CN117947210A - Preparation of lycoris radiata genome probe and genome in-situ hybridization method thereof - Google Patents
Preparation of lycoris radiata genome probe and genome in-situ hybridization method thereof Download PDFInfo
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Abstract
The invention provides a lycoris genome probe preparation and a genome in-situ hybridization method thereof, which comprises the following steps: uniformly mixing plant total genome DNA with MT nuclear type or AA nuclear type chromosome with water and biotin marked notch transcription mixture, heating, and fully reacting to obtain a probe; the volume ratio of DNA to water to biotin-labeled gap transcription mixture = 1:15:4. The invention improves the hybridization success rate and the identifiable specificity through the selection of the preparation materials of the lycoris genome probe and the improvement of the conditions and the comprehensive optimization and adjustment of the genome in-situ hybridization technical parameters, ensures the specificity of the genome in-situ hybridization technology (GISH) and improves the success rate of the GISH technology.
Description
Technical Field
The invention belongs to the technical field of genome in-situ hybridization methods, and particularly relates to a lycoris genome probe preparation and a genome in-situ hybridization method thereof.
Background
The chromosome in situ hybridization technique is a molecular cytogenetic technique for directly hybridizing a nucleic acid on a tissue section by using a labeled DNA or RNA probe with a known sequence to locate a specific target gene sequence. The accurate identification of the chromosome is carried out by combining the probe and the target DNA, and mainly comprises two kinds of fluorescence in situ hybridization and genome in situ hybridization, wherein the probe adopted by the fluorescence in situ hybridization technology (FISH) is mainly cloned rDNA and other main tandem repeat sequences, and the karyotype and ploidy of the chromosome are identified according to the position and the quantity of hybridization signals. At present, research shows that the distribution of lycoris plant signal sites on chromosomes is obtained through FISH, the structural variation of the chromosomes is recognized, rDNA is positioned on the chromosomes, and rRNA gene physical maps are constructed. However, the existing FISH technology cannot distinguish parent chromosomes of the filial generation, and cannot directly identify parents of the hybrid.
The Genome In Situ Hybridization (GISH) technology is to use the total genome DNA of plants as a probe to hybridize with the chromosomes of the hybrid seeds so as to identify the parents of the hybrid seeds, identify the recombinant fragments of the chromosomes and ascertain the source of the allopolyploid. The GISH technology is simple and easy to operate, does not need to separate probes, only needs to extract the total genome DNA of the plants to carry out marking as the probes, can be hybridized with the whole chromosome, and can observe hybridization sites at any period of cell division so as to rapidly and accurately detect target chromosomes (fragments) and genomes. The GISH technology is widely used for identifying plant heterologous chromatin and researching species origin evolution at present, and effectively identifying lily hybrid offspring chromosomes. However, the research work of the GISH technology in the aspects of the identification of the chromosomes of the lycoris hybrid parents, the identification of the relationships among the seeds and the genetic composition of the hybrid is slow, mainly because the lycoris is mainly natural hybrid, and under the condition that the hybrid parents are not clear, the suitable probes and the blockage are difficult to select, so that the application of the GISH technology in lycoris plants is limited.
In view of this, the present invention has been made.
Disclosure of Invention
The first object of the present invention is to provide a method for preparing lycoris genome probe, which can improve the success rate of hybridization and the identifiable specificity by screening the source of the probe and changing the preparation conditions.
The second object of the present invention is to provide a hybridization method of in-situ genome of the probe prepared in the above-mentioned lycoris genome in-situ hybridization method, which ensures the specificity of the genome in-situ hybridization technology and improves the success rate of the GISH technology by screening the blocking DNA and changing the reaction conditions in the hybridization process.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
a preparation method of lycoris genome probe comprises the following steps:
uniformly mixing plant total genome DNA with MT nuclear type or AA nuclear type chromosome with water and biotin marked notch transcription mixture, heating, and fully reacting to obtain the plant total genome DNA; the volume ratio of DNA to water to biotin-labeled gap transcription mixture = 1:15:4.
Preferably, as a further specific embodiment, the source of plant total genomic DNA of the MT karyotype chromosome is lycoris radiata or lycoris radiata; the source of the plant total genome DNA of the AA nuclear chromosome is brocade.
A probe prepared by the method for preparing the lycoris genome probe.
MT nuclear type chromosome or AA nuclear type chromosome is selected as a material of the probe in the invention. Lycoris karyotype can be classified into A, MT and MT-A, and the lycoris plant has higher chromosome homology among species, so that in the application of GISH, the hybridization specificity is low, and the hybridization signal is not obvious. In the invention, MT or AA karyotype chromosomes are used as materials, when parents of MT karyotype and A karyotype are hybridized, the MT karyotype is used as a probe for hybridization, so that the identification is easier, the length of the karyotype chromosomes is longer, the relativity with other species is farther, the interference of other homologous chromosomes can be eliminated in the hybridization process, the hybridization signals are easier to identify on the chromosomes, and the signals are clearer and more visible. In addition, after the DNA and water are mixed in the ratio of biotin-labeled notch transcription mixture=1:15:4, the signal value of hybridization signals can be increased to the maximum extent, and the success rate of the GISH technology is ensured.
A method for in situ hybridization of lycoris genome by using a probe prepared by the method for preparing the lycoris genome probe comprises the following steps:
Preparation and pretreatment of chromosome slide specimens, preparation of blocking DNA, co-denaturation of chromosomes and the probes and the blocking DNA, hybridization, tightness washing and signal detection.
Preferably, as a further specific embodiment, the preparation and pretreatment of the chromosome slide specimen comprises the following steps:
taking lycoris radicle, adding cycloheximide for treatment, and then fixing;
adding the fixed root tip into a mixed solution with the volume ratio of 1%, and carrying out enzymolysis treatment;
heating the root tip after enzymolysis, tabletting, and fixing for the second time.
Preferably, as a further specific embodiment, the blocking DNA is herring sperm DNA.
Preferably, as a further specific embodiment, the secondary fixing step needs to add paraformaldehyde with a volume ratio of 4% for fixing for 10min-12min.
Preferably, as a further specific embodiment, the enzymolysis time is 40min-90min.
The genome in situ hybridization method provided by the invention can be used for carrying out genome in situ hybridization by taking chromosomes in the metaphase of mitosis of the filial generation of lycoris plants as targets to be detected and taking the whole genome DNA of one parent species as probe DNA. The lycoris plant has high chromosome homology including near hybridization and distant hybridization, and the blocking DNA is difficult to block the nonspecific hybridization sites of the seeds by hybridization, wherein the whole genome DNA of the other parent species is used as the blocking DNA, and the specificity between the seeds is difficult to distinguish. In the prior art, proper blocking DNA cannot be selected for wild species, and the used blocking DNA is mostly used for identifying hybrid species by matching with probes.
The herring sperm DNA is used as the blocking DNA, and is matched with the prepared probe, so that the interference of homologous chromosomes with relatively close relativity is eliminated, and wild species can be better identified, so that unified herring sperm DNA is used as the blocking DNA, and the herring sperm DNA is simple, convenient and easy to distinguish.
Meanwhile, the preparation of chromosome slide specimens and the adjustment of pretreatment conditions strengthen the full hybridization of chromosomes, probe DNA and blocking DNA, and improve the success rate of hybridization and identifiable specificity. The fixation of paraformaldehyde with the volume ratio of 4% at normal temperature for 10-12 min can ensure that the chromosome is not eluted in the eluting process, thereby avoiding the possibility of reducing the success rate of the GISH because the concentration of the eluted chromosome is too low.
In the preparation and pretreatment condition process of the chromosome slide specimen, pectase and cellulase are mixed in a volume ratio of 1:1 to prepare mixed solution, and enzymolysis time is controlled to be 40-90 min, so that pectic substances among lycoris tissues can be fully decomposed, cell walls are softened and partially decomposed, cells and chromosomes are easier to disperse and flatten, and cell membranes are prevented from being broken too early due to overlong enzymolysis time, so that the chromosome is lost, signal values are influenced, and the subsequent experiments are facilitated.
Compared with the prior art, the invention has the beneficial effects that:
(1) Ensuring the specificity of the genome in situ hybridization technology and providing the success rate of the GISH technology.
(2) The invention ensures the success rate of the lycoris genome in-situ hybridization method by comprehensively optimizing and adjusting the condition parameters of genome in-situ hybridization, the in-situ hybridization of the lycoris genome is carried out by utilizing the method provided by the invention, the hybridization specificity is more convenient and rapid to distinguish, the hybridization signal is clearer and more visible on the chromosome, the parent chromosome of the hybrid is easier to identify, and the invention provides possibility for the genetic analysis of the germplasm identification and hybridization breeding of the lycoris.
Drawings
Fig. 1: chromosome fluorescence microscopy images of lycoris radiata obtained by the embodiment of example 1; wherein A is a microscopic image of the lycoris chromosome counterstained with DAPI in example 1, and B is a microscopic image of the hybridization signal of the lycoris chromosome successfully hybridized in example 1;
Fig. 2: chromosome fluorescence microscopy images of lycoris radiata shanxi obtained by using the embodiment of example 1; wherein C is a microscopic image of the lycoris shanxi chromosome counterstained with DAPI in example 1, and D is a microscopic image of the hybridization signal of the lycoris shanxi in example 1 after successful hybridization;
Fig. 3: chromosome fluorescence microscopy images of lycoris radiata shanxi obtained by using the embodiment of example 2; wherein A is a microscopic examination image of the chromosome of the lycoris shanxi obtained by counterstaining with DAPI in example 2, and B is a microscopic examination image of the hybridization signal obtained by successful hybridization of the lycoris shanxi in example 2;
Fig. 4: chromosome fluorescence microscopy images of lycoris radiata obtained by the embodiment of example 3; wherein A is a microscopic image obtained by counterstaining the chromosome of lycoris persica with DAPI in example 3, and B is a microscopic image of hybridization signals obtained after successful hybridization of lycoris persica in example 3;
fig. 5: chromosome hybridization signal fluorescence microscopy of lycoris obtained in the embodiment of comparative example 1 was used.
Detailed Description
The technical solution of the present invention will be clearly and completely described in conjunction with the specific embodiments, but it will be understood by those skilled in the art that the examples described below are some, but not all, examples of the present invention, and are intended to be illustrative only and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
The material of lycoris to be tested in the following examples is derived from root tips of lycoris radiata (l. Incarnata) and lycoris shanxi (l. Shaanaxiensis), which are natural hybrid seeds, and lycoris radiata (l. Chinensis) is a primary seed. The method comprises the steps of taking the Chinese lycoris with chromosome MT nucleus type as a probe total genome DNA and taking herring sperm DNA as a blocking DNA.
The embodiment provides a genome in situ hybridization method of lycoris radiata, which comprises the following specific steps:
(1) Preparation and pretreatment of chromosome slide specimens
Taking 1-2cm long young roots of lycoris radiata, dripping 200ppm of cycloheximide, and treating for 4 hours in a 25 ℃ artificial climatic chamber; fixing the treated root tip in Carnot fixing solution, and fixing at 4deg.C for 24 hr; dripping 20 μl of 1% (pectase: cellose=1:1) mixed solution into the fixed root tip, and performing enzymolysis at 37deg.C for 60min; dripping 20 mu L of 45% acetic acid into the root tip after enzymolysis, heating for 10s on a heating plate at 65 ℃, tabletting, putting into a refrigerator at-80 ℃, freezing to uncover tablets, dehydrating and drying by absolute ethyl alcohol, and drying at 37 ℃ for at least 7-12h for standby, wherein 4% paraformaldehyde is required to be added for fixing for 10min at normal temperature in order to ensure that the chromosome is not eluted.
Table 1 amounts of the substances
(2) Preparation of probes and selection of blocking DNA
DNA extraction: the total genome DNA of the lycoris radiata with chromosome MT karyotype and the lycoris radiata with chromosome AA karyotype are used as probe materials. The leaves of lycoris radiata and brocade are taken, the total genome DNA is extracted by adopting a CTAB method (broad-spectrum plant DNA genome extraction kit, D3161-01), the total genome DNA of lycoris radiata is uniformly mixed with water and biotin-marked notch transcription mixture=1:15:4, the mixture is subjected to light-shielding reaction at 15 ℃ for 90min, heated in a water bath at 65 ℃ for 10min, and stored at minus 20 ℃ to obtain probe DNA, and herring sperm DNA is selected as blocking DNA.
(3) Co-denaturation, hybridization of chromosome and probe, blocking DNA
After the slide glass is dried, 100. Mu.L of 70% deionized formamide is dripped into the slide glass, and the slide glass is denatured on a heating plate at 75 ℃ for 5min; denaturing the hybridization mixture at 100deg.C for 13min, and cooling in ice at 4deg.C for at least 5min; the denatured hybridization mixture was added to the denatured chromosome slide and hybridization was maintained at 37℃for 12 hours.
(4) Tightness wash
Taking out the glass slide after hybridization, washing the glass slide by adopting a 2X physiological saline sodium citrate buffer solution to remove the cover glass for 5min, and washing the glass slide by adopting a 0.1X physiological saline sodium citrate buffer solution at 42 ℃ for 30min; slides were washed three times for 5min each in 2 Xnormal saline sodium citrate buffer.
(5) Signal detection
The washed slide was washed in 1 Xbuffer 1 (0.1M Tris-HCl, 0.15M sodium chloride, pH 7.5) for 5min; 200 mu L of blocking buffer (1% blocker placed in buffer 1) is added dropwise to each slide, and incubated for 5min; 100 μl of blocking buffer containing 0.4 μl streptavidin-CY 3 (Invitrogen, camarillo, calif.) was added dropwise, and incubated for 1h at 37deg.C with wetting; washing three times with 1 Xbuffer 1 at 37℃for 5min each; each slide was added dropwise with 100 μl of a blocking buffer containing 4 μl of biotinylated anti-streptavidin (Vector Laboratories, burlingame, CA), and incubated at 37deg.C for 1h under wet incubation; the slide glass was washed with 2 x physiological saline sodium citrate buffer for 5min; dropping a drop of DAPI-containing caplet seal (Vectashield 1200); the results of the microscopic examination are shown in FIGS. 1 and 2 (lycoris chromosomes are counterstained blue with DAPI and hybridization signals are purple) using Zeiss axiophor fluorescence microscopy.
Example 2
The present example provides an in situ hybridization method of lycoris genome, and example 2 selects a chromosome AA nuclear type brocade flower (L. sprengeri) as a probe total genome DNA; the material of lycoris to be detected in the embodiment is derived from a chromosome slide pretreated by lycoris radiata of Shaanxi (L. shaanxiensis), herring sperm DNA is used as blocking DNA, and the embodiment provides an in-situ hybridization method of lycoris radiata genome, which is different from the method of the embodiment 1 only in that the heating temperature of the chromosome slide in the step (1) is adjusted to 75 ℃ from 65 ℃ of the embodiment 1, so that the method is helpful for enhancing the chromosome fixation of partial species and accelerating the splitting item splitting; the chromosomal denaturation time in the chromosomal hybridization of step (2) is changed from 5min of example 1 to 8min of example 2; the chromosome denaturation can be more thorough by prolonging the denaturation time of different species; the hybridization time of the hybridization solution in the step (4) and the chromosome is from 12h of the example 1 to 17h of the example 2, and the specific steps are as follows:
(1) Preparation and pretreatment of chromosome slide specimens
Taking the root tip of lycoris radiata young roots, dripping 200ppm of cycloheximide, and treating for 4 hours in a 25 ℃ artificial climatic box; fixing the treated root tip in Carnot fixing solution, and fixing at 4deg.C for 24 hr; dripping 1% (pectase: cellose=1:1) of mixed solution 20 mu L into the fixed root tip, and performing enzymolysis at 37 ℃ for 60min; dripping 20 mu L of 45% acetic acid into the root tip after enzymolysis, heating the root tip on a heating plate at 75 ℃ for 10 seconds, tabletting, putting the root tip into a refrigerator at-80 ℃, freezing and uncovering the tablets, dehydrating and drying the root tip by absolute ethyl alcohol, and drying the root tip at 37 ℃ for 7-12 hours for standby, wherein 4% paraformaldehyde is required to be added for fixing the root tip at normal temperature for 10 minutes in order to ensure that the chromosome is not eluted.
(2) Co-denaturation of chromosome and probe and blocking DNA
After the glass slide is dried, 100 mu L of 70% deionized formamide is dripped into the glass slide, and the glass slide is denatured on a heating plate at 75 ℃ for 8min; the hybridization mixture was denatured at 100℃for 13min and cooled in ice at 4℃for at least 5min.
(3) Hybridization
The denatured hybridization mixture was added to the denatured chromosome slide and hybridization was maintained at 37℃for 17 hours.
(4) Tightness wash
Taking out the glass slide after hybridization, washing the glass slide by adopting a 2X physiological saline sodium citrate buffer solution to remove the cover glass for 5min, and washing the glass slide by adopting a 0.1X physiological saline sodium citrate buffer solution at 42 ℃ for 30min; slides were washed three times for 5min each in 2 Xnormal saline sodium citrate buffer.
(5) Signal detection
The washed slide was washed in 1 Xbuffer 1 (0.1M Tris-HCl, 0.15M sodium chloride, pH 7.5) for 5min; 200 μl of blocking buffer (1% blocker placed in buffer 1,
Incubating for 5min; 100 μl of blocking buffer containing 0.4 μl streptavidin-CY 3 (Invitrogen, camarillo, calif.) was added dropwise, and incubated at 37deg.C for 1h with wet incubation; washing three times with 1 Xbuffer 1 at 37℃for 5min each; each slide was added dropwise with 100 μl of a blocking buffer containing 4 μl of biotinylated anti-streptavidin (Vector Laboratories, burlingame, CA), and incubated at 37deg.C for 1h under wet incubation; the slide glass was washed with 2 x physiological saline sodium citrate buffer for 5min; dropping a drop of DAPI-containing caplet seal (Vectashield 1200); the results of the microscopic examination using a Zeiss Axiophot fluorescence microscope are shown in FIG. 3 (the lycoris chromosome was counterstained with DAPI to appear blue and the hybridization signal to appear purple).
Example 3
The present example provides an in situ hybridization method of lycoris genome, differing from the method of example 1 only in the selection of probes; example 3A lycoris radiata (L.sp.th) was selected as the total genomic DNA of the probe, the chromosome karyotype of which was MT, and the material to be tested was lycoris radiata (L.sp.th), as shown in FIG. 4.
Comparative example 1
The specific embodiment corresponds to example 1, with the modification of the DNA: water: the volume ratio of biotin-labeled gap transcription mixture=2:7:11, (as shown in fig. 5), and the images revealed that the probe did not hybridize successfully to the chromosome.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (8)
1. The preparation method of the lycoris genome probe is characterized by comprising the following steps of:
uniformly mixing plant total genome DNA with MT nuclear type or AA nuclear type chromosome with water and biotin marked notch transcription mixture, heating, and fully reacting to obtain a probe;
The volume ratio of DNA to water to biotin-labeled gap transcription mixture = 1:15:4.
2. The method for preparing a lycoris genome probe according to claim 1, characterized in that the source of plant total genomic DNA of MT nuclear chromosome is lycoris radiata or lycoris radiata; the source of the plant total genome DNA of the AA nuclear chromosome is brocade.
3. A probe prepared by the method for preparing a lycoris genome probe according to any of claims 1-2.
4. A method of in situ hybridization of lycoris genome using the probe of claim 3 comprising the steps of:
preparation and pretreatment of chromosome slide specimens, selection of blocking DNA, co-denaturation of chromosomes and the probes and the blocking DNA, hybridization, elasticity washing and signal detection.
5. The method of in situ hybridization of lycoris genome according to claim 4, characterized in that the preparation and pretreatment of the chromosome slide specimen comprises the steps of:
taking lycoris radicle, adding cycloheximide for treatment, and then fixing;
adding the fixed root tip into a mixed solution with the volume ratio of 1%, and carrying out enzymolysis treatment;
heating the root tip after enzymolysis, tabletting, and fixing for the second time.
6. The method of in situ hybridization of lycoris genome according to claim 4, characterized in that the blocking DNA is herring sperm DNA.
7. The method according to claim 5, wherein the secondary immobilization step requires the addition of 4% by volume paraformaldehyde for immobilization for 10min to 12min.
8. The method of in situ hybridization of lycoris genome according to claim 5, characterized in that the enzymatic hydrolysis time is 40min-90min.
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