CN105085642B - Afriocan agapanthus auxin signal transcription modulin Aux/IAA2 and its encoding gene and probe - Google Patents
Afriocan agapanthus auxin signal transcription modulin Aux/IAA2 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/IAA2 and its encoding gene and probe, protein of the protein for following (a) or (b)：(a) protein being made of the amino acid sequence as shown in SEQ ID NO.4；(b) amino acid sequence shown in SEQ ID NO.4 is by replacing, lacking or add one or several amino acid and with the protein as derived from (a) of Afriocan agapanthus Aux/IAA2 protein active.The present invention also provides a kind of nucleic acid sequences for encoding above-mentioned protein, and the probe of 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 very big 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/IAA2 and its encoding gene and probe.
Background technique
Endogenous hormones develop Growth of Ornamental Plants and fancy points regulation plays an important role, and auxin is in plant
It is inside a kind of unique endogenous hormones with polar translocation feature.The content and growth and hair of the distribution relation to plant of auxin
Educate the polar structure and spatial shape of speed and each organ-tissue.The signal transduction path of auxin is studied more clear at present
Chu, wherein Aux/IAA is important auxin transcription inhibitory factor, can inhibit turning for auxin when it is in conjunction 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 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 and body embryo seedling have decisive role.In addition, interrupting Polar Transport of Auxin to 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 there is any document report relevant to Afriocan agapanthus Aux/IAA albumen and its coding gene sequence.
Summary of the invention
It is an object of the invention to fill up the clone of Afriocan agapanthus Aux/IAA2 gene, expression pattern analysis and Afriocan agapanthus
The blank of Aux/IAA2 albumen, provides a kind of Afriocan agapanthus auxin signal transcription modulin Aux/IAA2, and the present invention also mentions
A kind of nucleic acid sequence for encoding above-mentioned protein and the probe for detecting the nucleic acid sequence are supplied；The invention discloses Afriocan agapanthus
Physiological effect and expression pattern after Aux/IAA2 genetic transformation arabidopsis, for from now on using technique for gene engineering to Aux/IAA2
The space-time characterisation of gene expression is regulated and controled, to provide theoretical foundation for body embryo generation, regulation of plant form, has very big 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 of the composition of amino acid sequence shown in ID NO.4；Or the substitution of the process of the amino acid sequence as shown in SEQ ID NO.4,
Lack 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,
Insertion and/or substitution, 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 is 1~10 amino acid in amino acid sequence shown in SEQ ID NO.4 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 for encoding above-mentioned protein.
Preferably, the nucleic acid sequence is specially：(a) base sequence is as shown in SEQ ID NO.3 the 1st~480；Or
(b) there is the sequence of at least 70% homology with nucleic acid shown in SEQ ID NO.3 the 1st~480；Or it (c) can be with SEQ ID
The sequence that nucleic acid shown in NO.3 the 1st~480 is hybridized.
Preferably, the nucleic acid sequence is specially 1~90 in nucleic acid sequence shown in SEQ ID NO.3 the 1st~480
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 probe for detecting above-mentioned nucleic acid sequence, the probe is with above-mentioned
The nucleic acid molecules of 8~100 continuous nucleotides of nucleic acid sequence, the probe can be used in test sample with the presence or absence of coding Afriocan agapanthus
The relevant nucleic acid molecules of Aux/IAA2.
In the present invention, " isolated DNA ", " DNA of purifying " refer to that the DNA or segment are located under native state
It is separated in the sequence of its two sides, also refers to that the DNA or segment are separated with the component under native state with nucleic acid, and
Separated with the protein to accompany in cell.
In the present invention, term " Afriocan agapanthus auxin signal transcription modulin Aux/IAA2 albumen coded sequence " refers to volume
Code has the nucleotide sequence of the polypeptide of Afriocan agapanthus Aux/IAA2 protein active, the 1st~480 as shown in SEQ ID NO.3
Nucleotide sequence and its degenerate sequence.The degenerate sequence refers to, is located at the 1st~480 nucleotide shown in SEQ ID NO.3
In, the sequence of generation after thering are one or more codons to be encoded replaced the degenerate codon of same amino acid.Due to close
The degeneracy of numeral, so the letter with the 1st~480 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 column at least 70%.
The term further includes the identical function that can encode natural Afriocan agapanthus Aux/IAA2 albumen, sequence shown in SEQ ID NO.3
The variant form of column.These variant forms include (but being not limited to)：Usually the missing of 1~90 nucleotide, insertion and/or
Replace, and is added to 60 at 5 ' and/or 3 ' ends with inner nucleotide.
In the present invention, term " Afriocan agapanthus auxin signal transcription modulin Aux/IAA2 " refers to Afriocan agapanthus Aux/
The polypeptide of sequence shown in the SEQ ID NO.4 of IAA2 protein active.The term further includes having and natural Afriocan agapanthus Aux/IAA2
Albumen identical function, SEQ ID NO.4 sequence 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/IAA2 albumen.
The variant form of Afriocan agapanthus auxin signal transcription modulin Aux/IAA2 of the 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/IAA2 correlation DNA hybridization and the antiserum of utilization Afriocan agapanthus Aux/IAA2 albumen obtained
More peptide or proteins.
In the present invention, " Afriocan agapanthus Aux/IAA2 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 | It is preferred to replace |
| 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/IAA2 albumen or polypeptide.These analogs and Afriocan agapanthus Aux/IAA2
The difference of related polypeptide can be the difference on amino acid sequence, be also possible to not influence the difference on the modified forms of 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 pass through site-directed mutagenesis or other known moleculars are raw
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 polypeptide of the 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 internal or external 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.It further include being modified to improve its anti-proteolytic properties or optimization
The polypeptide of solubility property.
In the present invention, the expression of the method analysis Afriocan agapanthus Aux/IAA2 gene product of real-time fluorescence quantitative PCR can be used
Mode, i.e. presence or absence and quantity of the mRNA transcript of analysis Afriocan agapanthus Aux/IAA2 gene in cell.
With the presence or absence of the detection method of Afriocan agapanthus Aux/IAA2 related nucleotide sequences in test sample of the present invention, 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/IAA2 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/IAA2 nucleotide sequence 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/IAA2 associated homologous gene or homologous protein.
In order to obtain with the dot matrix of Afriocan agapanthus Aux/IAA2 related gene, Afriocan agapanthus cDNA text can be screened with DNA probe
Library, these probes are used under low high stringency conditions32P does radioactivity mark to the relevant all or part of Afriocan agapanthus Aux/IAA2
Obtained by note.The cDNA library for being suitable for screening is the library from Afriocan agapanthus.Building 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 family of Aux/IAA2.
Afriocan agapanthus Aux/IAA2 associated nucleotide full length sequence of the invention or its segment usually can with PCR amplification method,
Recombination method or artificial synthesized method obtain.For PCR amplification method, can disclosed related nucleotide sequence according to the present invention,
Especially open reading frame sequence carrys out design primer, and with the commercially available library cDNA or presses routine side well known by persons skilled in the art
The library cDNA prepared by method expands as template and obtains 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 obtain related sequence in large quantity with recombination method.This is usually 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 is also can be used in the segment of albumen of the present invention, passes through 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).Synthetic proteins matter can in vitro
To carry out by hand or automatically.For example, the 431A type peptide synthesizer of Applied Biosystems can be used
(Foster City, CA) is automatically synthesized peptide.Each segment of chemical synthesis albumen of the present invention can be distinguished, then use chemistry side
Method is connected to generate the molecule of overall length.
Using Afriocan agapanthus auxin signal transcription modulin Aux/IAA2 of the invention, pass through various traditional screening methods
Method can filter out the substance to interact related to Afriocan agapanthus Aux/IAA2 albumen or inhibitor and antagonist etc..
Afriocan agapanthus ornamental value is high, is widely used, and tall and straight scape is excellent fresh-cut flower variety, 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/IAA2, and be transformed into model plant arabidopsis, using fluorescence
Physiological effect and expression pattern of the method analysis Aux/IAA2 gene of real-time quantitative PCR in arabidopsis, to utilize base from now on
Because of the spatial and temporal expression of engineering technology regulation Aux/IAA2 gene, to provide theory in terms of for the fast numerous, breeding of new variety of body embryo
Foundation has very big application value.
Detailed description of the invention
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 nucleotide sequence of Afriocan agapanthus Aux/IAA2 gene and potato Aux/IAA13 gene mRNA of the invention
Homology search (GAP) result；
Fig. 2 is the homologous of Afriocan agapanthus Aux/IAA2 albumen of the invention and the amino acid sequence of nipa palm Aux/IAA22 albumen
Compare (FASTA) result, wherein identical amino acid is marked between two sequences with amino acid monocase.
Fig. 3 is wild type and Aux/IAA2 transgenic Arabidopsis plants upgrowth situation Phenotypic Observation；
Fig. 4 is wild type and Aux/IAA2 transgenic arabidopsis Aux/IAA2 Quantitative analysis of gene expression.
Specific embodiment
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
The molecular clonings such as Sambrook：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/IAA2 gene
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 reagent box " extracted total RNA (Trizol：Invitrogen), first is used
Aldehyde is denaturalized the integrality of gel electrophoresis identification RNA, then in spectrophotometer (Thermo Scientific NANODROP 1000
Spectrophotometer 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/IAA2 base according to Afriocan agapanthus transcript profile
Because of core fragment.Using RACE method (SMARTerTMRACE cDNA Amplification Kit：Clonetech it) carries out
CDNA full-length clone divides 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, utilize primer Aux/IAA2 F (SEQ ID NO.1)
PCR is carried out with Aux/IAA2 R (SEQ ID NO.2), amplification obtains 254bp segment, recycles and be connected to pMD19-T Simple
On vector carrier, use RV-M and M13-47 as universal primer, using termination object fluorescent marker (Big-Dye, Perkin-
Elmer, USA) method, be sequenced on ABI377 sequenator (Perkin-Elmer, USA), sequencing result by
The website NCBI carries out BLAST (http://blast.ncbi.nlm.nih.gov/) existing database (GenBank) is compared, know
Its nucleic acid sequence and coding albumen and known oil palm, nipa palm, Musa acuminata Aux/IAA gene homology it is very high, just
Step thinks that it is an Aux/IAA gene；
(2)3′RACE
Two wheel nest-type PRCs complete the amplification of 3 ' end sequences.
The first round：UPM+3'-GSP1(5′-GGTCAAGGTCAACCACGCAGGAAAAT-3′)(SEQ ID NO.5)
Second wheel：NUP+3'-GSP2(5′-GAGGACAAAGATGGGGATTGGATGCT-3′)(SEQ ID NO.6)
UPM and NUP provide for kit.3 ' RACE obtain the 3 ' end sequences (475 bp) of Afriocan agapanthus Aux/IAA2, return
It receives, is connected on pMD19-T Simple vector carrier, 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 sequenator (Perkin-Elmer, USA)
Sequencing, sequencing result is by carrying out BLAST (http in the website NCBI://blast.ncbi.nlm.nih.gov/) compare it is existing
Database (GenBank), know its nucleic acid sequence and encode albumen and known Musa acuminata, rice, oil palm Aux/IAA
The homology of gene 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'-GSP1(5′-AGCATCCAATCCCCATCTTTGTCCTC-3′)(SEQ ID NO.7)
Second wheel：NUP+5'-GSP2(5′-GGTTGACCTTGACCGTCCTTGCTTTA-3′)(SEQ ID NO.8)
UPM and NUP provide for kit.5 ' RACE obtain the 5 ' end sequences (360bp) of Afriocan agapanthus Aux/IAA2 gene,
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 analysis, as a result proves that the Aux/IAA2 gene newly obtained from Afriocan agapanthus is one really by row splicing
The relevant gene of a 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/IAA2 gene
Son designs specific primer ORF-F (5 '-according to the sequence of acquisition from initiation codon and terminator codon respectively
ATGAGAGATCCAATGGAAGGCGAAAC-3 ') (SEQ ID NO.9), ORF-R (5 '-
TTACCCCCATGGAACATCTCCAACA-3 ') (SEQ ID NO.10), PCR is carried out by template of Afriocan agapanthus cDNA, is expanded
To the complete encoding sequence (SEQ ID NO.3) of 480bp Afriocan agapanthus Aux/IAA2 albumen.
The sequence information and homology analysis of embodiment 2, Afriocan agapanthus Aux/IAA2 gene
The new Afriocan agapanthus Aux/IAA2 full length gene opening code-reading frame sequence of the present invention is 480bp, and detailed sequence is shown in SEQ
Sequence shown in ID NO.3.The amino acid sequence of Afriocan agapanthus Aux/IAA2 albumen is derived according to opening code-reading frame sequence, totally 159
A amino acid residue, molecular weight 18.07kDa, isoelectric point (pI) are 6.15, and detailed sequence is shown in sequence shown in SEQ ID NO.4；
The opening code-reading frame sequence of Afriocan agapanthus Aux/IAA2 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 database, as a result, it has been found that it with
Potato Aux/IAA13 gene (accession number：XM_006342346.1) there is on nucleotide level the 96% phase same sex, such as scheme
(Query shown in 1：The coding gene sequence of Afriocan agapanthus Aux/IAA2；Sbjct：The mRNA sequence of potato Aux/IAA13)；?
On amino acid levels, it and nipa palm Aux/IAA22 gene (accession number：XP_008788132.1) also have 61% consistency and
68% similitude, (Query as shown in Figure 2：The amino acid sequence of Afriocan agapanthus Aux/IAA2 albumen；Sbjct：Nipa palm Aux/
The amino acid sequence of IAA22 albumen).It can be seen that the Aux/IAA gene of Afriocan agapanthus Aux/IAA2 gene and other known species
No matter higher homology all there is from nucleic acid or protein level.
Embodiment 3, Afriocan agapanthus Aux/IAA2 genetic transformation model plant arabidopsis
1. the building of the expression vector containing target gene (Afriocan agapanthus Aux/IAA2 gene)
According to Afriocan agapanthus Aux/IAA2 full length gene 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 of selection) 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/IAA2 base
The coding region sequence of cause is connected in intermediate vector (such as pMD19-T) and is sequenced, then correct Afriocan agapanthus Aux/ will be sequenced
The coding region sequence of IAA2 gene 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 identification is carried out to the Agrobacterium after conversion, to guarantee to contain Afriocan agapanthus
The plant expression vector successful conversion of Aux/IAA2 gene 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 gentamicin, 25mg/L
In the YEP fluid nutrient medium 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 medium of Kan containing 400mL
In, 28 DEG C, 200rpm, 13-16h is cultivated, culture reaches to absorbance OD600 receives bacterium between 1.5-2.0, receiving bacterium condition is 23
DEG C, 5000rpm, 8min；
(3) transformed plant：(need before conversion one day or the conversion same day cut off 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 L 6-BA (mother liquor 1mg/ of 0.04% (v/v) are added into remaining sucrose solution
ML), stir evenly, before conversion mix the two, 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, guarantees that plant moisture is sufficient.
3. the screening of transgenic positive strain
Plant after conversion sowing after silique is all mature, is placed at room temperature for one in the desiccation culture ware for being lined with filter paper
Week keep seed all dry, sieve filter seed with the stainless steel of 50 mesh later, remove silique, collection transgenosis T0 for seed simultaneously
It is seeded in hole tray, carries out Resistance of Seedling screening with 0.05% (v/v) glyphosate, obtain T1 for transgenic plant, lasting screening
Until obtaining T3 for homozygote transgenic plant.Wild type and the phenotype of Aux/IAA2 transgenic Arabidopsis plants have conspicuousness
Difference (Fig. 3)；Aux/IAA2 growth of transgenic plants obviously slowly and WT lines, illustrates that Aux/IAA2 has auxin
Negative regulation effect.
4. transgenic Arabidopsis plants Aux/IAA2 gene expression difference
The blade 0.2g of arabidopsis wild type and Aux/IAA2 transgenic plant is sheared, RNA, preparation cDNA are extracted and is carried out
Real-time PCR Analysis.The specific primer that Aux/IAA2 gene quantification is analyzed in Real-time PCR is qAUX/IAA2 F
(5 '-GACGATTGTGGTGTTGAT-3 ') (SEQ ID NO.11), primer qAUX/IAA2 R (5 '-
CCATTGGAGCAAGTTCTT-3 ') (SEQ ID NO.12, reference gene are arabidopsis UBQ5 gene, and primer is UBQ5-F (5 '-
GACGCTTCATCTCGTCC-3 ') (SEQ ID NO.13), UBQ5-R (5 '-CCACAGGTTGCGTTAG-3 ') (SEQ ID
NO.14).Using 2-ΔΔCtMethod makees relative quantitative assay, the results showed that the table of Aux/IAA2 in transgenic arabidopsis
It is higher up to measuring, it is 3.4 times of reference gene UBQ5, is 5838 times (Fig. 4) of wild-type plant.Show Aux/IAA2
Do not expressed in WT lines.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to 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 substantive content of the invention.
Claims (3)
1. a kind of protein of following (a)：
(a) protein being made of the amino acid sequence as shown in SEQ ID NO.4.
2. a kind of nucleic acid of protein described in 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~480.
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