CN101603079A - Peony ACC synthase gene Ps-ACS 1 specific probe - Google Patents

Abstract

The invention provides a kind of peony ACC synthase gene Ps-ACS 1 specific probe, it has the nucleotide sequence shown in the sequence table SEQ ID NO.1, perhaps its useful length fragment.Experiment shows, probe of the present invention can be used in the specific detection of Ps-ACS1 gene, avoided other gene family member's of in gene expression analysis research, bringing expression interference, helped studying on this basis Regulation Mechanism and the binding mode of ACS different members in the tree peony ethylene reaction because homology is higher.For overall understanding ethene and tree peony cut-flower bloom old and feeble relation, announcement ethene is bloomed after it is adopted and senescence process in binding mode and regulatory mechanism provide the foundation.

Description

Peony ACC synthase gene Ps-ACS 1 specific probe

Technical field

The present invention relates to biological technical field, be specifically related to the specific probe of peony ACC synthase gene Ps-ACS 1.

Background technology

Tree peony (Paeonia suffruticosa) is the elaboration in Chinese traditional famous flower, have " aromatic ", " king's in spending " good reputation, its large flower and brilliant color, not only be subjected to liking of China people deeply, and meet westerner's aesthetic conceptions, therefore become at present one of most popular flowers and trees in the world gradually.In recent years, along with market growing to the fresh cutting flower demand, except gardens with seedling and the potted flower, the potential world market of tree peony cut-flower also enlarges day by day.Yet, do not pass a test because the tree peony nature florescence concentrates, the Dan Duohua florescence is short, adopt the back fresh-keeping technology during storage and transportation, cause the fresh cutting flower difficult quality guarantee, can't realize the long-term and remote supply of material, this not only becomes one of important factor of restriction China tree peony cut-flower industrialization production, and has also lost the good opportunity of much earning foreign exchange for this reason.

The ethene that is called as old and feeble hormone with cut flower withering in close relations, be the focus of research of cutting flower withering for many years always.At present, a large amount of generations of having found ethene are to impel one of important physiological reason that tree peony cuts flower withering (history Guoan etc., 1997,1999); On this basis, we analyze discovery to 13 tree peony kind endogenous ethylene metabolic characteristicss, with tree peony ' Luoyang is red ' is several cut-flower acetate releasing quantities and closely related (the Jiaet al. of flowering senescence process of representative, 2006), but it is to the reaction of exogenous ethylene very complicated (Zhou et al., 2006).Therefore, in order to verify the mechanism of action of ethene in the open senescence process of tree peony cut-flower, thereby, also need more deep research for the exploitation of its post-harvest fresh-keeping technology provides theoretical foundation.

Since the ethene biosynthetic pathway discloses (Yang and Hoffman, 1984), the research of cut-flower senescence mechanism of ethylene progresses into molecular level.In the ethene biosynthetic pathway, ACC synthetic enzyme (ACS) and two key enzymes of acc oxidase (ACO) are arranged.Wherein, ACS is considered to the rate-limiting enzyme in the ethene biosynthesizing, and it is by a multigene family encode (Johnson and Ecker, 1998; Ge et al., 2000), and different members is subjected to inducing of the different factors (comprise and grow signal, plant hormone and environmental stimulus etc.) and produces ethene (Zarembinski and Theologis, 1997; Bui and O ' Neill, 1998).At present, Dianthus caryophyllus L. (Park et al., 1992; Van Altvorst and Bovy, 1995), the molecular regulation mechanism research of ACS gene in the flowering senescence process in Chinese rose (Wanget al., 2004) and the orchid many ornamental plants such as (O ' Neil et al., 1993) is carried out in succession.But in tree peony, even in the whole Paeoniaceae, the clone of ACS gene and the research of Regulation Mechanism thereof also do not have report.

Summary of the invention

The object of the present invention is to provide the specific probe of peony ACC synthase gene Ps-ACS 1, for the follow-up functional study of Ps-ACS1 gene and application provide the basis.

Specific probe of the present invention has the nucleotide sequence shown in the sequence table SEQ ID NO.1 or its useful length fragment.Those skilled in the art will be understood that useful length fragment described here be the finger that produces according to SEQ ID NO.1 can with the sequence of PS-ACS1 specific hybridization.More than the preferred 60bp of these sequence lengths.

The marker of probe can be radio-labeling or nonradioactive labeling, for example adopts fluorescent mark.

Above-mentioned probe and suitable reagent composition test kit can be used with convenient.

Show that by experiment probe of the present invention has good specificity, can be used in the specific detection of Ps-ACS1 gene.

The present invention separates the Ps-ACS1 gene first from tree peony ' Luoyang is red ' petal, and synthesized the specific probe of this gene, avoided other gene family member's of in gene expression analysis research, bringing expression interference, helped studying on this basis Regulation Mechanism and the binding mode of ACS different members in the tree peony ethylene reaction because homology is higher.For overall understanding ethene and tree peony cut-flower bloom old and feeble relation, announcement ethene is bloomed after it is adopted and senescence process in binding mode and regulatory mechanism provide the foundation.

Description of drawings

(continuous Fig. 1 is the Ps-ACS1 gene cDNA full length nucleotide and the aminoacid sequence of inferring a) for Fig. 1 a, Fig. 1 b, wherein lowercase is represented non-translational region, terminator represented in asterisk, 7 conserved regions of ACC synthetic enzyme are represented in the shadow zone, it in the square frame the conservative amino-acid residue that exists in all transaminases, the line part is the active centre of ACC synthetic enzyme, and the amino-acid residue of overstriking is represented the lysine residue avtive spot in conjunction with pyridoxal phosphate and methionine(Met);

Fig. 2 tree peony Ps-ACS1 gene Southern hybridization analysis.With the 30 μ g DNA that extract in ' Luoyang is red ' young leaflet tablet, carrying out enzyme with EcoRI (a), EcoRV (b), HindIII (c) cuts, after 0.8% (w/v) agarose gel electrophoresis separates, be transferred on the nylon membrane, hybridize with the intermediate segment probe (A) and the specific probe (B) of digoxigenin labeled respectively;

Fig. 3 ethene and 1-MCP handle the influence to Ps-ACS1 genetic expression in ' Luoyang is red ' petal.Cut-flower is used for the petal sampling immediately and extracts total RNA after measuring the ethene growing amount, each swimming lane is the total RNA of 20 μ g, is that confidential reference items are weighed total RNA applied sample amount with rRNA.The index of blooming of digitized representation tree peony cut-flower.Each hybridization all repeats at least twice.

Embodiment

Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.

If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.

The clone of embodiment 1Ps-ACS1 gene

1. the extraction of the total RNA of peony petal

(1) RNA extracts used plastics, glassware and reagent preparation and all removes the Rnase processing by " molecular cloning experiment guide " (third edition).

(2) get the petal sample 1.0g that tree peony ' Luoyang is red ' blooms the phase flower, after being ground to powder in the liquid nitrogen, change over to rapidly in the 1.5mL centrifuge tube, the CTAB extracting solution 600 μ L (2%CTAB that add 65 ℃ of preheatings, 2%PVP, 100mM TrisCl (pH8.0), 10mMNaCl, 25mM EDTA (pH8.0), 2% beta-mercaptoethanol (adding before using)), cover tight lid, violent immediately vortex vibration 30s is dispersed in the solution powder fully, and 65 ℃ of temperature are bathed 2-5min, violent during this time vortex vibration 3-5 time, after being cooled to room temperature, in above-mentioned mixed solution, add isopyknic chloroform/primary isoamyl alcohol (24: 1) extracting twice, behind each vortex vibration 5min, room temperature or 18 ℃ down 10, the centrifugal 15min of 000rpm shifts supernatant to new centrifuge tube, adds 1/4 volume 10M LiCl, mixing, 4 ℃ of post precipitations that spend the night, 4 ℃, 10, the centrifugal 20min collecting precipitation of 000rpm, with 500 μ L SSTE (1.0M NaCl, 0.5%SDS, 10mM TrisCl, 1mM EDTA) damping fluid dissolution precipitation adds extracting 1-2 time again of isopyknic chloroform/primary isoamyl alcohol.Add 2 times of volume dehydrated alcohols after shifting supernatant, place 2h or-70 ℃ of placement 30min, 4 ℃ for-20 ℃, 10, the centrifugal 20min of 000rpm, precipitated rna, successively with 500 μ L, 70% ethanol, 500 μ L dehydrated alcohol rinsings precipitation, after drying up on the super Jing Tai, with 30 μ L RNase-free ddH ₂The O dissolving.Agarose gel electrophoresis and spectrophotometer detect its quality and calculate total rna content (rna content=D260 * 0.04 * extension rate), are stored in-80 ℃ of Ultralow Temperature Freezers standby.

2. the segmental clone of tree peony Ps-ACS1 dna homolog

(1) reverse transcription

Get the above-mentioned total RNA of 2 μ g, add DEPC ddH ₂O to 9.5 μ L, 70 ℃ of sex change 5min are centrifugal a little behind the ice bath 2min immediately.Add each component successively by table 1.42 ℃ of water-bath 60min ,-20 ℃ of preservations are standby.

Table 1 reverse transcription system

(2) pcr amplification

Add the laggard performing PCR amplification of each component successively by table 2.The primer is:

ACS-F：5′-CGGGATCCGTATCAAGATTATCATGG-3’

ACS-R：5′-AACTGCAGGAGAGGCTGTAGAGAATATG-3’

Table 2PCR reaction system

(3) PCR product sepharose reclaims

The PCR product is carried out electrophoretic analysis in 1.0% sepharose, cut under the ultraviolet lamp and expect that the consistent band of clip size reclaims, working method sees the biochemical company limited of sky, Beijing root product sepharose for details and reclaims the test kit specification sheets.

(4) connect

Get PCR product 7 μ L, add 1 μ L T4DNA ligase enzyme, 10 * Buffer respectively, 1 μ L T4DNA ligase enzyme and 1 μ L pGEM-T carrier are to total system 10 μ L, and mixing is put to connect under 16 ℃ of conditions and spent the night, and puts-20 ℃ of preservations then.

(5) transformed into escherichia coli DH5a competent cell and positive colony screening

A. the calcium chloride preparation method is adopted in the preparation of escherichia coli DH5a competent cell, and method is with reference to " molecular cloning experiment guide " (third edition).

B. prepare LB solid medium (adding 100mg/L Amp), wherein contain the ammonia benzyl of 50 μ g/mL, in the dark 4 μ L IPTG (200mg/mL) and 40 μ L X-gal (20mg/mL) are evenly coated on the flat board, forward absorbs 1-3h.

C. draw 200 μ L competent cells (operation on ice) with aseptic suction nozzle and put in the aseptic Eppendorf pipe of 1.5mL precooling, add 10 μ L and connect product, mixing (under light finger bomb tube wall 3-4) places 30min on ice immediately gently.

D. pipe is put heat shock 90s in 42 ℃ of waters bath with thermostatic control, during prevent to shake as far as possible, and put back to 2min on ice immediately.

E. add 800 μ L and do not contain antibiotic LB liquid nutrient medium, put upside down mixing, in 37 ℃ of shaking culture 1-2h (150r/min).

F.5000rpm behind the centrifugal 3min, inhale and remove 600 μ L supernatant liquors, will precipitate suspension again, with the bacterium spreader above-mentioned culture is evenly coated on the LB solid medium down in aseptic condition.

G. dull and stereotyped after 37 ℃ of forwards are placed to liquid and are absorbed by substratum fully, be inverted overnight incubation to growing single bacterium colony (12-16h).

H. use the single bacterium colony of aseptic toothpick picking white in LB (Am+) liquid nutrient medium, shaken overnight under 37 ℃ of conditions is extracted plasmid DNA and is carried out bacterium liquid PCR evaluation.

(6) plasmid enzyme restriction of positive colony is identified

Single bacterium colony bacterium liquid that the PCR that learns from else's experience identifies is inoculated in LB (Am+) liquid nutrient medium, is cultured to logarithmic phase, and alkaline process extracts plasmid DNA in a small amount.Get plasmid 7 μ L, add 2 μ L enzyme cutting buffering liquids and 1 μ L EcoRI enzyme, ddH ₂O supplies 20 μ L, puts to 37 ℃ of following enzymes to cut the laggard row agarose gel electrophoresis detection of 1-2h, entrusts Beijing AudioCodes biotech firm to finish examining order with connecting correct mono-clonal.

3. the acquisition (Fig. 1) of tree peony Ps-ACS1 gene cDNA full length sequence

According to Race test kit SMART ^TMRACE cDNA Amplification Kit specification sheets is synthetic 5 ' and 3 ' RACE cDNA, first chain, carry out the RACE amplification respectively as masterplate.Wherein 3 ' RACE Auele Specific Primer be 5 '-AGCCTCGTTTCATTAGCATTGCAG-3 ', 5 ' RACE Auele Specific Primer is 5 '-CTCATGACAATGCGGTCTGGATCGA-3 ', at 94 ℃ of 30s, 57 ℃ of 30s finish 28 circulations under 72 ℃ of 2.5min conditions.Steps such as the recovery of PCR product gel, connection, conversion and evaluation are the same.After the positive colony order-checking, two end sequences and the resulting intermediate sequence of RT-PCR are spliced acquisition Ps-ACS1 gene cDNA full length sequence mutually.

4.Ps-ACS1 cDNA full length sequence

The method that adopts RT-PCR and RACE-PCR to combine, obtaining length is the tree peony Ps-ACS1cDNA full length sequence (Fig. 1) of 1766bp.The ORFFinder that utilizes NCBI to provide carries out sequential analysis and finds that this cDNA total length includes the opening code-reading frame of a 1479bp, 492 amino acid of encoding, 5 ' untranslated head of district 146bp, 3 ' untranslated head of district 141bp.This proteic molecular weight of ProtParam (http://au.expasy.org/tools/protparam.html) prediction is 54.91kD, and theoretical iso-electric point is 7.15.Do you utilize prosite software (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl? page=npsa_prosite.html) the proteic specific function of predictive coding site is found; Ps-ACS1 full-length cDNA proteins encoded contains 1 N-glycosylation site (N-glycosylation site) (being positioned at 179-182 amino-acid residue NFTV); the protein kinase phosphorylation site (cAMP-and cGMP-dependent protein kinase phosphorylation site) (being positioned at the amino-acid residue RKMS of 302-305 position and the amino-acid residue KKQS of 445-448 position) that 2 cyclic monophosphates and cyclic guanosine monophosphate rely on; 4 protein kinase C phosphorylation sites (Protein kinase C phosphorylationsite) (amino-acid residue TLK of 221-223 position; the amino-acid residue SFK of 457-459 position; the amino-acid residue SGK of 460-462 position; the amino-acid residue SPR of 469-471 position); 3 casein kinase i I phosphorylation sites (Casein kinase II phosphorylation site) (amino-acid residue SALE of 185-188 position; the amino-acid residue SIAE of 254-257 position; the amino-acid residue SYND of 292-295 position); 10 N-Semen Myristicae acidylate sites (N-myristoylation site) (are positioned at the amino-acid residue GNGHGE of 19-24 position; the amino-acid residue GLAENL of 53-58 position; the amino-acid residue GGRVTF of 113-118 position, the amino-acid residue GLVSTQ of 308-313 position etc.) and 1 transaminase I class phosphoric acid pyrrole diindyl aldehyde prothetic group binding site (Aminotransferases class-I pyridoxal-phosphateattachment site) (being positioned at the amino-acid residue SLSKDMGFPGFRVG of 275-288 position).

The aminoacid sequence that the Ps-ACS1cDNA total length is inferred is analyzed discovery (Fig. 1), have all conserved regions (Zarembinski andTheologis, 1997 in the coded open reading frame; Bleeker and Kende, 2000), also comprised the lysine residue avtive spot (SLS that has in conjunction with pyridoxal phosphate and methionine(Met) ability in one of them conserved regions KDMGFPGFRVG) (Yip et al., 1990).In addition, also contain 11 amino-acid residues that in all transaminases, all exist in this aminoacid sequence.

The preparation of embodiment 2 probes and hybridization detect

1. probe preparation

(Roche, Cat.No.1636090) specification sheets carry out digoxigenin labeled with the Ps-ACS1 intermediate segment that RT-PCR obtains, preparation probe A according to PCR DIG probe synthesis Kit.In addition, according to the sequence of Ps-ACS1 near 3 ' end, design a pair of special primer, Ps-ACS1F:5 '-CAGGTCTCT TCTTATGGATGG-3 ', Ps-ACS1R:5 '-GTAATTAAGCTCGAAGAAGGG-3 ', product with Ps-ACS13 ' RACE is a template, carries out the digoxigenin labeled of amplified fragments, preparation specific probe B.After reaction finishes, get 3 μ l PCR product certification mark situations, remaining probe be put in preserve in-20 ℃ of refrigerators standby.

2.Southern hybridization

(1) main agents and preparation thereof

A. sex change liquid: 0.5M NaOH, 1.5M NaCl.

B. neutralizer: 0.5M Tris alkali, 1.5M NaCl, adjust pH to 7.5.

C.20 * and SSC:NaCl 175.3g, trisodium citrate 88.2g, add ddH ₂O to 800ml transfers pH to 7.0 with 2N NaOH, uses ddH again ₂O is settled to 1000ml, autoclaving (3M NaCl, 3M Trisodium Citrate).

D. prehybridization solution: 7%SDS, 50% deionized formamide, 5 * SSC, 2%Blockingreagent, 50mM Ph7.0 NaH ₂PO ₄

E. film washing liquid I:2 * SSC, 0.1%SDS.

F. film washing liquid II:0.1 * SSC, 0.1%SDS.

G. maleic acid solution: the 0.1M toxilic acid, 0.15M NaCl regulates pH to 7.5 (20 ℃) with the NaOH solid.

H.Washing buffer: add 0.3% (v/v) Tween 20 in the maleic acid solution.

I.10 * blocking: with the blocker powder be dissolved in the maleic acid solution to final concentration be 10% (W/V), vibration is the heating hydrotropy also, behind the autoclaving, is kept at-20 ℃.

J. confining liquid: in 10: 1 ratios with maleic acid solution dilution 10 * blocking, matching while using.

K. detect liquid: 0.1M Tris-HCl, 0.1M NaCl pH 9.5 (20 ℃).

L. antibody-solutions: take out antibody A P (Anti-Digoxingenin), 10, the centrifugal 5min of 000rpm is from surperficial imbitition.Press 10000-20000 with confining liquid: 1 dilution, final concentration is 75-37.5mU/ml, matching while using.

M. liquid develops the color: diluted CDP-Star in detecting liquid by 1: 100.

(2) the genomic dna enzyme is cut

Extracting genome DNA is pressed the method for document (Murray and Thompson, 1980) and is carried out.Getting genomic dna 30 μ g carries out enzyme through EcoR I, EcoRV, HindIII etc. according to the enzyme specification sheets respectively and cuts and spend the night.Get 5 μ l enzymes and cut product 80V electrophoresis 1h on 0.8% sepharose, whether the ultraviolet detection enzyme is cut thorough.As thoroughly, then remaining enzyme is cut sample electrophoresis on the product, be used to change film hybridization.

(3) electrophoresis

Cut in the product sample-loading buffer that adds 1/10V to enzyme, abundant mixing, centrifugal collection, 65 ℃ of sex change 10min, after ice swashed 5min, (voltage 1～2V/cm) 8h migrated to 2/3 place of glue to bromjophenol blue to carry out 0.8% agarose gel electrophoresis.

(4) changeing film is undertaken by " molecular cloning experiment guide " (third edition).

(5) hybridization

A. get an amount of prehybridization solution (20ml/100cm ₂) in hybrid pipe, after 42 ℃ of preheatings, film is placed in the pipe, do not contain DNA and simultaneously be close to tube wall, drain bubble, sealing, 42 ℃ of prehybridizations are 1h at least.

B. according to 2-4 μ l PCR probe mark product/100cm ₂, get an amount of probe in the 1.5ml centrifuge tube, after the sealing, put 5-10min sex change in the boiling water, be put in 5min on ice then immediately, with an amount of prehybridization solution (3.5ml/100cm of preheating ₂) dilution, mixing.

C. outwell prehybridization solution, along the hybridization solution of tube wall adding mixing, note directly not being added on the film and avoiding producing bubble, 42 ℃ of hybridization are spent the night.

D. after hybridization is finished, but with hybridization solution be recovered in one can the test tube of low temperature resistant boiling water bath again in, be stored in-20 ℃ in order to reusing.During repeated use, thaw and at 68 ℃ of following sex change 10min.

(6) wash film

A. Hybond membrane takes out and is placed in the plastics casing, uses film washing liquid I, washes film under the room temperature 2 times, and each 5-10min removes non-binding probe to reduce background.

B. film washing liquid II washes film 2 times for 60-65 ℃, each 15min.

(7) detection of hybridization signal is according to CDP-Star ^TM(available from German Roche company) specification sheets carries out.

3.Northern hybridization

(1) main agents and preparation thereof

A.0.5M EDTA:EDTA 16.61g adds ddH ₂O to 80ml transfers pH to 8.0, is settled to 100ml, adds DEPC 100 μ l, vibration, and 37 ℃ are spent the night autoclaving.

B.1M NaAc:NaAc13.6g adds ddH ₂O to 100ml adds DEPC 100 μ l, vibration, and 37 ℃ are spent the night autoclaving.

C.10 * and the MOPS damping fluid: accurately take by weighing 41.86g MOPS and be dissolved in 700mlDEPC-H ₂Among the O,, add 20ml 1M NaAc and 20ml 0.5M EDTA (pH8.0) that DEPC handles, DEPC-H with 2mol/L NaOH adjust pH to 7.0 ₂O is settled to 1L, and (200mM MOPS, pH 7.0,50mM NaAc, 10mM EDTA) keeps in Dark Place behind the filtration sterilization.

D. formaldehyde gel sample loading buffer: 50% glycerine, 1mM EDTA (pH 8.0), 0.25% (m/v) tetrabromophenol sulfonphthalein, 0.2mg/ml ethidium bromide.

E.RNA sample buffer: 10%10 * MOPS damping fluid, 17% formaldehyde, 45% deionized formamide.

F.10 * and formaldehyde gel electrophoresis sample loading buffer: 50% glycerine (being diluted in the DEPC treating water), 10mM EDTA (Ph8.0), 0.25% (m/v) tetrabromophenol sulfonphthalein.

G.20 * and SSC:NaCl 175.3g, trisodium citrate 88.2g, add ddH ₂O to 800ml transfers pH to 7.0 with 2N NaOH, uses ddH again ₂O is settled to 1000ml.DEPC handles, autoclaving (3M NaCl, 3M Trisodium Citrate).

The preparation of all the other reagent is with reference to the Southern hybridization portion.

(2) denaturing formaldehyde agarose gel electrophoresis

A. get other tree peony of different flowering level ' Luoyang is red ' petal and extract RNA.

B. glue: with 70% alcohol flushing one time, dry standby the glue apparatus.Prepare 1.2% denaturing formaldehyde glue: take by weighing the 1.2g agarose, place clean flask, add 80ml DEPC treating water, low fiery heat fused is even in the microwave oven.When treating the glue cold to 60-70 ℃, successively to wherein adding 10ml formaldehyde and 10ml 10 * MOPS damping fluid, pour into immediately behind the mixing in the glue groove (attention avoids producing bubble), cover preservative film, room temperature is placed 1h at least.

C. the RNA sample is dissolved on ice, be diluted to 2 μ g/ μ l with the DEPC treating water.Get 10 μ l and dilute good RNA sample in the little centrifuge tube of 500 μ l that DEPC handled, add 30 μ l RNA sample buffers and 1 μ l EB, mixing places 65 ℃ of water-baths to be incubated 10min centrifuge tube, places 2min on ice again; Add 4 μ l formaldehyde gel sample loading buffers, mixing in every pipe.

D. go up sample: prepared gel is put into electrophoresis chamber, add electrophoretic buffer (1 * MOPS damping fluid), the high plastic emitting face 1-2mm of liquid level carefully extracts comb sample well is remained intact.With micropipet the sample for preparing is added well.

E. electrophoresis: cover electrophoresis chamber, connect power supply, sample termination negative pole, in under the voltage of 7.5V/ml about electrophoresis 1h, put upside down positive and negative electrode and gel direction, to guarantee the pH value stabilization of damping fluid, continue about electrophoresis 1h, when tetrabromophenol sulfonphthalein arrive glue long 3/4 the time stop electrophoresis.After electrophoresis finishes, can be in detected result under the ultraviolet lamp.

G. observe and finish, glue is excised one jiao, DEPC treating water drip washing 3 times, 5min at least with thorough removal formaldehyde, uses 20 * SSC of 10V to soak 20min, in order to changeing film more at every turn.

(3) change film, hybridize, wash steps such as film, detection and hybridize with Southern.

4.Ps-ACS1 specific probe sequence and specific detection result

The Ps-ACS1 specific probe sequence that amplification obtains is across 3 of coding region ' end and part 3 ' non-translational region, and long is 399bp, and its nucleotide sequence is shown in sequence table SEQ ID NO.1.

Southern hybridization.With the 30 μ g DNA that extract in ' Luoyang is red ' young leaflet tablet, carrying out enzyme with EcoRI (a), EcoRV (b), HindIII (c) cuts, after 0.8% (w/v) agarose gel electrophoresis separates, be transferred on the nylon membrane, hybridize with the intermediate segment probe A and the specific probe B of digoxigenin labeled respectively, wherein, the initiation site of probe A in total length is 410-986bp, and the initiation site of probe B in total length is 1231-1630bp.By Southern hybridization, with Ps-ACS1 intermediate sequence synthetic probe in contrast, detect the specificity (Fig. 2) of this probe.The result shows: utilize intermediate segment for after probe hybridizes, the swimming lane that the EcoRI enzyme is cut occurs that two bands appear in the swimming lane that 4 bands, EcoRV enzyme cut, the HindIII enzyme is cut the back and 1 master tape and the weak band of 3-4 bar are occurred, the different members that has the ACS gene family in the genome is described, this probe can be hybridized with different members, can cause the expression between the different members to disturb in gene expression analysis research.1 band (Fig. 2 all only appears on each swimming lane and adopt the Ps-ACS1 specific probe to hybridize discovery, B), thereby got rid of this gene in genome for multiple copied may, and prove that the specific probe that this probe can be used as Ps-ACS1 is used for Northern hybridization.

Northern hybridization.The index of will blooming is that 1 grade tree peony spray bottle inserts in the container that fills 200mL distilled water, is divided into three groups, 50 every group, is placed on respectively in the glass box of 100L.Inject ethene in the glass box of good seal with syringe to one of them, make that the ethene final concentration reaches 10 μ LL in the case ^-1Place an amount of 1-MCP powder in another case, the 1-MCP gas concentration that the back that is dissolved in water is discharged reaches 1.0 μ LL ^-1, sealing rapidly; Do not inject any gas in the glass box of sealing contrast flower material, place 6h for 20 ℃.During the processing for preventing CO ₂Place the NaOH solution of 100mL 1N concentration in the accumulation, case.Processing after finishing is taken out spray, regularly respectively get 4 (every as repetitions) by open levels every day, measure the endogenous ethylene growing amount of cut-flower respectively, be that a bag is taken a sample according to every 3g petal then, masking foil is wrapped, be stored in behind the liquid nitrogen flash freezer in-80 ℃ of Ultralow Temperature Freezers, be used for sampling and extract total RNA.Each swimming lane is the total RNA of 20 μ g, is that confidential reference items are weighed total RNA applied sample amount with rRNA.The index of blooming of digitized representation tree peony cut-flower.Each hybridization all repeats at least twice.Utilize this specific probe to carry out Northern hybridization.The result as shown in Figure 4, Ps-ACS1 never detected in contrast open early stage of cut-flower (1-3 level), just began to express when cut-flower reaches 4 grades, and strengthened strongly in the open later stage.Ethene has significantly promoted the expression of Ps-ACS1, processing just can detect the accumulation of its transcription product, along with the opening of flower after finishing when cut-flower still is in 1 grade, the expression amount of Ps-ACS1 descends gradually at 2,3 grades, returns to 1 grade level then when being open into 4 grades again.Similar to contrast is that the cut-flower Ps-ACS1 that ethene is handled also strengthens in the time of 5 grades strongly, and its expression amount obviously is subjected to inducing of exogenous ethylene.In the whole opening advancement of cut-flower, Ps-ACS1 is subjected to the remarkable inhibition that 1-MCP handles, and only just detects a small amount of expression when blossom to 5 grade.Illustrate that this probe can be used to carry out gene expression analysis research.

5. the 40th to 260 fragment on the employing SEQ ID NO.1 is carried out Sorthern and Northern hybridization and detection respectively according to the method described above as probe.The result shows, in Southern hybridization, different enzymes are cut on each swimming lane of processing and single band all occurred, and Northern hybridization can detect the expression of Ps-ACS1 in the peony petal after the different treatment, illustrate that this probe can detect the Ps-ACS1 gene specifically.

6. the 100th to 320 fragment on the employing SEQ ID NO.1 is carried out Southern and Northern hybridization and detection respectively according to the method described above as probe.The result shows, in Southern hybridization, different enzymes are cut on each swimming lane of processing and single band all occurred, and Northern hybridization can detect the expression of Ps-ACS1 in the peony petal after the different treatment, illustrate that this probe can detect the Ps-ACS1 gene specifically.

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Park?KY，Drory?A，Woodson?WR.Molecular?cloning?of?an?1-aminocyclopropane-1-carboxylatesynthase?from?senescence?carnation?flower?petals[J].Plant?Mol?Biol，1992，18：377-386.

Van?Altvorst?A?C，Bovy?A?G.The?role?of?ethylene?in?the?senescence?of?carnation?flowers[J].PlantGrowth?Regul，1995，16：43-53.

Wang?Y，Kumar?PP.Heterologous?expression?of?Arabidopsis?ERS1?causes?delayed?senescence?incoriander[J].Plant?Cell?Rep，2004，22：678-684.

Yang?SF，Hoffman?N?E.Ethylene?biosynthesis?and?its?regulation?in?higher?plants[J].Annu.Rev.Plant?Physiol，1984，35：155-189.

Yip?WK，Dong?JG，Kenny?JW，Thompson?GA，Yang?S?F.Characterization?and?sequencing?of?theactive?site?of?1-aminocyclopropane-1-carboxylate?synthase[J].Proc?Natl?Acad?Sci?USA，1990，87：7930-7934.

Zarembinski?TI，Theologis?A.Expression?characteristics?of?OS-ACS1?and?OS-ACS2，twomembers?of?the?1-aminocyclopropane-1-carboxylate?family?in?rice(Oryza?sativa?L.cv.Habiganj?Aman?II)during?partial?submergence[J].Plant?Molecular?Biology，1997，33：71-77.

Zhou?L，Jia?P?Y，Guo?W?W，et?al.Influence?of?ethylene?on?postharvest?behavior?of‘Luo?YangHong’tree?peony?cut?flower[C].International?Society?for?Horticultural?Science.27 ^thInternational?Horticultural?Congress&Exhibition?Abstracts.Seoul?Korea，2006：289.

Sequence table

＜110〉Beijing Forestry University

＜120〉peony ACC synthase gene Ps-ACS 1 specific probe

<130>

<160>5

<170>PatentIn?version?3.3

<210>1

<211>399

<212>DNA

＜213〉tree peony

<400>1

caggtctctt?cttatggatg?gatttaagct?cgctgctcaa?ggagaagacg?gtcgaagcag 60

agataacact?ttggcgagtg?ataatcaatg?aagttaaact?caatgtttca?cctggttcat 120

cttttcattg?ctcggagcct?ggatggtttc?gggtttgctt?tgctaacata?gatgatgcca 180

ccatggacat?tgctcttcga?aggattctaa?catttgcact?caaggccaag?gaagcagatg 240

tgccaaggaa?gaaacaaagt?tggcaaaaca?acaaccttag?actcagcttc?aaatctggga 300

aatatgatga?tgtcaagttg?tctcctcgta?tgatgtcccc?ttgcatgagg?tcccctcact 360

cccctatacc?acaatcaccc?cttgttcgag?cttaattac 399

<210>2

<211>1766

<212>DNA

＜213〉tree peony

<220>

<221>5’UTR

<222>(1)..(146)

<220>

<221>CDS

<222>(147)..(1625)

<220>

<221>3’UTR

<222>(1426)..(1766)

<400>2

acgcggggac?aaaaacataa?cagcatacgc?aatcaagcaa?gaccaaactt?cctataaatt 60

ctgcttctgc?tattcgatca?tcattattac?attcttctct?acaaacctcc?cctgtttttt 120

tcttcaaatt?tctctagtca?cataaa?atg?gga?ttc?atg?tcc?aca?gat?caa?caa 173

Met?Gly?Phe?Met?Ser?Thr?Asp?Gln?Gln

1 5

aag?caa?ttg?ctg?tca?aag?atg?gca?acc?ggc?aat?ggc?cat?gga?gaa?gac 221

Lys?Gln?Leu?Leu?Ser?Lys?Met?Ala?Thr?Gly?Asn?Gly?His?Gly?Glu?Asp

10 15 20 25

tct?cct?tac?ttt?gat?ggt?tgg?aag?gca?tat?gac?agc?aat?cca?ttt?cat 269

Ser?Pro?Tyr?Phe?Asp?Gly?Trp?Lys?Ala?Tyr?Asp?Ser?Asn?Pro?Phe?His

30 35 40

ctt?aat?aac?aat?cct?aat?ggg?gtt?atc?caa?atg?gga?ctt?gca?gaa?aat 317

Leu?Asn?Asn?Asn?Pro?Asn?Gly?Val?Ile?Gln?Met?Gly?Leu?Ala?Glu?Asn

45 50 55

ctg?ctt?tcc?ttt?gat?gtg?att?caa?gaa?tgg?gtt?ctg?aat?aat?cca?aaa 365

Leu?Leu?Ser?Phe?Asp?Val?Ile?Gln?Glu?Trp?Val?Leu?Asn?Asn?Pro?Lys

60 65 70

gcc?tcc?att?tgc?acg?cca?gaa?gga?att?aat?gaa?ttc?aga?gat?act?gct 413

Ala?Ser?Ile?Cys?Thr?Pro?Glu?Gly?Ile?Asn?Glu?Phe?Arg?Asp?Thr?Ala

75 80 85

att?ttt?cag?gat?tat?cat?ggg?ttt?cca?gag?ttc?aga?aat?gct?gtt?gca 461

Ile?Phe?Gln?Asp?Tyr?His?Gly?Phe?Pro?Glu?Phe?Arg?Asn?Ala?Val?Ala

90 95 100 105

aaa?ttt?atg?gga?aaa?gtg?gga?gga?gga?aga?gtc?aca?ttc?gat?cca?gac 509

Lys?Phe?Met?Gly?Lys?Val?Gly?Gly?Gly?Arg?Val?Thr?Phe?Asp?Pro?Asp

110 115 120

cgc?att?gtc?atg?agt?ggt?ggg?gca?act?gga?gct?cat?gag?att?ctg?gcc 557

Arg?Ile?Val?Met?Ser?Gly?Gly?Ala?Thr?Gly?Ala?His?Glu?Ile?Leu?Ala

125 130 135

ttc?tgc?ttg?gct?gac?ccc?ggt?gat?gca?ttt?ctg?gtg?cca?act?cca?tat 605

Phe?Cys?Leu?Ala?Asp?Pro?Gly?Asp?Ala?Phe?Leu?Val?Pro?Thr?Pro?Tyr

140 145 150

tat?cca?gga?tat?gat?cgc?gat?ttg?aga?tgg?cga?aca?gga?gct?caa?ctg 653

Tyr?Pro?Gly?Tyr?Asp?Arg?Asp?Leu?Arg?Trp?Arg?Thr?Gly?Ala?Gln?Leu

155 160 165

ctt?ccc?gtt?caa?tgc?gac?agc?tct?aac?aat?ttc?acg?gtt?acc?ata?agc 701

Leu?Pro?Val?Gln?Cys?Asp?Ser?Ser?Asn?Asn?Phe?Thr?Val?Thr?Ile?Ser

170 175 180 185

gcc?cta?gaa?ttg?gcg?tac?gag?aag?gct?caa?gct?gca?aac?att?aaa?gta 749

Ala?Leu?Glu?Leu?Ala?Tyr?Glu?Lys?Ala?Gln?Ala?Ala?Asn?Ile?Lys?Val

190 195 200

aag?ggt?ttg?atc?ata?aac?aac?ccg?tca?aat?cca?tta?ggc?act?gtc?tta 797

Lys?Gly?Leu?Ile?Ile?Asn?Asn?Pro?Ser?Asn?Pro?Leu?Gly?Thr?Val?Leu

205 210 215

aat?gga?gag?aca?cta?aaa?act?ata?gtg?aac?ttc?atc?aat?gaa?aag?aac 845

Asn?Gly?Glu?Thr?Leu?Lys?Thr?Ile?Val?Asn?Phe?Ile?Asn?Glu?Lys?Asn

220 225 230

atc?cac?ctt?gtt?tgt?gat?gag?att?tac?gcg?gcc?act?gtc?ttt?agc?cat 893

Ile?His?Leu?Val?Cys?Asp?Glu?Ile?Tyr?Ala?Ala?Thr?Val?Phe?Ser?His

235 240 245

cct?cgt?ttc?att?agc?att?gca?gaa?ata?gta?aac?gac?atg?aat?ggt?gtt 941

Pro?Arg?Phe?Ile?Ser?Ile?Ala?Glu?Ile?Val?Asn?Asp?Met?Asn?Gly?Val

250 255 260 265

aat?cga?aat?ctc?atc?cac?gtt?gtc?tac?agt?ctc?tca?aag?gac?atg?ggg 989

Asn?Arg?Asn?Leu?Ile?His?Val?Val?Tyr?Ser?Leu?Ser?Lys?Asp?Met?Gly

270 275 280

ttc?cct?gga?ttt?agg?gtt?ggc?att?gtg?tat?tca?tac?aat?gat?gcc?gtt 1037

Phe?Pro?Gly?Phe?Arg?Val?Gly?Ile?Val?Tyr?Ser?Tyr?Asn?Asp?Ala?Val

285 290 295

gtc?aat?tgt?gcg?cgc?aag?atg?tct?agc?ttt?ggg?cta?gtt?tca?act?caa 1085

Val?Asn?Cys?Ala?Arg?Lys?Met?Ser?Ser?Phe?Gly?Leu?Val?Ser?Thr?Gln

300 305 310

acc?caa?cac?cta?att?gcg?tca?atg?cta?tca?gat?gaa?cac?ttc?atc?gag 1133

Thr?Gln?His?Leu?Ile?Ala?Ser?Met?Leu?Ser?Asp?Glu?His?Phe?Ile?Glu

315 320 325

aga?tat?att?gtg?gag?agt?gca?aat?aaa?tta?gca?gaa?agg?cag?agg?ctc 1181

Arg?Tyr?Ile?Val?Glu?Ser?Ala?Asn?Lys?Leu?Ala?Glu?Arg?Gln?Arg?Leu

330 335 340 345

ttc?act?agg?gga?ctt?tct?caa?gta?ggc?att?aat?ttt?ttg?aag?agc?aat 1229

Phe?Thr?Arg?Gly?Leu?Ser?Gln?Val?Gly?Ile?Asn?Phe?Leu?Lys?Ser?Asn

350 355 360

gca?ggt?ctc?ttc?tta?tgg?atg?gat?tta?agc?tcg?ctg?ctc?aag?gag?aag 1277

Ala?Gly?Leu?Phe?Leu?Trp?Met?Asp?Leu?Ser?Ser?Leu?Leu?Lys?Glu?Lys

365 370 375

acg?gtc?gaa?gca?gag?ata?aca?ctt?tgg?cga?gtg?ata?atc?aat?gaa?gtt 1325

Thr?Val?Glu?Ala?Glu?Ile?Thr?Leu?Trp?Arg?Val?Ile?Ile?Asn?Glu?Val

380 385 390

aaa?ctc?aat?gtt?tca?cct?ggt?tca?tct?ttt?cat?tgc?tcg?gag?cct?gga 1373

Lys?Leu?Asn?Val?Ser?Pro?Gly?Ser?Ser?Phe?His?Cys?Ser?Glu?Pro?Gly

395 400 405

tgg?ttt?cgg?gtt?tgc?ttt?gct?aac?ata?gat?gat?gcc?acc?atg?gac?att 1421

Trp?Phe?Arg?Val?Cys?Phe?Ala?Asn?Ile?Asp?Asp?Ala?Thr?Met?Asp?Ile

410 415 420 425

gct?ctt?cga?agg?att?cta?aca?ttt?gca?ctc?aag?gcc?aag?gaa?gca?gat 1469

Ala?Leu?Arg?Arg?Ile?Leu?Thr?Phe?Ala?Leu?Lys?Ala?Lys?Glu?Ala?Asp

430 435 440

gtg?cca?agg?aag?aaa?caa?agt?tgg?caa?aac?aac?aac?ctt?aga?ctc?agc 1517

Val?Pro?Arg?Lys?Lys?Gln?Ser?Trp?Gln?Asn?Asn?Asn?Leu?Arg?Leu?Ser

445 450 455

ttc?aaa?tct?ggg?aaa?tat?gat?gat?gtc?aag?ttg?tct?cct?cgt?atg?atg 1565

Phe?Lys?Ser?Gly?Lys?Tyr?Asp?Asp?Val?Lys?Leu?Ser?Pro?Arg?Met?Met

460 465 470

tcc?cct?tgc?atg?agg?tcc?cct?cac?tcc?cct?ata?cca?caa?tca?ccc?ctt 1613

Ser?Pro?Cys?Met?Arg?Ser?Pro?His?Ser?Pro?Ile?Pro?Gln?Ser?Pro?Leu

475 480 485

gtt?cga?gct?taa?ttactcagtc?catctgtaat?taagatgagg?aaagttagtg 1665

Val?Arg?Ala

490

tatgtaattt?agaacaaatt?gatgcattct?ttcctgtggt?aatagccaat?aaaacatgga 1725

ccatttaatt?gactgtagac?aagaggaaga?aattcgaaag?t 1766

<210>3

<211>492

<212>PRT

＜213〉tree peony

<400>3

Met?Gly?Phe?Met?Ser?Thr?Asp?Gln?Gln?Lys?Gln?Leu?Leu?Ser?Lys?Met

1 5 10 15

Ala?Thr?Gly?Asn?Gly?His?Gly?Glu?Asp?Ser?Pro?Tyr?Phe?Asp?Gly?Trp

20 25 30

Lys?Ala?Tyr?Asp?Ser?Asn?Pro?Phe?His?Leu?Asn?Asn?Asn?Pro?Asn?Gly

35 40 45

Val?Ile?Gln?Met?Gly?Leu?Ala?Glu?Asn?Leu?Leu?Ser?Phe?Asp?Val?Ile

50 55 60

Gln?Glu?Trp?Val?Leu?Asn?Asn?Pro?Lys?Ala?Ser?Ile?Cys?Thr?Pro?Glu

65 70 75 80

Gly?Ile?Asn?Glu?Phe?Arg?Asp?Thr?Ala?Ile?Phe?Gln?Asp?Tyr?His?Gly

85 90 95

Phe?Pro?Glu?Phe?Arg?Asn?Ala?Val?Ala?Lys?Phe?Met?Gly?Lys?Val?Gly

100 105 110

Gly?Gly?Arg?Val?Thr?Phe?Asp?Pro?Asp?Arg?Ile?Val?Met?Ser?Gly?Gly

115 120 125

Ala?Thr?Gly?Ala?His?Glu?Ile?Leu?Ala?Phe?Cys?Leu?Ala?Asp?Pro?Gly

130 135 140

Asp?Ala?Phe?Leu?Val?Pro?Thr?Pro?Tyr?Tyr?Pro?Gly?Tyr?Asp?Arg?Asp

145 150 155 160

Leu?Arg?Trp?Arg?Thr?Gly?Ala?Gln?Leu?Leu?Pro?Val?Gln?Cys?Asp?Ser

165 170 175

Ser?Asn?Asn?Phe?Thr?Val?Thr?Ile?Ser?Ala?Leu?Glu?Leu?Ala?Tyr?Glu

180 185 190

Lys?Ala?Gln?Ala?Ala?Asn?Ile?Lys?Val?Lys?Gly?Leu?Ile?Ile?Asn?Asn

195 200 205

Pro?Ser?Asn?Pro?Leu?Gly?Thr?Val?Leu?Asn?Gly?Glu?Thr?Leu?Lys?Thr

210 215 220

Ile?Val?Asn?Phe?Ile?Asn?Glu?Lys?Asn?Ile?His?Leu?Val?Cys?Asp?Glu

225 230 235 240

Ile?Tyr?Ala?Ala?Thr?Val?Phe?Ser?His?Pro?Arg?Phe?Ile?Ser?Ile?Ala

245 250 255

Glu?Ile?Val?Asn?Asp?Met?Asn?Gly?Val?Asn?Arg?Asn?Leu?Ile?His?Val

260 265 270

Val?Tyr?Ser?Leu?Ser?Lys?Asp?Met?Gly?Phe?Pro?Gly?Phe?Arg?Val?Gly

275 280 285

Ile?Val?Tyr?Ser?Tyr?Asn?Asp?Ala?Val?Val?Asn?Cys?Ala?Arg?Lys?Met

290 295 300

Ser?Ser?Phe?Gly?Leu?Val?Ser?Thr?Gln?Thr?Gln?His?Leu?Ile?Ala?Ser

305 310 315 320

Met?Leu?Ser?Asp?Glu?His?Phe?Ile?Glu?Arg?Tyr?Ile?Val?Glu?Ser?Ala

325 330 335

Asn?Lys?Leu?Ala?Glu?Arg?Gln?Arg?Leu?Phe?Thr?Arg?Gly?Leu?Ser?Gln

340 345 350

Val?Gly?Ile?Asn?Phe?Leu?Lys?Ser?Asn?Ala?Gly?Leu?Phe?Leu?Trp?Met

355 360 365

Asp?Leu?Ser?Ser?Leu?Leu?Lys?Glu?Lys?Thr?Val?Glu?Ala?Glu?Ile?Thr

370 375 380

Leu?Trp?Arg?Val?Ile?Ile?Asn?Glu?Val?Lys?Leu?Asn?Val?Ser?Pro?Gly

385 390 395 400

Ser?Ser?Phe?His?Cys?Ser?Glu?Pro?Gly?Trp?Phe?Arg?Val?Cys?Phe?Ala

405 410 415

Asn?Ile?Asp?Asp?Ala?Thr?Met?Asp?Ile?Ala?Leu?Arg?Arg?Ile?Leu?Thr

420 425 430

Phe?Ala?Leu?Lys?Ala?Lys?Glu?Ala?Asp?Val?Pro?Arg?Lys?Lys?Gln?Ser

435 440 445

Trp?Gln?Asn?Asn?Asn?Leu?Arg?Leu?Ser?Phe?Lys?Ser?Gly?Lys?Tyr?Asp

450 455 460

Asp?Val?Lys?Leu?Ser?Pro?Arg?Met?Met?Ser?Pro?Cys?Met?Arg?Ser?Pro

465 470 475 480

His?Ser?Pro?Ile?Pro?Gln?Ser?Pro?Leu?Val?Arg?Ala

485 490

<210>4

<211>26

<212>DNA

＜213〉artificial sequence

<400>4

CGGGATCCGT?ATCAAGATTA?TCATGG 26

<210>5

<211>28

<212>DNA

＜213〉artificial sequence

<400>5

AACTGCAGGA?GAGGCTGTAG?AGAATATG 28

Claims (5)

1, peony ACC synthase gene Ps-ACS 1 specific probe, it has the nucleotide sequence shown in the SEQ IDNO.1, perhaps its useful length fragment.

2, probe as claimed in claim 1 is characterized in that, described useful length fragment is greater than 60bp.

3, probe as claimed in claim 2 is characterized in that, described useful length fragment is near 3 of SEQ ID NO.1 sequence ' end.

4, the test kit that contains each described probe of claim 1～3.

5, each described probe of claim 1～3 or the described test kit of claim 4 application in detecting peony ACC synthase gene Ps-ACS 1.

Images