity mark and obtains.The cDNA library being suitable for screening is the library from Agipanthus.The method built from the cDNA library of interested cell or tissue is that biology field is well-known.In addition, many such cDNA libraries also can buy, such as, purchased from Clontech, Stratagene, Palo Alto, Cal..This screening method can identify the nucleotide sequence of the gene family relevant to Agipanthus Aux/IAA3.
Agipanthus Aux/IAA3 associated nucleotide full length sequence of the present invention or its fragment can obtain by the method for pcr amplification method, recombination method or synthetic usually.For pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the eDNA storehouse prepared by ordinary method well known by persons skilled in the art as template, amplification and relevant sequence.When sequence is longer, usually needs to carry out twice or repeatedly pcr amplification, and then the fragment that each time amplifies is stitched together by proper order.
After obtaining relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This is normally cloned into carrier, then proceeds to cell, is then separated from the host cell after propagation by ordinary method and obtains relevant sequence.
In addition, also by chemosynthesis, sudden change is introduced in protein sequence of the present invention.
Except producing with recombination method, the fragment also available solid phase technique of albumen of the present invention, is produced (people such as Stewart, (1969) Solid phase peptide synthssis, WH Freeman Co., San Francisco by direct peptide synthesis; MerrifieldJ. (1963) J.Am Chem.Soc 85:2149-2154).Synthetic protein can carry out by hand or automatically in vitro.Such as, can with the 431A type peptide synthesizer (Foster City, CA) of Applied Biosystems from dynamic synthetic peptide.Each fragment of chemosynthesis albumen of the present invention can be distinguished, then chemically connected the molecule producing total length.
Utilize Agipanthus growth hormone signal transcription modulin Aux/IAA3 of the present invention, by various conventional screening assays, can filter out and relevant to Agipanthus Aux/IAA3 albumen interactional material occur, or inhibitor and antagonist etc.
Agipanthus ornamental value is high, is widely used, and its scape is tall and straight is excellent fresh cutting flower kind, and be also the agapanthus expressing love except rose, its market requirement is also increasing.The present invention clones the encoding sequence of growth hormone signal transcription modulin Aux/IAA3 in Agipanthus plant materials first, and be transformed in model plant Arabidopis thaliana, adopt physiological effect in Arabidopis thaliana of the methods analyst Aux/IAA3 gene of fluorescence real-time quantitative PCR and expression pattern, for the spatial and temporal expression utilizing genetic engineering technique to regulate and control Aux/IAA3 gene from now on, thus provide theoretical foundation in numerous, breeding of new variety aspect soon for body embryo, there is very large using value.
Accompanying drawing explanation
By reading the detailed description done non-limiting example with reference to the following drawings, other features, objects and advantages of the present invention will become more obvious:
Fig. 1 is Homology search (GAP) result of the nucleotide sequence of Agipanthus Aux/IAA3 gene of the present invention and castor-oil plant Aux/IAA13 gene mRNA;
Fig. 2 is Homology search (FASTA) result of the aminoacid sequence of Agipanthus Aux/IAA3 albumen of the present invention and Musa acuminata Aux/IAA9 albumen, and wherein, identical amino acid marks with amino acid monocase between two sequences.
Fig. 3 is wild-type and Aux/IAA3 transgenic Arabidopsis plants upgrowth situation Phenotypic Observation;
Fig. 4 is wild-type and Aux/IAA3 transgenic arabidopsis Aux/IAA3 Quantitative analysis of gene expression.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.These embodiments are only not used in for illustration of the present invention and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, such as Sambrook equimolecular clone: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.
the clone of embodiment 1, Agipanthus Aux/IAA3 gene
1. the acquisition of vegetable material
Get Agipanthus to grow up seedling leaf tissue, for extracting RNA;
The extracting of 2.RNA
By " RNA prep pure plant total RNA extraction reagent box " extracted total RNA (Trizol:Invitrogen), by the integrity of denaturing formaldehyde gel electrophoresis qualification RNA, then in upper purity and the concentration measuring RNA of spectrophotometer (Thermo Scientific NANODROP1000Spectrophotometer);
3. the full-length clone of gene
According to the protein function annotation result of Agipanthus transcript profile order-checking (RNA-seq), obtain Agipanthus Aux/IAA3 gene core fragment.Adopt RACE method (SMARTer
tMrACE cDNA Amplification Kit:Clonetech) carry out cDNA full-length clone, a point three phases carries out:
(1) RT-PCR obtains gene intermediate segment
The RNA of extraction is carried out reverse transcription (Prime Script II 1st Strand cDNA Synthesis Kit: precious biotechnology (Dalian) company limited), with the first chain cDNA for template, primer Aux/IAA3F (SEQ ID NO.1) and Aux/IAA3R (SEQ ID NO.2) is utilized to carry out PCR, amplification obtains 403bp fragment, reclaim and be connected on pMD19-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) check order on, sequencing result is by carrying out the BLAST existing database of (http://blast.ncbi.nlm.nih.gov/) comparison (GenBank) in NCBI website, know its nucleotide sequence and proteins encoded and known Musa acuminata, nipa palm, the homology of the Aux/IAA3 gene of oil palm is very high, tentatively think that it is an Aux/IAA3 gene,
(2)3′RACE
Two take turns the amplification that nest-type PRC completes 3 ' end sequence.
The first round: UPM+3 '-GSP1 (5 '-AAGATGGAGGGTGTGGCAATAGGGAG-3 ') (SEQ IDNO.5)
Second takes turns: NUP+3 '-GSP2 (5 '-TTACGAGGATGAAGAAGGGGACTGGA-3 ') (SEQ IDNO.6)
UPM and NUP provides for test kit.3 ' RACE obtains the 3 ' end sequence (422bp) of Agipanthus Aux/IAA3, reclaim, be connected on pMD19-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) check order on, sequencing result is by carrying out the BLAST existing database of (http://blast.ncbi.nlm.nih.gov/) comparison (GenBank) in NCBI website, know its nucleotide sequence and proteins encoded and known Musa acuminata, paddy rice, the homology of the Aux/IAA3 gene of oil palm is very high,
(3)5′RACE
With 5 ' RACE ready cDNA for template, take turns by two the amplification that nest-type PRC completes 5 ' end sequence,
The first round: UPM+5 '-GSP 1 (5 '-TCCAGTCCCCTTCTTCATCCTCGTAA-3 ') (SEQ IDNO.7)
Second takes turns: NUP+5 '-GSP2 (5 '-CTCCCTATTGCCACACCCTCCATCTT-3 ') (SEQ IDNO.8)
UPM and NUP provides for test kit.5 ' RACE obtains the 5 ' end sequence (512bp) of Agipanthus Aux/IAA3 gene, reclaim after connecting and check order with the method above, the sequencing result of the sequence obtained by above-mentioned 3 kinds of methods is spliced, to splice sequence submits to BLAST to analyze, result proves that the Aux/IAA3 gene newly obtained from Agipanthus is the gene that a growth hormone transcription inhibitory factor is relevant really, the ORF Finding (http://www.ncbi.nlm.nih.gov/gorf) of sequencing result in conjunction with NCBI is predicted, initiator codon and the terminator codon of Agipanthus Aux/IAA3 gene are found, according to the sequence obtained, respectively from initiator codon and terminator codon design Auele Specific Primer ORF-F (5 '-ATGGAGCTAGAGTTAGGCCTTGCTCTCAAT-3 ') (SEQ ID NO.9), ORF-R (5 '-CTATGCAGGTCTCTCTCGTTTGTTACATCG-3 ') (SEQ ID NO.10), with Agipanthus cDNA for template carries out PCR, amplification obtains the complete encoding sequence (SEQID NO.3) of 549bp Agipanthus Aux/IAA3 albumen.
the sequence information of embodiment 2, Agipanthus Aux/IAA3 gene and homology analysis
The new Agipanthus Aux/IAA3 full length gene opening code-reading frame sequence of the present invention is 549bp, and detailed sequence is shown in sequence shown in SEQ IDNO.3.Derive the aminoacid sequence of Agipanthus Aux/IAA3 albumen according to opening code-reading frame sequence, totally 182 amino-acid residues, molecular weight is 20.73kDa, and iso-electric point (pI) is 6.44, and detailed sequence is shown in sequence shown in SEQ ID NO.4;
The opening code-reading frame sequence of Agipanthus Aux/IAA3 and the aminoacid sequence blast program of proteins encoded thereof are carried out Nucleotide and protein homology search in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR database, found that it and castor-oil plant Aux/IAA13 gene (accession number: XM_002526495.1) have the homogeny of 83% on nucleotide level, (Query: the coding gene sequence of Agipanthus Aux/IAA3 as shown in Figure 1; The mRNA sequence of Sbjct: castor-oil plant Aux/IAA13); On amino acid levels, it and Musa acuminata Aux/IAA9 gene (accession number: XP_009419087.1) also have the consistence of 45% and the similarity of 58%, as shown in Figure 2 (the aminoacid sequence of Query: Agipanthus Aux/IAA3 albumen; The aminoacid sequence of Sbjct: Musa acuminata Aux/IAA9 albumen).As can be seen here, all there is higher homology in the Aux/IAA gene of Agipanthus Aux/IAA3 gene and other known species from nucleic acid or protein level.
embodiment 3, Agipanthus Aux/IAA3 gene transformation model plant Arabidopis thaliana
1. containing the structure of the expression vector of goal gene (Agipanthus Aux/IAA3 gene)
According to Agipanthus Aux/IAA3 full length gene encoding sequence (SEQ ID NO.3), design amplification is at the primer of complete coding reading frame, and on upstream and downstream primer, introduce restriction endonuclease sites (this is determined by the carrier selected) respectively, so that construction of expression vector.With the amplified production obtained in embodiment 1 for template, after pcr amplification, the coding region sequence of Agipanthus Aux/IAA3 gene is connected in intermediate carrier (as pMD19-T) and checks order, again the coding region sequence of Agipanthus Aux/IAA3 gene correct for order-checking is cloned into (as pHB) in expression vector further, (as GV3101) in agrobacterium tumefaciens is proceeded to having identified under the prerequisite that reading frame is correct, and PCR qualification is carried out to the Agrobacterium after transforming, to ensure that the plant expression vector successful conversion containing Agipanthus Aux/IAA3 gene enters in agrobacterium tumefaciens.
2. Agrobacterium-Mediated Transformation Arabidopis thaliana
(1) Agrobacterium is shaken in advance: choose positive monoclonal and contain in the YEP liquid nutrient medium of 50mg/L Kan, 50mg/L gentamicin, 25mg/LRif to 25ml, 28 DEG C, 200rpm shakes bacterium 24h;
(2) spread cultivation Agrobacterium: spread cultivation to containing in 400mL Kan resistance YEP substratum by the Agrobacterium bacterium liquid shaken in advance with 1: 100,28 DEG C, 200rpm, cultivate 13-16h, be cultured to absorbancy OD600 and reach between 1.5-2.0 and receive bacterium, receiving bacterium condition is 23 DEG C, 5000rpm, 8min;
(3) transformed plant: (needing transforming the day before yesterday or transforming the little Hua cutting off angle fruits all on plant the same day and bloomed and show money or valuables one carries unintentionally) prepares the 1/2MS solution of 500mL containing 5% sucrose, with a small amount of MS solution, the Agrobacterium of collecting precipitation is hanged, shake up, Silwet L-77 and the 10 μ L6-BA (mother liquor is 1mg/mL) of 0.04% (v/v) are added in remaining sucrose solution, stir evenly, before conversion, the two is mixed, base of the plant and inflorescence are immersed in 50s in bacterium liquid, taking-up drains bacterium liquid, put into disposable plastic bag, sealing, moisturizing.After all plant transformation, black box on cover, lucifuge cultivates 24h.Take out plant afterwards, erect plants is placed, waters Aquaponic, ensure that plant moisture is sufficient.
3. the screening of transgenic positive strain
Plant after conversion is sowing after angle fruit all maturation, in the desiccation culture ware being lined with filter paper, room temperature is placed one week, make seed all dry, use 50 object stainless steel sift filter seed afterwards, except chamfer fruit, collect transgenosis T0 for seed and be seeded in cave dish in, carry out Resistance of Seedling screening with 0.05% (v/v) glyphosate, obtain T1 for transfer-gen plant, continue screening until obtain T3 for homozygote transfer-gen plant.The phenotype of wild-type and Aux/IAA3 transgenic Arabidopsis plants has significant difference (Fig. 3); Aux/IAA3 growth of transgenic plants is slow and WT lines obviously, illustrates that Aux/IAA3 has negative regulation effect to growth hormone.
4. transgenic Arabidopsis plants Aux/IAA3 gene expression difference
Shear the blade 0.2g of Arabidopis thaliana wild-type and Aux/IAA3 transfer-gen plant, extract RNA, preparation cDNA and carry out Real-time PCR Analysis.In Real-time PCR, the Auele Specific Primer of Aux/IAA3 gene quantification analysis is qAUX/IAA3F (5 '-CAATAACCATATCGCCTATCC-3 ') (SEQ ID NO.11), primer qAUX/IAA3R (5 '-ATCGTCCTCCTTGTCATC-3 ') (SEQ ID NO.12), reference gene is Arabidopis thaliana UBQ5 gene, primer is UBQ5-F (5 '-GACGCTTCATCTCGTCC-3 ') (SEQ ID NO.13), UBQ5-R (5 '-CCACAGGTTGCGTTAG-3 ') (SEQ ID NO.14).Adopt
method makes relative quantitative assay, and result shows that in transgenic arabidopsis, the expression amount of Aux/IAA3 is higher, is reference gene
3.2 times of UBQ5 are wild-type plant 5738 times (Fig. 4).Show that Aux/IAA3 does not express in WT lines.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (7)
1. the protein of one kind following (a) or (b):
A protein that () is made up of the such as aminoacid sequence shown in SEQ ID NO.4;
B the aminoacid sequence shown in () SEQ ID NO.4 passes through replacement, lacks or adds one or several amino acid and has the protein derivative by (a) of Agipanthus Aux/IAA3 protein-active.
2. protein as claimed in claim 1, it is characterized in that, described protein for aminoacid sequence shown in SEQ ID NO.4 is through 1 ~ 50 amino acid whose disappearance, insertion and/or replacement, or within C-terminal and/or N-terminal add 1 ~ 20 amino acid and the sequence that obtains.
3. protein as claimed in claim 2, is characterized in that, described protein for 1 ~ 10 amino acid in aminoacid sequence shown in SEQ ID NO.4 replace by the similar or close amino acid of character and the sequence that formed.
4. nucleic acid sequences to proteins described in a coding claim 1.
5. nucleotide sequence as claimed in claim 4, it is characterized in that, described nucleotide sequence is specially:
A () base sequence is as shown in SEQ ID NO.3 1st ~ 549;
Or the nucleic acid shown in (b) and SEQ ID NO.3 1st ~ 549 has the sequence of the homology of at least 70%;
Or (c) can carry out the sequence of hybridizing with the nucleic acid shown in SEQ ID NO.3 1st ~ 549.
6. nucleotide sequence as claimed in claim 4, it is characterized in that, described nucleotide sequence is specially the disappearance of 1 ~ 90 Nucleotide in the nucleotide sequence shown in SEQ ID NO.3 1st ~ 549, insertion and/or replacement, or 5 ' and/or 3 ' sequence of being formed with inner nucleotide of end interpolation 60.
7. for detecting a probe for nucleotide sequence as claimed in claim 4, it is characterized in that, described probe is the nucleic acid molecule including described nucleotide sequence 8 ~ 100 continuous nucleotides.
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| CN106084021A (en) * | 2016-05-06 | 2016-11-09 | 上海交通大学 | Afriocan agapanthus auxin response factor ApARF1 and encoding gene thereof and probe |
| CN105837670B (en) * | 2016-05-06 | 2019-06-21 | 上海交通大学 | Afriocan agapanthus auxin response factor ApARF2 and its encoding gene and probe |
| CN106084021B (en) * | 2016-05-06 | 2019-06-21 | 上海交通大学 | Afriocan agapanthus auxin response factor ApARF1 and its encoding gene and probe |
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