CN103525922A - Endogenous reference gene suitable for brachypodium distachyon exogenous gene detection and copy number analysis, and application thereof - Google Patents

Endogenous reference gene suitable for brachypodium distachyon exogenous gene detection and copy number analysis, and application thereof Download PDF

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CN103525922A
CN103525922A CN201310454983.1A CN201310454983A CN103525922A CN 103525922 A CN103525922 A CN 103525922A CN 201310454983 A CN201310454983 A CN 201310454983A CN 103525922 A CN103525922 A CN 103525922A
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储昭庆
朱宏
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SHANGHAI CHEN SHAN BOTANICAL GRADEN
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Abstract

The invention discloses an endogenous reference gene suitable for brachypodium distachyon exogenous gene detection and copy number analysis, and application thereof. Through bioinformatics analysis and molecular biology experimental verification, a BDFIM gene serving as the endogenous reference gene for transgenic brachypodium distachyon qualitative and quantitative polymerase chain reaction (PCR) detection and copy number analysis is found from the brachypodium distachyon, and the nucleotide sequence of the gene is as shown in SEQ ID NO.1. The endogenous reference gene can be applied to qualitative and quantitative detection of the exogenous gene of the transgenic brachypodium distachyon, and also can be applied to analysis on the copy number of the exogenous gene in the transgenic brachypodium distachyon species.

Description

A kind of internal standard gene and application thereof that is suitable for two fringe false bromegrass foreign genes detections and copy number analysis
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of applicable transgenosis two fringe false bromegrass foreign genes detect and copy number is analyzed internal standard gene and application thereof.
Background technology
So-called internal standard gene is in certain kind of plant, to have a genoid non-specific in specificity between kind, kind, low copy number feature.Between kind, to refer to selected gene be special for these species to specificity, and in other species, have very low homology; In kind, the non-specific internal standard gene that refers to has very high homology and similarity in the different cultivars of same species.For example, in the detection by quantitative of Genetically Modified Plant, internal standard gene is often used as the internal reference of detection by quantitative, requires internal standard gene in different cultivars, to have the feature of kind of internal specific.Transgenic product is being carried out in detection by quantitative, because the foreign gene of detection by quantitative is generally low copy, therefore requiring the internal standard gene to have the feature of low copy number, general internal standard gene is 1～3 copy, and optimal situation is 1 copy.
In the detection of plant transgene and converted products thereof, the primary effect of internal standard gene is to determine the source of species that detects sample.In transgenosis detects, if sample to be detected is plant material, the plant origin of that product is can be easy to know from outward appearance.But for plant converted products, as flour, food, vegetable oil product, be to be difficult to from distinguishing in appearance the source of vegetable material.At this moment just need internal standard gene to identify the source of plant prod.Internal standard gene is species specificity, can be used for these species and other species to come down to the kind difference of nearly source.Therefore by setting up the PCR detection system of internal standard gene, can judge accurately that the plant constituent detecting in sample originates, for further analyzing and lay the foundation.
Plant internal standard gene can be evaluated the effect of nucleic acid extraction.In transgenic product testing process, the quality of nucleic acid extraction has very large impact for PCR result.Owing to there being very large difference between vegetable material, processing stage is also not quite similar, so method for extracting nucleic acid also should improve according to different needs.Before the nucleic acid of sample extraction is carried out to pcr analysis, be necessary the effect of DNA extraction adequately and reasonably to evaluate, to guarantee the accuracy of pcr analysis.Evaluation for DNA quality can be undertaken by spectrophotometer method, agarose gel electrophoresis detection and fluorometry.But these methods can only be carried out quantity or qualitative guestimate to the nucleic acid of institute's extracting, can not judge the supression factor that whether contains PCR reaction in the DNA solution of extracting.Setting up internal standard gene is the effective ways that address this problem as a contrast of experiment.Can whether the normal operation of internal standard gene PCR system in contrast, can indicate the sample DNA of extracting for carrying out PCR, detect analysis.Therefore, internal standard gene is the effective means of getting rid of false negative result.
Internal standard gene plays the effect of internal reference in Genetically Modified Plant quantitative PCR detection.The quantitative PCR detection of transgenic plant and products thereof can be divided into two kinds of methods, i.e. absolute quantitation method and relative quantification method.In simple terms, the result that absolute quantitation method obtains, is to detect Plant Genome in sample or absolute mass or the copy number of external source goal gene.The result that relative quantification method obtains is to detect the percentage composition of external source goal gene in sample gene group DNA in sample, the copy number of external source goal gene and the ratio value representation of Plant Genome copy number for result.When actual transgenosis detection and the enforcement of various countries' labeling system, the standard of foundation is the concrete transgenosis relative content in sample, it must obtain by the analysis of relative quantification method, and absolute quantitation can only be as a process of relative quantification method in reality detects.Internal standard gene is for the quality of plant genome DNA or the quantitative analysis of copy number.And internal standard gene plays conclusive effect for genetically modified detection by quantitative, do not have suitable internal standard gene not carry out quantitative analysis for transgenic plant and converted products thereof, can not determine the content of transgenosis accurately of transgenic plant and converted products thereof.
In transgenic plant, copy number of foreign gene often affects expression level and the genetic stability of goal gene.Therefore, in transgenic research, a crucial step is exactly to detect the copy number of foreign gene, to filter out copy number transfer-gen plant few or single copy, supplies further research or breeding utilization.It is mainly Southern hybrid method that traditional transfer-gen plant copy number detects, but the method workload is large, and the DNA material needing is more, wastes time and energy, and often will use the proemial medicine of the tools such as radio isotope.In recent years, utilize high-throughput, quick, sensitive quantifying PCR method to detect the favor that transgenosis copy number is subject to scientific research personnel gradually.The method is according to Ct(cycle thresholds) the be inversely proportional to principle of linear relationship of the logarithmic value of value and starting template number, production standard curve, obtain the dependency equation of Ct value and starting template number, this equation of Ct value substitution of sample just can be obtained to the starting template number of goal gene, by the comparison with internal standard gene starting template number, with regard to this copy number of foreign gene in known dao gene group.
Internal standard gene detects and copy number has very important effect in detecting at the quantitative and qualitative analysis PCR of Genetically Modified Plant, and what for the research of plant internal standard gene, also develop in the world is very fast.Up to the present, the internal standard gene detecting for transgenosis of bibliographical information has 27.Wherein the Lectin gene of soybean is the internal standard gene detecting for the anti-careless glycosides phosphine soybean GTS40-3-2 of transgenosis the earliest, Lectin gene is distinctive agglutinin gene in soybean, by the detection to Lectin gene, determine to detect and in sample, whether to contain the quality that soybean source and judgement detect sample DNA, and in genetically engineered soybean detection by quantitative as quantitative internal reference.The Zein of corn, zssIIb, hmgA, Invertase and Adh1 gene were all once used as internal standard gene and carried out transgenic corns PCR detection, also there is article to compare these internal standard genes of corn, find that above-mentioned several gene is all suitable for the qualitative PCR detection of transgenic corns, but also do not find about the report to two fringe false bromegrass internal standard genes at present.
Two fringe false bromegrasses are a kind of cold-season-type temperate zone grasses, in some places also usually as a kind of herbage, plant type is short and small owing to possessing for this kind of plant, self-pollination, seed are not scattered, the features such as life cycle is short, growth conditions is simple, be used as in recent years a kind of novel Gramineae model plant, so transgenosis two fringe false bromegrass technology also develop rapidly.Because the copy number of foreign gene is larger on the stably express impact of foreign gene, therefore when taking a transfer-gen plant, first to analyze its copy number of foreign gene, traditional copy number analysis is to utilize Southern detection technique, this kind of method wastes time and energy, and cost is higher, if a large amount of materials is analyzed, workload is larger.Therefore in recent years occurred utilizing quantitative PCR method to the analysis of transgenic plant gene copy number of foreign gene, this kind of technology can make up the deficiency of Southern detection technique, is especially applicable to lot of materials to carry out high throughput testing.In addition two fringe false bromegrasses also can be used as a kind of herbage, so transgenosis two fringe false bromegrasses also may be processed to feed, also need the internal standard gene of this kind of plant when feed is carried out to transgenosis detection.But the report that not yet has in the world the internal standard gene that is applicable to two fringe false bromegrass foreign genes detections and copy number analysis at present.Therefore, the internal standard gene pairs of the applicable two fringe false bromegrass foreign genes detections of screening and copy number analysis promotes that two fringe false bromegrass transgenic research meanings are very great.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of internal standard gene and application thereof that is suitable for two fringe false bromegrass foreign genes detections and copy number analysis, overcomes the present situation that not yet has the internal standard gene that is applicable to two fringe false bromegrass foreign genes detections and copy number analysis at present.
The internal standard gene that is suitable for two fringe false bromegrass foreign genes detections and copy number analysis of the present invention, two fringe false bromegrass internal standard gene BDFIM genes, its sequence is as shown in SEQ ID NO1, and according to BDFIM gene order design qualitative, the primer pair BDFIM-F/BDFIM-R of quantitative PCR detection, design Southern probe carries out the analysis of BDFIM gene copy number, for transgenosis two fringe false bromegrasses, detect, wherein, qualitative PCR detects identical with the primer pair of quantitative PCR detection, its sequence is as shown in SEQ ID NO2 and shown in SEQ ID NO3, Southern probe sequence is as shown in SEQ ID NO4.
The application that is suitable for the internal standard gene of two fringe false bromegrass foreign genes detections and copy number analysis of the present invention, for the foreign gene of qualitative and quantitative analysis transgenosis two fringe false bromegrasses.
The application that is suitable for the internal standard gene of two fringe false bromegrass foreign genes detections and copy number analysis of the present invention, for analyzing foreign gene at the copy number of transgenosis two fringe false bromegrasses.
Further, the present invention has designed the primer pair HPT-F/HPT-R that utilizes internal standard BDFIM genetic testing two fringe false bromegrass copy number of foreign gene, and its sequence is respectively as shown in SEQ ID NO5 and shown in SEQ ID NO6.
Further, internal standard gene-BDFIM gene of the present invention possesses kind of an interior consistence, and two all fringe false bromegrass kinds all contain BDFIM gene; Specificity between described BDFIM gene also possesses kind, i.e. this gene not in other species; By Southern, hybridize and also proved that the copy number of BDFIM gene in diploid two fringe false bromegrass genomes is 1.
The present invention utilizes information biology means to analyze BDFIM gene order, has obtained the native gene BDFIM sequence of two fringe false bromegrasses, and its sequence is as shown in SEQ ID NO1., quantitative PCR method qualitative by utilizing detects two fringe false bromegrasses of 14 kinds of different varietiess and 16 kinds of other species respectively, proves specificity between two fringe false bromegrass BDFIM gene kinds, plants interior nonspecific feature; By utilizing Southern hybridizing method to carry out the analysis of BDFIM gene copy number to 7 kinds of different varietiess, two fringe false bromegrasses, result shows that in 3 kinds of different varieties diploids, two fringe false bromegrasses, BDFIM gene is all 1 copy, and in 4 kinds of different varieties polyploids, two fringe false bromegrasses, BDFIM gene is multiple copied.Owing to take diploid as main for the acceptor material of two fringe false bromegrass transgenic researches at present, so BDFIM gene meets the low copy feature of internal standard gene, shows: BDFIM gene meets the feature of internal standard gene by these experimental results.
Of the present inventionly by the foreign gene HPT gene to two fringe false bromegrasses, carry out copy number analysis, further proved that this BDFIM gene can be used as the internal standard gene of two fringe false bromegrasses.
Beneficial effect of the present invention:
The present invention verifies by bioinformatic analysis and molecular biology experiment, in two fringe false bromegrasses, searched out the internal standard gene-BDFIM gene as transgenosis two fringe false bromegrass foreign genes detect and copy number is analyzed, this BDFIM gene has the feature of specificity, low copy number between kind of an interior consistence, kind, the standard-required that meets internal standard gene, is suitable for the internal standard gene that two fringe false bromegrass foreign genes detect and copy number is analyzed.
Accompanying drawing explanation
Fig. 1 is nucleotide sequence and qualitative, the quantification PCR primer position of BDFIM fragment.
Fig. 2 is non-specific qualification result in BDFIM gene kind, and wherein, M is marker, 1 is BD21, and 2 is BD21-3, and 3 is BD1-1,4 is BD29-1, and 5 is BD18-1, and 6 is BD23-1,7 is BD26-1, and 8 is BD13-1, and 9 is BD2-3,10 is BD20-1, and 11 is BD10-1, and 12 is BD28,13 is BD25-1, and 14 is BD19-2,15 negative contrasts.
Fig. 3 is specificity identification result between BDFIM gene kind, and wherein M is Marker, and 1 is two fringe false bromegrasses, 2 is Chinese rose, and 3 is cotton, and 4 is rye grass, 5 is carnation, and 6 is the tuber of dwarf lilyturf, and 7 is African chrysanthemum, 8 is Bermuda grass, and 9 is tobacco, and 10 is corn, 11 is paddy rice, and 12 is soybean, and 13 is wheat, 14 is barley, and 15 is Arabidopis thaliana.
Fig. 4 is non-specific Quantitative measurement result in BDFIM gene kind.
Fig. 5 is specificity Quantitative measurement result between BDFIM gene kind.
Fig. 6 is the copy number that Southern-Blot analyzes internal standard gene, and wherein 1 is Bd21-3, and 2 is Bd21, and 3 is Bd29-1, and 4 is Bd19-2, and 5 is Bd20-1, and 6 is Bd28, and 7 is Bd10-1, and 8 is blank.
Fig. 7 is BdFIM gene real-time quantitative PCR amplification curve.
Fig. 8 is BdFIM gene canonical plotting.
Fig. 9 is HPT gene real-time quantitative PCR amplification curve.
Figure 10 is HPT gene canonical plotting.
Embodiment
Below in conjunction with specific embodiment, technical scheme of the present invention is described in further detail, but described embodiment does not limit the scope of the invention.Should be noted that, following examples are only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement the technical scheme of invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in claim scope of the present invention.
Embodiment 1
1, experiment material
1.1 vegetable material
Two fringe false bromegrasses: the two fringe false bromegrass materials of selecting are liploid plant and polyploid plant, refer to table 1.
Two selected fringe false bromegrass materials in table 1 experiment
Figure BDA0000389849070000051
Figure BDA0000389849070000061
Other common crops: 1. Chinese rose; 2. cotton; 3. rye grass; 4. carnation; 5. tuber of dwarf lilyturf; 6. African chrysanthemum; 7. Bermuda grass; 8. tobacco; 9. corn; 10. paddy rice; 11. soybean; 12. wheats; 13. barleys; 14. Arabidopis thalianas.
1.2 enzymes and reagent
Restriction enzyme, dNTPs, DL2000Markers, Taq archaeal dna polymerase and damping fluid thereof are purchased from Takara.Primer is synthesized by bio-engineering corporation, and other biochemical reagents are import packing or domestic analytical pure.
1.3 laboratory apparatus
EPS-300 type nucleic acid electrophoresis apparatus (Shanghai Tian Neng Science and Technology Ltd.), T100 type pcr amplification instrument (Bio-Rad, USA), Eon nucleic acid and the protein analyzer of BioTek company, CFX96 quantitative PCR instrument (Bio-Rad, USA), other instruments comprise: whizzer, thermostat water bath, incubator, day equality.
2, experimental technique and process
Searching of 2.1 candidate's internal standard genes
During transgenic plant PCR detects, need the internal standard gene using must meet 3 conditions: non-specific in planting, plant between specificity, copy property (for improving detection sensitivity, being preferably single copy gene) surely.Secondly first by By consulting literatures, choose corresponding single copy gene, by online BLAST, determine the homology of this gene and other crops, finally by quantitative and qualitative PCR detection and Southern, detect the operability of determining this internal standard gene.
The extraction of 2.2 DNA of plants and detection
2.2.1 the extraction of DNA of plants
A. get two appropriate fringe false bromegrass samples and be placed in mortar, add liquid nitrogen by sample grind into powder, take the sample that about 70mg～100mg grinds and proceed in the Eppendorf pipe of 1.5ml.
B. look quantity of material number, add 600～700 μ l CTAB damping fluid of preheating, after mixing gently, 65 ℃ of water bath heat preservations 1 hour, during between or vibration mix.
C. in pipe, add equal-volume phenol/chloroform (600～700 μ l), turn upside down and fully mix, standing extracting 10min.
D.12000rpm * 10 minutes centrifugal, draws in the Eppendorf pipe that supernatant to is new.
E. add and the isopyknic Virahol of supernatant (approximately 600 μ l), mix, normal temperature is placed after 10min, and the centrifugal 10min of 12000g, removes supernatant, retains precipitation.
F. in precipitation, add 60 μ l TER, after with rifle head, it fully being mixed after 37 ℃ of placement 2min, in 37 ℃, place approximately 1 hour.
G. after taking out, add 140 μ l TE, then add 200 μ l phenol/chloroforms, mix standing a moment.
H.12000rpm * 5min is centrifugal, gets supernatant (approximately 180 μ l), adds 1/10 volume 3M NaAc and 2 times of volume dehydrated alcohols, places 30min for-20 ℃.
I.12000rpm * 10min is centrifugal, removes supernatant, adds 75% ethanol 200 μ l, and 12000rpm * 5min is centrifugal, abandons supernatant, and room temperature is dried, and is dissolved in the aseptic ddH20 of 50 μ l.
2.2.2DNA detect: get the DNA solution electrophoresis that 3 μ l extract, the sepharose that the running gel of selecting is 1%, judges the quality of DNA according to its brightness and diffusion, utilize ultraviolet spectrophotometer to measure concentration and the purity of the DNA that puies forward.
The design of 2.3 primers, probe
Design 1 pair for the Auele Specific Primer of two fringe false bromegrass internal standard genes, wherein, designed identical primer pair in qualitative PCR reaction and quantitative PCR reaction, is BDFIM-F/BDFIM-R, and its sequence is as shown in SEQ ID NO2 and shown in SEQ ID NO3.Designed southern hybridization probe, its sequence is respectively as shown in SEQ ID NO4 simultaneously.Also designed the primer pair HPT-F/HPT-R that utilizes internal standard BDFIM genetic testing two fringe false bromegrass copy number of foreign gene, its sequence is respectively as shown in SEQ ID NO5 and shown in SEQ ID NO6, referring specifically to table 2.
Table 2 quantitative and qualitative analysis PCR reacts primer
Figure BDA0000389849070000071
The qualitative detection of 2.4 internal standard genes is identified
1) non-specific evaluation in internal standard gene kind
Non-specific evaluation in BDFIM gene kind: take non-transgenic two fringe false bromegrass kind BD21, BD21-3, BD1-1, BD29-1, BD18-1, BD23-1, BD26-1, BD13-1, BD2-3, BD20-1, BD10-1, BD28, BD25-1, BD19-2 genomic dna is PCR reaction template, with BDFIM-F/BDFIM-R primer pair, carry out pcr amplification reaction, its PCR reaction conditions is: 94 ℃ of denaturations 5 minutes; 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ are extended 30 seconds, carry out altogether 35 circulations; Finally, 72 ℃ are extended end after 5 minutes, and reaction finishes rear absorption 10 μ l products and on 2% agarose, carries out electrophoresis detection.
2) specificity identification between internal standard gene kind
Specificity identification between BDFIM gene kind: get Chinese rose, cotton. the genomic dna of the plants such as rye grass, carnation, the tuber of dwarf lilyturf, African chrysanthemum, Bermuda grass, tobacco, corn, paddy rice, soybean, wheat, barley, Arabidopis thaliana is PCR reaction template, with BDFIM-F/BDFIM-R primer pair, carry out pcr amplification reaction, its PCR reaction conditions is: 94 denaturations 5 minutes; 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ are extended 30 seconds, carry out altogether 35 circulations; Finally, 72 ℃ are extended end after 5 minutes, and reaction finishes rear absorption 10 μ l products and on 2% agarose, carries out electrophoresis detection.
The detection by quantitative of 2.5 internal standard genes is identified
1) non-specific evaluation in BDFIM kind: take non-transgenic kind BD21, BD21-3, BD1-1, BD29-1, BD18-1, BD23-1, BD26-1, BD13-1, BD2-3, BD20-1, BD10-1, BD28, BD25-1, BD19-2 genomic dna is PCR reaction template, carries out quantitative pcr amplification reaction with BDFIM-F/BDFIM-R primer pair.Its PCR reaction conditions is: 94 ℃ of denaturations 5 minutes; 94 ℃ of sex change 15 seconds, 60 ℃ of annealing 45 seconds, carries out 45 circulations altogether.
2) specificity identification between internal standard gene kind
Specificity identification between BDFIM gene kind: get Chinese rose, cotton. the genomic dna of the plants such as rye grass, carnation, the tuber of dwarf lilyturf, African chrysanthemum, Bermuda grass, tobacco, corn, paddy rice, soybean, wheat, barley, Arabidopis thaliana is PCR reaction template, with BDFIM-F/BDFIM-R primer pair, carry out quantitative pcr amplification reaction, its PCR reaction conditions is: 94 ℃ of denaturations 5 minutes; 94 ℃ of sex change 15 seconds, 60 ℃ of annealing 45 seconds, carries out 45 circulations altogether.
2.6 Southern hybridization
2.6.1 DNA probe mark
The reaction of each standard can mark 10ng to 3 μ g linearity DNA, also can the more substantial DNA of mark, but all compositions and volume are wanted corresponding increase.DNA probe thermally denature, boils 10 minutes, cooling in 5 minutes on ice, stand-by rapidly.
Get Eppendorf pipe (1.5ml) and be placed on ice, add following reagent:
Figure BDA0000389849070000081
At 37 ℃, be incubated at least 60min～20h.
Boil termination reaction 5 minutes.
The centrifugal 30s of 15000rpm, is placed in stand-by on ice.
Note: it is standby that the probe that mark is good also can be placed on-20 ℃ of refrigerators, with before to first carry out denaturing treatment.
2.6.2 plant genome DNA enzyme is cut
Get different varieties two fringe false bromegrass BD21-3, BD21, BD29-1, BD19-2, BD20-1, BD28, BD10-1 genomic dna carries out enzyme with HindIII and cuts.
The about 10ug of DNA 25 μ l()
ddH 2O 17μl
Restriction enzyme (NEB, 10u/ μ l) 3 μ l
10*Buffer 5μl
2.6.3 transferring film
(1) utilize 1v/cm to carry out electrophoresis, after electrophoresis, gel is moved in the dry roasting ware of a glass, repair the nonuseable part of gel with sharp cutter, in the gel upper left corner, (well one end is upper) cuts one jiao, serves as a mark.
(2) the 1.5mol/L NaCL, the 0.5mol/L NaOH that gel are placed in to several times volume soak 45 minutes and gentle continuous jolting, and DNA is deformed.
(3) gel being placed in to deionized water and pausing rinsing, be soaked in subsequently the 1mol/L Tris.CL(PH7.4 of several times volume) in 1.5mol/L NaCL, under room temperature, leniently constantly jolting, makes it neutralization.Change neutralizer, make gel continue to soak 15 minutes.
(4), when gel is still in neutralizer, with a sheet glass of a Whatman3MM filter paper parcel, make a gel platform.Platform is put into a large dry roasting ware, pour transfering buffering liquid (10 * SSC) into, make liquid level a little less than platform surface.After the 3MM filter paper of platform top drenches, with glass stick, drive all bubbles (shift fragment and be less than 500bp, use 20 * SSC transfering buffering liquid) out of.
(5) with new scissors, cut out a nitrocellulose filter, the length of filter membrane and width should be respectively than the large 1mm of gel, need wear gloves or with blunt-ended forceps during contact filter membrane, can not be with there being greasy hand to contact filter membrane.
(6) till nitrocellulose filter being floated over to deionized water surface and soaking, use subsequently 20 * SSC to soak filter membrane at least 5 minutes, deduct one jiao of filter membrane, make it corresponding with the corner cut of gel.
(7) from neutralizer, take out gel, make its back side upwards gel upset; Gel is put in to 3MM filter paper central authorities moistening on platform, between filter paper and gel, can not be detained bubble.
(8) use Parafilm film around gel periphery, using this as barrier.
(9) above gel, place moistening nitrocellulose filter, and make both corner cut overlaids.Between filter membrane and gel, should not leave bubble.
(10) with 2 * SSC solution, soak two 3MM filter paper onesize with gel, moistening filter paper is placed on moistening nitrocellulose filter top, drives the bubble being detained with glass stick out of therebetween.
Cut a folded paper handkerchief that is slightly less than 3MM filter paper, place it in the top of 3MM filter paper, and above paper handkerchief, put a sheet glass, then use the weight compacting of a 500g.
(11) above-mentioned DNA is shifted and continue to carry out 8-24 hour, after paper handkerchief soaks, the paper handkerchief that should renew.
(12) after shifting, throw off paper handkerchief and the filter paper of gel top, upset gel and nitrocellulose filter,, be placed on a dry 3MM filter paper upper with the one side of gel, with pencil or the ballpoint pen of a dead-soft, and the position of mark gel well on filter membrane.
(13) from nitrocellulose filter, peel off gel, with 6 * SSC solution in soaking at room temperature filter membrane 5 minutes.
(14) from 6 * SSC solution, take out filter membrane, lie on a piece of paper towel after the solution on filter membrane is dripped to the greatest extent.In room temperature, dry more than 30 minutes.
(15) filter membrane drying is placed in the middle of two 3MM filter paper, with 80 ℃ of dry roasting 30min～2h of vacuum oven.
2.6.4 prehybridization
With scissors, the part below film tetrabromophenol sulfonphthalein is cut off.Put into hybrid pipe, sealing.The efficient hybridization solution 5ml of Hyb that adds 65 ℃ of preheatings, drains bubble, sealing.Put into 42 ℃ of hybrid heater hybridization 1-2 hour.
2.6.5 hybridization:
(1) 100 ℃ of the good probes of mark are boiled 10 minutes, ice bath is cooling immediately.
(2) hybrid pipe is taken out from hybrid heater, pour out prehybridization solution, get the efficient hybridization solution of 20mlHyb, add the probe of sex change, mix.Join in hybrid pipe, put into 42 ℃ of hybrid heater vibration hybridization and spend the night.
2.6.6 wash film
(1) take out hybrid pipe, with tweezers, take out film, put into the plate of 2 * SSC0.1%SDS solution that 20ml is housed, the washed twice of at room temperature vibrating, each 5min.
(2) with tweezers, film is proceeded in 0.1 * SSC, 0.1%SDS solution (first putting 50 ℃ of water-bath preheatings) to 50 ℃ of water-bath vibration washed twice, each 15min.
(3) with tweezers, film taking-up being proceeded to vibration in the plate that 20ml lavation buffer solution is housed washs 5 minutes.
2.6.7 signal detection
(1) after hybridization and rigorous washing, in lavation buffer solution, infiltrate 1-5 minute.
(2) in 30ml blocking-up liquid, hatch 30 minutes.
(3) in 10ml antibody liquid, hatch 30 minutes.
(4) with 30ml washings washing 2 * 15 minutes.
(5) at 15ml, detect balance 2-5 minute in damping fluid.
(6) abandon detection damping fluid, film faces up (containing DNA face), is placed on transparent plastic bag, takes out CSPD-ready-to-use(substrate), drop in film surface and evenly smear.
(7) with sealing machine, film is encapsulated in transparent plastic bag, is put in 37 ℃ and strengthens reaction 10 minutes.
(8) film DNA is faced up and is put in compressing tablet box, X film is taken out in darkroom, puts on film and compresses, and spends the night to take out and develops.
(9) film is taken out, in developing solution, develop, after having developed, put into stop bath and stop developing.Then put into clear water and rinse well, naturally dry.
The analysis of 2.7 foreign genes (HPT) copy number
Need to be first respectively with BDFIM-F/BDFIM-R and HPT-F/HPT-R primer pair two fringe false bromegrass genomic dnas and the plasmid quantitative pcr amplification reaction that contains HPT gene according to test, two fringe false bromegrass genomic dnas and the plasmid quantitative pcr amplification reaction that contains HPT gene carry out respectively 5 times and 10 times of dilutions, and its PCR reaction conditions is: 94 ℃ of denaturations 5 minutes; 94 ℃ of sex change 15 seconds, 60 ℃ of annealing 45 seconds, carries out 45 circulations altogether, sets up BDFIM and HPT typical curve.Then according to formula X0/R0=10 [(CT, X – IX)/SX] – [(CT, R – IR)/SR]calculate copy number of foreign gene, wherein R0 represents the copy number of internal standard gene, and X0 represents the copy number of foreign gene.IX and IR represent the intercept of typical curve, the slope that SX and SR are typical curve.C t,Rand C t,XcT value for internal standard gene and foreign gene.Then select 5 homozygote transfer-gen plants that copy number of foreign gene is known, the BDFIM of take carries out copy number of foreign gene analysis as internal standard gene utilizes above-mentioned formula.
3, experimental result
Searching and online Blast result of 3.1 candidate's internal standard genes
BDFIM (LOC100831539) gene is two fringe false bromegrass native genes, and this gene is carried out to Blast analysis, and result shows: can not find the DNA fragmentation of continuous 50bp and its homology, Fig. 1 is qualitative, quantification PCR primer sequence and position.
The qualitative detection of 3.2 internal standard genes is identified
Non-specific evaluation in BDFIM gene kind: the Auele Specific Primer of use table 2 design carries out pcr amplification reaction to BDFIM-F/BDFIM-R to two fringe false bromegrasses of different varieties, result is referring to Fig. 2, electrophoresis result is presented at and in 14 different varietiess, can amplifies size for 231bp, single-minded band, stripe size is consistent with expection, explanation all contains candidate gene BDFIM in the different varieties of two fringe false bromegrasses, therefore, non-specific in having kind of two fringe false bromegrass BDFIM genes.
Specificity identification between 3.3 internal standard gene kinds
Specificity identification between BDFIM kind: the Auele Specific Primer of use table 2 design carries out pcr amplification reaction to BDFIM-F/BDFIM-R to common cut-flower plant and other plant, and result is referring to Fig. 3.The electrophoretic analysis of PCR product is presented in common cut-flower plant and other plant and does not amplify specific band, and in positive, have size for 231bp, single-minded band, illustrate in other plant and do not contain the BDFIM gene of two fringe false bromegrasses and the gene higher with BDFIM DNA homolog, prove that two fringe false bromegrass BDFIM genes have the specificity between planting.
The detection by quantitative of 3.4 internal standard genes is identified
1) non-specific Quantitative measurement in internal standard gene kind
Select respectively 14 different varietiess two fringe false bromegrass DNA as template, select corresponding primer, reaction system and reaction conditions to carry out quantitative PCR reaction, result is referring to Fig. 4.The primer pair that shows the internal standard gene specific of two fringe false bromegrasses all can detect respectively similar fluorescent signal in two fringe false bromegrasses of different varieties, and non-specific in all having in two fringe false bromegrass kinds kind of BDFIM gene is described thus.
2) specificity Quantitative measurement between internal standard gene kind
Select respectively the genomic dna of the plants such as two fringe false bromegrasses, Chinese rose, cotton, rye grass, carnation, the tuber of dwarf lilyturf, African chrysanthemum, Bermuda grass, tobacco, corn, paddy rice, soybean, wheat, barley, Arabidopis thaliana as template, select corresponding primer, reaction system and reaction conditions to carry out quantitative PCR reaction, result is referring to Fig. 5, shows primer for the two fringe false bromegrass BDFIM designs two fringe false bromegrass genomic dnas that can only increase.Explanation thus, two fringe false bromegrass BDFIM genes have the specificity between planting.
3.5Southern detected result
Southern results of hybridization shows that two selected fringe false bromegrass BDFIM genes are single copy in liploid plant, is multiple copied in polyploid plant, and result is referring to Fig. 6.
3.6 utilize BDFIM genetic testing transgenosis two fringe false bromegrass copy number of foreign gene
According to experiment needs, the typical curve of model internal standard gene and foreign gene, result is referring to Fig. 7 and Fig. 8.Then according to formula X0/R0=10 [(CT, X – IX)/SX] – [(CT, R – IR)/SR]calculate copy number of foreign gene, wherein R0 represents the copy number of internal standard gene, and X0 represents the copy number of foreign gene.IX and IR represent the intercept of typical curve, the slope that SX and SR are typical curve.C t,Rand C t,XcT value for internal standard gene and foreign gene.Select the known transfer-gen plant of copy number of foreign gene, utilize BDFIM gene as internal standard gene, utilize above-mentioned formula to record copy number of foreign gene consistent with the copy number having recorded.Proof BDFIM gene can be used as the copy number of internal standard gene test foreign gene, and result is referring to table 3.
The calculation result of table 3 transfer-gen plant foreign gene HPT gene copy number
Figure BDA0000389849070000131
Figure IDA0000389849150000011
Figure IDA0000389849150000021

Claims (5)

1. be suitable for the internal standard gene that two fringe false bromegrass foreign genes detect and copy number is analyzed, it is characterized in that, described internal standard gene is BDFIM gene, and its nucleotide sequence is as shown in SEQ ID NO1.
2. be according to claim 1ly suitable for the internal standard gene that two fringe false bromegrass foreign genes detect and copy number is analyzed, it is characterized in that, primer for described internal standard gene order design qualitative, quantitative PCR detection, design Southern probe carries out the analysis of BDFIM gene copy number, for transgenosis two fringe false bromegrasses, detect, wherein, primer pair qualitative, quantitative PCR detection is identical, its sequence is as shown in SEQ ID NO2 and shown in SEQ ID NO3, and Southern probe sequence is as shown in SEQ ID NO4.
3. the application that is suitable for the internal standard gene of two fringe false bromegrass foreign genes detections and copy number analysis as claimed in claim 1, is characterized in that, for the foreign gene of qualitative and quantitative analysis transgenosis two fringe false bromegrasses.
4. the application that is suitable for the internal standard gene of two fringe false bromegrass foreign genes detections and copy number analysis as claimed in claim 1, is characterized in that, for analyzing foreign gene at the copy number of transgenosis two fringe false bromegrasses.
5. the application that is suitable for the internal standard gene of two fringe false bromegrass foreign genes detections and copy number analysis as claimed in claim 4, it is characterized in that, analyzing foreign gene is HPT-F/HPT-R at the copy number primer pair used of transgenosis two fringe short handle grass seeds, and its sequence is as shown in SEQ ID NO5 and shown in SEQ ID NO6.
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