CN103205500A - Multicolor fluorescence in situ hybridization (MFISH) method for quickly analyzing and identifying alien chromosome of wheat - Google Patents
Multicolor fluorescence in situ hybridization (MFISH) method for quickly analyzing and identifying alien chromosome of wheat Download PDFInfo
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Abstract
The invention provides a multicolor fluorescence in situ hybridization (MFISH) method for quickly analyzing and identifying alien chromosome of wheat. The method comprises the following steps: (1) preprocessing actively dividing tissues in meristematic zone of wheat root tip by N2O and conducting enzymolysis on the actively dividing tissues to obtain a metaphase specimen with clear images and uniform distribution of chromosomes; (2) marking a fluorescent probe of fundamental genome of wheat by nick translation method; (3) conducting fluorescence in situ hybridization on chromosomes at mitosis metaphase obtained in step (1) by using the constructed fluorescent probe in step (2); and (4) analyzing the fluorescence in situ hybridization results obtained in step (3). According to the method provided by the invention, not only can alien chromosomes and chromosome segments in distant hybrid wheat be identified, but also alien chromosomes and chromosome segments of distant hybrids of other crops can be identified.
Description
Technical field
The invention belongs to the Celluar and Molecular Biology research field.The present invention relates to the method for the multi-color fluorescence in situ hybridization of a kind of real-time analysis and evaluation wheat exogenous chromosome, the invention still further relates to a kind of method of the plant chromosome sample that obtains being carried out the recross analysis, the invention still further relates to a kind of method for preparing plant chromosome metacinesis phase sample, can obtain clear picture, the finely disseminated metacinesis phase of karyomit(e) sample.
Background technology
Wheat is one of important crops in the world, supply with socio-economic development, grain security and human nutrition health closely related.Genetic improvement is the key link that improves wheat yield and quality, and conventional hybridization breeding approach is still mainly adopted in wheat breed improvement at present, but traditional breeding way exists long, character improvement of cycle not significantly and shortcoming such as narrow range.Hereditary basis is narrow to have become the key factors that Main Agronomic Characters such as restriction crop varieties unit yield, resistance, adaptability further promote (leaf make the country prosperous et al.2011).Therefore, the hereditary basis of widening kind has become the active demand of crop breeding development.
Distant hybirdization is the important channel that species form, it is one of important factor of organic evolution, distant hybirdization can be broken kind the boundary between (or section, genus), genetic material between different plant species is exchanged or combination, two or more species are combined through the beneficial characteristics that long-term evolution accumulates, double and natural selection through genome is natural again, form the stronger new species of vitality.Import foreign gene by distant hybirdization, significant to modern wheat improvement.
The GENE SOURCES of common wheat is wideer, and more than 300 species great majority can both be hybridized with common wheat in the wheat family, and existing 5 15 kinds of genus (5 kinds that comprise Triticum) have shifted disease-resistant gene to common wheat.China the 1950's just with wheat and middle couchgrass, long fringe couchgrass distant hybirdization success, bred a collection of kind so far in the production popularization.As spring wheat varieties such as " imperial wheat No. 1 ", " the imperial wheat No. 2 " bred by middle couchgrass, " No. 6, Neoma Foams ".Simultaneously, 1 to No. 7 high-quality, disease-resistant of the octoploid of wheat-wheatgrass of breeding " in far away " has become the good breeding parent.Breed a collection of wheat breed by the long fringe couchgrass of decaploid, year cultivated area of wherein " laying down for a short time No. 6 " once reached 66.67 ten thousand hm2, and reached 20 years (Dong Yu treasure 2001) in the production use.
Chromosome engineering had once been brought into play important effect on polyploid crop breeding histories such as wheat.The seventies in 20th century, the 1RS/1BL translocation line material of rye has been found in Europe in the filial generation of wheat and rye, and for the wheat breed improvement, cultivate some row high yields, disease-resistant, dwarf wheat kinds such as Luo Fulin, and the rapid big area popularization in each wheat main producing region in the world, each country is according to oneself breeding objective subsequently, transformation thousands of 1RS/1BL kind, for outstanding contribution has been made in world wheat production.The 1BL/1RS transposition extensively distributes in world's wheat breed, by specific chromosome translocation and replacement between planting, the galianconism of chromosomes of rye 1R (1RS) has been present in the genome of a large amount of common wheats, and many have the gene of vital role and resistant gene to economical character and change over to the wheat cdna group from rye thus and (appoint swallow and Wang Tao 2006).
Traditional chromosome engineering exists that efficient is low, the cycle is long, recombinant chromosome is difficult to accurate and effective and follows the tracks of and Rapid identification, and is difficult to and a series of problems such as modern molecular biology technique is connected mutually, has seriously limited further developing of chromosome engineering research.So the achievement of analysis genome rapidly and efficiently seems particularly important.Progress along with the karyomit(e) operative technique, especially the development of DNA hybridization in situ technique, for the evaluation of heterochromosome system, the identification of transposition breakpoint provide useful instrument, can identify size, position and the breakpoint of transposition fragment exactly, lay a good foundation for the useful gene of autotelic transfer carries out the wheat breed improvement.
Genomic in situ hybridization (Genomic in situ hybridization GISH) is a kind of hybridization in situ technique that grows up late 1980s.Its adopts total genomic dna from the species probe that serves as a mark, and blockades with suitable concentration with the total genomic dna of another species, carries out in situ hybridization at target chromosome.Blockading between DNA and the labeled DNA probe, the preferential and general sequence hybridization of the DNA that blockades, remaining specific sequence mainly is labeled probe and hybridizes.Mukai usefulness biotin labeling diploid AA genome ancestors such as (Mukai and Gill 1991) plant T rotoci urartu total genomic dna, digoxigenin labeled diploid DD genome ancestors plant Aegilops squarrosa total genomic dna, unlabelled total genomic dna is that possible BB genome ancestors plant Aegilops speltoides, hexaploid common wheat (Triticum aestivum) Metaphase Chromosome is carried out in situ hybridization, only just A, B and D genome are shown as Huang, palm fibre and orange simultaneously with two kinds of fluoresceins.After this Sanchez-Moran (Sanchez-Moran et al.1999) etc. utilizes this technology to show A, B, D and the R genome of wheat-rye in deriving kind simultaneously with different colours.(Han et al.2003 such as Korea Spro; Han et al.2004) utilize digoxin and biotin labeled genome GISH to identify the karyomit(e) composition of partial amphidiploid and the transposition between the analysis karyomit(e), but the probe mark method of using mostly is indirect method greatly, namely use non-fluorescent substance such as label probes such as vitamin H or digoxin, bring fluorescent substance into by affine connection or immune response again, has the hybridization signal amplification, although indirect method FISH can amplify hybridization signal, increase hybridization susceptibility, but to hatch to amplify hybridization signal through loaded down with trivial details wash-out and antibody after the hybridization, waste time and energy, and in elution process, be easy to cause problems such as chromosome elimination distortion, this experiment is by direct method FISH, namely when the nick-translation method label probe, directly mix fluorescein ribonucleoside triphosphote marker, but after the hybridization through simple flushing microscopy just, greatly simplified the in situ hybridization program, keep chromosomal optimal morphology, can both well observe identifying the mutual transposition between exogenous chromosome composition and analysis and the karyomit(e).
Summary of the invention
An object of the present invention is to provide the multi-color fluorescence in situ hybridization method of a kind of real-time analysis and evaluation wheat exogenous chromosome.
Particularly, the method for analysis of the present invention and evaluation wheat exogenous chromosome comprises the steps:
1) passes through N
2The vigorous tissue of the pre-treatment of O gas and the cell fission of enzymolysis wheat tip of a root meristematic zone obtains clear picture, the finely disseminated metacinesis phase of karyomit(e) sample;
2) utilize nick translation method mark labeling wheat family basis genome fluorescent probe;
3) utilizing step 2) fluorescent probe that makes up carries out fluorescence in situ hybridization to utilizing step 1) to divide phase karyomit(e) mitosis metaphase;
4) analytical procedure 3) the fluorescence in situ hybridization result that obtains.
Wherein step 1) comprises the steps: (a) N
2O gas pre-treatment: get the suitable wheat tip of a root, be placed in the moistening centrifuge tube, put into N
2Handled 0.5-5 hour N in the O air chamber
2The pressure of O is 10ATM (1.01Mpa); (b) with N
2The pretreated tip of a root of O gas is fixed; (c) enzymolysis processing: the tip of a root that fixes is washed with water, and the meristematic tissue that cuts off root tips is with polygalacturonase and cellulose treatment 30min-2h.
Step 2 wherein) the wheat family of mark basis genome is selected from the AA genome, DD genome, RR rye genome, the long fringe couchgrass of 10X genome, or transgenosis purpose fragment carrier.
The step of most critical is the blending ratio of the fluorescent probe of the used nick translation method mark of step 3) hybridization in the above-mentioned steps, and the genomic mass ratio of AA genome and DD can not be less than 1: 4.For example, fluorescence in situ hybridization is carried out in division mitosis metaphase of the wheat of laying down for a short time (couchgrass and common wheat filial generation) and triticale (rye and common wheat filial generation) mutually; Probe blending ratio wherein should be the mixed probe that how much comes according to differing materials (exogenous chromosome derives from rye or couchgrass) exogenous chromosome, AA genome and DD genome homology are than higher, so AA genome and DD genome have cross hybridization, chromosomal color is inconsistent (during probe mark with the color of probe mark, the AA genome is green, the DD genome is red, after having hybridized, the AA genome is green, the DD genome is red), so probe ratio (the genomic ratio of AA genome and DD) can not be less than 1: 4, the BB genome is as blockading, with AA genome and DD genome certain homology is arranged, but and BB genome (own) homology own is the highest, separate four genomes so be conducive to a hybridization region to blockading with an a high proportion of BB genome of blockading, BB is genomic to be blockaded than (mass ratio) up to 160-200 doubly.
Another purpose of the present invention provides a kind of method of the plant chromosome sample that obtains being carried out the recross analysis.
Particularly, the present invention comprises the steps: the method that the plant chromosome sample that obtains carries out the recross analysis
1) passes through N
2The pre-treatment of O gas and enzymolysis of plants tip of a root meristematic zone or the vigorous tissue of bud point meristematic zone cell fission obtain clear picture, the finely disseminated metacinesis phase of karyomit(e) sample;
2) utilize nick translation method mark fluorescent probe;
3) utilizing step 2) fluorescent probe that makes up carries out fluorescence in situ hybridization to utilizing step 1) to divide phase karyomit(e) mitosis metaphase;
4) through simply flushing, on the basis that keeps chromosome morphology, use multiple hybridization probe to carry out one or many hybridization repeatedly, carry out evaluation and the analysis of exogenous chromosome.
The step of most critical is in the step 4) in the above-mentioned steps, through simply flushing, uses multiple hybridization probe repeatedly on the basis that keeps chromosome morphology, carries out one or many hybridization successively, carries out evaluation and the analysis of exogenous chromosome.After having hybridized for the first time; have the anti-quencher that contains DAPI on the chromosome specimen; compare thickness; only need 2 * SSC (protein on the protection karyomit(e); preventing karyomit(e) distortion) wash-out just can directly (for example carry out follow-up hybridization successively under the room temperature; hybridization for the second time, hybridization for the third time etc.), problem such as chromosome elimination distortion in the loaded down with trivial details wash-out before having avoided and the elution process.
In a specific embodiments of the present invention, the invention provides the multi-color fluorescence in situ hybridization method of a kind of real-time analysis and evaluation wheat exogenous chromosome, it is characterized in that:
1) passes through N
2The pre-treatment of O gas and enzymolysis Gansu Province spring 23, the tip of a root meristematic zone cell of octoploid of wheat-wheatgrass XY784 obtains clear picture, the finely disseminated metacinesis phase of karyomit(e) sample;
2) utilize nick translation method mark A genome, D genome, the long fringe couchgrass of R genome and decaploid fluorescent probe;
3) utilizing step 2) fluorescent probe that makes up carries out fluorescence in situ hybridization to utilizing step 1) to divide phase karyomit(e) mitosis metaphase;
4) analytical procedure 3) the fluorescence in situ hybridization result that obtains.
In a specific embodiments of the present invention, described karyomit(e) to be detected and chromosome segment are selected from Gansu Province spring 23, the transgenic line of octoploid of wheat-wheatgrass XY784 and Pwy86.
Though one or more in the above-mentioned technology have obtained application in relevant research, but several like this technology of integrated application constitute complete experimental program, and are applied in the evaluation of exogenous chromosome in the plant distant hybrids or chromosome segment and do not see the correlative study report.The present invention utilizes this cover research approach successfully with the external source euchromosome in the high-sensitivity detection distant hybirdization plant first, overcome the undesirable problem of plant euchromosome detection effect in the prior art, be a kind of technological breakthrough, embodied this research method and had advance, novelty and practicality preferably.
In sum, the invention provides following:
1. a real-time analysis and identify the multi-color fluorescence in situ hybridization method of exogenous chromosome, it comprises the steps:
1) passes through N
2The vigorous tissue of the pre-treatment of O gas and the cell fission of enzymolysis of plants tip of a root meristematic zone obtains clear picture, the finely disseminated metacinesis phase of karyomit(e) sample;
2) utilize the nick translation method labeling wheat family genomic fluorescent probe in basis;
3) utilizing step 2) fluorescent probe that makes up carries out fluorescence in situ hybridization to utilizing step 1) to divide phase karyomit(e) mitosis metaphase;
4) analytical procedure 3) the fluorescence in situ hybridization result that obtains.
2. the 1st described method, wherein step 1) comprises the steps: (a) N
2O gas pre-treatment: get the suitable wheat tip of a root, be placed in the moistening centrifuge tube, put into N
2Handled 0.5-5 hour N in the O air chamber
2The pressure of O is 10ATM (1.01Mpa); (b) with N
2The pretreated tip of a root of O gas is fixed; (c) enzymolysis processing: the tip of a root that fixes is washed with water, and the meristematic tissue that cuts off root tips is with polygalacturonase and cellulose treatment 30min-2h.
3. the 1st described method, wherein step 2) in the wheat family basis genome of mark be selected from the AA genome, DD genome, RR rye genome, 10X long fringe couchgrass genome or transgenosis purpose fragment carrier.
4. the 1st described method, wherein the mass ratio that mixes of the genomic probe of diploid Urartu AA genome and diploid aegilops tauschii D D can not be less than 1: 4.
5. the 1st described method, wherein diploid intend the genomic mass ratio of blockading of this inferior you holder BB be 160-200 doubly.
6. the 1st described method wherein carried out the recross analysis to the chromosome of wheat sample that obtains, and wherein per step recross analysis wash-out is with wash-out under 2 * SSC room temperature.
7. one kind is carried out the method that recross is analyzed to the plant chromosome sample that obtains, and described method comprises the steps:
1) passes through N
2The pre-treatment of O gas and enzymolysis of plants tip of a root meristematic zone or the vigorous tissue of bud point meristematic zone cell fission obtain clear picture, the finely disseminated metacinesis phase of karyomit(e) sample;
2) utilize nick translation method mark fluorescent probe;
3) utilizing step 2) fluorescent probe that makes up carries out fluorescence in situ hybridization to utilizing step 1) to divide phase karyomit(e) mitosis metaphase;
4) through simply flushing, on the basis that keeps chromosome morphology, use multiple hybridization probe to carry out one or many hybridization repeatedly, carry out evaluation and the analysis of exogenous chromosome.
8. the 7th described method, wherein the simple flushing of step 4) is with wash-out under 2 * SSC room temperature.
9. method for preparing plant chromosome metacinesis phase sample, described method comprises the steps:
(a) N
2O gas pre-treatment: get the suitable tip of a root of described plant or bud point, be placed in the moistening centrifuge tube, put into N
2Handled 0.5-5 hour N in the O air chamber
2The pressure of O is 10ATM (1.01Mpa); (b) with N
2The pretreated tip of a root of O gas or bud point are fixing; (c) enzymolysis processing: the tip of a root or the bud point that fix are washed with water, cut off root tips or bud point meristematic tissue, with polygalacturonase and cellulose treatment 30min-2h.
10. the 9th described method, wherein said plant is monocotyledons.
The accompanying drawing summary
Fig. 1 .1RS/1BL translocation line karyomit(e) is formed.Arrow is depicted as 1RS karyomit(e).The green rye R that represents among the figure, yellow represents wheat A genome, and brown represents wheat B genome, the red wheat D genome that represents.
Fig. 2. octoploid of wheat-wheatgrass karyomit(e) is formed.Arrow and asterisk are depicted as two pairs of translocation chromosomes of wheat A-D group.Green represents the long fringe couchgrass of decaploid, and yellow represents wheat A genome, and brown represents wheat B genome, the red wheat D genome that represents.
Fig. 3. three in situ hybridization A of chromosome of wheat sample: the fluorescence in situ hybridization detection of transgenosis signal, arrow are depicted as the insertion site of goal gene; B: multi-color fluorescence in situ hybridization, detect the chromosome of wheat group at place, transgenosis site, green represents wheat A chromosome group, and brown represents wheat B chromosome group, redness represents wheat D genome, and arrow is depicted as the A chromosome group that goal gene has inserted wheat; C: the concrete karyomit(e) at tumor-necrosis factor glycoproteins pSC119.2 and pAS1 testing goal gene place, green is shown tumor-necrosis factor glycoproteins pSC119.2 signal, and redness is shown tumor-necrosis factor glycoproteins pAS1 signal, and arrow is depicted as the 1A karyomit(e) that goal gene has inserted wheat.
Embodiment
Come further to illustrate the present invention by the following examples.But should be appreciated that described embodiment is illustrational purpose, and be not intended to limit the scope of the invention and spirit.
In addition, specify unless those skilled in the art be also to be understood that in addition, used reagent is commercially available other reagent of analytical pure level among the following embodiment.
The evaluation of embodiment 1. Gansu Province spring, 23 spring wheat materials
1. Gansu Province spring 23 spring wheat preparation of dividing phase mitosis metaphase
Used Gansu Province spring 23 spring wheat, the germ plasm resource of preserving from the general laboratory of Inst. of Genetics and Development Biology, CAS South Korea (can referring to Yang Wen hero 2005).
1) N
2O handles the tip of a root
When treating tip of a root length to 2-3cm, cut, be placed in the moistening centrifuge tube, put into the N2O air chamber behind the cover lid and handle 1-3h, pressure is 10ATM (1.01Mpa);
2) tip of a root is fixed
90% acetic acid is 5-10 minute (being no more than 1h) fixedly, distillation washing 2 times.The tip of a root after fixing can change over to be put into-20 ℃ in 70% the ethanol and can preserve for many years;
3) enzymolysis
With filter paper the water on the tip of a root is blotted a little, tip of a root meristematic tissue is downcut, put into 20 μ L mixed solutions (the enzymolysis solution preparation a: plastic culture dish is put on the balance of polygalacturonase and cellulase, add 1 * citrate buffer solution (10mM Trisodium Citrate+10mM EDTA to the inside, pH5.5) 9.7g, taking-up places on ice, take by weighing 0.1g polygalacturonase Y-23 (available from Japanese Japan Yakult company, 1%w/w) and 0.2g cellulase Onozuka R-10 (available from Japanese Japan Yakult company, 2%w/w) add in the damping fluid, with rifle head pressure-vaccum hydrotropy, enzyme dissolving back installs in the 0.5ml thin-walled PCR pipe with the skimmer branch,-20 ℃ of preservations) in, 37 ℃ water-bath 40-60 minute
4) drip sheet
Wash 2 times with 70% alcohol, stay some alcohol when washing for the second time, approximately 200ul is in centrifuge tube, with dissecting needle the tip of a root is smashed to pieces, centrifugal about 10 seconds of low speed (＜2000 change) falls to do with alcohol, ice acetic acid (each tip of a root 20-40 μ l looks the plant root tip size) suspension root-tip cells.
5) microscopy
Clean slide glass is placed in the moistening box, and every slide glass drips 6-7 μ l root-tip cells suspension, covers lid, and microscopy after 5 minutes is kept in the refrigerator chromosome sectioning sample standby.
2.AA group, the probe mark of DD group and RR group
1) the CTAB method is extracted plant genome DNA
Get plant (rye, the long fringe couchgrass of decaploid, Triticum tauschii, Urartu and speltoides, the tender blade of (these seeds are available from Germany IPK institute) children, reference literature (Kidwell et al.1992) extracts the DNA of plant, makes the concentration of DNA finally reach 200ng/ul, label probe then.Wherein speltoides will do and blockade, and concentration will reach 2000ng/ul.
2) add following component in the centrifuge tube on ice:
Add good after, blow and beat mixing repeatedly with the rifle head, can not vortex, 15 ℃ are incubated 2 hours.Place 15 ℃ of reaction 2h in the metal bath instrument (H2O3-PRO, China).(mark-dNTP is Alexa Fluor-488-dUTP (green glow), Texas
-5-dCTP (ruddiness) is available from Invitrogen company; Dna polymerase i is available from Invitrogen company, and concentration is 10U/ μ L, can directly use 10x Nick translation buffer for 100ml Tris 6.05g, MgCl
2, 0.47g, HCl transfers pH7.8); Wherein rye, long fringe couchgrass and Urartu are with green fluorescence mark (Alexa Fluor-488-dUTP (green glow)), and Triticum tauschii is carried out mark (Texas with red fluorescence
-5-dCTP), speltoides does to blockade and (that is, speltoides genomic dna high pressure is interrupted, 115 ℃, 8 minutes, it was about 200-300bp that genomic dna is broken into fragment), do blockade before, genomic fragment need be broken into 200-300bp, such effect of blockading is best.
3) 13000 leave heart 30-40 minute, abandon supernatant liquor, wash primary sedimentation with 70% ethanol and dehydrated alcohol respectively, lucifuge is dried.
4) add 10 μ l, 2 * SSC, 1 * TE damping fluid (pH7.0), making the probe final concentration is 200ng/ul.
3. utilize the good genomic probe of mark that Gansu Province spring 23 spring wheat is carried out the evaluation of fluorescence in situ hybridization
1) method with program 1 prepares Gansu Province spring 23 spring wheat Metaphase Chromosome sample.
2) it is crosslinked sample be hybridized (film-making) to put in the UV-crosslinked instrument (code-No.CL-1000 is available from U.S. UVP company) 0.125J/cm2.
3) film-making is placed on ice, taking out the good probe of a small amount of mark organizes by rye: AA: the BB group: DD group=1: 1: 4: 4 or the long fringe couchgrass of decaploid: AA group: BB group: DD group=1: 1.5: 4: 4 dilution proportion, add the good hybridization solution 6 μ l of dilution on every slide glass, covered, 90 ℃ of sex change 5 minutes, 55 ℃ of hybridization are more than 6 hours.
4) film-making is taken out from wet box, put into 2 * SSC and make the cover glass landing, with thieving paper the slice, thin piece back side is dried, drip a last anti-quencher (H-1200 who contains DAPI, available from U.S. Vector Labs company), covering the cover glass of 24 * 50mm, is that available fluorescent microscope (Olympus BX61) carries out microscopy, photograph (Magnafier CCD camera) after several minutes.
4. result and discussion
Result of study is seen Fig. 1.
As shown in Figure 1, demonstration is that 1RS/1BL translocation line karyomit(e) is formed.Arrow is depicted as 1RS karyomit(e).The green rye R that represents among the figure, article two, green the short arm of a chromosome, be from rye 1RS, yellow represents wheat A genome, and 14 karyomit(e)s are arranged, brown represents wheat B genome, 14 karyomit(e)s are arranged, and redness represents wheat D genome, and 14 karyomit(e)s are arranged, 42 karyomit(e)s are arranged in the whole cell, are that the karyomit(e) of 1RS/1BL translocation line is formed.
This research utilizes two fluorescent probes of four genomes first, Gansu Province spring 23 spring wheat is carried out genome karyomit(e) to be identified, just can clearly analyze by an in situ hybridization and to tell four genomes, and four genomic karyomit(e)s are formed cheer and bright, show that the method for utilizing the present invention to adopt successfully detects and identify existence and the structure of exogenous chromosome, this experiment finishes just to need 3 day time to identifying from sending out seed, not only rapidly and efficiently but also sensitive.
The karyomit(e) compositional analysis of embodiment 2. octoploid of wheat-wheatgrass XY784
1. octoploid of wheat-wheatgrass XY784 divides the preparation of phase mitosis metaphase
Used octoploid of wheat-wheatgrass XY784 is from the germ plasm resource (Zhang Xueyong et al.1995) of Inst. of Genetics and Development Biology, CAS's Li Zhensheng laboratory preservation.
1) N2O handles the tip of a root with embodiment 1.1
2) tip of a root is fixing with embodiment 1.2
3) enzymolysis is with embodiment 1.3
4) drip sheet with embodiment 1.4
5) microscopy is with embodiment 1.5
2.AA group, the probe mark of the long fringe couchgrass group of DD group and decaploid
1) the CTAB method is extracted plant genome DNA with embodiment 2.1
2) probe mark is with embodiment 2.2
3) with embodiment 2.3
4) with embodiment 2.4
3. utilize the good genomic probe of mark octoploid of wheat-wheatgrass XY784 to be carried out the evaluation of fluorescence in situ hybridization
1) method with program 1 prepares octoploid of wheat-wheatgrass XY784 Metaphase Chromosome sample.
2) with embodiment 3.2
3) film-making is placed on ice, taking out the good probe of a small amount of mark organizes by the long fringe couchgrass of decaploid: AA: the BB group: DD group=1: 1: 4: 4 or the long fringe couchgrass of decaploid: AA group: BB group: DD group=1: 1.5: 4: 4 dilution proportion, add the good hybridization solution 6 μ l of dilution on every slide glass, covered, 90 ℃ of sex change 5 minutes, 55 ℃ of hybridization are more than 6 hours.
4) with embodiment 3.4
4. result and discussion
Result of study is seen Fig. 2.
As shown in Figure 1, demonstration is that octoploid of wheat-wheatgrass karyomit(e) is formed.Green represents the long fringe couchgrass of decaploid, chromosome number is 14, the yellow wheat A genome that represents, chromosome number is 12, has lacked 2 than normal A group chromosome, and brown represents wheat B genome, chromosome number is 14, redness represents wheat D genome, and chromosome number is 16, has Duoed 2 than normal D group chromosome.Arrow and asterisk are depicted as two pairs of translocation chromosomes of wheat A-D group.56 karyomit(e)s are arranged in the whole cell, are octoploid of wheat-wheatgrass.
This research utilizes two fluorescent probes of four genomes first, octoploid of wheat-wheatgrass XY784 is carried out genome karyomit(e) to be identified, just can clearly analyze by an in situ hybridization and to tell four genomes, and four genomic karyomit(e)s are formed cheer and bright, two pairs of translocation chromosomes of A-D group have also been analyzed simultaneously, for later economical character evaluation and molecule marker provide the basis, show that the method for utilizing the present invention to adopt successfully detects and identify existence and the composition of exogenous chromosome, this experiment finishes just to need 3 day time to identifying from sending out seed, not only rapidly and efficiently but also sensitive.
The Metaphase Chromosome preparation of embodiment 3. different plants
Further describe the present invention below with reference to specific embodiment, but it should be appreciated by those skilled in the art that the present invention is not limited to these specific embodiments.
1. seed germination
(1) seed is placed in the culture dish that is lined with moistening filter paper, sprouts in the 21-23 ℃ of thermostat container.
2. plant chromosome sample preparations
1) with embodiment 1.1.
2) with embodiment 1.2.
3) with embodiment 1.3.
4) with embodiment 1.4.
5) with embodiment 1.5.
3. in situ hybridization
1) method with program 1 prepares chromosome specimen mitosis metaphase.
2) with embodiment 3.2
3) with embodiment 3.3
When 4) carrying out second time in situ hybridization, the chromosome specimen of hybridizing is put into the anti-quencher that 2 * SSC flush away contains DAPI, with thieving paper the slice, thin piece back side is dried, carry out secondary in situ hybridization, subsequent step is with embodiment 3.4, and in situ hybridization is the same for the third time.
3. result and discussion
Result of study is seen Fig. 3.
As shown in Figure 3, three in situ hybridization figure A of the same sample of chromosome of wheat: the fluorescence in situ hybridization detection of transgenosis signal, arrow are depicted as the insertion site of goal gene; Figure B: multi-color fluorescence in situ hybridization, detect the chromosome of wheat group at place, transgenosis site, green represents wheat A chromosome group, and brown represents wheat B chromosome group, redness represents wheat D genome, and arrow is depicted as the A chromosome group that goal gene has inserted wheat; Figure C: the concrete karyomit(e) at tumor-necrosis factor glycoproteins pSC119.2 and pAS1 testing goal gene place, green is shown tumor-necrosis factor glycoproteins pSC119.2 signal, and redness is shown tumor-necrosis factor glycoproteins pAS1 signal, and arrow is depicted as the 1A karyomit(e) that goal gene has inserted wheat.
This research is carried out three in situ hybridization to the same sample of chromosome of wheat first and is analyzed, but after the hybridization through simple flushing microscopy just, and can on the basis that keeps chromosome morphology, use multiple hybridization probe repeatedly, greatly simplify the in situ hybridization program, be highly suitable for evaluation and the analysis of exogenous chromosome.
Should be appreciated that, although with reference to its exemplary embodiment, the present invention is shown particularly and describe, but will be understood by those skilled in the art that, under the condition that does not deviate from by the defined the spirit and scope of the present invention of accompanying Claim, the variation of various forms and details can be carried out therein, the arbitrary combination of various embodiments can be carried out.
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Claims (10)
1. a real-time analysis and identify the multi-color fluorescence in situ hybridization method of exogenous chromosome, it comprises the steps:
1) passes through N
2The vigorous tissue of the pre-treatment of O gas and the cell fission of enzymolysis wheat tip of a root meristematic zone obtains clear picture, the finely disseminated metacinesis phase of karyomit(e) sample;
2) utilize the nick translation method labeling wheat family genomic fluorescent probe in basis;
3) utilizing step 2) fluorescent probe that makes up carries out fluorescence in situ hybridization to utilizing step 1) to divide phase karyomit(e) mitosis metaphase;
4) analytical procedure 3) the fluorescence in situ hybridization result that obtains.
2. the described method of claim 1, wherein step 1) comprises the steps: (a) N
2O gas pre-treatment: get the suitable wheat tip of a root, be placed in the moistening centrifuge tube, put into N
2Handled 0.5-5 hour N in the O air chamber
2The pressure of O is 10ATM (1.01Mpa); (b) with N
2The pretreated tip of a root of O gas is fixed; (c) enzymolysis processing: the tip of a root that fixes is washed with water, and the meristematic tissue that cuts off root tips is with polygalacturonase and cellulose treatment 30min-2h.
3. the described method of claim 1, wherein step 2) in the wheat family basis genome of mark be selected from the AA genome, DD genome, RR rye genome, 10X long fringe couchgrass genome or transgenosis purpose fragment carrier.
4. the described method of claim 1, wherein diploid Urartu AA genome and the genomic probe mixing quality of diploid aegilops tauschii D D ratio can not be less than 1: 4.
5. the described method of claim 1, wherein diploid intend the genomic mass ratio of blockading of this inferior you holder BB be 160-200 doubly.
6. the described method of claim 1 is wherein carried out the recross analysis to the chromosome of wheat sample that obtains, and wherein per step recross analysis wash-out is with wash-out under 2 * SSC room temperature.
7. one kind is carried out the method that recross is analyzed to the plant chromosome sample that obtains, and described method comprises the steps:
1) passes through N
2The pre-treatment of O gas and enzymolysis of plants tip of a root meristematic zone or the vigorous tissue of bud point meristematic zone cell fission obtain clear picture, the finely disseminated metacinesis phase of karyomit(e) sample;
2) utilize nick translation method mark fluorescent probe;
3) utilizing step 2) fluorescent probe that makes up carries out fluorescence in situ hybridization to utilizing step 1) to divide phase karyomit(e) mitosis metaphase;
4) through simply flushing, on the basis that keeps chromosome morphology, use multiple hybridization probe to carry out one or many hybridization repeatedly, carry out evaluation and the analysis of exogenous chromosome.
8. the described method of claim 7, wherein the simple flushing of step 4) is with wash-out under 2 * SSC room temperature.
9. method for preparing plant chromosome metacinesis phase sample, described method comprises the steps:
(a) N
2O gas pre-treatment: get the suitable tip of a root of described plant or bud point, be placed in the moistening centrifuge tube, put into N
2Handled 0.5-5 hour N in the O air chamber
2The pressure of O is 10ATM (1.01Mpa); (b) with N
2The pretreated tip of a root of O gas or bud point are fixing; (c) enzymolysis processing: the tip of a root or the bud point that fix are washed with water, cut off root tips or bud point meristematic tissue, with polygalacturonase and cellulose treatment 30min-2h.
10. the described method of claim 9, wherein said plant is monocotyledons.
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