CN103278502B - The detection method of promoter activity - Google Patents
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Abstract
The present invention relates to a kind of detection method of promoter activity, comprising: Agrobacterium infects plant embryos; Dual culture at least 2 days; GUS identifies.The detection method of promoter activity of the present invention only needs plant embryos (particularly maize) Dual culture 3 days, without the need to the tissue culture procedures of experience a few weeks longer even several months, just by GUS staining analysis and/or gus protein assay, promoter activity is identified, avoid maize genotype and transform specificity, the differentiation-inducing and links such as difficulty of taking root, save the time, improve screening effeciency, reduce production cost, experimental result is directly perceived, accurate simultaneously.
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 the promoter be operably connected having and work in plant host.The selection of promoter sequence will determine the time that allogeneic dna sequence is expressed in plant host and position.Therefore, when needs are expressed in plant is preferably organized, using-system specificity promoter.On the contrary, when needs are expressed in whole vegetable cell, use constitutive promoter.The plant of conversion can be obtained by the separate living tissue directly processing plant embryos, by the analysis of transformed plant to determine whether promoter sequence starts object allogeneic dna sequence in targeted tissue and express.
Corn gene technology obtains very large development in recent years, has established 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 pathway.Comparatively speaking, agrobacterium-mediated transformation is higher and become the plant transgenic technology be most widely used at present with its efficiency, and the basic procedure of the method is: explant → callus → agrobacterium mediation converted → Multiple Buds → screen → take root → transformant.Therefore, when utilizing agriculture bacillus mediated maize genetic conversion to carry out promoter sequence Activity determination, there is following defect:
(1) the Callus formation time often reaches even several months a few weeks longer, very consuming time, and workload is heavy;
(2) crop such as the differentiation-inducing comparatively paddy rice difficulty of corn embryo callus, the differentiation rate of Multiple Buds is very unstable;
(3) difficulty of taking root of plant is broken up;
(4) due to the genotypic difference of transgene receptor plant, diversity kind or strain can not be suitable for, show comparatively significantly maize genotype and transform specificity.
Summary of the invention
The object of this invention is to provide a kind of detection method of promoter activity, effectively overcome that prior art is consuming time, workload is heavy, differentiation-inducing and difficulty of taking root, genotype transform the technological deficiencies such as specificity.
For achieving the above object, the invention provides a kind of detection method of promoter activity, comprising:
Agrobacterium infects plant embryos;
Dual culture at least 2 days;
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.
Described Dual culture is specially Dual culture 2-5 days at least 2 days.Further, described Dual culture within least 2 days, be specially and Agrobacterium infected after maize Dual culture 3 days.
On the basis of technique scheme, described GUS qualification comprises GUS staining analysis and/or GUS determination of activity.
Further, described GUS staining analysis is specially and carries out qualitative analysis according to the size of GUS stained area to promoter activity.Described GUS determination of activity is specially carries out GUS determination of activity to the protein extract of maize.
In the present invention, term " promoter " refers to DNA regulatory region, usually starts the TATA box of RNA synthesis containing the applicable transcription initiation site that rna plymerase ii can be guided in specific coding sequence.Promoter can in addition containing other recognition sequence, and be generally positioned at upstream or the 5 ' end of TATA box, be called upstream promoter element, they affect transcription initiation rate.Admittedly, because promoter region nucleotide sequence is own through determining, in 5 ' non-translational region of concrete promoter region upstream, separation andpreconcentration regulating element belongs to prior art further.Therefore, promoter region comprises upstream regulatory elements further, and it is given any heterologous nucleotide sequence be operably connected with promoter sequence and expresses.
In the present invention, term " gene " to refer under suitable regulation and control region (such as plant expressible promoter region) controls in cell containing any DNA fragmentation in region of DNA territory (" the region of DNA territory of transcribing ") being transcribed into RNA molecule (such as mRNA).Therefore, the DNA fragmentation that gene may connect containing several operability, such as promoter, 5 ' untranslated leader, coding region and 3 ' the untranslated region containing polyadenylation site.Endogenous plant gene is the gene of natural discovery in plant species.Mosaic gene is any gene usually do not found in plant species, or under its natural environment its promoter and the region of DNA territory of partly or entirely transcribing or with this gene at least another regulate and control any gene that region has nothing to do.
In the present invention, term " constitutive promoter " refers under the control of such promoter, and the expression somewhat constant of object heterologous nucleotide sequence, on certain level, does not have notable difference at the different tissues of plant and/or different growth and development stage expression.It is described that to be organized as origin source in plant identical and perform the structural units of one or more cell type set of same function; such as 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 different organs, more effectively complete organic whole vital movement process.Described growth and development stage can be divided into embryo stage, plantlet stage, the stage of ripeness and ageing phase according to the difference of phytomorph, function.The 35SRNA gene promoter deriving from cauliflower mosaic virus (CaMV) is used as the major promoter of dicotyledon; And the actin promoter of rice plants and the ubiquitin promoter of cereal are suitable for monocotyledon usually.
Promoter sequence of the present invention and fragment thereof, when being assembled into DNA structure and making promoter sequence be operably connected with object heterologous nucleotide sequence, manipulate any plant for heredity.Described " being operably connected " to refer between promoter sequence of the present invention with second sequence functional is connected, and wherein promoter sequence starts and regulates transcribing of the DNA sequence dna that corresponds to second sequence.Usually, be operably connected and refer to that the nucleotide sequence be connected is continuous print, be adjacent to if desired in conjunction with two protein-coding regions, and in same reading frame.In this way, promoter nucleotide sequence and object heterologous nucleotide sequence form described mosaic gene in expression cassette together with provide, to express in object 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 and qualitative analysis is carried out to promoter activity by GUS dyeing, especially constitutive promoter in the detection method of promoter activity of the present invention.
2, quick.Only need plant embryos (particularly maize) Dual culture 3 days in the detection method of promoter activity of the present invention, just by GUS staining analysis and/or gus protein assay, promoter activity is identified, without the need to the tissue culture procedures of experience a few weeks longer even several months, avoid maize genotype and transform specificity, the differentiation-inducing and links such as difficulty of taking root, save the time, improve screening effeciency.
3, economical.Although the detection method of promoter activity of the present invention make use of transgenic approach, do not experience the process of follow-up tissue cultures, decrease the required consumption because obtaining transfer-gen plant, thus significantly reduce production cost.
4, accurate.Although the detection method of promoter activity of the present invention only utilizes plant embryos (particularly maize), by GUS staining analysis and/or GUS determination of activity, promoter activity is identified, but compared with the transfer-gen plant of experience tissue culture procedures, the trend of promoter activity is identical, and namely testing result is accurately.
Below by drawings and Examples, technical scheme of the present invention is described in further detail.
Accompanying drawing explanation
Fig. 1 is that the recombinant cloning vector pT-prZmUbi of the detection method of promoter activity of the present invention builds process flow diagram;
Fig. 2 is that the recombinant expression carrier p90001 of the detection method of promoter activity of the present invention builds process flow diagram;
Fig. 3 is the recombinant expression carrier p90030 schematic diagram of the detection method of promoter activity of the present invention;
Fig. 4 is the recombinant expression carrier p90031 schematic diagram of the detection method of promoter activity of the present invention;
Fig. 5 is the recombinant expression carrier p90039 schematic diagram of the detection method of promoter activity of the present invention;
Fig. 6 is the recombinant expression carrier p80000 schematic 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
The technical scheme of the detection method of promoter activity of the present invention is further illustrated below by specific embodiment.
The structure of the first embodiment, recombinant expression carrier containing promoter sequence
1, the recombinant cloning vector containing promoter sequence to be measured is built
PrZmUbi nucleotide sequence is connected into cloning vector pGEM-T(Promega, Madison, USA, CAT:A3600) on, operation steps is undertaken by Promega Products pGEM-T carrier instructions, obtain recombinant cloning vector pT-prZmUbi, it builds 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 LacZ initiation codon; SP6 is SP6RNA polymerase promoter; T7 is t7 rna polymerase promoter; PrZmUbi is the Ubiqutin(ubiquitin of corn variety B73) gene promoter (SEQIDNO:1); MCS is multiple clone site).
Then by recombinant cloning vector pT-prZmUbi heat shock method transformation of E. 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 DEG C of water-baths 30 seconds; Cultivate 1 hour (under 200rpm rotating speed shaking table shake) for 37 DEG C, scribble IPTG(isopropylthio-β-D-galactoside on surface) and the chloro-3-indoles of the bromo-4-of X-gal(5--β-D-galactoside) LB flat board (the tryptone 10g/L of ampicillin (100mg/L), yeast extract 5g/L, NaCl10g/L, agar 15g/L, adjusts pH to 7.5 with NaOH) upper grow overnight.Picking white colony, LB fluid nutrient medium (tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, ampicillin 100mg/L, with NaOH adjust pH to 7.5) under temperature 37 DEG C of conditions overnight incubation.Its plasmid of alkalinity extraction: by bacterium liquid centrifugal 1min under 12000rpm rotating speed, remove supernatant, the precipitation thalline solution I (25mMTris-HCl, 10mMEDTA(ethylenediamine tetraacetic acid) of 100 μ l ice precoolings, 50mM glucose, pH8.0) suspend; Add the solution II (0.2MNaOH, 1%SDS(lauryl sodium sulfate) that 150 μ l newly prepare), pipe is put upside down 4 times, mixing, puts 3-5min on ice; Add the ice-cold solution III of 150 μ l (4M potassium acetate, 2M acetic acid), fully mix immediately, place 5-10min on ice; Centrifugal 5min under temperature 4 DEG C, rotating speed 12000rpm condition, adds 2 times of volume absolute ethyl alcohols in supernatant, and after mixing, room temperature places 5min; Centrifugal 5min under temperature 4 DEG C, rotating speed 12000rpm condition, abandons supernatant, and precipitation concentration (V/V) is dry after the ethanol washing of 70%; Add 30 μ l containing RNase(20 μ g/ml) TE(10mMTris-HCl, 1mMEDTA, PH8.0) dissolution precipitation; Water-bath 30min at temperature 37 DEG C, digestion RNA; Save backup in temperature-20 DEG C.
The plasmid extracted is after NcoI and PstI enzyme cuts qualification, carry out sequence verification to positive colony, result shows that the Ubiqutin gene promoter sequence of the corn variety B73 inserted in recombinant cloning vector pT-prZmUbi is for the nucleotide sequence shown in SEQ ID NO:1.
According to the method for above-mentioned structure recombinant cloning vector pT-prZmUbi, prOsAPX nucleotide sequence is connected on cloning vector pGEM-T, obtain recombinant cloning vector pT-prOsAPX, wherein, prOsAPX is the Ascorbate peroxidase gene promoter (SEQIDNO:2) that rice varieties is Japanese fine.Enzyme is cut and is correctly inserted with prOsAPX nucleotide sequence described in sequence verification recombinant cloning vector pT-prOsAPX.
According to the method for above-mentioned structure recombinant cloning vector pT-prZmUbi, prOsAct1 nucleotide sequence is connected on cloning vector pGEM-T, obtain recombinant cloning vector pT-prOsAct1, wherein, prOsAct1 is the actin gene promotor (SEQIDNO:3) of rice varieties.Enzyme is cut and is correctly inserted with prOsAct1 nucleotide sequence described in sequence verification recombinant cloning vector pT-prOsAct1.
According to the method for above-mentioned structure recombinant cloning vector pT-prZmUbi, prOsPGD1 nucleotide sequence is connected on cloning vector pGEM-T, obtain recombinant cloning vector pT-prOsPGD1, wherein, prOsPGD1 is the glucose phosphate dehydrogenase gene promoter (SEQIDNO:4) that rice varieties is Japanese fine.Enzyme is cut and is correctly inserted with prOsPGD1 nucleotide sequence described in sequence verification recombinant cloning vector pT-prOsPGD1.
2, the recombinant expression carrier containing promoter sequence to be measured is built
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)), between BamHI and the HindIII site prZmUbi nucleotide sequence cut being inserted into expression vector p90000, conventional enzymatic cleavage methods carrier construction is utilized to be well-known to those skilled in the art, be built into recombinant expression carrier p90001, it builds flow process (Kan: kanamycin gene as shown in Figure 2; RB: right margin; The Ubiqutin(ubiquitin of prZmUbi: corn variety B73) gene promoter (SEQIDNO:1); GUS: beta-glucosiduronatase gene (SEQIDNO:5); Nos: the terminator (SEQIDNO:6) of rouge alkali synthetase gene; LB: left margin).
By recombinant expression carrier p90001 heat shock method transformation of E. coli T1 competent cell, its hot shock condition is: 50 μ l Escherichia coli T1 competent cells, 10 μ l plasmid DNA (recombinant expression carrier p90001), 42 DEG C of water-baths 30 seconds; Cultivate 1 hour (under 200rpm rotating speed shaking table shake) for 37 DEG C; Then at LB solid plate (the tryptone 10g/L containing 50mg/L kanamycins (Kanamycin), yeast extract 5g/L, NaCl10g/L, agar 15g/L, adjust pH to 7.5 with NaOH) upper cultivation 12 hours under temperature 37 DEG C of conditions, picking white colony, at LB fluid nutrient medium (tryptone 10g/L, yeast extract 5g/L, NaCl10g/L, kanamycins 50mg/L, with NaOH adjust pH to 7.5) under temperature 37 DEG C of conditions overnight incubation.Its plasmid of alkalinity extraction.The plasmid restriction enzyme BamHI of extraction and HindIII enzyme are cut rear qualification, and positive colony is carried out order-checking qualification, result shows that the nucleotides sequence of recombinant expression carrier p90001 between BamHI and HindIII site is classified as nucleotide sequence shown in SEQ ID NO:1, i.e. the Ubiqutin(ubiquitin of corn variety B73) gene promoter sequence.
According to the method for above-mentioned structure recombinant expression carrier p90001, BamHI and HindIII enzyme is cut the described prOsAPX nucleotide sequence insertion expression vector p90000 that recombinant cloning vector pT-prOsAPX cuts, 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 is cut the described prOsAct1 nucleotide sequence insertion expression vector p90000 that recombinant cloning vector pT-prOsAct1 cuts, 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 is cut the described prOsPGD1 nucleotide sequence insertion expression vector p90000 that recombinant cloning vector pT-prOsPGD1 cuts, 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.
Expression vector p90000 is utilized to obtain recombinant expression carrier p80000(positive control), as shown in Figure 6 (carrier framework: pCAMBIA2301(CAMBIA mechanism can provide); Kan: kanamycin gene; RB: right margin; LB: left margin).
3, recombinant expression carrier transformation Agrobacterium
To oneself through building correct recombinant expression carrier p90001, p90030, p90031, p90039 and p80000(empty vector control) be transformed into Agrobacterium LBA4404 (Invitrgen by liquid nitrogen method, Chicago, USA, CAT:18313-015) in, its conversion condition is: 100 μ L Agrobacterium LBA4404s, 3 μ L plasmid DNA (recombinant expression carrier); Be placed in liquid nitrogen 10 minutes, 37 DEG C of tepidarium 10 minutes; Agrobacterium LBA4404 after conversion is inoculated in LB test tube and cultivates 2 hours under temperature 28 DEG C, rotating speed are 200rpm condition, be applied on the LB flat board containing the rifampin (Rifampicin) of 50mg/L and the kanamycins (Kanamycin) of 100mg/L until grow positive monoclonal, picking Colony Culture also extracts its plasmid, and digestion verification result shows recombinant expression carrier p90001, p90030, p90031, p90039 and p80000(empty vector control) structure is entirely true.
Second embodiment, proceed to the acquisition of the maize of promoter sequence to be measured
The Agrobacterium infestation method conveniently adopted, the corn variety of axenic cultivation is combined 31(Z31) rataria and the first embodiment in Agrobacterium Dual culture described in 3, with the recombinant plant expression vector p90001 by 2 structures in the second embodiment, p90030, p90031, p90039 and p80000(empty vector control) in T-DNA(comprise prZmUbi promoter sequence, prOsAPX promoter sequence, prOsAct1 promoter sequence, prOsPGD1 promoter sequence and gus gene) be transferred in maize chromosome group, obtain the maize proceeding to prZmUbi promoter sequence, proceed to the maize of prOsAPX promoter sequence, proceed to the maize of prOsAct1 promoter sequence, proceed to the maize of prOsPGD1 promoter sequence and proceed to empty vector control maize, simultaneously using wild-type corn embryo as negative contrast.
For agriculture bacillus mediated corn transformation, briefly, be separated immature rataria, contact rataria with agrobacterium suspension from corn, wherein promoter to be measured can be passed at least one cell (step 1: infect step) of one of rataria by Agrobacterium.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, 2,4-dichlorphenoxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH5.3)) in start inoculation.Rataria and Agrobacterium Dual culture one period (3 days) (step 2: Dual culture step).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, 2,4-dichlorphenoxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH5.8) upper cultivation.
The determination of activity of the 4th embodiment, promoter sequence to be measured
Experiment one, GUS dyeing
Get the maize that 50-100 proceeds to prZmUbi promoter sequence respectively, proceed to the maize of prOsAPX promoter sequence, proceed to the maize of prOsAct1 promoter sequence, proceed to the maize of prOsPGD1 promoter sequence and positive control maize as sample, with reference to (JeffersonR.A. such as Jefferson, BurgessS.M., HirshD.Beta-glucuronidasefromEscherichiacoliasagenefusio nmarker.Proc.Natl.Acad.Sci., 1986, method 83:8447-8454) has also done suitable improvement, by 37 DEG C of sealing dyeing two days in staining solution, the expression way of GUS is checked from histochemistry.The principal ingredient of described staining solution is: 0.1MNaPO
4, 0.01MEDTA(pH8.0), the 0.5mM potassium ferricyanide, 0.5mM potassium ferrocyanide, the chloro-3-indoles of the bromo-4-of 0.5mg/ml5--β-D-glucosiduronic acid (X-gluc).X-gluc decomposition in situ can be produced blue precipitate by the GUS enzyme produced in the cell of express transgenic, thus can locate transgenosis.
Simultaneously using wild-type corn embryo as negative contrast, carry out detection according to the method described above and analyze.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 corn embryo (WT) and empty vector control maize (NG) do not develop the color, proceed to the maize of prZmUbi promoter sequence, proceed to the maize of prOsAPX promoter sequence, proceed to the maize of prOsAct1 promoter sequence and all develop the color, wherein, the maize proceeding to prZmUbi promoter sequence is larger with GUS stained area in the maize proceeding to prOsAct1 promoter sequence, next is the maize proceeding to prOsAPX promoter sequence, and the maize GUS proceeding to prOsPGD1 promoter sequence dyes not obvious.
The qualification of experiment two, GUS activity
Get the maize proceeding to prZmUbi promoter sequence respectively, proceed to the maize of prOsAPX promoter sequence, proceed to the maize of prOsAct1 promoter sequence, proceed to the maize of prOsPGD1 promoter sequence and empty vector control maize as sample, with reference to (Jefferson such as Jefferson, R.A., Kavanagh, T.A.andBevan, M.W.GUSfusions:beta-glucuronidaseasasensitiveandversatil egenefusionmarkerinhigherplants.EMBOJ., 1987, method 3901-3907), active by the GUS of fluorescence spectrometry promoters driven to be measured with 4-methyl umbelliferone (4-MU).Simultaneously using wild-type corn embryo as negative contrast, carry out detection according to the method described above and analyze.3 repetitions are established in experiment, average.
The concrete grammar measuring GUS activity is as follows:
Step 1, take the maize proceeding to prZmUbi promoter sequence, the maize proceeding to prOsAPX promoter sequence, the maize proceeding to prOsAct1 promoter sequence, the maize proceeding to prOsPGD1 promoter sequence and each 20mg of positive control maize respectively, add 500 μ l Extraction buffer (50mMNaPO
4(pH7.0), 1mM dithiothreitol (DTT) (DTT), 10mMEDTA, 0.1% sarcosyl (Sarcosyl), 0.1% Triton X-100 (TritonX-100)) in mortar, be ground into homogenate;
Step 2, under temperature 4 DEG C, rotating speed 4000rpm after centrifugal 10min, collect supernatant, preserve at juxtaposition 4 DEG C;
Step 3, reaction buffer (50mMNaPO in 250 μ l preheatings (37 DEG C, at least 30min)
4(pH7.0), 1mM dithiothreitol (DTT) (DTT), 10mMEDTA, 0.1% sarcosyl (Sarcosyl), 0.1% Triton X-100 (TritonX-100), 1mM4-methylumbelliferyl keto acyl-beta-glucosidase acid (MUG)) in add supernatant described in 50 μ l, mixing, react 15min at temperature 37 DEG C after, 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 SpectraMaxM2;
Step 5, get described supernatant 30 μ l, be settled to 360 μ l with water, after taking out 30 μ l, add 100 μ lBCA(bicinchoninic acids) reactant (BCA protein quantification kit), react 30min at temperature 37 DEG C after, then cool 10min under room temperature;
Step 6, simultaneously with bovine serum albumin(BSA) (BSA) production standard curve (0,25,50,75,100,150,200 and 250 μ g/ml), measure the 560nm absorbance value of each sample with SpectraMaxM2;
Step 7, represent that GUS is active with the picomole number of 4-methyl umbelliferone (4-MU) and the ratio of protein content and time (pmol4-MU/mg albumen/min).
The experimental result of transgenic corns embryo gus gene activity is as shown in table 1.The GUS activity value (pmol4-MU/mg albumen/min) recording the maize proceeding to prZmUbi promoter sequence, the maize proceeding to prOsAPX promoter sequence, the maize proceeding to prOsAct1 promoter sequence, the maize proceeding to prOsPGD1 promoter sequence, empty vector control maize (NG) and wild-type corn embryo (WT) is respectively 49935 ± 83,1008 ± 23,18322 ± 33,0 ± 20,0 ± 13 and 0 ± 11.Simultaneously above-mentioned experimental result also shows, proceeds to the maize of prZmUbi promoter sequence, proceeds to the maize of prOsAPX promoter sequence, proceeds to the maize of prOsAct1 promoter sequence, the maize proceeding to prOsPGD1 promoter sequence, empty vector control maize (NG) are corresponding with the sequence of its GUS stained area with the sequence of the GUS activity value of wild-type corn embryo (WT).
GUS determination of activity average result in table 1, transgenic corns embryo containing promoter to be measured
In sum, the detection method of promoter activity of the present invention only needs plant embryos (particularly maize) Dual culture 3 days, just by GUS staining analysis and/or gus protein assay, promoter activity is identified, without the need to the tissue culture procedures of experience a few weeks longer even several months, avoid maize genotype and transform specificity, the differentiation-inducing and links such as difficulty of taking root, save the time, improve screening effeciency, more decrease the required consumption because obtaining transfer-gen plant, thus significantly reduce production cost; Can with the naked eye directly observe by GUS dyeing and qualitative analysis be carried out to promoter activity simultaneously, especially constitutive promoter, and corresponding with GUS determination of activity result, also coincide with the trend of promoter activity in the transfer-gen plant of experience tissue culture procedures, testing result is accurate.
It should be noted last that, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not depart from the spirit and scope of technical solution of the present invention.
Claims (3)
1. a detection method for promoter activity, is characterized in that, comprising:
Agrobacterium infects plant embryos;
Dual culture at least 2 days;
GUS identifies, particularly, comprises the steps:
Immature rataria is separated from corn, with the agrobacterium suspension contact rataria comprising promoter sequence, Agrobacterium is enable promoter to be measured to be passed at least one cell of one of rataria, rataria and Agrobacterium Dual culture 3 days, wherein, rataria is cultivated after infecting step on solid medium, described solid medium is MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetosyringone 100mg/L, 2,4-dichlorphenoxyacetic acid 1mg/L, agar 8g/L, pH5.8;
Carry out GUS determination of activity to the protein extract of the maize proceeding to promoter sequence, concrete steps are as follows:
Step 1, take the maize that proceeds to promoter sequence and each 20mg of positive control maize respectively, add 500 μ l Extraction buffers and be ground into homogenate in mortar, described Extraction buffer is the 50mMNaPO of pH7.0
4, 1mM dithiothreitol (DTT), 10mMEDTA, 0.1% sarcosyl, 0.1% Triton X-100;
Step 2, under temperature 4 DEG C, rotating speed 4000rpm after centrifugal 10min, collect supernatant, preserve at juxtaposition 4 DEG C;
Step 3, at 250 μ l37 DEG C, add supernatant described in 50 μ l in the reaction buffer of at least 30min preheating, mixing, described reaction buffer is the 50mMNaPO of pH7.0
4, 1mM dithiothreitol (DTT), 10mMEDTA, 0.1% sarcosyl, 0.1% Triton X-100, the acid of 1mM4-methylumbelliferyl keto acyl-beta-glucosidase, react 15min at temperature 37 DEG C after, taking out 30 μ l, to join 270 μ l mass/volume be cessation reaction in the sodium carbonate reaction terminating liquid of 2%;
The typical curve of step 4, making 4-MU, measures the Ex365/Em455 value of each sample with SpectraMaxM2;
Step 5, get described supernatant 30 μ l, be settled to 360 μ l with water, add 100 μ lBCA reactants after taking out 30 μ l, react 30min at temperature 37 DEG C after, then cool 10min under room temperature;
Step 6, simultaneously with bovine serum albumin(BSA) production standard curve, measure the 560nm absorbance value of each sample with SpectraMaxM2.
2. the detection method of promoter activity according to claim 1, it is characterized in that, described promoter is constitutive promoter.
3. the detection method of promoter activity according to claim 2, it is characterized in that, described constitutive promoter comprises Ubi gene promoter, APX gene promoter, Actin1 gene promoter or PGD1 gene promoter.
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| WO1997048814A2 (en) * | 1996-06-21 | 1997-12-24 | Monsanto Company | Methods for the production of stably-transformed, fertile wheat employing agrobacterium-mediated transformation and compositions derived therefrom |
| PT1306440E (en) * | 2000-08-03 | 2007-02-28 | Japan Tobacco Inc | Method of improving gene transfer efficiency into plant cells |
| US7598430B2 (en) * | 2002-03-20 | 2009-10-06 | J.R. Simplot Company | Refined plant transformation |
| CN102517285B (en) * | 2011-12-23 | 2014-05-21 | 北京大北农科技集团股份有限公司 | Tissue-specific promoter and application thereof |
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2013
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