CN101603078A - Peony ACC oxidase gene Ps-ACO 1 specific probe - Google Patents

Abstract

The invention provides a kind of peony ACC oxidase gene Ps-ACO 1 specific probe, it has the nucleotide sequence shown in the sequence table SEQ ID NO.1, perhaps its useful length fragment.Experiment shows that probe of the present invention can be used in the specific detection of Ps-ACO1 gene, and whether the ACO gene exists regulating and controlling effect in the open senescence process of tree peony cut-flower in order to verify, for the exploitation and even the transgenic breeding of its post-harvest fresh-keeping technology provides theoretical foundation.

Description

Peony ACC oxidase gene Ps-ACO 1 specific probe

Technical field

The present invention relates to biological technical field, be specifically related to the specific probe of peony ACC oxidase gene Ps-ACO 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, ACO generates ethene with the ACC oxidation in plant ethene biosynthetic pathway, be last enzyme in the whole process.Early stage studies show that, the ACC of external source is supplied with plant tissue the growing amount of ethene is increased, thereby think that it is a composition, does not constitute the Ethylene Biosynthesis rate-limiting enzyme.But also there are some to discover in recent years, the increase that IAA can promote ethylene yield by ethene auto-induction the ACO gene transcription realize, therefore, ACO also might be a rate-limiting enzyme that influences in the ethene biosynthetic pathway, and the ACO expression of gene is non-composing type, regulation and control on its transcriptional level may controlled the generating rate (ten Have and Woltering, 1997) of ethene.At present, avocado (McGarvey et al., 1990), Dianthus caryophyllus L. (Woodson et al., 1992), peach (Callahan et al., 1992), apple (Dong et al., 1992), butterfly orchid (O ' Noill et al., 1993), Arabidopis thaliana (Kieberet al., 1997) etc. the ACO gene cDNA clone of species obtains in succession, relevant ACO expression of gene analysis and research are also carried out in multiple cut-flower, wherein study the most deeply with the model plant Dianthus caryophyllus L., now successfully suppressed the expression of himself by importing antisense ACO gene, made ethylene yield significantly descend, petal is crispaturaed and is suppressed, cutting flower life obviously prolongs (Savin et al., 1995).

Up to now, the relevant Study on mechanism of ethene in tree peony cut-flower postharvest senescence still is blank, more do not have the correlative study on the molecular level.Whether the ACO gene exists regulating and controlling effect in the open senescence process of tree peony cut-flower in order to verify, for the exploitation and even the transgenic breeding of its post-harvest fresh-keeping technology provides theoretical foundation, Ps-ACO1 in tree peony ' Luoyang the is red ' petal is separated first, 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 ACO different members in the tree peony ethylene reaction because homology is higher.

Summary of the invention

The object of the present invention is to provide the specific probe of peony ACC oxidase gene Ps-ACO 1, for the follow-up functional study of Ps-ACO1 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-ACO1 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-ACO1 gene.

The present invention separates the Ps-ACO1 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 ACO different members in the tree peony ethylene reaction because homology is higher.Whether the ACO gene exists regulating and controlling effect in the open senescence process of tree peony cut-flower in order to verify, for the exploitation and even the transgenic breeding of its post-harvest fresh-keeping technology provides theoretical foundation.

Description of drawings

Fig. 1 Ps-ACO1 gene cDNA full length nucleotide and the aminoacid sequence of inferring;

The Ps-ACO1 aminoacid sequence conserved regions prediction that Fig. 2 BlastP derives;

Fig. 3 tree peony Ps-ACO1 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 as probe with the specific fragment of digoxigenin labeled;

Fig. 4 ethene and 1-MCP handle the influence to Ps-ACO1 genetic expression in ' Luoyang is red ' petal, and cut-flower is used for the petal sampling immediately and extracts total RNA after measuring the ethene growing amount, and 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-ACO1 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-ACO1 dna homolog

(1) reverse transcription

Get the above-mentioned total RNA of 2 μ g, add DEPCddH 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:

ACO-F：5′-CGAAAATTGGGGTTTGTATGAG-3′

ACO-R：5′-TCGAAGCGCGGTTCCTTGG-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 carried out bacterium liquid PCR and is identified.

(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-ACO1 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 '-AGCAGCTCCTCGGCCAGTGTCTCT-3 ', 5 ' RACE Auele Specific Primer is 5 '-GATGCACCGTGTGATCGCTCAAAC-3 ', at 94 ℃ of 30s, 59 ℃ 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-ACO1 gene cDNA full length sequence mutually.

4.Ps-ACO1cDNA full length sequence

The tree peony Ps-ACO1 gene intermediate segment of RT-PCR, 3 ' RACE, 5 ' RACE amplification gained is spliced mutually with end sequence, and obtaining length is the tree peony Ps-ACO1cDNA full length sequence (Fig. 1) of 1221bp.Discover at present, in other plant, separate the ACO full length gene obtain 306-326 amino acid (the Pogson et al. that generally encodes, 1995), the ORF Finder that utilizes NCBI to provide carries out sequential analysis and finds, this cDNA total length includes opening code-reading frame and a poly (A) tail of a 939bp, 5 ' untranslated head of district 65bp, 3 ' untranslated head of district 217bp, 312 amino acid of encoding.ProtParam ( Http:// au.expasy.org/tools/protparam.html) predict that this proteic molecular weight is 35.22kD, theoretical iso-electric point is 5.32.

With BlastP the coded albumen conserved regions of Ps-ACO1 is predicted discovery, with totally two of the albumen conserved regions of this gene coupling, be respectively the PcbC and the 2-OG-FeII_Oxy (Fig. 2) that depends on the dioxygenase family of ferrous ion and 2-oxoglutaric acid of dioxygenase family, this and the present result of study unanimity (Ververdis and John, 1991) of relevant ACO property of protein.Utilize prosite software ( Http:// npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl? Page=npsa_prosite.html) discovery of the proteic specific function of predictive coding site; Ps-ACO1 full-length cDNA proteins encoded contains 1 protein kinase C phosphorylation site (the amino-acid residue SVK of 303-305 position), 2 casein kinase i I phosphorylation site (amino-acid residue SKIE of 116-119 position; the amino-acid residue TLAE of 120-123 position) and 4 N-Semen Myristicae acidylate sites (be positioned at the amino-acid residue GGERAA of 14-19 position; the amino-acid residue GLEKGY of 134-139 position; the amino-acid residue GTKVSN of 153-158 position, the amino-acid residue GNRMSL of 239-244 position).

The preparation of embodiment 2 probes and hybridization detect

1. probe preparation

According to the sequence of Ps-ACO1 near 3 ' end, design a pair of special primer, Ps-ACO1F:5 '-ATGGTAATAGGATGTCGCTTG-3 ', Ps-ACO1R:5 '-ACCCCAACATTCTAAACACAC-3 ', product with Ps-ACO13 ' RACE is a template, according to PCR DIG probe synthesis Kit (Roche, Cat.No.1636090) specification sheets, carry out the digoxigenin labeled of amplified fragments, the preparation specific probe.After reaction finishes, get 3 μ LPCR 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 the sample-loading buffer that adds 1/10V in the product to enzyme, abundant mixing, centrifugal collection, 65 ℃ of sex change 10min, ice carries out 0.8% agarose gel electrophoresis (voltage 1-2V/cm) 8h after swashing 5min, migrates to 2/3 place of glue to bromjophenol blue.

(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, each 5-10min.Remove 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-ACO1 specific probe sequence and specific detection result

The Ps-ACO1 specific probe sequence that amplification obtains is across 3 of coding region ' end and part 3 ' non-translational region, and long is 338bp, 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, 0.8% (w/v) agarose gel electrophoresis is transferred on the nylon membrane after separating, and hybridizes as probe with the specific fragment of digoxigenin labeled.By Southern hybridization, detect the specificity (Fig. 3) of this probe.The result shows and all only occurs a band on each swimming lane, proves that the specific probe that this probe can be used as Ps-ACO1 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, and is stored in behind the liquid nitrogen flash freezer in-80 ℃ of Ultralow Temperature Freezers, is used for sampling and extracts 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, the expression of Ps-ACO1 does not change with open old and feeble process in the cut-flower that contrast or ethene, 1-MCP are handled, but is subjected to the slight promotion or the inhibition of ethene and 1-MCP processing.Illustrate that this probe can be used to carry out gene expression analysis research.

5. the 65th to 240 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-ACO1 in the peony petal after the different treatment, illustrate that this probe can detect the Ps-ACO1 gene specifically.

6. the 105th to 310 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-ACO1 in the peony petal after the different treatment, illustrate that this probe can detect the Ps-ACO1 gene specifically.

Reference

Sa nurse Brooker J, Ritchie E F not, Manny A Disi T, work. the molecular cloning experiment guide. the 3rd edition. Beijing: Science Press, 2002.

History Guoan, Guo Xiangfeng, Han Jianguo is bloomed and the research of ethene and lipid peroxidation between senescence phase etc. tree peony. Northwest Agricultural University's journal 1999,27 (5): 50-53.

History Guoan, Yang Zhengshen, Wang Changzhong, etc. temperature and chemical agent discharge tree peony cut-flower ethene and the storage quality influence. northern gardening, 1997,6:62-63.

Callahan?A?M，Morgens?P?H，Wright?P，Nichols?Jr?K?E.Comparison?of?pch313(pTOM?13homolog)RNA?accumulation?during?fruit?softening?and?wounding?of?two?phenotypicallydifferent?peach?cultivars.Plant?Physiol，1992，100：482-488.

Dong?JG，Olson?D，Silverstone?A?et?al.Sequence?of?a?cDNA?coding?for?a1-aminocyclopropane-1-carboxylate?oxidase?homolog?from?apple?fruit.Plant?Physiol，1992，98：1530.

Jia?P?Y，Zhou?L，Guo?W?W，et?al.Postharvest?behavior?and?endogenous?ethylene?pattern?oftree-peony?cut?flowers.International?Society?for?Horticultural?Science.27 ^th?InternationalHorticultural?Congress&Exhibition?Abstracts.Seoul?Korea，2006：267.

Kieber?J?J.The?ethylene?response?pathway?in?Arabidopsis.Annu?Rev?Plant?Mol?Biol，1997，48：277-296.

McGarvey?D?J，Yu?H，Christoffersen?R?E.Nucleotide?sequence?of?a?ripening-related?cDNA?fromavocado?fruit.Plant?Mol?Biol，1990，15：165-167.

O’Neil?S?D，Nadeau?JA，Zhang?X?S，BuiA?Q，Halevy?A?H.Interorgan?regulation?of?ethtlenebiosynthetic?genes?by?pollination.Plant?Cell，1993，5：419-432..

Pogson?B?J，Downs?C?G，Davies?K?M..Differential?expression?of?two?1-aminocyclopropane-1-carboxylic?acid?oxidase?genes?in?broccoli?after?harvest.Plant?Physiol.，1995，108：651-657.

Savin?K?W，Baudinette?S?C，Graham?M?W?et?al.Antisense?ACC?oxidase?RNA?delays?carnationpetal?senescence，1995，30：970-972.

ten?Have?A，Woltering?EJ.Ethylene?biosynthetic?genes?are?differently?expressed?during?carnation(Dianthus?caryopgyllus?L.)flower?senescence.Plant?Mol.Bio.，1997，84：89-97.

Ververidis?P，John?P.Complete?recovery?in?vitro?of?ethylene?forming?enzyme?activity.Phytochemistry，1991，30：：725-727.

Woodson?WR，Park?KY，Drory?A，Larsen?PB，Wang?H.Expression?of?ethylene?biosyntheticpathway?transcripts?in?senescence?carnation?flowers.Plant?Physiol，1992，99：526-532.

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

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

Sequence table

＜110〉Beijing Forestry University

＜120〉peony ACC oxidase gene Ps-ACO 1 specific probe

<130>

<160>5

<170>PatentIn?version?3.3

<210>1

<211>338

<212>DNA

＜213〉tree peony

<400>1

atggtaatag?gatgtcgctt?gcttcgtttt?acaacccggg?aagcgatgcc?gttatctatc 60

cggcaccggc?tttgctaggg?aaagaggcac?aagtgtaccc?caaattcatt?tttgaagact 120

acatgaagct?gtatgctggg?ttgaagtttc?aggccaagga?gcctagattt?gaggccatga 180

aagccgtgga?agaaagtgtc?aagttgggtc?caattgcaac?tgcttgagag?gtgtttttaa 240

tagtgttgtg?ttggttgttg?ttgtttgttg?gtgtgtgtgt?gtgaccaaag?ctagtttaag 300

tcaaaatgga?aaaataagtg?tgtttagaat?gttggggt 338

<210>2

<211>1221

<212>DNA

＜213〉tree peony

<220>

<221>5’UTR

<222>(1)..(65)

<220>

<221>CDS

<222>(66)..(1004)

<220>

<221>3’UTR

<222>(1005)..(1221)

<400>2

acgcggggag?atcagtaaca?cacaactcag?agattgagag?agagagagag?agagaaagag 60

agaaa?atg?gca?aac?ttc?cca?gtg?atc?aac?tta?gag?aaa?cta?aat?ggt?ggg 110

Met?Ala?Asn?Phe?Pro?Val?Ile?Asn?Leu?Glu?Lys?Leu?Asn?Gly?Gly

1 5 10 15

gag?aga?gca?gct?acc?atg?gag?atc?atc?aac?gat?gct?tgt?gag?aac?tgg 158

Glu?Arg?Ala?Ala?Thr?Met?Glu?Ile?Ile?Asn?Asp?Ala?Cys?Glu?Asn?Trp

20 25 30

ggt?ttc?ttt?gag?ata?gtg?aac?cat?gga?atc?tct?cct?gag?ttc?atg?gac 206

Gly?Phe?Phe?Glu?Ile?Val?Asn?His?Gly?Ile?Ser?Pro?Glu?Phe?Met?Asp

35 40 45

aca?gta?gaa?aag?ttg?aca?aag?gag?cat?tac?aag?aag?tgt?atg?gag?cag 254

Thr?Val?Glu?Lys?Leu?Thr?Lys?Glu?His?Tyr?Lys?Lys?Cys?Met?Glu?Gln

50 55 60

aag?ttc?aag?gaa?ctg?gtg?gca?agc?aaa?gct?cta?gag?aca?gaa?atc?aat 302

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

65 70 75

gat?atg?gat?tgg?gaa?agc?act?ttc?cac?ttg?cgc?cat?ctc?ccc?ata?tcc 350

Asp?Met?Asp?Trp?Glu?Ser?Thr?Phe?His?Leu?Arg?His?Leu?Pro?Ile?Ser

80 85 90 95

aac?atg?gct?gaa?att?cct?gat?ctt?gat?gat?gaa?tac?agg?aaa?gtc?atg 398

Asn?Met?Ala?Glu?Ile?Pro?Asp?Leu?Asp?Asp?Glu?Tyr?Arg?Lys?Val?Met

100 105 110

aag?gaa?ttt?gca?tca?aaa?ata?gag?aca?ctg?gcc?gag?gag?ctg?cta?gac 446

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

115 120 125

ttg?ata?tgt?gaa?aat?ctc?gga?cta?gag?aaa?ggc?tac?tta?aag?aag?gcc 494

Leu?Ile?Cys?Glu?Asn?Leu?Gly?Leu?Glu?Lys?Gly?Tyr?Leu?Lys?Lys?Ala

130 135 140

ttt?tat?gga?tcc?aag?ggt?cca?acc?ttt?ggc?acc?aag?gtc?agc?aac?tac 542

Phe?Tyr?Gly?Ser?Lys?Gly?Pro?Thr?Phe?Gly?Thr?Lys?Val?Ser?Asn?Tyr

145 150 155

cct?cca?tgc?ccc?aaa?cca?gac?ttg?att?aag?gga?ctc?cga?gct?cac?act 590

Pro?Pro?Cys?Pro?Lys?Pro?Asp?Leu?Ile?Lys?Gly?Leu?Arg?Ala?His?Thr

160 165 170 175

gat?gcc?ggt?ggc?ctt?atc?tta?ctc?ttc?caa?gat?gat?cgt?gtt?agc?ggt 638

Asp?Ala?Gly?Gly?Leu?Ile?Leu?Leu?Phe?Gln?Asp?Asp?Arg?Val?Ser?Gly

180 185 190

ctt?cag?cta?ctc?aaa?gac?gat?cag?tgg?gtc?gat?gtt?cct?cct?atg?cgt 686

Leu?Gln?Leu?Leu?Lys?Asp?Asp?Gln?Trp?Val?Asp?Val?Pro?Pro?Met?Arg

195 200 205

cat?tcc?att?gtg?atc?aac?att?ggt?gat?cag?ctt?gag?gtg?atc?acc?aat 734

His?Ser?Ile?Val?Ile?Asn?Ile?Gly?Asp?Gln?Leu?Glu?Val?Ile?Thr?Asn

210 215 220

gga?aag?tac?aag?agt?gtg?atg?cac?cgt?gtg?atc?gct?caa?acc?gat?ggt 782

Gly?Lys?Tyr?Lys?Ser?Val?Met?His?Arg?Val?Ile?Ala?Gln?Thr?Asp?Gly

225 230 235

aat?agg?atg?tcg?ctt?gct?tcg?ttt?tac?aac?ccg?gga?agc?gat?gcc?gtt?830

Asn?Arg?Met?Ser?Leu?Ala?Ser?Phe?Tyr?Asn?Pro?Gly?Ser?Asp?Ala?Val

240 245 250 255

atc?tat?ccg?gca?ccg?gct?ttg?cta?ggg?aaa?gag?gca?caa?gtg?tac?ccc?878

Ile?Tyr?Pro?Ala?Pro?Ala?Leu?Leu?Gly?Lys?Glu?Ala?Gln?Val?Tyr?Pro

260 265 270

aaa?ttc?att?ttt?gaa?gac?tac?atg?aag?ctg?tat?gct?ggg?ttg?aag?ttt 926

Lys?Phe?Ile?Phe?Glu?Asp?Tyr?Met?Lys?Leu?Tyr?Ala?Gly?Leu?Lys?Phe

275 280 285

cag?gcc?aag?gag?cct?aga?ttt?gag?gcc?atg?aaa?gcc?gtg?gaa?gaa?agt 974

Gln?Ala?Lys?Glu?Pro?Arg?Phe?Glu?Ala?Met?Lys?Ala?Val?Glu?Glu?Ser

290 295 300

gtc?aag?ttg?ggt?cca?att?gca?act?gct?tga?gaggtgtttt?taatagtgat 1024

Val?Lys?Leu?Gly?Pro?Ile?Ala?Thr?Ala

305 310

gtgttggttg?ttgttgtttg?ttggtgtgtg?tgtgtgacca?aagctagttt?aagtcaaaat 1084

ggaaaaataa?gtgtgtttag?aatgttgggg?tcagagccta?ctataagggg?tatcagtatt 1144

actatttgta?taaagtgacc?aaaattactt?cttataaagt?attattcgaa?aaaaaaaaaa 1204

aaaaaaaaaa?aaaaaaa 1221

<210>3

<211>312

<212>PRT

＜213〉tree peony

<400>3

Met?Ala?Asn?Phe?Pro?Val?Ile?Asn?Leu?Glu?Lys?Leu?Asn?Gly?Gly?Glu

1 5 10 15

Arg?Ala?Ala?Thr?Met?Glu?Ile?Ile?Asn?Asp?Ala?Cys?Glu?Asn?Trp?Gly

20 25 30

Phe?Phe?Glu?Ile?Val?Asn?His?Gly?Ile?Ser?Pro?Glu?Phe?Met?Asp?Thr

35 40 45

Val?Glu?Lys?Leu?Thr?Lys?Glu?His?Tyr?Lys?Lys?Cys?Met?Glu?Gln?Lys

50 55 60

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

65 70 75 80

Met?Asp?Trp?Glu?Ser?Thr?Phe?His?Leu?Arg?His?Leu?Pro?Ile?Ser?Asn

85 90 95

Met?Ala?Glu?Ile?Pro?Asp?Leu?Asp?Asp?Glu?Tyr?Arg?Lys?Val?Met?Lys

100 105 110

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

115 120 125

Ile?Cys?Glu?Asn?Leu?Gly?Leu?Glu?Lys?Gly?Tyr?Leu?Lys?Lys?Ala?Phe

130 135 140

Tyr?Gly?Ser?Lys?Gly?Pro?Thr?Phe?Gly?Thr?Lys?Val?Ser?Asn?Tyr?Pro

145 150 155 160

Pro?Cys?Pro?Lys?Pro?Asp?Leu?Ile?Lys?Gly?Leu?Arg?Ala?His?Thr?Asp

165 170 175

Ala?Gly?Gly?Leu?Ile?Leu?Leu?Phe?Gln?Asp?Asp?Arg?Val?Ser?Gly?Leu

180 185 190

Gln?Leu?Leu?Lys?Asp?Asp?Gln?Trp?Val?Asp?Val?Pro?Pro?Met?Arg?His

195 200 205

Ser?Ile?Val?Ile?Asn?Ile?Gly?Asp?Gln?Leu?Glu?Val?Ile?Thr?Asn?Gly

210 215 220

Lys?Tyr?Lys?Ser?Val?Met?His?Arg?Val?Ile?Ala?Gln?Thr?Asp?Gly?Asn

225 230 235 240

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

245 250 255

Tyr?Pro?Ala?Pro?Ala?Leu?Leu?Gly?Lys?Glu?Ala?Gln?Val?Tyr?Pro?Lys

260 265 270

Phe?Ile?Phe?Glu?Asp?Tyr?Met?Lys?Leu?Tyr?Ala?Gly?Leu?Lys?Phe?Gln

275 280 285

Ala?Lys?Glu?Pro?Arg?Phe?Glu?Ala?Met?Lys?Ala?Val?Glu?Glu?Ser?Val

290 295 300

Lys?Leu?Gly?Pro?Ile?Ala?Thr?Ala

305 310

<210>4

<211>22

<212>DNA

＜213〉artificial sequence

<400>4

CGAAAATTGG?GGTTTGTATG?AG 22

<210>5

<211>19

<212>DNA

＜213〉artificial sequence

<400>5

TCGAAGCGCG?GTTCCTTGG 19

Claims (5)

1, peony ACC oxidase gene Ps-ACO 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 oxidase gene Ps-ACO 1.

Images