CN105837670B - Agativin auxin response factor ApARF2 and its encoding gene and probe - Google Patents
Agativin auxin response factor ApARF2 and its encoding gene and probe Download PDFInfo
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- C07K—PEPTIDES
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- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8291—Hormone-influenced development
- C12N15/8294—Auxins
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- 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
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Abstract
The present invention relates to a kind of Afriocan agapanthus auxin response factor ApARF2 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 ApARF2 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
Afriocan agapanthus auxin response factor ApARF2 and its encoding gene and probe of the present invention, and in particular to a kind of Afriocan agapanthus
Auxin response factor ApARF2 and its encoding gene and probe.
Background technique
Hormone develops Growth of Ornamental Plants and fancy points regulation plays an important role, and auxin is in plant
A kind of unique endogenous hormones with polar translocation feature.The content of auxin and the growth and development of distribution relation to plant are fast
The polar structure and spatial shape of degree and each organ-tissue.The signal transduction path of auxin is studied more clear at present,
Middle auxin response factor (ARF) is the transcription factor of a kind of regulation auxin responsive genes expression, is passed in the signal of auxin
In center during leading, it can specifically be tied with the auxin response element in auxin responsive genes promoter region
It closes, promotion or the expression of suppressor.
Afriocan agapanthus is tropical flowering perennial, and flower amount is big, the florescence is long, ornamental value is high, has underground stem tuber tissue.We
The Afriocan agapanthus body embryo system that early period establishes shows auxin signal to the induction of Afriocan agapanthus body embryo, body embryo form, body embryo quantity and body
Embryo seedling has decisive role.In addition, interrupting Polar Transport of Auxin to Afriocan agapanthus florescence, plant using external source regulation and control substance
It downgrades, morphology of terminal inflorescence has apparent regulating and controlling effect.Therefore, auxin signal works to Flower Industrialization production with breeding improvement
It is of crucial importance.
The encoding gene of ARF is cloned from various plants to be come, comprising: arabidopsis, rice, corn, grape, castor-oil plant
Deng.But in ornamental plant, especially flowering bulb, the clone of ARF, expression pattern and protein sequence are unclear.Currently,
There is not any document report relevant to Afriocan agapanthus ARF albumen and its coding gene sequence.
Summary of the invention
In view of the drawbacks of the prior art, the purpose of the present invention is to provide a kind of Afriocan agapanthus auxin response factor ApARF2
And its encoding gene and probe, purpose also reside in the clone for filling up Afriocan agapanthus auxin response factor ApARF2 gene, expression mould
The blank of formula analysis and Afriocan agapanthus ApARF2 albumen, provides a kind of Afriocan agapanthus ApARF2 albumen, the present invention also provides one
Kind encodes the nucleic acid sequence of above-mentioned protein and the probe of the detection nucleic acid sequence；The invention discloses Afriocan agapanthus ApARF2
Physiological effect and expression pattern after genetic transformation arabidopsis, for from now on using technique for gene engineering to ApARF2 gene expression
Space-time characterisation is regulated and controled, to provide theoretical foundation for body embryo generation, regulation of plant form, has very big application value.
In a first aspect, the present invention provides a kind of (a) as follows or protein (b):
(a) protein of the composition of the amino acid sequence as shown in SEQ ID NO.4；
(b) amino acid sequence shown in SEQ ID NO.4 through substitution, lack or add one or several amino acid and
The protein as derived from (a) with Afriocan agapanthus ApARF2 protein active.
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 specifically: (a) base sequence is as shown in SEQ ID NO.3 the 1st~2349；Or
(b) there is the sequence of at least 70% homology with nucleic acid shown in SEQ ID NO.3 the 1st~2349；Or it (c) can be with SEQ ID
The sequence that nucleic acid shown in NO.3 the 1st~2349 is hybridized.
Preferably, the nucleic acid sequence is specially 1~90 in nucleic acid sequence shown in SEQ ID NO.3 the 1st~2349
Missing, insertion and/or the substitution of a 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 auxin response factor ApARF2.
Fourth aspect, a kind of specific primer pair of expansion in said nucleic acid sequences, such as SEQ ID NO.9 and SEQ ID
Shown in NO.10.
5th aspect, the present invention also provides a kind of application of nucleic acid sequence in the expression of regulating plant growth element.
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 response factor ApARF2 albumen coded sequence " refers to that coding has the ancient philosophers
The nucleotide sequence of the polypeptide of lotus ApARF2 protein active, the 1st~2349 nucleotide sequence as shown in SEQ ID NO.3 and
Its degenerate sequence.The degenerate sequence refers to, is located in the 1st~2349 nucleotide shown in SEQ ID NO.3, there is one or more
The sequence that a codon generates after being encoded replaced the degenerate codon of same amino acid.Due to the degeneracy of codon,
So can also be compiled with the 1st~2349 nucleotide sequence homology shown in SEQ ID NO.3 down to about 70% degenerate sequence
Code goes out sequence shown in SEQ ID NO.4.The term further include with the homology of nucleotide sequence shown in SEQ ID NO.3 extremely
Few 70% nucleotide sequence.
The term further includes identical function, the SEQ ID that can encode natural Afriocan agapanthus auxin response factor ApARF2 albumen
The variant form of sequence shown in NO.3.These variant forms include (but being not limited to): being usually lacking for 1~90 nucleotide
It loses, be inserted into and/or replace, and be added to 60 at 5 ' and/or 3 ' ends with inner nucleotide.
In the present invention, term " Afriocan agapanthus auxin response factor ApARF2 " refers to Afriocan agapanthus ApARF2 protein active
SEQ ID NO.4 shown in sequence polypeptide.The term further include with natural Afriocan agapanthus ApARF2 albumen identical function,
The variant form of SEQ ID NO.4 sequence.These variant forms include (but being not limited to): being usually 1~50 amino acid
Missing, insertion and/or substitution, and in C-terminal and/or N-terminal addition one or be amino acid within 20.For example, at this
In field, when being substituted with similar nature or similar amino acid, the function of protein is not usually changed.For another example, in C
End and/or N-terminal, which add one or several amino acid generally also, will not change the function of protein.The term further includes the ancient philosophers
The active fragment and reactive derivative of lotus ApARF2 albumen.
The variant form of Afriocan agapanthus auxin response factor ApARF2 of the invention includes: homologous sequence, conservative variation
Body, allelic variant, natural mutation, induced mutants, can be related to Afriocan agapanthus ApARF2 under high or low high stringency conditions
The encoded albumen of the DNA of DNA hybridization and more peptide or proteins of the antiserum acquisition using Afriocan agapanthus ApARF2 albumen.
In the present invention, " Afriocan agapanthus ApARF2 conservative variation's polypeptides " refer to and amino acid sequence shown in SEQ ID NO.4
Column are compared, and have at most 10 amino acid to be replaced by amino acid with similar or analogous properties and form polypeptide.These conservatives become
Different polypeptide is replaced preferably based on table 1 and is generated.
Table 1
Invention further includes the analog of Afriocan agapanthus ApARF2 albumen or polypeptide.These analogs are related to Afriocan agapanthus ApARF2
The difference of 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, such as
Random mutagenesis is generated and radiating or being exposed to mutagens, can also pass through site-directed mutagenesis or other known molecular biology
Technology.Analog further includes the analog with the residue (such as D- amino acid) different from natural L-amino acids, and is had non-
The analog of natural or synthetic amino acid (such as β, gamma-amino acid).It should be understood that polypeptide of the invention is not limited to
State the representative polypeptide enumerated.
Modification (not changing primary structure usually) form includes: the chemical derivative form such as acetyl of internal or external polypeptide
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.
It in the present invention, can be with the method analysis Afriocan agapanthus ApARF2 gene of real-time fluorescence quantitative PCR in arabidopsis
Physiological effect and expression pattern, the i.e. protein function of analysis Afriocan agapanthus ApARF2 gene coding.
With the presence or absence of the detection method of Afriocan agapanthus ApARF2 related nucleotide sequences in test sample of the present invention, including with
The probe stated is hybridized with sample, and then whether detection probe has occurred combination.The sample is the product after PCR amplification,
Middle PCR amplification primer correspond to Afriocan agapanthus ApARF2 related nucleosides coding sequences, and can be located at the coded sequence two sides or
It is intermediate.Primer length is generally 15~50 nucleotide.
In addition, Afriocan agapanthus ApARF2 nucleotide sequence according to the present invention and amino acid sequence, it can be in nucleic acid homology
Or on the homology basis of expression protein, screen Afriocan agapanthus ApARF2 associated homologous gene or homologous protein.
In order to obtain with the dot matrix of Afriocan agapanthus ApARF2 related gene, Afriocan agapanthus cDNA library can be screened with DNA probe,
These probes are used under low high stringency conditions32P does radioactivity label to the relevant all or part of Afriocan agapanthus ApARF2 and obtains
's.The cDNA library for being suitable for screening is the library from Afriocan agapanthus.Construct the cDNA from interested cell or tissue
The method in library is that molecular biology field is well-known.In addition, many such cDNA libraries are also commercially available, such as
Purchased from Clontech, Stratagene, Palo Alto, Cal.This screening technique can identify related to Afriocan agapanthus ApARF2
Gene family nucleotide sequence.
Afriocan agapanthus ApARF2 associated nucleotide full length sequence of the invention or its segment usually can with PCR amplification method, again
Group method or artificial synthesized method obtain.For PCR amplification method, can disclosed related nucleotide sequence according to the present invention, especially
It is that open reading frame sequence carrys out design primer, and with the commercially available library cDNA or presses conventional method well known by persons skilled in the art
The prepared library cDNA 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).In vitro synthetic proteins matter can by hand or from
It is dynamic to carry out.For example, dynamic circuit connector can be come from the 431A type 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.
It can be filtered out using Afriocan agapanthus auxin response factor ApARF2 of the invention by various conventional screening assays
The substance to interact related to Afriocan agapanthus auxin response factor ApARF2 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 response factor ApARF2, and be transformed into model plant arabidopsis, using fluorescence real-time quantitative PCR
Physiological effect and expression pattern of the method analysis ApARF2 gene in arabidopsis, to be regulated and controled from now on using technique for gene engineering
The spatial and temporal expression of ApARF2 gene has very big answer to provide theoretical foundation in terms of for the fast numerous, breeding of new variety of body embryo
With 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 Homology search of the nucleotide sequence of Afriocan agapanthus ApARF2 gene and mulberry tree ARF gene mRNA of the invention
(GAP) result；
Fig. 2 is the Homology search of the amino acid sequence of Afriocan agapanthus ApARF2 albumen and oil palm ARF23 albumen of the invention
(FASTA) result, wherein identical amino acid is marked between two sequences with amino acid monocase.
Fig. 3 is wild type and ApARF2 transgenic Arabidopsis plants upgrowth situation Phenotypic Observation；
Fig. 4 is wild type and ApARF2 transgenic arabidopsis ApARF2 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 ApARF2 gene
1. the acquisition of vegetable material
Take Afriocan agapanthus (Agapanthus praecox ssp.Orientalis) (Zhang D., Zhuo L.H., Shen
X.H.Sporogenesis and gametogenesis in Agapanthus praecox Willd.orientalis
(Leighton)Leighton and their systematic implications.Plant Syst.Evol.2010,
288:1-11.) adult seedling leaf tissue, 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
The purity and concentration of RNA are measured on 1000Spectrophotometer)；
3. the full-length clone of gene
The protein function annotation of (RNA-seq) is sequenced as a result, obtaining Afriocan agapanthus ApARF2 gene according to Afriocan agapanthus transcript profile
Core fragment.Using RACE method (SMARTerTMRACE cDNA Amplification Kit:Clonetech) carry 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 (II 1st Strand cDNA Synthesis Kit of Prime Script: precious
Bioengineering (Dalian) Co., Ltd), using the first chain cDNA as template, PCR is carried out using following primer:
ARF2-F (SEQ ID NO.1): 5 '-AGGCAAATGTTCCGTCTT-3 '
ARF2-R (SEQ ID NO.2): 5 '-TCCCTGCTTATGTACCTTAGTG-3 '
Amplification obtains 1274bp segment, recycles and is connected on pMD19-T Simple vector carrier, with RV-M and
M13-47 is as universal primer, using the method for terminating object fluorescent marker (Big-Dye, Perkin-Elmer, USA),
It is sequenced on ABI377 sequenator (Perkin-Elmer, USA), sequencing result is by carrying out BLAST in the website NCBI
(http://blast.ncbi.nlm.nih.gov/) compares existing database (GenBank), knows its nucleic acid sequence and coding
Albumen and known oil palm, water lily, grape ARF gene homology it is very high, it was initially believed that it is an ApARF2 gene；
(2)3′RACE
Two wheel nest-type PRCs complete the amplification of 3 ' end sequences.
The first round: UPM+3 '-GSP1 (SEQ ID NO.5)
5′-ATGGTAATGAATCAGAGACCGCCCCT-3′
Second wheel: NUP+3 '-GSP2 (SEQ ID NO.6)
5′-TAGCGAGTGGGACAAGGGTCAGCATA-3′
UPM and NUP provide for kit.3 ' RACE obtain the 3 ' end sequences (479bp) of Afriocan agapanthus ApARF2, recycling,
It is connected on pMD19-T Simple vector carrier, uses RV-M and M13-47 as universal primer, using termination object fluorescence mark
The method for remembering (Big-Dye, Perkin-Elmer, USA), is surveyed on ABI377 sequenator (Perkin-Elmer, USA)
Sequence, sequencing result are existing by carrying out BLAST (http://blast.ncbi.nlm.nih.gov/) comparison in the website NCBI
Database (GenBank) knows that its nucleic acid sequence and coding albumen and the homology of known oil palm, the ARF gene of grape are 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 (SEQ ID NO.7)
5′-ATGCTGACCCTTGTCCCACTCGCTAC-3′
Second wheel: NUP+5 '-GSP2 (SEQ ID NO.8)
5′-TTTGGTCTAGGAACTGGAAGGGGGTT-3′
UPM and NUP provide for kit.5 ' RACE obtain the 5 ' end sequences (1209bp) of Afriocan agapanthus ApARF2 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 ApARF2 gene newly obtained from Afriocan agapanthus is one really by row splicing
Auxin response factor gene, by the ORF Finding of sequencing result combination NCBI (http: //
Www.ncbi.nlm.nih.gov/gorf it) predicts, it was found that the initiation codon and termination codon of Afriocan agapanthus ApARF2 gene
Son designs specific primer according to the sequence of acquisition from initiation codon and terminator codon respectively:
ORF-F (SEQ ID NO.9): 5 '-ATGGAGCTAGAGTTAGGCCTTGCTCTCAAT-3 ',
ORF-R (SEQ ID NO.10): 5 '-CTATGCAGGTCTCTCTCGTTTGTTACATCG-3 ',
PCR is carried out by template of Afriocan agapanthus cDNA, amplification obtains the overall length code sequence of 2349bp Afriocan agapanthus ApARF2 albumen
It arranges (SEQ ID NO.3).
The sequence information and homology analysis of embodiment 2, Afriocan agapanthus ApARF2 gene
The new Afriocan agapanthus ApARF2 full length gene opening code-reading frame sequence of the present invention is 2349bp, and detailed sequence is shown in SEQ ID
Sequence shown in NO.3.The amino acid sequence of Afriocan agapanthus ApARF2 albumen is derived according to opening code-reading frame sequence, totally 782 amino
Sour residue, molecular weight 87.78kDa, isoelectric point (pI) are 6.36, and detailed sequence is shown in sequence shown in SEQ ID NO.4；
The opening code-reading frame sequence of Afriocan agapanthus ApARF2 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
Mulberry tree ARF gene (accession number: XM_010093503.1) has the 91% phase same sex on nucleotide level, as shown in Figure 1
(Query: the coding gene sequence of Afriocan agapanthus ApARF2；Sbjct: the mRNA sequence of mulberry tree ARF)；On amino acid levels, it
Also there are 62% consistency and 73% similitude with oil palm ARF23 gene (accession number: XP_010942377.1), such as Fig. 2 institute
Show (Query: the amino acid sequence of Afriocan agapanthus ApARF2 albumen；Sbjct: the amino acid sequence of oil palm ARF23 albumen).Thus may be used
See, it is higher no matter the ARF gene of Afriocan agapanthus ApARF2 gene and other known species all exists from nucleic acid or protein level
Homology.
Embodiment 3, Afriocan agapanthus ApARF2 genetic transformation model plant arabidopsis
1. the building of the expression vector containing target gene (Afriocan agapanthus ApARF2 gene)
According to Afriocan agapanthus ApARF2 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, so as to
Construction of expression vector.Using the amplified production obtained in embodiment 1 as template, after PCR amplification, by Afriocan agapanthus ApARF2 gene
Coding region sequence is connected in intermediate vector (such as pMD19-T) and is sequenced, then correct Afriocan agapanthus ApARF2 gene will be sequenced
Coding region sequence be further cloned into expression vector (such as pHB), be transferred under the premise of having identified that reading frame is correct
In Agrobacterium tumefaciems (such as GV3101), and PCR identification is carried out to the Agrobacterium after conversion, to guarantee to contain Afriocan agapanthus ApARF2 base
The plant expression vector successful conversion of cause enters in Agrobacterium tumefaciems.
2. Agrobacterium-Mediated Transformation arabidopsis
(1) Agrobacterium is shaken in advance: choosing positive monoclonal to 25ml kanamycins containing 50mg/L, 50mg/L gentamicin, 25mg/
In the YEP fluid nutrient medium of L rifampin, 28 DEG C, 200rpm shakes bacterium for 24 hours；
(2) spread cultivation Agrobacterium: the Agrobacterium bacterium solution shaken in advance being spread cultivation with 1:100 to the YEP of kalamycin resistance containing 400mL and is trained
It supports in base, 28 DEG C, 200rpm, cultivates 13-16h, 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 30s 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 ApARF2 transgenic Arabidopsis plants have conspicuousness poor
Different (Fig. 3)；ApARF2 growth of transgenic plants is significantly faster than that WT lines, illustrates that ApARF2 there is positive regulation to make auxin
With.
4. transgenic Arabidopsis plants ApARF2 gene expression difference
The blade 0.2g of arabidopsis wild type and ApARF2 transgenic plant is sheared, RNA is extracted, prepares cDNA and carry out reality
When quantitative PCR analysis.The specific primer that ApARF2 gene quantification is analyzed in Real-time PCR are as follows:
QT-F (SEQ ID NO.11): 5 '-ACTCAGATGGATTACTCACAA-3 ',
QT-R (SEQ ID NO.12): 5 '-GGCTCATAGGTAGGATTCAA-3 ',
Reference gene is arabidopsis UBQ5 gene, primer are as follows:
UBQ5-F (SEQ ID NO.13): 5 '-GACGCTTCATCTCGTCC-3 ',
UBQ5-R (SEQ ID NO.14): 5 '-CCACAGGTTGCGTTAG-3 '.
Using 2-△△CtMethod makees relative quantitative assay, the results showed that the expression quantity of ApARF2 is higher in transgenic arabidopsis, is
3.6 times of reference gene UBQ5 are 4632 times (Fig. 4) of wild-type plant.Show the ApARF2 not table in WT lines
It reaches.
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 (4)
1. a kind of following protein:
The protein of the composition of the amino acid sequence as shown in SEQ ID NO.4.
2. a kind of nucleic acid sequence of protein described in coding claim 1.
3. the nucleic acid sequence as described in claim 2, characterized in that the nucleic acid sequence specifically: base sequence such as SEQ ID
Shown in NO.3 the 1st~2349.
4. a kind of application of nucleic acid sequence as claimed in claim 2 in the expression of regulating plant growth element.
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| CN103333233A (en) * | 2013-06-28 | 2013-10-02 | 上海交通大学 | Agapanthus praecox auxin receptor protein TIR1 and coding gene and probe thereof |
| CN104961814A (en) * | 2015-06-25 | 2015-10-07 | 上海交通大学 | Agapanthus auxin signal transcriptional control protein Aux/IAA3 and encoding gene and probe thereof |
| CN104961815A (en) * | 2015-06-25 | 2015-10-07 | 上海交通大学 | Agapanthus auxin signal transcriptional control protein Aux/IAA1 and encoding gene and probe thereof |
| CN105085642A (en) * | 2015-06-25 | 2015-11-25 | 上海交通大学 | Agapanthus africanus auxin signal transcriptional control egg white Aux/IAA2 and encoding gene and probe thereof |
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| CN103333233A (en) * | 2013-06-28 | 2013-10-02 | 上海交通大学 | Agapanthus praecox auxin receptor protein TIR1 and coding gene and probe thereof |
| CN104961814A (en) * | 2015-06-25 | 2015-10-07 | 上海交通大学 | Agapanthus auxin signal transcriptional control protein Aux/IAA3 and encoding gene and probe thereof |
| CN104961815A (en) * | 2015-06-25 | 2015-10-07 | 上海交通大学 | Agapanthus auxin signal transcriptional control protein Aux/IAA1 and encoding gene and probe thereof |
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