CN104961814B - Afriocan agapanthus auxin signal transcription modulin Aux/IAA3 and its encoding gene and probe - Google Patents
Afriocan agapanthus auxin signal transcription modulin Aux/IAA3 and its encoding gene and probe Download PDFInfo
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Abstract
The invention discloses a kind of Afriocan agapanthus auxin signal transcription modulin Aux/IAA3 and its encoding gene and probe, protein of the protein for following (a) or (b)：(a) by amino acid sequence forms as shown in SEQ ID NO.4 protein；(b) amino acid sequence shown in SEQ ID NO.4 is by substitution, the protein derived from (a) for lacking or adding one or several amino acid and have Afriocan agapanthus Aux/IAA3 protein actives.The present invention also provides the probes of a kind of nucleic acid sequence of the above-mentioned protein of coding, and the above-mentioned nucleic acid sequence of detection；The present invention is to regulate and control Afriocan agapanthus Auxin Signal Tranducation approach using technique for gene engineering, to achieve the purpose that control its growth and development, organ morphology are built up, provides theoretical foundation for molecular breeding, has prodigious application value.
Description
Technical field
The present invention relates to Afriocan agapanthus auxin signal transcription modulin and its encoding genes and probe, and in particular to a kind of
Afriocan agapanthus auxin signal transcription modulin Aux/IAA3 and its encoding gene and probe.
Background technology
Endogenous hormones develop Growth of Ornamental Plants and fancy points regulation and control play an important role, and auxin is in plant
It is inside a kind of unique endogenous hormones with polar translocation feature.The content of auxin and growth and hair of the distribution relation to plant
Educate speed and the polar structure and spatial shape of each organ-tissue.The signal transduction path of auxin is studied more clear at present
Chu, wherein Aux/IAA are important auxin transcription inhibitory factor, can inhibit turning for auxin when it is combined with auxin
Record reduces growth cellulose content.
It is plant molecular breeding and the most effective technical system of rapid propagation in vitro, existing research that (body embryo), which occurs, for somatic embryo
Show that plant embryonal induction depends on exogenous auxin regulation and control, and exogenous auxin substance picloram is to unifacial leaf napiform root
Flowers body embryo induction tool wholesomeness.Afriocan agapanthus is tropical flowering perennial, and flower amount is big, the florescence is long, ornamental value is high, has underground
Stem tuber tissue.The Afriocan agapanthus body embryo system that we establish early period show auxin signal to the induction of Afriocan agapanthus body embryo, body embryo form,
Body embryo quantity has decisive role with body embryo seedling.In addition, interrupting Polar Transport of Auxin pair hundred using external source regulation and control substance
Sub- lotus flower phase, plant are downgraded, morphology of terminal inflorescence has apparent regulating and controlling effect.Therefore, auxin signal produces Flower Industrialization
It is of crucial importance with breeding improvement work.
The encoding gene of Aux/IAA is cloned from various plants to be come, including：Arabidopsis, rice, corn, grape, castor
Fiber crops etc..But in ornamental plant, especially flowering bulb, the clone of Aux/IAA, expression pattern and protein sequence are still unclear
Chu.Currently, not having any and Afriocan agapanthus Aux/IAA3 albumen and its relevant document report of coding gene sequence.
Invention content
It is an object of the invention to fill up the clone of Afriocan agapanthus Aux/IAA3 genes, expression pattern analysis and Afriocan agapanthus
The blank of Aux/IAA3 albumen, provides a kind of Afriocan agapanthus auxin signal transcription modulin Aux/IAA3, and the present invention also carries
A kind of nucleic acid sequence encoding above-mentioned protein and the probe for detecting the nucleic acid sequence are supplied；The invention discloses Afriocan agapanthus
Physiological effect after Aux/IAA3 genetic transformation arabidopsis and expression pattern, to utilize technique for gene engineering to Aux/IAA3 from now on
The space-time characterisation of gene expression is regulated and controled, and to occur for body embryo, regulation of plant form provides theoretical foundation, has prodigious answer
With value.
In a first aspect, the present invention provides Afriocan agapanthus auxin signal transcription modulin, the protein is by such as SEQ
The protein that amino acid sequence shown in IDNO.4 forms；Or the amino acid sequence shown in SEQ IDNO.4 is by replacing, lacking
Lose or add one or several amino acid and the protein with Afriocan agapanthus auxin signal transcription modulin feature.
Preferably, the protein be SEQ ID NO.4 shown in amino acid sequence by 1~50 amino acid missing,
It is inserted into and/or replaces, or sequence obtained from amino acid within 1~20 is added in C-terminal and/or N-terminal.
It is further preferred that the protein be shown in SEQ ID NO.4 in amino acid sequence 1~10 amino acid by property
The sequence that matter is similar or similar amino acid is replaced and is formed.
Second aspect, the present invention provides a kind of nucleic acid sequences of the above-mentioned protein of coding.
Preferably, the nucleic acid sequence is specially：(a) base sequence is as shown in SEQ ID NO.3 the 1st~549；Or
(b) there is the sequence of at least 70% homology with nucleic acid shown in SEQ ID NO.3 the 1st~549；Or it (c) can be with SEQ ID
The sequence that nucleic acid is hybridized shown in NO.3 the 1st~549.
Preferably, the nucleic acid sequence is specially 1~90 in nucleic acid sequence shown in SEQ ID NO.3 the 1st~549
Missing, insertion and/or the substitution of nucleotide, and in 5 ' and/or 3 ' 60 sequences formed with inner nucleotide of end addition.
The third aspect, the present invention also provides a kind of probes of the above-mentioned nucleic acid sequence of detection, and the probe is with above-mentioned
The nucleic acid molecules of 8~100 continuous nucleotides of nucleic acid sequence, the probe can be used for detecting in sample with the presence or absence of coding Afriocan agapanthus
The relevant nucleic acid molecules of Aux/IAA3.
In the present invention, " DNA of separation ", " DNA of purifying " refer to that the DNA or segment are located under native state
It is separated in the sequence of its both sides, also refers to the DNA or segment and separated with the component with nucleic acid under native state, and
Separated with the protein to accompany in cell.
In the present invention, term " Afriocan agapanthus auxin signal transcription modulin Aux/IAA3 albumen coded sequences " refers to volume
Code has the nucleotide sequence of the polypeptide of Afriocan agapanthus Aux/IAA3 protein actives, the 1st~549 as shown in SEQ ID NO.3
Nucleotide sequence and its degenerate sequence.The degenerate sequence refers to being located at the 1st~549 nucleotide shown in SEQ ID NO.3
In, there are one or multiple codons be encoded same amino acid degenerate codon replace after generation sequence.Due to close
The degeneracy of numeral, so the letter with the 1st~549 nucleotide sequence homology shown in SEQ ID NO.3 down to about 70%
And sequence can also encode out sequence shown in SEQ ID NO.4.The term further includes and nucleotides sequence shown in SEQ ID NO.3
The nucleotide sequence of the homology of row at least 70%.
The term further includes the identical function that can encode natural Afriocan agapanthus Aux/IAA3 albumen, sequence shown in SEQ ID NO.3
The variant form of row.These variant forms include (but being not limited to)：Usually the missing of 1~90 nucleotide, be inserted into and/or
Substitution, and 60 are added to inner nucleotide at 5 ' and/or 3 ' ends.
In the present invention, term " Afriocan agapanthus auxin signal transcription modulin Aux/IAA3 " refers to Afriocan agapanthus Aux/
The polypeptide of sequence shown in the SEQ ID NO.4 of IAA3 protein actives.The term further includes having and natural Afriocan agapanthus Aux/IAA3
Albumen identical function, SEQ ID NO.4 sequences variant form.These variant forms include (but being not limited to)：Usually 1
Missing, insertion and/or the substitution of~50 amino acid, and in C-terminal and/or N-terminal addition one or be ammonia within 20
Base acid.For example, in the art, when being substituted with similar nature or similar amino acid, not usually changing protein
Function.For another example, the function of protein will not be changed by adding one or several amino acid generally also in C-terminal and/or N-terminal.
The term further includes the active fragment and reactive derivative of Afriocan agapanthus Aux/IAA3 albumen.
The variant form of Afriocan agapanthus auxin signal transcription modulin Aux/IAA3 of the present invention includes：Homologous sequence,
Conservative variant, allelic variant, natural mutation, induced mutants, energy and Afriocan agapanthus under high or low high stringency conditions
What the encoded albumen of DNA of Aux/IAA3 correlation DNA hybridizations and the antiserum of utilization Afriocan agapanthus Aux/IAA3 albumen obtained
More peptide or proteins.
In the present invention, " Afriocan agapanthus Aux/IAA3 conservative variation's polypeptides " refer to and amino acid shown in SEQ ID NO.4
Sequence is compared, and has at most 10 amino acid to be replaced by amino acid with similar or analogous properties and form polypeptide.These conservatives
Variant polypeptides are replaced preferably based on table 1 and are generated.
Table 1
| Initial residue | Representative substitution | Preferred substitution |
| 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 further includes the analog of Afriocan agapanthus Aux/IAA3 albumen or polypeptide.These analogs and Afriocan agapanthus Aux/IAA3
The difference of related polypeptide can be the difference on amino acid sequence, can also be the difference on the modified forms for do not influence sequence,
Or have both at the same time.These polypeptides include natural or induction genetic variant.Induction variant can be obtained by various technologies
It arrives, such as generates random mutagenesis by radiating or being exposed to mutagens, can also be given birth to by site-directed mutagenesis or other known moleculars
The technology of object.Analog further includes the analog with the residue (such as D- amino acid) different from natural L-amino acids, and
Analog with non-naturally occurring or synthesis amino acid (such as β, gamma-amino acid).It should be understood that the polypeptide of the present invention is not
It is limited to the above-mentioned representative polypeptide enumerated.
Modification (not changing primary structure usually) form include：The chemical derivative form of in vivo or in vitro polypeptide such as acetyl
Change or carboxylated.Modification further includes glycosylation, is carried out in the synthesis and processing of polypeptide or in further processing step such as those
Glycosylation modified and generation polypeptide.This modification can carry out glycosylated enzyme (such as mammal by the way that polypeptide to be exposed to
Glycosylase or deglycosylation enzyme) and complete.Modified forms further include with phosphorylated amino acid residue (such as phosphoric acid junket ammonia
Acid, phosphoserine, phosphothreonine) sequence.Further include being modified to improve its anti-proteolytic properties or optimization
The polypeptide of solubility property.
Using the Afriocan agapanthus auxin signal transcription modulin Aux/IAA3 of the present invention, pass through various traditional screening methods
Method can filter out the substance to interact related to Afriocan agapanthus Aux/IAA3 albumen or inhibitor and antagonist etc..
With the presence or absence of the detection method of Afriocan agapanthus Aux/IAA3 related nucleotide sequences in present invention detection sample, including with
Above-mentioned probe is hybridized with sample, and then whether detection probe has occurred combination.The sample is the product after PCR amplification,
Wherein PCR amplification primer corresponds to Afriocan agapanthus Aux/IAA3 related nucleosides coding sequences, and can be located at the two of the coded sequence
Side or centre.Primer length is generally 15~50 nucleotide.
In addition, Afriocan agapanthus Aux/IAA3 nucleotide sequences according to the present invention and amino acid sequence, it can be homologous in nucleic acid
Property or expression protein homology basis on, screen Afriocan agapanthus Aux/IAA3 associated homologous gene or homologous protein.
In order to obtain with the dot matrix of Afriocan agapanthus Aux/IAA3 related genes, Afriocan agapanthus cDNA texts can be screened with DNA probe
Library, these probes are used under low high stringency conditions32P does radioactivity mark to the relevant all or parts of Afriocan agapanthus Aux/IAA3
Obtained by note.The cDNA library for being suitable for screening is the library from Afriocan agapanthus.Structure comes from interested cell or tissue
The method of cDNA library be that molecular biology field is well-known.In addition, many such cDNA libraries can also be bought
It arrives, such as purchased from Clontech, Stratagene, Palo Alto, Cal..This screening technique can identify and Afriocan agapanthus
The nucleotide sequence of the relevant gene families of Aux/IAA3.
The present invention Afriocan agapanthus Aux/IAA3 associated nucleotides full length sequence or its segment usually can use PCR amplification method,
Recombination method or artificial synthesized method obtain.For PCR amplification method, can according to related nucleotide sequence disclosed in this invention,
Especially open reading frame sequence carrys out design primer, the commercially available libraries cDNA is used in combination or by routine side well known by persons skilled in the art
The libraries eDNA prepared by method expand as template and obtain related sequence.When sequence is longer, it is often necessary to carry out twice or repeatedly
Then the segment that each time amplifies is stitched together by PCR amplification by proper order again.
After obtaining related sequence, so that it may to obtain related sequence in large quantity with recombination method.This is typically by it
It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.
In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.
Other than being generated with recombination method, solid phase technique also can be used in the segment of albumen of the present invention, by direct synthetic peptide
Produced (Stewart et al., (1969) Solid phase peptide synthssis, WH Freeman Co., San Francisco；
Merrifield J.(1963)J.Am Chem.Soc 85：2149-2154).In vitro synthetic protein can by hand or from
It is dynamic to carry out.For example, dynamic circuit connector can be come from the 431A types peptide synthesizer (Foster City, CA) of Applied Biosystems
At peptide.Each segment of chemical synthesis albumen of the present invention can be distinguished, then chemically connected to generate point of overall length
Son.
Using the Afriocan agapanthus auxin signal transcription modulin Aux/IAA3 of the present invention, pass through various traditional screening methods
Method can filter out the substance to interact related to Afriocan agapanthus Aux/IAA3 albumen or inhibitor and antagonist etc..
Afriocan agapanthus ornamental value is high, is widely used, and it is excellent fresh-cut flower variety that scape is tall and straight, again in addition to rose
The agapanthus of love can be most expressed in addition, and the market demand is also increasing.The present invention clones raw in Afriocan agapanthus plant for the first time
The coded sequence of long element signal transcription modulin Aux/IAA3, and be transformed into model plant arabidopsis, using fluorescence
Physiological effect and expression pattern of the method analysis Aux/IAA3 genes of real-time quantitative PCR in arabidopsis, to utilize base from now on
Because engineering technology regulates and controls the spatial and temporal expression of Aux/IAA3 genes, to provide theory for fast numerous, the breeding of new variety aspect of body embryo
Foundation has prodigious application value.
Description of the drawings
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is the Afriocan agapanthus Aux/IAA3 genes of the present invention and the nucleotide sequence of castor-oil plant Aux/IAA13 gene mRNAs
Homology search (GAP) result；
Fig. 2 is the Afriocan agapanthus Aux/IAA3 albumen of the present invention and the amino acid sequence of Musa acuminata Aux/IAA9 albumen
Homology search (FASTA) result, wherein identical amino acid is marked between two sequences with amino acid monocase.
Fig. 3 is wild type and Aux/IAA3 transgenic Arabidopsis plants upgrowth situation Phenotypic Observations；
Fig. 4 is wild type and Aux/IAA3 transgenic arabidopsis Aux/IAA3 Quantitative analysis of gene expression.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.These embodiments are merely to illustrate the present invention and do not have to
In limiting the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as
Sambrook equimoleculars are cloned：Laboratory manual (New York：Cold Spring Harbor Laboratory Press,
1989) condition described in, or according to the normal condition proposed by manufacturer.
The clone of embodiment 1, Afriocan agapanthus Aux/IAA3 genes
1. the acquisition of vegetable material
Afriocan agapanthus adult seedling leaf tissue is taken, for extracting RNA；
The extracting of 2.RNA
With " RNA prep pure plant total RNA extraction reagents box " extracted total RNA (Trizol：Invitrogen), first is used
Aldehyde is denaturalized the integrality of gel electrophoresis identification RNA, then in spectrophotometer (Thermo Scientific
NANODROP1000Spectrophotometer the purity and concentration of RNA are measured on)；
3. the full-length clone of gene
The protein function annotation of (RNA-seq) is sequenced as a result, obtaining Afriocan agapanthus Aux/IAA3 bases according to Afriocan agapanthus transcript profile
Because of core fragment.Using RACE methods (SMARTerTMRACE cDNA Amplification Kit：Clonetech it) carries out
CDNA full-length clones divide three phases to carry out：
(1) RT-PCR obtains gene intermediate segment
The RNA of extraction is subjected to reverse transcription (Prime Script II 1st Strand cDNA Synthesis Kit：
Precious bioengineering (Dalian) Co., Ltd), using the first chain cDNA as template, using primer Aux/IAA3F (SEQ ID NO.1) and
Aux/IAA3R (SEQ ID NO.2) carries out PCR, and amplification obtains 403bp segments, recycles and be connected to pMD19-T Simple
On vector carriers, use RV-M and M13-47 as universal primer, using termination object fluorescent marker (Big-Dye, Perkin-
Elmer, USA) method, be sequenced on ABI377 sequenators (Perkin-Elmer, USA), sequencing result by
The websites NCBI carry out BLAST (http：//blast.ncbi.nlm.nih.gov/) existing database (GenBank) is compared, know
Its nucleic acid sequence and the homology for encoding albumen and known Musa acuminata, the Aux/IAA3 genes of nipa palm, oil palm are very high, just
Step thinks that it is an Aux/IAA3 gene；
(2)3′RACE
Two wheel nest-type PRCs complete the amplification of 3 ' end sequences.
The first round：UPM+3'-GSP1(5′-AAGATGGAGGGTGTGGCAATAGGGAG-3′)(SEQ ID NO.5)
Second wheel：NUP+3'-GSP2(5′-TTACGAGGATGAAGAAGGGGACTGGA-3′)(SEQ ID NO.6)
UPM and NUP provide for kit.3 ' RACE obtain the 3 ' end sequences (422bp) of Afriocan agapanthus Aux/IAA3, return
It receives, is connected on pMD19-T Simple vector carriers, use RV-M and M13-47 as universal primer, object is glimmering using terminating
The method of signal (Big-Dye, Perkin-Elmer, USA) carries out on ABI377 sequenators (Perkin-Elmer, USA)
Sequencing, sequencing result in the websites NCBI by carrying out BLAST (http：//blast.ncbi.nlm.nih.gov/) compare have
Database (GenBank), know its nucleic acid sequence and encode albumen and known Musa acuminata, rice, oil palm Aux/
The homology of IAA3 genes is very high；
(3)5′RACE
Using 5 ' RACE ready cDNA as template, the amplification of 5 ' end sequences is completed by two wheel nest-type PRCs,
The first round：UPM+5'-GSP 1(5′-TCCAGTCCCCTTCTTCATCCTCGTAA-3′)(SEQ ID NO.7)
Second wheel：NUP+5'-GSP2(5′-CTCCCTATTGCCACACCCTCCATCTT-3′)(SEQ ID NO.8)
UPM and NUP provide for kit.5 ' RACE obtain the 5 ' end sequences (512bp) of Afriocan agapanthus Aux/IAA3 genes,
Recycling connection after be sequenced with method as above, by the sequencing result of the sequence obtained by above-mentioned 3 kinds of methods into
Splicing sequence is submitted BLAST analyses by row splicing, and it is one as a result to prove the Aux/IAA3 genes newly obtained from Afriocan agapanthus really
A relevant gene of auxin transcription inhibitory factor, by the ORF Finding (http of sequencing result combination NCBI：//
Www.ncbi.nlm.nih.gov/gorf it) predicts, it was found that the initiation codon and termination codon of Afriocan agapanthus Aux/IAA3 genes
Son designs specific primer ORF-F (5 '-from initiation codon and terminator codon respectively according to the sequence of acquisition
ATGGAGCTAGAGTTAGGCCTTGCTCTCAAT-3 ') (SEQ ID NO.9), ORF-R (5 '-
CTATGCAGGTCTCTCTCGTTTGTTACATCG-3 ') (SEQ ID NO.10), PCR is carried out by template of Afriocan agapanthus cDNA, is expanded
Increasing obtains the complete encoding sequence (SEQ ID NO.3) of 549bp Afriocan agapanthus Aux/IAA3 albumen.
The sequence information and homology analysis of embodiment 2, Afriocan agapanthus Aux/IAA3 genes
The new Afriocan agapanthus Aux/IAA3 full length gene opening code-reading frame sequences of the present invention are 549bp, and detailed sequence is shown in SEQ
Sequence shown in ID NO.3.The amino acid sequence of Afriocan agapanthus Aux/IAA3 albumen is derived according to opening code-reading frame sequence, totally 182
A amino acid residue, molecular weight 20.73kDa, isoelectric point (pI) are 6.44, and detailed sequence is shown in sequence shown in SEQ ID NO.4；
The opening code-reading frame sequence of Afriocan agapanthus Aux/IAA3 and its amino acid sequence blast program for encoding albumen are existed
Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+
Nucleotide and protein homology search are carried out in PDB+SwissProt+Superdate+PIR databases, as a result, it has been found that it with
Castor-oil plant Aux/IAA13 gene (accession number：XM_002526495.1) with the 83% phase same sex, such as Fig. 1 on nucleotide level
Shown (Query：The coding gene sequence of Afriocan agapanthus Aux/IAA3；Sbjct：The mRNA sequence of castor-oil plant Aux/IAA13)；In amino
On sour water is flat, it and Musa acuminata Aux/IAA9 gene (accession number：XP_009419087.1) also have 45% consistency and
58% similitude, (Query as shown in Figure 2：The amino acid sequence of Afriocan agapanthus Aux/IAA3 albumen；Sbjct：Musa acuminata
The amino acid sequence of Aux/IAA9 albumen).It can be seen that the Aux/IAA of Afriocan agapanthus Aux/IAA3 genes and other known species
No matter higher homology all there is from nucleic acid or protein level in gene.
Embodiment 3, Afriocan agapanthus Aux/IAA3 genetic transformation model plant arabidopsis
1. the structure of the expression vector containing target gene (Afriocan agapanthus Aux/IAA3 genes)
According to Afriocan agapanthus Aux/IAA3 full length genes coded sequence (SEQ ID NO.3), design amplification is read in complete coding
The primer of frame, and introduce restriction endonuclease sites (this can be depending on the carrier selected) respectively in upstream and downstream primer, with
Just construction of expression vector.Using the amplified production obtained in embodiment 1 as template, after PCR amplification, by Afriocan agapanthus Aux/IAA3 bases
The coding region sequence of cause is connected in intermediate carrier (such as pMD19-T) and is sequenced, then correct Afriocan agapanthus Aux/ will be sequenced
The coding region sequence of IAA3 genes is further cloned into expression vector (such as pHB), under the premise of having identified that reading frame is correct
It is transferred in Agrobacterium tumefaciems (such as GV3101), and PCR identifications is carried out to the Agrobacterium after conversion, to ensure to contain Afriocan agapanthus
The plant expression vector successful conversion of Aux/IAA3 genes enters in Agrobacterium tumefaciems.
2. Agrobacterium-Mediated Transformation arabidopsis
(1) Agrobacterium is shaken in advance：Positive monoclonal is chosen to 25ml Kan containing 50mg/L, 50mg/L gentamicins, 25mg/L
In the YEP fluid nutrient mediums of Rif, 28 DEG C, 200rpm shakes bacterium for 24 hours；
(2) spread cultivation Agrobacterium：The Agrobacterium bacterium solution shaken in advance is spread cultivation with 1: 100 to the resistance YEP culture mediums of Kan containing 400mL
In, 28 DEG C, 200rpm, 13-16h is cultivated, bacterium is received in culture between reaching 1.5-2.0 to absorbance OD600, it is 23 to receive bacterium condition
DEG C, 5000rpm, 8min；
(3) transformed plant：(need to cut off on the day of conversion the previous day or conversion silique all on plant and it is in full bloom with
And the little Hua to show money or valuables one carries unintentionally) the 1/2MS solution that 500mL contains 5% sucrose is prepared, the Agrobacterium of collection is precipitated with a small amount of MS solution and is hanged
It rises, shakes up, the Silwet L-77 and 10 μ L6-BA (mother liquor 1mg/ of 0.04% (v/v) are added into remaining sucrose solution
ML), stir evenly, by the two mixing before conversion, base of the plant and inflorescence are immersed in 50s in bacterium solution, taking-up drains bacterium solution, is put into
In disposable plastic bag, sealing, moisturizing.After by all plant transformations, flight data recorder on cover is protected from light culture for 24 hours.It takes out later
Plant places erect plants, pours Aquaponic, ensures that plant moisture is sufficient.
3. the screening of transgenic positive strain
Plant after conversion sowing after silique is all ripe, one is placed at room temperature in the desiccation culture ware for being lined with filter paper
Week keep seed all dry, use the stainless steel of 50 mesh to sieve filter seed later, remove silique, collection transgenosis T0 for seed simultaneously
It is seeded in hole tray, Resistance of Seedling screening is carried out with 0.05% (v/v) glyphosate, obtain T1 for transfer-gen plant, lasting screening
Until obtaining T3 for homozygote transfer-gen plant.Wild type and the phenotype of Aux/IAA3 transgenic Arabidopsis plants have conspicuousness
Difference (Fig. 3)；Aux/IAA3 growth of transgenic plants obviously slowly and WT lines, illustrates that Aux/IAA3 has auxin
Negative regulation acts on.
4. transgenic Arabidopsis plants Aux/IAA3 gene expression differences
The blade 0.2g of arabidopsis wild type and Aux/IAA3 transfer-gen plants is sheared, extraction RNA, cDNA is prepared and carries out
Real-time PCR Analysis.The specific primer that Aux/IAA3 gene quantifications are analyzed in Real-time PCR is qAUX/IAA3F
(5 '-CAATAACCATATCGCCTATCC-3 ') (SEQ ID NO.11), primer qAUX/IAA3R (5 '-
ATCGTCCTCCTTGTCATC-3 ') (SEQ ID NO.12), reference gene is arabidopsis UBQ5 genes, primer UBQ5-F
(5 '-GACGCTTCATCTCGTCC-3 ') (SEQ ID NO.13), UBQ5-R (5 '-CCACAGGTTGCGTTAG-3 ') (SEQ ID
NO.14).UsingMethod makees relative quantitative assay, the results showed that and the expression quantity of Aux/IAA3 is higher in transgenic arabidopsis,
It is reference gene
3.2 times of UBQ5 are 5738 times (Fig. 4) of wild-type plant.Show Aux/IAA3 not in WT lines
Expression.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (3)
1. a kind of protein of following (a)：
(a) by amino acid sequence forms as shown in SEQ ID NO.4 protein.
2. the nucleic acid of protein described in a kind of coding claim 1.
3. nucleic acid as claimed in claim 2, characterized in that the sequence of the nucleic acid is specially：Base sequence such as SEQ ID
Shown in NO.3 the 1st~549.
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