CN108977565A - A kind of probe combinations and method suitable for plant chromosome rDNA physical positioning - Google Patents
A kind of probe combinations and method suitable for plant chromosome rDNA physical positioning Download PDFInfo
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Abstract
The invention discloses a kind of probe combinations and method suitable for plant chromosome rDNA physical positioning；It is related to molecular and cytogenetic techniques field.The probe combinations include the probe of SEQ ID NO.1-4, are 5S rDNA probe, 5.8S rDNA probe, 18S rDNA probe and 25S rDNA probe respectively；Chromosome in Situ Hybridization of Plants is carried out using the probe combinations and method, the chromosome and fluorescence in situ hybridization signal of high-resolution, the Chromosomal in situ hybridization suitable for various plants can be captured；And the probe combinations are oligonucleotide probe, have the characteristics that low manufacture cost, easy to use, easy to operate.
Description
Technical field
The present invention relates to molecular and cytogenetic techniques fields, are suitable for plant chromosome rDNA in particular to one kind
The probe combinations and method of physical positioning.
Background technique
Fluorescence in situ hybridization technique (Fluorescence in situ hybridization, FISH) is that handle uses biotin
Etc. fluorescence labeling materials label DNA fragmentation be used as probe (probe), with chromosomal DNA carry out molecule hybridize, fluorescence show
The Present site with the DNA fragmentation of probes complementary is directly detected under micro mirror.
By fluorescence in situ hybridization technique can by rDNA (Ribosomal DNA, rDNA) on chromosome into
Row physical positioning.Currently, rDNA has carried out extensive physical set in important mode crop and the genome of industrial crops
Position is that the chromosome of these species identifies, genome structure analysis, physical map building and the research of species affiliation
Provide direct information.
The preparation method of common rDNA probe: the double chain DNA sequence of rDNA is obtained by extracting plasmid, is then passed through
Shifting method label probe is incised, probe applies detected through gel electrophoresis probe size after marking, and the probe of suitable size just may be used
For fluorescence in situ hybridization.The probe of digoxin and biotin labeling is since stability is relatively high, thus in research work
It is used widely.
The shortcomings that probe: probe label it is higher to DNA quality requirement, from obtain high quality DNA to probe mark and
Size detection needs to spend higher reagent expense；And need to save bacterium solution, it shakes bacterium and expands numerous Escherichia coli, process is more numerous
It is trivial, expend time and manpower；The probe for incising shifting method label is double-strand and length is inconsistent, and hybridization efficiency is lower.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of probe combinations suitable for plant chromosome rDNA physical positioning, using this
Probe combinations carry out Chromosome in Situ Hybridization of Plants, can produce more visible fluorescence signal, the dyeing suitable for various plants
Body in situ hybridization；And the probe combinations are oligonucleotide probe, have the spies such as low manufacture cost, easy to use, easy to operate
Point.
Another object of the present invention is to provide a kind of methods for Chromosome in Situ Hybridization of Plants, using this method
Chromosome in Situ Hybridization of Plants is carried out, can produce clearly fluorescence signal, carries out physics convenient for the rDNA to plant chromosome
Positioning；The rDNA probe that this method uses is oligonucleotide probe, easy to use, easy to operate.
The present invention is implemented as follows:
On the one hand, the present invention provides a kind of probe combinations suitable for plant chromosome rDNA physical positioning comprising 5S
RDNA probe and/or 45S rDNA probe；
The nucleotide sequence of above-mentioned 5S rDNA probe is as shown in SEQ ID NO.1；
Above-mentioned 45S rDNA probe is made of 5.8S rDNA probe, 18S rDNA probe and 25S rDNA probe, above-mentioned
The nucleotide sequence of 5.8S rDNA probe is as shown in SEQ ID NO.2, the nucleotide sequence such as SEQ of above-mentioned 18S rDNA probe
Shown in ID NO.3, the nucleotide sequence of above-mentioned 25S rDNA probe is as shown in SEQ ID NO.4.
The scientific and reasonable design of inventor through the invention, the SEQ ID that probe combinations provided by the invention include
The 5S rDNA probe of NO.1, according to the 1-59 at the end arabidopsis 5S rDNA (GenBank AJ307346.2) transcriptional domain 5'
Bit base designs, and obtains the 5S rDNA probe as shown in SEQ ID NO.1 of length 59nt.
In addition, be different from traditional 45S rDNA plasmid sequence, the present inventor is according to 3 of arabidopsis 45S rDNA
The oligonucleotide probe that transcriptional domain devises 3 59nt altogether (is named as 5.8S rDNA probe, 18S rDNA probe and 25S
RDNA probe).Wherein, 5.8S rDNA probe provided by the invention is according to arabidopsis 5.8S rDNA (GenBank NR_
141643.1) the 1-59 bit base at the end 5' designs, and 18S rDNA probe provided by the invention is according to arabidopsis 18S
The 1-59 bit base at the end 5' rDNA (GenBank NR_141642.1) designs.By comparison, it was found that arabidopsis 25S rDNA
The base in the region 25-84 at the end probe 5' is more conservative, and designing length according to the region is 59nt such as SEQ ID NO.4
Shown 25S rDNA probe.
Above-mentioned 5S rDNA probe (SEQ ID NO.1), 5.8S rDNA probe (SEQ ID NO.2), 18S rDNA are visited
Needle (SEQ ID NO.3) and 25S rDNA probe (SEQ ID NO.4) are oligonucleotide probe, with traditional FISH system institute
Plasmid probe is compared, and oligonucleotides fluorescence in situ hybridization does not need to use Plasmid DNA label probe, and denatured probe is also not required to
Add antibody, both save the cost, also saves the time.
In addition, the length of each probe is 59nt, each probe is both selected from the suitable region arabidopsis rDNA, such to set
In respect of the popularity conducive to the sensitivity of raising probe, reliability and application, allow to former for the chromosome of various plants
Position hybridization, generates more visible fluorescence signal, carries out physical positioning convenient for the rDNA to plant chromosome.
Further, in some embodiments of the present invention, it is glimmering to be marked with first for the both ends of above-mentioned 5S rDNA probe
Light group；The both ends of above-mentioned 45S rDNA probe are marked with the second fluorophor i.e. 5.8S rDNA probe, 18S rDNA probe
Marking with the both ends of 25S rDNA probe has fluorophor.Above-mentioned first fluorophor and above-mentioned second fluorophor
It is different.
It was found by the inventors of the present invention that compared to the fluorophor labelling strategies of traditional one end, using the two of probe
The strategy of end mark fluorescent group can increase fluorescence signal, be to show clearer fluorescence signal after probe hybridizes, be easy to
Detection and observation.
Further, in some embodiments of the present invention, above-mentioned first fluorophor is Fluoresceincarboxylic acid (FAM)；
Above-mentioned second fluorophor is carboxyl tetramethylrhodamine (TAMRA).
It should be noted that the classification of fluorophor is not limited to above-mentioned classification, other fluorophors or it is interpreted as fluorescence
Dyestuff such as Alexa 350, Alexa 405, Alexa 430, Alexa 488, Alexa 555, Alexa 647, AMCA, ammonia
Base acridine, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-
TRX, 5- carboxyl -4 ', 5 '-two chloro- 2 ', 7 '-dimethoxyfluoresceins, 5- carboxyl -2 ', 4 ', 5 ', 7 '-tetrachlorofluoresceins, 5-
Fluoresceincarboxylic acid, 5- carboxyrhodamine, 6- carboxyrhodamine, 6- carboxyl tetramethylrhodamine, Cascade Blue, Cy2,
Cy3, Cy5, Cy7,6-FAM, dansyl Cl, fluorescein, HEX, 6-JOE, NBD (7- nitro benzo -2- oxa- -1,3- diazole),
It is Oregon Green 488, Oregon Green 500, Oregon Green514, Pacific Blue, phthalic acid, right
Phthalic acid, M-phthalic acid, cresols consolidate purple, cresols royal purple, brilliant cresyl blue, p-aminobenzoic acid, erythrosine, phthalocyanine, azo
Methine, cyanine, xanthine, succinylfluoresceins, rare earth metal cryptate, three pairs of pyridyl group diamines europiums, the cave-shaped chemical combination of europium
Object or chelate, diamines, dicyanin, La Jolla indigo plant dyestuff, allophycocyanin, allococyanin B, phycocyanin C,
Phycocyanin R, thiamines, algae red green white, phycoerythrin R, REG, rhodamine be green, rhodamine isothiocyanates, rhodamine are red,
ROX, TAMRA, TET, TRIT (the different mercaptan of tetramethylrhodamine), tetramethylrhodamine and texas Red etc. are also can be by
For marking probe provided by the present invention.
No matter which kind of fluorescent dye is selected, as long as marking probe provided by the present invention such as 5S rDNA probe
(SEQ ID NO.1), 5.8S rDNA probe (SEQ ID NO.2), 18S rDNA probe (SEQ ID NO.3) or 25S rDNA
Probe (SEQ ID NO.4), all belongs to the scope of protection of the present invention.
Further, in some embodiments of the present invention, above-mentioned plant be tulip, lily, corn, morning glory,
Begonia etc..
In another aspect, the present invention provides a kind of methods of Chromosome in Situ Hybridization of Plants comprising following steps:
In situ hybridization step: hybridization solution is added on toward plant chromosome film-making, is hybridized；
Wherein, above-mentioned hybridization solution contains above-mentioned probe combinations.
Further, in some embodiments of the present invention, 5S rDNA concentration and probe concentration is 1- in above-mentioned hybridization solution
2ng/μl。
Further, in some embodiments of the present invention, the concentration of 45S rDNA probe is 1- in above-mentioned hybridization solution
2ng/μl.I.e. the sum of concentration of 5.8S rDNA probe, 18S rDNA probe and 25S rDNA three kinds of probes of probe is 1-2ng/ μ
l。
The concentration of probe be influence it is subsequent should fluorescence signal a key factor, probe is in suitable concentration range
It is interior, it not only can produce obvious fluorescence signal, but also can save the cost.Concentration and probe concentration is too low, is easy to cause echo signal not
Obviously, concentration and probe concentration is excessively high, will lead to the increase of use cost, and the increase of fluorescence signal intensity is not obvious.
By the control of 5S rDNA concentration and probe concentration in the range of 1-2ng/ μ l, and by each probe in 45S rDNA probe
(5.8S rDNA probe, 18S rDNA probe and 25S rDNA probe) concentration controls in the range of 0.3-0.7 ng/ μ l
To generate apparent Observable signal, concentration and probe concentration is further increased on this scope, the enhancing of fluorescence signal is not shown
It writes.
It should be noted that 5S rDNA concentration and probe concentration can be 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,
In 1.9 and 2ng/ μ l any one or both range.
5.8S rDNA concentration and probe concentration can be any one in 0.3,0.4,0.5,0.6 and 0.7ng/ μ l or both
Range.
18S rDNA concentration and probe concentration can be any one in 0.3,0.4,0.5,0.6 and 0.7ng/ μ l or both
Range.
25S rDNA concentration and probe concentration can be any one in 0.3,0.4,0.5,0.6 and 0.7ng/ μ l or both
Range.
Further, in some embodiments of the present invention, above-mentioned hybridization solution also contains: deionized formamide
(dFA), SSC buffer and dextran sulfate (DS).
Further, in some embodiments of the present invention, in situ before hybridization step, the above method further includes
Pre-treatment step；
Above-mentioned pre-treatment step includes: to impregnate the plant tip of a root of acquirement at room temperature with ring acetamide, uses distilled water
It is transferred to after flushing in the fixer of Kano.
Further, in some embodiments of the present invention, above-mentioned plant be tulip, lily, corn, morning glory,
Begonia etc..
The good split coil method of form is the premise for carrying out in situ hybridization, can make chromosome using above-mentioned pre-treatment step
Good form is kept, is conducive to subsequent in situ hybridization and generates clearly signal.
In short, not only can produce clearly fluorescence using the method for Chromosome in Situ Hybridization of Plants provided by the invention
Signal, and also have the characteristics that operating procedure is simple and convenient.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment
Attached drawing is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not to be seen as
It is the restriction to range, it for those of ordinary skill in the art, without creative efforts, can be with
Other relevant attached drawings are obtained according to these attached drawings.
Fig. 1 is the fluorescence in situ hybridization detection result of the T.iliensis chromosome in the embodiment of the present invention 3；In figure: A:
The chromosome testing result figure of fluorescence in situ hybridization is not carried out；B: 5S rDNA (green) after having carried out fluorescence in situ hybridization and
The composite diagram of 45S rDNA (red) signal；C: cutting out from C2 and believes according to chromosome morphology and oligonucleotides rDNA probe
Number pairing arrangement chromosome；Scale bar is 10 μm.
Fig. 2 is the fluorescence in situ hybridization detection result of the vertical flower bud tulip chromosome in the embodiment of the present invention 4；In figure: A:
The chromosome testing result figure of fluorescence in situ hybridization is not carried out；B: 5S rDNA (green) after having carried out fluorescence in situ hybridization and
The composite diagram of 45S rDNA (red) signal；C: cutting out from G2 and believes according to chromosome morphology and oligonucleotides rDNA probe
Number pairing arrangement chromosome；Scale bar is 10 μm.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer
It is recommended that condition carry out.Reagents or instruments used without specified manufacturer is the routine that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
Present embodiments provide a kind of probe combinations for Chromosome in Situ Hybridization of Plants comprising 5S rDNA probe
With 45S rDNA probe；
The nucleotide sequence of 5S rDNA probe is as shown in SEQ ID NO.1；
45S rDNA probe is made of 5.8S rDNA probe, 18S rDNA probe and 25S rDNA probe.
The nucleotide sequence of 5.8S rDNA probe is as shown in SEQ ID NO.2, the nucleotide sequence of 18S rDNA probe
As shown in SEQ ID NO.3, the nucleotide sequence of 25S rDNA probe is as shown in SEQ ID NO.4.
Wherein, 5S rDNA probe both ends flag F AM fluorophor；
5.8S rDNA probe, 18S rDNA probe and 25S rDNA probe these three probes both ends mark TAMRA
Fluorophor.
The nucleotide sequence of each rDNA oligonucleotide probe and modification are as shown in table 1 below:
The nucleotide sequence of each rDNA probe of table 1 and modification
Embodiment 2
A kind of method of the Chromosome in Situ Hybridization of Plants present embodiments provided comprising following steps:
The preparation of 1 metaphase chromosome
(1) draw materials: the tip of a root (0.5-1.5cm) for taking plant growth vigorous is used as material.
(2) it pre-processes: the tip of a root of fresh acquirement is pre-processed 4-8 hours (no with the ring acetamide of 0.7mM at room temperature
With difference the time required to species, determines according to actual conditions), after distilled water flushing, (3:1 second is transferred in the fixer of Kano
Alcohol: acetic acid, v/v).
(3) digest: it is clean that the tip of a root fixed is placed in cleaning down in distilled water, then cuts Root apical meristem,
And be put into the enzymatic mixture containing 4% cellulose and 2% pectase, about 0.5- is digested in 37 DEG C of insulating box
1.5h (different the time required to different plant species, determines according to actual conditions).
(4) film-making:
Root apical meristem after enzymatic hydrolysis is placed in water, then shifts on 1-2 group separate living tissue to glass slide, uses tweezer
Son gently squeezes out apical meristem cell, and 60% acetic acid of about 20 μ l is added dropwise, is mixed with dissecting needle, glass slide is placed in
In the thermal station that temperature is 55 DEG C, with dissecting needle smear cell suspension, is rinsed and air-dried with fresh Kano fixer immediately.
2 metaphase chromosomes are taken pictures and film-making pretreatment
(1) it takes pictures: after film-making adds the Vectashield containing DAPI, covered, in fluorescence microscopy microscopic observation
And good chromosome separation phase of taking pictures, while recording the coordinate position of clapped split coil method.
(2) clean: the film-making with 2 or more good split coil methods steeped in 1 × PBS and falls coverslip, in 42 DEG C 1
It is washed in × PBS 10 minutes, 70%, 90% and 100% ethyl alcohol is dehydrated step by step, each 5 minutes, is then air-dried.
(3) it pre-fixes: film-making is fixed 15 minutes in 4% formalin；2 × SSC is washed 3 times, every time 5 minutes；
70%, 90% and 100% ethyl alcohol is dehydrated step by step, each 5 minutes, is then air-dried.
3 in situ hybridizations
(1) chromosome sectioning is denaturalized: it is (different that chromosome sectioning is denaturalized 2-6min in 85 DEG C, 70% deionized formamide
It is different the time required to species), it is immediately transferred to be dehydrated step by step in the ice ethyl alcohol of the 70%, 90% and 100% of -20 DEG C, every time
Then 5min is air-dried.
(2) preparing hybrid liquid: according to 2 preparing hybrid liquid of table.
Hybridization solution ingredient is as shown in table 2 below:
The ingredient and proportion of 2 20 μ l hybridization solution of table
Wherein, 45SrDNA probe is by 5.8S rDNA probe (20ng/ μ l), 18S rDNA probe (20ng/ μ l), 25S
RDNA probe (20ng/ μ l) mixes in equal volume.5SrDNA probe and 45SrDNA probe are the spy that embodiment 1 provides
Needle.
(3) in situ hybridization: the hybridization solution of 20 μ l is added dropwise in every film-making, then chromosome sectioning is placed in wet box, 37 DEG C
Hybridized overnight.
(4) post-hybridization washing and mounting: getting rid of coverslip, and at room temperature, 2 × SSC rinses 5min, and 1 × TNT rinses 5min.
After being rinsed with deionized water, film-making is dried up, finally with Vectashield (containing DAPI) mounting of 20 μ l.
5 photograph and image procossing
According to the split coil method coordinate position recorded, (the Model BX51 under fluorescence microscope；Olympus, Tokyo,
Japan film-making) is observed, and shoots image using subsidiary CCD.With ImagJ software by the gray level image photomontage of shooting,
And picture is handled using ADOBE PHOTOSHOP 5.0 (Adobe Systems, http: // www.adobe.com),
It is final using the chromosome and fluorescence signal synthesising picture that are shot before in situ hybridization, ensured optimum dyeing volume morphing and clear
Hybridization signal.
Embodiment 3
The present embodiment with the T.iliensis (T.iliensis Regel) of Tulipa be material, according to embodiment 2
Method carries out fluorescence in situ hybridization, as a result as shown in Figure 1.
It will be seen from figure 1 that chromosome morphology is good, fluorescence signal is very clear, there is 5 5S in T.iliensis
The site rDNA (green), wherein 4 are located at nearly centric region on the galianconism of No. 4 and No. 10 homologues, in addition 1 is located at
In No. 11 homologues item chromosome it is long-armed on.T.iliensis shares 15 sites 45S rDNA (red), big portion
Quartile is in long-armed end, wherein 2 are distributed in the galianconism end of No. 11 chromosomes, 12 sites are distributed in 5,6,8,9,10 and
The long-armed end of No. 12 homologues.There is the long-armed end of item chromosome there are 45S rDNA in No. 7 homologues
Point, in addition one does not have signal.
Embodiment 4
The present embodiment, for material, is pressed with the vertical flower bud tulip (T.patens Agardh.ex Schult.) of Tulipa
Fluorescence in situ hybridization is carried out according to the method for embodiment 2, as a result as shown in Figure 2.
Figure it is seen that fluorescence signal is very clear, flower bud tulip of hanging down has two pairs of sites 5S rDNA (green), point
Not Wei Yu the nearly centromere of 1 and No. 10 the short arm of a chromosome position.45S rDNA is predominantly located in the galianconism of chromosome.Flower bud of hanging down is strongly fragrant
Jin Xiang has 22 sites 45S rDNA (red), wherein 16 are located at galianconism end, 6 are located at long-armed end.3,4,6,8,9
There is 1 loci to be located at galianconism end with No. 12 homologues.There is item chromosome in 2 and No. 11 homologue chromosomes
There is no signal, in addition a homologue has the site of 45S rDNA in galianconism end.5 and No. 7 homologues it is long-armed
There is 1 pair of signaling point in end.The long galianconism of No. 10 homologues respectively has 1 pair of site 45SrDNA.No. 1 homologue galianconism
End is it sometimes appear that extremely weak 45S rDNA signaling point.
To sum up, it can be seen that tulip chromosome is carried out using probe combinations provided in an embodiment of the present invention in situ miscellaneous
It hands over, produces gem-pure fluorescence signal.Thus illustrate, carry out plant using probe combinations provided by the invention and method
Chromosomal in situ hybridization can capture the chromosome and fluorescence in situ hybridization signal of high-resolution, be applicable to a variety of
The Chromosomal in situ hybridization of plant such as lily, corn, morning glory, begonia etc..And the probe combinations are oligonucleotides spy
Needle has the characteristics that low manufacture cost, easy to use, easy to operate.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any
Modification, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Nanjing Forestry University
<120>a kind of probe combinations and method suitable for plant chromosome rDNA physical positioning
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 59
<212> DNA
<213>artificial sequence
<400> 1
ggatgcgatc ataccagcac taatgcaccg gatcccatca gaactccgca gttaagcgt 59
<210> 2
<211> 59
<212> DNA
<213>artificial sequence
<400> 2
aaaacgactc tcggcaacgg atatctcggc tctcgcatcg atgaagaacg tagcgaaat 59
<210> 3
<211> 59
<212> DNA
<213>artificial sequence
<400> 3
tacctggttg atcctgccag tagtcatatg cttgtctcaa agattaagcc atgcatgtg 59
<210> 4
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<213>artificial sequence
<400> 4
cccgctgagt ttaagcatat caataagcgg aggaaaagaa actaacaagg attccctta 59
Claims (10)
1. a kind of probe combinations suitable for plant chromosome rDNA physical positioning, which is characterized in that it includes 5S rDNA probe
And/or 45S rDNA probe；
The nucleotide sequence of the 5S rDNA probe is as shown in SEQ ID NO.1；
The 45S rDNA probe is made of 5.8S rDNA probe, 18S rDNA probe and 25S rDNA probe, the 5.8S
The nucleotide sequence of rDNA probe is as shown in SEQ ID NO.2, the nucleotide sequence of the 18S rDNA probe such as SEQ ID
Shown in NO.3, the nucleotide sequence of the 25S rDNA probe is as shown in SEQ ID NO.4.
2. probe combinations according to claim 1, which is characterized in that the both ends of the 5S rDNA probe are marked with first
Fluorophor；The both ends of the 45S rDNA probe are marked with the second fluorophor.
3. probe combinations according to claim 2, which is characterized in that first fluorophor is Fluoresceincarboxylic acid；
Second fluorophor is carboxyl tetramethylrhodamine.
4. probe combinations according to claim 1-3, which is characterized in that the plant be selected from tulip, lily,
Corn, morning glory and begonia.
5. a kind of method of Chromosome in Situ Hybridization of Plants, which is characterized in that it includes the following steps:
In situ hybridization step: hybridization solution is added on toward plant chromosome film-making, is hybridized；
Wherein, the hybridization solution contains the described in any item probe combinations of claim 1-4.
6. according to the method described in claim 5, it is characterized in that, 5S rDNA concentration and probe concentration is 1-2ng/ μ in the hybridization solution
l。
7. according to the method described in claim 5, it is characterized in that, the concentration of 45S rDNA probe is 1- in the hybridization solution
2ng/μl。
8. according to the method described in claim 5, it is characterized in that, the hybridization solution also contains: dFA, SSC buffer and DS.
9. according to the described in any item methods of claim 5-8, which is characterized in that in situ before hybridization step, the method
It further include pre-treatment step；
The pre-treatment step includes: to impregnate the plant tip of a root of acquirement at room temperature with ring acetamide solution, uses distilled water
It is transferred to after flushing in the fixer of Kano.
10. according to the described in any item methods of claim 5-8, which is characterized in that the plant is selected from tulip, lily, jade
Rice, morning glory and begonia.
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| CN201810928031.1A CN108977565B (en) | 2018-08-14 | 2018-08-14 | A kind of probe combinations and method suitable for plant chromosome rDNA physical positioning |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218499A (en) * | 2020-02-20 | 2020-06-02 | 南京林业大学 | Frozen slice-based 3D fluorescence in situ hybridization method for poplar root tips |
| CN111323288A (en) * | 2020-04-03 | 2020-06-23 | 贵州省烟草科学研究院 | Winged tobacco monosome identification method |
| CN111534513A (en) * | 2019-09-11 | 2020-08-14 | 广东美格基因科技有限公司 | Reverse transcription primer pool and kit for removing ribosomal RNA and method for removing ribosomal RNA |
| CN113025742A (en) * | 2021-03-25 | 2021-06-25 | 广州王老吉大健康产业有限公司 | Molecular identification method for medicinal material microcos paniculata and southern Hainan Broussonetia papyrifera |
| CN113957166A (en) * | 2020-07-20 | 2022-01-21 | 南京农业大学 | Ribosome DNA oligonucleotide mixed probe sleeve based on chrysanthemum kindred plant reference genome and development method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102787166A (en) * | 2012-07-16 | 2012-11-21 | 北京林业大学 | Fluorescence in situ hybridization method of prunus mume chromosome |
| CN103409524A (en) * | 2013-08-12 | 2013-11-27 | 南开大学 | Fluorescence in situ hybridization method for positioning 45S rDNA on plant chromosome |
| CN103409523A (en) * | 2013-08-12 | 2013-11-27 | 南开大学 | Fluorescence in situ hybridization method of 5S rDNA on plant chromosome |
| CN106399499A (en) * | 2016-09-18 | 2017-02-15 | 新乡医学院 | Fluorescence in-situ hybridization method for asparagus fern medium-term chromosomes |
| CN106987590A (en) * | 2017-05-25 | 2017-07-28 | 河南省农业科学院 | One cultivates peanut oligonucleotide probe and its design method and application method |
-
2018
- 2018-08-14 CN CN201810928031.1A patent/CN108977565B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102787166A (en) * | 2012-07-16 | 2012-11-21 | 北京林业大学 | Fluorescence in situ hybridization method of prunus mume chromosome |
| CN103409524A (en) * | 2013-08-12 | 2013-11-27 | 南开大学 | Fluorescence in situ hybridization method for positioning 45S rDNA on plant chromosome |
| CN103409523A (en) * | 2013-08-12 | 2013-11-27 | 南开大学 | Fluorescence in situ hybridization method of 5S rDNA on plant chromosome |
| CN106399499A (en) * | 2016-09-18 | 2017-02-15 | 新乡医学院 | Fluorescence in-situ hybridization method for asparagus fern medium-term chromosomes |
| CN106987590A (en) * | 2017-05-25 | 2017-07-28 | 河南省农业科学院 | One cultivates peanut oligonucleotide probe and its design method and application method |
Non-Patent Citations (2)
| Title |
|---|
| 兰添颖等: "菠菜 rDNA 及端粒多色荧光原位杂交分析", 《遗传》 * |
| 牛凯等: "rDNA序列在多种蔬果类植物染色体上的定位", 《南开大学学报（自然科学版）》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111534513A (en) * | 2019-09-11 | 2020-08-14 | 广东美格基因科技有限公司 | Reverse transcription primer pool and kit for removing ribosomal RNA and method for removing ribosomal RNA |
| CN111218499A (en) * | 2020-02-20 | 2020-06-02 | 南京林业大学 | Frozen slice-based 3D fluorescence in situ hybridization method for poplar root tips |
| CN111323288A (en) * | 2020-04-03 | 2020-06-23 | 贵州省烟草科学研究院 | Winged tobacco monosome identification method |
| CN113957166A (en) * | 2020-07-20 | 2022-01-21 | 南京农业大学 | Ribosome DNA oligonucleotide mixed probe sleeve based on chrysanthemum kindred plant reference genome and development method |
| CN113957166B (en) * | 2020-07-20 | 2023-10-27 | 南京农业大学 | Ribosome DNA oligonucleotide mixed probe sleeve based on chrysanthemum related species plant reference genome and development method |
| CN113025742A (en) * | 2021-03-25 | 2021-06-25 | 广州王老吉大健康产业有限公司 | Molecular identification method for medicinal material microcos paniculata and southern Hainan Broussonetia papyrifera |
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Application publication date: 20181211 Assignee: Nanjing peptide crystal Biotechnology Co., Ltd Assignor: Nanjing Forestry University Contract record no.: X2019320000336 Denomination of invention: Probe combination and method suitable for physically positioning plant chromosome rDNA (ribosomal Deoxyribonucleic Acid) Granted publication date: 20190618 License type: Common License Record date: 20191206 Application publication date: 20181211 Assignee: Nanjing runke Biotechnology Co., Ltd Assignor: Nanjing Forestry University Contract record no.: X2019320000337 Denomination of invention: Probe combination and method suitable for physically positioning plant chromosome rDNA (ribosomal Deoxyribonucleic Acid) Granted publication date: 20190618 License type: Common License Record date: 20191206 Application publication date: 20181211 Assignee: Nanjing dehetang Biotechnology Co., Ltd Assignor: Nanjing Forestry University Contract record no.: X2019320000335 Denomination of invention: Probe combination and method suitable for physically positioning plant chromosome rDNA (ribosomal Deoxyribonucleic Acid) Granted publication date: 20190618 License type: Common License Record date: 20191206 |