CN103278502A - Detection method for promoter activity - Google Patents
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- CN103278502A CN103278502A CN2013101428163A CN201310142816A CN103278502A CN 103278502 A CN103278502 A CN 103278502A CN 2013101428163 A CN2013101428163 A CN 2013101428163A CN 201310142816 A CN201310142816 A CN 201310142816A CN 103278502 A CN103278502 A CN 103278502A
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Abstract
The invention relates to a detection method for promoter activity. The detection method comprises the steps of infecting a plant embryo by agrobacterium, co-culturing for at least two days, and carrying out GUS identification. The detection method for the promoter activity can identify the promoter activity through GUS staining analysis and/or GUS protein content determination only by co-culturing the plant embryo (particularly corn embryo) for three days, without a tissue culture process lasting for a plurality of weeks or even a plurality of months, and omits links such as corn genotype transformed specificity, induced differentiation, difficulty to take roots, etc. Therefore, time is saved; save time, screening efficiency is increased; production cost is reduced; and experimental results are intuitive and accurate.
Description
Technical field
The present invention relates to a kind of detection method of promoter activity, particularly relate to a kind of method for quick of constitutive promoter.
Background technology
The expression of allogeneic dna sequence in plant host depends on has the promoter that is operably connected that works in plant host.The selection of promoter sequence will determine time and the position that allogeneic dna sequence is expressed in plant host.Therefore, when needs are expressed in plant is preferably organized, the using-system specificity promoter.On the contrary, when needs are expressed, use constitutive promoter in whole vegetable cells.Can obtain plant transformed by the separate living tissue of direct processing plant embryos, whether in destination organization, start the purpose allogeneic dna sequence by the analysis to transformed plant with definite promoter sequence and express.
The corn gene technology has obtained very big development in recent years, has set up a series of transformation technologies such as agrobacterium-mediated transformation, particle bombardment, PEG method, electric shocking method, silicon carbide fibre mediated method and pollen tube introductory technique.Comparatively speaking, agrobacterium-mediated transformation is higher and become the plant transgenic technology that is most widely used at present with its efficient, and the basic procedure of this method is: the explant → callus → agrobacterium mediation converted → bud of growing thickly → screen → take root → transformant.Therefore, when utilizing agriculture bacillus mediated maize genetic conversion to carry out the active detection of promoter sequence, there is following defective:
(1) the callus formation time often reaches a few weeks longer even several months, and is very consuming time, and workload is heavy;
(2) the maize callus induce differentiation than crop difficulties such as paddy rice, the differentiation rate of the bud of growing thickly is very unstable;
(3) difficulty of taking root of differentiation plant;
(4) because the genotypic difference of transgene receptor plant can not be suitable for diversity kind or strain, showing comparatively significantly, maize genotype transforms specificity.
Summary of the invention
The detection method that the purpose of this invention is to provide a kind of promoter activity overcomes effectively that prior art is consuming time, workload is heavy, induces differentiation and the difficulty of taking root, genotype to transform technological deficiencies such as specificity.
For achieving the above object, the invention provides a kind of detection method of promoter activity, comprising:
Agrobacterium is infected plant embryos;
Cultivated at least 2 days altogether;
GUS identifies.
On the basis of technique scheme, described Agrobacterium comprises promoter sequence.
Preferably, described promoter is constitutive promoter.
Further, described constitutive promoter comprises Ubi gene promoter, APX gene promoter, Actin1 gene promoter or PGD1 gene promoter.
On the basis of technique scheme, described plant is corn.
The described cultivation altogether was specially common cultivation 2-5 days at least 2 days.Further, the described maize that was specially at least 2 days after Agrobacterium infected of cultivating was altogether cultivated 3 days altogether.
On the basis of technique scheme, described GUS identifies and comprises GUS staining analysis and/or GUS determination of activity.
Further, described GUS staining analysis is specially according to the size of GUS dyeing area promoter activity is carried out qualitative analysis.The protein extract that described GUS determination of activity is specially maize carries out the GUS determination of activity.
Term among the present invention " promoter " refers to the DNA regulatory region, contains usually to guide rna plymerase ii to start the synthetic TATA box of RNA at the suitable transcription initiation site of specific coding sequence.Promoter can contain other recognition sequence in addition, generally is positioned at upstream or the 5 ' end of TATA box, is called upstream promoter element, and they influence transcription initiation speed.Admittedly, because the own warp of promoter region nucleotide sequence is definite, belong to prior art from the interior further separation of 5 ' non-translational region of concrete promoter region upstream and evaluation regulating element.Therefore, promoter region further comprises the upstream regulation element, and it is given any heterologous nucleotide sequence that is operably connected with promoter sequence and expresses.
Term among the present invention " gene " refers to contain at cell any dna fragmentation in the DNA zone (" the DNA zone of transcribing ") that is transcribed into RNA molecule (for example mRNA) under suitable regulation and control zone (for example plant expressiveness promoter region) control.Therefore, gene may contain the dna fragmentation that several operability connect, for example promoter, 5 ' untranslated leader, coding region and contain 3 ' untranslated zone of polyadenylation site.The endogenous plant gene is the gene of natural discovery in plant species.Mosaic gene is any gene of usually not finding in plant species, or under natural situation its promoter and the DNA zone of partly or entirely transcribing or with the irrelevant any gene in another regulation and control zone at least of this gene.
Term among the present invention " constitutive promoter " refers under the control of such promoter, and the expression of purpose heterologous nucleotide sequence is constant on certain level substantially, do not have notable difference at different tissues and/or the different growth and development stage expression of plant.It is described that to be organized as origin source in the plant identical and carry out the structural units that one or more cell types set of same function form; for example protective tissue, conducting tissue, nutritive issue, mechanical tissue, separate living tissue; severally different organize organic cooperation, be closely connected; form different organs (organ); work in coordination between the different organs, more effectively finish organic whole vital movement process.Described growth and development stage can be divided into embryo stage, seedling stage, the stage of ripeness and old and feeble stage according to the difference of phytomorph, function.The 35S rna gene promoter that derives from cauliflower mosaic virus (CaMV) is used as the main promoter of dicotyledon; And the ubiquitin promoter of the actin promoter of rice plants and cereal is suitable for monocotyledon usually.
Promoter sequence of the present invention and fragment thereof when being assembled into dna structure promoter sequence is operably connected with the purpose heterologous nucleotide sequence, are used for heredity and control any plant.Described " being operably connected " refers to functional connection the between promoter sequence of the present invention and second sequence, and wherein promoter sequence starts and regulates transcribing corresponding to the dna sequence dna of second sequence.Usually, being operably connected refers to that the nucleotide sequence that is connected is continuous, be adjacent in case of necessity in conjunction with two protein-coding regions, and in same reading frame.In this way, the promoter nucleotide sequence constitutes described mosaic gene with the purpose heterologous nucleotide sequence to be provided in expression cassette, to express in the purpose plant.
The invention provides a kind of detection method of promoter activity, have the following advantages:
1, directly perceived.Can with the naked eye directly observe by GUS dyeing in the detection method of promoter activity of the present invention and promoter activity is carried out qualitative analysis, especially constitutive promoter.
2, quick.Only need in the detection method of promoter activity of the present invention plant embryos (particularly maize) was cultivated 3 days altogether, just can identify promoter activity by GUS staining analysis and/or gus protein assay, need not to experience the tissue culture procedures of a few weeks longer even several months, avoid maize genotype to transform specificity, induced links such as differentiation and the difficulty of taking root, save the time, improved screening effeciency.
3, economy.Though the detection method of promoter activity of the present invention has been utilized the transgenosis means, do not experience the process that follow-up tissue is cultivated, reduced because of the essential consumption of acquisition transfer-gen plant, thereby greatly reduced production cost.
4, accurate.Though the detection method of promoter activity of the present invention is only utilized plant embryos (particularly maize), by GUS staining analysis and/or GUS determination of activity promoter activity is identified, but compare with the transfer-gen plant of experience tissue culture procedures, the trend of promoter activity is coincide, and namely testing result is accurately.
Below by drawings and Examples, technical scheme of the present invention is described in further detail.
Description of drawings
Fig. 1 makes up process flow diagram for the recombinant cloning vector pT-prZmUbi of the detection method of promoter activity of the present invention;
Fig. 2 makes up process flow diagram for the recombinant expression carrier p90001 of the detection method of promoter activity of the present invention;
Fig. 3 is the recombinant expression carrier p90030 synoptic diagram of the detection method of promoter activity of the present invention;
Fig. 4 is the recombinant expression carrier p90031 synoptic diagram of the detection method of promoter activity of the present invention;
Fig. 5 is the recombinant expression carrier p90039 synoptic diagram of the detection method of promoter activity of the present invention;
Fig. 6 is the recombinant expression carrier p80000 synoptic diagram of the detection method of promoter activity of the present invention;
Fig. 7 is the GUS colored graph of the transgenic corns embryo of the detection method of promoter activity of the present invention.
Embodiment
Further specify the technical scheme of the detection method of promoter activity of the present invention below by specific embodiment.
First embodiment, contain the structure of the recombinant expression carrier of promoter sequence
1, makes up the recombinant cloning vector that contains promoter sequence to be measured
The prZmUbi nucleotide sequence is connected into cloning vector pGEM-T(Promega, Madison, USA, CAT:A3600) on, operation steps is undertaken by the product pGEM-T of Promega company carrier instructions, obtain recombinant cloning vector pT-prZmUbi, it makes up flow process, and (wherein, Amp represents ampicillin resistance gene as shown in Figure 1; F1 represents the origin of replication of bacteriophage f1; LacZ is the LacZ initiation codon; SP6 is the SP6RNA polymerase promoter; T7 is the t7 rna polymerase promoter; PrZmUbi is the Ubiqutin(ubiquitin of corn variety B73) gene promoter (SEQ ID NO:1); MCS is multiple clone site).
Then recombinant cloning vector pT-prZmUbi is used heat shock method transformed into escherichia coli T1 competent cell (Transgen, Beijing, China, CAT:CD501), its hot shock condition is: 50 μ l Escherichia coli T1 competent cells, 10 μ l plasmid DNA (recombinant cloning vector pT-prZmUbi), 42 ℃ of water-baths 30 seconds; Cultivate 1 hour (shaking table shakes under the 200rpm rotating speed) for 37 ℃, scribble the IPTG(isopropylthio-on the surface) and X-gal(5-bromo-4-chloro-3-indoles-β-D-galactoside) LB flat board (the tryptone 10g/L of ampicillin (100mg/L), yeast extract 5g/L, NaCl10g/L, agar 15g/L transfers pH to 7.5 with NaOH) last grow overnight.The picking white colony, in LB fluid nutrient medium (NaCl10g/L, ampicillin 100mg/L transfers pH to 7.5 with NaOH for tryptone 10g/L, yeast extract 5g/L) under 37 ℃ of conditions of temperature overnight incubation.Alkaline process extracts its plasmid: with bacterium liquid centrifugal 1min under the 12000rpm rotating speed, remove supernatant, the precipitation thalline is with the solution I (25mM Tris-HCl, 10mM EDTA(ethylenediamine tetraacetic acid) of 100 μ l ice precooling, 50mM glucose, pH8.0) suspension; The solution II (0.2M NaOH, 1%SDS(lauryl sodium sulfate) that adds the new preparation of 150 μ l), pipe is put upside down 4 times, mixed, put 3-5min on ice; Add the ice-cold solution III of 150 μ l (4M potassium acetate, 2M acetic acid), abundant mixing is placed 5-10min on ice immediately; Centrifugal 5min under 4 ℃ of temperature, rotating speed 12000rpm condition adds 2 times of volume absolute ethyl alcohols in supernatant, room temperature is placed 5min behind the mixing; Centrifugal 5min under 4 ℃ of temperature, rotating speed 12000rpm condition abandons supernatant, and precipitation is to dry after 70% ethanol washs with concentration (V/V); Add 30 μ l and contain RNase(20 μ g/ml) TE(10mM Tris-HCl, 1mM EDTA, PH8.0) dissolution precipitation; In 37 ℃ of following water-bath 30min of temperature, digestion RNA; Standby in temperature-20 ℃ preservation.
The plasmid that extracts is after NcoI and PstI enzyme are cut evaluation, positive colony is carried out sequence verification, and the result shows that the Ubiqutin gene promoter sequence of the corn variety B73 that inserts among the recombinant cloning vector pT-prZmUbi is the nucleotide sequence shown in the SEQ ID NO:1 in the sequence table.
Method according to above-mentioned structure recombinant cloning vector pT-prZmUbi, the prOsAPX nucleotide sequence is connected on the cloning vector pGEM-T, obtain recombinant cloning vector pT-prOsAPX, wherein, prOsAPX is the fine ascorbate peroxidase enzyme gene promoter (SEQ ID NO:2) of rice varieties Japan.Enzyme is cut with prOsAPX nucleotide sequence described in the sequence verification recombinant cloning vector pT-prOsAPX and is correctly inserted.
Method according to above-mentioned structure recombinant cloning vector pT-prZmUbi, the prOsAct1 nucleotide sequence is connected on the cloning vector pGEM-T, obtain recombinant cloning vector pT-prOsAct1, wherein, prOsAct1 is the actin gene promotor (SEQ ID NO:3) of rice varieties.Enzyme is cut with prOsAct1 nucleotide sequence described in the sequence verification recombinant cloning vector pT-prOsAct1 and is correctly inserted.
Method according to above-mentioned structure recombinant cloning vector pT-prZmUbi, the prOsPGD1 nucleotide sequence is connected on the cloning vector pGEM-T, obtain recombinant cloning vector pT-prOsPGD1, wherein, prOsPGD1 is the fine glucose phosphate dehydrogenase gene promoter (SEQ ID NO:4) of rice varieties Japan.Enzyme is cut with prOsPGD1 nucleotide sequence described in the sequence verification recombinant cloning vector pT-prOsPGD1 and is correctly inserted.
2, make up the recombinant expression carrier that contains promoter sequence to be measured
With restriction enzyme BamHI and HindIII respectively enzyme cut recombinant cloning vector pT-prZmUbi and expression vector p90000(carrier framework: pCAMBIA2301(CAMBIA mechanism can provide)), the prZmUbi nucleotide sequence that downcuts is inserted between the BamHI and HindIII site of expression vector p90000, it is well-known to those skilled in the art utilizing conventional enzyme blanking method carrier construction, be built into recombinant expression carrier p90001, it makes up flow process (Kan: kanamycin gene as shown in Figure 2; RB: right margin; PrZmUbi: gene promoter (SEQ ID NO:1) the Ubiqutin(ubiquitin of corn variety B73); GUS: beta-glucosiduronatase gene (SEQ ID NO:5); Nos: the terminator of rouge alkali synthetase gene (SEQ ID NO:6); LB: left margin).
With heat shock method transformed into escherichia coli T1 competent cell, its hot shock condition is with recombinant expression carrier p90001: 50 μ l Escherichia coli T1 competent cells, 10 μ l plasmid DNA (recombinant expression carrier p90001), 42 ℃ of water-baths 30 seconds; Cultivate 1 hour (shaking table shakes under the 200rpm rotating speed) for 37 ℃; Containing LB solid plate (the tryptone 10g/L of 50mg/L kanamycins (Kanamycin) then, yeast extract 5g/L, NaCl10g/L, agar 15g/L transfers pH to 7.5 with NaOH) go up under 37 ℃ of conditions of temperature and cultivated 12 hours, the picking white colony, at LB fluid nutrient medium (tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, kanamycins 50mg/L transfers pH to 7.5 with NaOH) under 37 ℃ of conditions of temperature overnight incubation.Alkaline process extracts its plasmid.The plasmid that extracts is cut the back with restriction enzyme BamHI and HindIII enzyme to be identified, and with the positive colony evaluation of checking order, the result shows that the nucleotides sequence of recombinant expression carrier p90001 between BamHI and HindIII site classify nucleotide sequence shown in the SEQ ID NO:1, i.e. the Ubiqutin(ubiquitin of corn variety B73 in the sequence table as) gene promoter sequence.
According to the method for above-mentioned structure recombinant expression carrier p90001, BamHI and HindIII enzyme are cut the described prOsAPX nucleotide sequence insertion expression vector p90000 that recombinant cloning vector pT-prOsAPX downcuts, obtain recombinant expression carrier p90030.Enzyme is cut and sequence verification recombinant expression carrier p90030 is described prOsAPX nucleotide sequence between BamHI and HindIII site, as shown in Figure 3.
According to the method for above-mentioned structure recombinant expression carrier p90001, KpnI and NotI enzyme are cut the described prOsAct1 nucleotide sequence insertion expression vector p90000 that recombinant cloning vector pT-prOsAct1 downcuts, obtain recombinant expression carrier p90031.Enzyme is cut and sequence verification recombinant expression carrier p90031 is described prOsAct1 nucleotide sequence between KpnI and NotI site, as shown in Figure 4.
According to the method for above-mentioned structure recombinant expression carrier p90001, KpnI and NotI enzyme are cut the described prOsPGD1 nucleotide sequence insertion expression vector p90000 that recombinant cloning vector pT-prOsPGD1 downcuts, obtain recombinant expression carrier p90039.Enzyme is cut and sequence verification recombinant expression carrier p90039 is described prOsPGD1 nucleotide sequence between KpnI and NotI site, as shown in Figure 5.
Utilize expression vector p90000 to obtain recombinant expression carrier p80000(over against shining), (carrier framework: pCAMBIA2301(CAMBIA mechanism can provide) as shown in Figure 6; Kan: kanamycin gene; RB: right margin; LB: left margin).
3, recombinant expression carrier transforms Agrobacterium
Oneself is contrasted through making up correct recombinant expression carrier p90001, p90030, p90031, p90039 and p80000(empty carrier) be transformed into Agrobacterium LBA4404(Invitrgen with the liquid nitrogen method, Chicago, USA, CAT:18313-015) in, its conversion condition is: 100 μ L Agrobacterium LBA4404,3 μ L plasmid DNA (recombinant expression carrier); Placed liquid nitrogen 10 minutes, 37 ℃ of tepidarium 10 minutes; It is to cultivate 2 hours under the 200rpm condition that Agrobacterium LBA4404 after transforming is inoculated in the LB test tube in 28 ℃ of temperature, rotating speed, be applied on the LB flat board of kanamycins (Kanamycin) of the rifampin (Rifampicin) that contains 50mg/L and 100mg/L until growing positive monoclonal, the picking monoclonal is cultivated and also to be extracted its plasmid, and enzyme is cut the checking result and shown recombinant expression carrier p90001, p90030, p90031, p90039 and the contrast of p80000(empty carrier) structure is entirely true.
Second embodiment, change the acquisition of the maize of promoter sequence to be measured over to
Agrobacterium infestation method according to the routine employing, the corn variety of axenic cultivation is combined 31(Z31) rataria and first embodiment in 3 described Agrobacteriums cultivate altogether, with the recombinant plant expression vector p90001 with 2 structures among second embodiment, p90030, p90031, the contrast of p90039 and p80000(empty carrier) T-DNA(in comprises the prZmUbi promoter sequence, the prOsAPX promoter sequence, the prOsAct1 promoter sequence, prOsPGD1 promoter sequence and gus gene) be transferred in the maize chromosome group, obtained to change over to the maize of prZmUbi promoter sequence, change the maize of prOsAPX promoter sequence over to, change the maize of prOsAct1 promoter sequence over to, change the maize of prOsPGD1 promoter sequence over to and change empty carrier contrast maize over to, contrast as negative with the wild type maize simultaneously.
Transform for agriculture bacillus mediated corn, briefly, separate immature rataria from corn, contact rataria with agrobacterium suspension, wherein Agrobacterium can be passed to promoter to be measured at least one cell (step 1: infect step) of one of rataria.In this step, rataria preferably immerses agrobacterium suspension (OD
660=0.4-0.6, infect nutrient culture media (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2, in the 4-dichlorphenoxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH5.3)) to start inoculation.Rataria and Agrobacterium are cultivated one period (3 days) (step 2: be total to incubation step) altogether.Preferably, rataria after infecting step at solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 100mg/L, 2, (2,4-D) 1mg/L, agar 8g/L pH5.8) go up cultivation to the 4-dichlorphenoxyacetic acid.
The determination of activity of the 4th embodiment, promoter sequence to be measured
Experiment one, GUS dyeing
Get the maize that 50-100 changes the prZmUbi promoter sequence over to respectively, change the maize of prOsAPX promoter sequence over to, change the maize of prOsAct1 promoter sequence over to, change over to the prOsPGD1 promoter sequence maize and over against according to maize as sample, with reference to (Jefferson R.A. such as Jefferson, Burgess S.M., Hirsh D.Beta-glucuronidase from Escherichia coliasa gene fusion marker.Proc.Natl.Acad.Sci., 1986, method 83:8447-8454) has also been done suitable improvement, dyeed two days by 37 ℃ of sealings in staining solution, from the expression way of the check GUS of histochemistry.The principal ingredient of described staining solution is: 0.1M NaPO
4, 0.01M EDTA(pH8.0), the 0.5mM potassium ferricyanide, 0.5mM potassium ferrocyanide, 0.5mg/ml5-bromo-4-chloro-3-indoles-β-D-glucosiduronic acid (X-gluc).The GUS enzyme that produces in the cell of express transgenic can produce the X-gluc decomposition in situ blue precipitation, thereby can locate transgenosis.
Contrast as negative with the wild type maize simultaneously, detect analysis according to the method described above.3 repetitions are established in experiment.
The experimental result of the GUS dyeing of transgenic corns embryo as shown in Figure 8.Experimental result shows except wild type maize (WT) and empty carrier contrast maize (NG) does not develop the color, changing the maize of prZmUbi promoter sequence, the maize that changes the prOsAPX promoter sequence over to, the maize that changes the prOsAct1 promoter sequence over to over to all develops the color, wherein, change the maize of prZmUbi promoter sequence over to and change in the maize of prOsAct1 promoter sequence GUS dyeing area over to bigger, next is the maize that changes the prOsAPX promoter sequence over to, and it is not obvious to change the maize GUS dyeing of prOsPGD1 promoter sequence over to.
The evaluation of experiment two, GUS activity
Get the maize that changes the prZmUbi promoter sequence over to respectively, change the maize of prOsAPX promoter sequence over to, change the maize of prOsAct1 promoter sequence over to, the maize and the empty carrier that change the prOsPGD1 promoter sequence over to contrast maize as sample, with reference to (Jefferson such as Jefferson, R.A., Kavanagh, T.A.and Bevan, M.W.GUS fusions:beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.EMBO J., 1987, method 3901-3907) is passed through fluorescence spectrometry promoter-driven GUS activity to be measured with 4-methyl umbelliferone (4-MU).Contrast as negative with the wild type maize simultaneously, detect analysis according to the method described above.3 repetitions are established in experiment, average.
The concrete grammar of measuring the GUS activity is as follows:
Step 1, take by weighing maize, the maize that changes the prOsAPX promoter sequence over to, the maize that changes the prOsAct1 promoter sequence over to that changes the prZmUbi promoter sequence over to, the maize that changes the prOsPGD1 promoter sequence over to and over against according to each 20mg of maize respectively, add 500 μ l and extract damping fluid (50mM NaPO
4(pH7.0), 1mM dithiothreitol (DTT) (DTT), 10mM EDTA, 0.1% sarcosyl (Sarcosyl), 0.1% Triton X-100 (Triton X-100)) in mortar, be ground into homogenate;
Step 2, behind centrifugal 10min under 4 ℃ of temperature, the rotating speed 4000rpm, collect supernatant, and put 4 ℃ and preserve down;
Step 3, at reaction buffer (the 50mM NaPO of 250 μ l preheatings (37 ℃, 30min) at least
4(pH7.0), 1mM dithiothreitol (DTT) (DTT), 10mM EDTA, 0.1% sarcosyl (Sarcosyl), 0.1% Triton X-100 (Triton X-100), 1mM4-methyl umbelliferone acyl-beta-glucosidase acid (MUG)) the middle described supernatant of 50 μ l that adds, mixing, after 37 ℃ of temperature are reacted 15min down, take out 30 μ l and join 270 μ l reaction terminating liquid (2%(m/v) sodium carbonate) middle cessation reaction;
Step 4, make the typical curve (0,50,100,250,500,1000,1500 be diluted in 2% sodium carbonate with 2000pmol4-MU) of 4-MU, measure the Ex365/Em455 value of each sample with SpectraMax M2;
Step 5, get described supernatant 30 μ l, water is settled to 360 μ l, adds 100 μ l BCA(bicinchoninic acids after taking out 30 μ l) reactant (BCA protein quantification kit), down behind the reaction 30min, cool off 10min in 37 ℃ of temperature again under room temperature;
Step 6, while are measured the 560nm absorbance value of each sample with bovine serum albumin(BSA) (BSA) production standard curve (0,25,50,75,100,150,200 and 250 μ g/ml) with SpectraMax M2;
Step 7, (pmol4-MU/mg albumen/min) is represented the GUS activity with picomole number and the ratio of protein content and time of 4-methyl umbelliferone (4-MU).
The experimental result of transgenic corns embryo gus gene activity is as shown in table 1.(pmol4-MU/mg albumen/min) is 49935 ± 83,1008 ± 23,18322 ± 33,0 ± 20,0 ± 13 and 0 ± 11 to record the GUS activity value of maize, the maize that changes the prOsAPX promoter sequence over to that changes the prZmUbi promoter sequence over to, the maize that changes the prOsAct1 promoter sequence over to, the maize that changes the prOsPGD1 promoter sequence over to, empty carrier contrast maize (NG) and wild type maize (WT) respectively.Above-mentioned experimental result also shows simultaneously, and the ordering that changes the maize of prZmUbi promoter sequence, the maize that changes the prOsAPX promoter sequence over to, the maize that changes the prOsAct1 promoter sequence over to, the maize that changes the prOsPGD1 promoter sequence over to, empty carrier contrast maize (NG) and the GUS activity value of wild type maize (WT) over to is corresponding with the ordering of its GUS dyeing area.
Table 1, contain GUS determination of activity average result in the transgenic corns embryo of promoter to be measured
In sum, the detection method of promoter activity of the present invention only needs plant embryos (particularly maize) was cultivated 3 days altogether, just can identify promoter activity by GUS staining analysis and/or gus protein assay, need not to experience the tissue culture procedures of a few weeks longer even several months, avoid maize genotype to transform specificity, induced links such as differentiation and the difficulty of taking root, saved the time, improved screening effeciency, more reduced because of the essential consumption of acquisition transfer-gen plant, thereby greatly reduced production cost; Can with the naked eye directly observe by GUS dyeing simultaneously and promoter activity is carried out qualitative analysis, especially constitutive promoter, and the result is corresponding with the GUS determination of activity, also with the transfer-gen plant of experience tissue culture procedures in the trend of promoter activity coincide, testing result is accurate.
It should be noted last that, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although with reference to preferred embodiment the present invention is had been described in detail, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement technical scheme of the present invention, and not break away from the spirit and scope of technical solution of the present invention.
Claims (10)
1. the detection method of a promoter activity is characterized in that, comprising:
Agrobacterium is infected plant embryos;
Cultivated at least 2 days altogether;
GUS identifies.
2. according to the detection method of the described promoter activity of claim 1, it is characterized in that described Agrobacterium comprises promoter sequence.
3. according to the detection method of the described promoter activity of claim 2, it is characterized in that described promoter is constitutive promoter.
4. according to the detection method of the described promoter activity of claim 3, it is characterized in that described constitutive promoter comprises Ubi gene promoter, APX gene promoter, Actin1 gene promoter or PGD1 gene promoter.
5. according to the detection method of the described promoter activity of claim 1, it is characterized in that described plant is corn.
6. according to the detection method of the described promoter activity of claim 1, it is characterized in that described the cultivation altogether was specially common cultivation 2-5 days at least 2 days.
7. according to the detection method of claim 5 or 6 described promoter activities, it is characterized in that the described maize that was specially at least 2 days after Agrobacterium infected of cultivating was altogether cultivated 3 days altogether.
8. according to the detection method of the described promoter activity of claim 1, it is characterized in that described GUS identifies and comprises GUS staining analysis and/or GUS determination of activity.
9. the detection method of described promoter activity according to Claim 8 is characterized in that, described GUS staining analysis is specially according to the size of GUS dyeing area carries out qualitative analysis to promoter activity.
10. according to the detection method of claim 5 or 8 described promoter activities, it is characterized in that the protein extract that described GUS determination of activity is specially maize carries out the GUS determination of activity.
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| CN103555716A (en) * | 2013-11-06 | 2014-02-05 | 北京大北农科技集团股份有限公司 | Intron sequence for enhancing expression and application of intron sequence |
| CN103555716B (en) * | 2013-11-06 | 2016-01-20 | 北京大北农科技集团股份有限公司 | Intron sequences of Enhanced expressing and uses thereof |
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| CN103278502B (en) | 2015-12-23 |
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