CN109988846B - Method suitable for in situ hybridization of red swamp crayfish gonad tissue mRNA paraffin section - Google Patents
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Abstract
The invention provides a method suitable for in situ hybridization of red swamp crayfish gonad tissue mRNA paraffin section, which belongs to the technical field of in situ hybridization, and comprises gonad tissue embedding, paraffin section, in situ hybridization and photographing record, wherein the in situ hybridization adopts a probe sequence shown as SEQ ID NO. 3. The probe has definite specificity on tissue localization of dsx genes, and realizes the display effect on dsx gene mRNA level; the invention establishes a paraffin section mRNA in situ hybridization method in the red swamp crayfish to study the expression mode of the related genes in the gonad, and can clearly describe the expression and positioning of the sex related genes of the red swamp crayfish in the gonad; the method of the invention has good dewaxing effect of paraffin sections, and simultaneously avoids tissue contraction and cell deformation, so that the tissue is still kept in situ, the tissue morphology is kept complete, the enzymolysis effect can be improved, the cells are well colored, the nuclear mass is distinct, and finally the accuracy of in situ hybridization is improved.
Description
Technical Field
The invention belongs to the technical field of in situ hybridization, and particularly relates to a method suitable for in situ hybridization of red swamp crayfish gonad tissue mRNA paraffin sections.
Background
The red-chelating crayfish (Cherax quadricariratus), commonly called Australian freshwater lobster, belongs to the genus of crayfish in the tropical region and the south of new guinea in the original Australia, has the characteristics of fast growth, strong stress resistance, tender meat quality, high meat yield and the like, and is one of rare and economic shrimps in the world freshwater. As with other freshwater decapod animals, the male individuals of the red swamp crayfish grow faster and have a larger body size than the female ones. Red rape has been widely cultivated, especially in recent decades, due to its remarkable biological properties, the breeding industry of red rape has developed rapidly. However, the mechanisms underlying this shrimp phenotype differentiation and binary development are currently unknown. In situ hybridization tissue (or cell) chemistry (In situ Hybridization Histochemistry, ISHH) is abbreviated as in situ hybridization (In Situ Hybridization), belongs to the category of solid phase molecular hybridization, and is a method for in situ detection of specific nucleic acid sequences in tissue cells by using labeled DNA or RNA as a probe, and is widely applied to laboratory analysis of mRNA distribution and enrichment degree in biological tissues. Therefore, the paraffin section mRNA in situ hybridization technology suitable for the gonad tissue of the red swamp crayfish is established, the tissue positioning and function research is carried out on the sex determination related genes, and the method is helpful for accelerating the disclosure and elucidation of the molecular action mechanism of sex determination and differentiation of the red swamp crayfish, thereby promoting the development of the sex control breeding technology of the red swamp crayfish.
Disclosure of Invention
The invention aims to provide a method which has definite specificity on the display of dsx genes and realizes the display effect on the mRNA level of the dsx genes and is suitable for the probe for the in situ hybridization of the red swamp crayfish gonad tissue mRNA paraffin section.
The technical scheme adopted by the invention for achieving the purpose is as follows:
the invention provides a method for a probe suitable for in situ hybridization of red swamp crayfish gonad tissue mRNA paraffin section, which comprises the following steps:
s1, step: using the cDNA of the gonad tissue of the red swamp crayfish obtained by reverse transcription as a template, and using a primer to amplify to obtain a partial ORF sequence of the dsx gene by a PCR reaction, wherein the sequence is used as a specific probe sequence of dsx gene mRNA;
s2, step: constructing a probe plasmid for dsx gene mRNA in-situ hybridization, transforming the probe plasmid into bacteria, culturing the bacteria, and sequencing;
s3, step: the dsx gene mRNA in situ hybridization probe was prepared. The sex-determining double genes of the red swamp crayfish are all homologous genes of dsx, the double genes dsx are positioned at the most downstream of the sex-determining signal path, and are quite conservative relative to the important genes for the sex-determining at the upstream, and the splicing forms of the double genes are changed; and the splice enhancement or inhibition factors for regulating and controlling the splice and the trans-acting elements combined with the splice are hardly changed in the same subgrade, and the splice enhancement or inhibition factors are regularly circulated among different subgrades.
Preferably, the dsx gene is expressed in the testis, but not in the ovary.
Preferably, the primers
The forward primer is: 5'-GAATTCTAATACGACTCACTATAGGGA-3';
the reverse primer is as follows: 5'-CCTATAGTGAGTCGTATTAAAGCTT-3'.
The probe has definite specificity on the display of dsx genes, and realizes the display effect on the mRNA level of the dsx genes.
The probe for in situ hybridization of the mRNA paraffin section of the gonad tissue of the red swamp crayfish is prepared by adopting the method.
Preferably, the probe sequence is as shown in SEQ ID NO. 3.
The invention further aims to provide a method which has the advantages of good dewaxing effect of paraffin sections, good enzymolysis effect, strong hybridization signal and good cell staining, can clearly describe the expression and positioning of sex-related genes of the culter ilishaeformis in gonads, and is suitable for in-situ hybridization of red-chelating crayfish gonad tissue mRNA frozen sections, and has high accuracy.
The technical scheme adopted by the invention for achieving the purpose is as follows:
the invention provides a method suitable for in situ hybridization of red swamp crayfish gonad tissue mRNA paraffin section, which comprises gonad tissue embedding, paraffin section, in situ hybridization and photographing record, wherein the probe is adopted for in situ hybridization.
Preferably, the in situ hybridization comprises the steps of:
1) Dewaxing paraffin sections: sequentially washing the slices with xylene, absolute ethyl alcohol, 85% alcohol, 75% alcohol and DEPC for dewaxing;
2) Digestion: slicing the tissue after water washing, boiling in a repairing liquid, naturally cooling, circling the gene pen, dripping proteinase K for digestion according to different index characteristics of different tissues, and flushing;
3) Prehybridization: dripping prehybridization solution on the digested slices, and incubating;
4) Hybridization: pouring out the prehybridization solution, dripping the hybridization solution containing the probe, hybridizing overnight, and washing;
3) Closing: dripping and sealing serum BSA, pouring off sealing liquid, dripping anti-DIG-HRP, and incubating;
4) DAB color development: after the slices are slightly dried, dripping freshly prepared DAB color development liquid into the circles, controlling the color development time under a microscope, and washing the slices with pure water to terminate color development, wherein the positive color is brown;
5) Counterstaining the nuclei: counterstaining with hematoxylin dye, washing with water, differentiating with 1% hydrochloric acid alcohol, washing with water, returning to blue with ammonia water, and washing;
6) Sealing piece: and (5) sealing the neutral resin.
Preferably, xylene contains 0.03-0.06% chloral and 2.8-3.0% lumefantrine. On one hand, the existence of chloral and benzofluorenol promotes toluene to rapidly dissolve paraffin for supporting tissues and cells, and the paraffin section has good dewaxing effect and simultaneously avoids tissue shrinkage and cell deformation, so that the tissues are still kept in situ, and the tissue morphology is kept complete; on the other hand, the permeability of the antibody to cell membranes can be increased, the cells are well colored, the nucleoplasm is clear, the structure is clear, the tissues and the cells are more transparent and clear, the observation is convenient, and the in situ hybridization accuracy is improved in the DAB color development and hematoxylin dye solution counterstain process; in addition, the toughness of the DNA chain can be increased, the complex formed by the DNA chain and protein is avoided to prevent protease from being digested, the enzymolysis effect is improved, and finally the accuracy of in-situ hybridization is improved.
Preferably, the concentration of probe hybridization solution is 50ng/ul.
Preferably, the embedding of gonadal tissue comprises the steps of:
the tissue is taken out and washed, then is immediately put into a fixing solution for fixing for 2-12h, and is soaked in wax and embedded after the fixation is completed and the gradient alcohol is dehydrated.
Compared with the prior art, the invention has the beneficial effects that:
the probe has definite specificity on the display of dsx genes, and realizes the display effect on the mRNA level of the dsx genes; the invention establishes a paraffin section mRNA in situ hybridization method in the red swamp crayfish to study the expression mode of the related genes in the gonad, can clearly describe the expression and positioning of the sex related genes of the red swamp crayfish in the gonad, and has important significance for the identification of sex regulation genes; the method of the invention has good dewaxing effect of paraffin sections, and simultaneously avoids tissue shrinkage and cell deformation, so that the tissue is still kept in situ, the tissue morphology is kept complete, the enzymolysis effect can be improved, the cells are well colored, the nuclear mass is distinct, and finally the accuracy of in situ hybridization is improved.
The technical scheme is adopted to provide the method suitable for in-situ hybridization of the red swamp crayfish gonad tissue mRNA paraffin section, so that the defects of the prior art are overcome, and the method is reasonable in design and convenient to operate.
Drawings
FIG. 1 is a schematic representation of an in situ hybridization analysis of paraffin sections of the dsx gene of red swamp crayfish in gonadal tissue in example 1 of the present invention.
Detailed Description
A method suitable for in situ hybridization of mRNA paraffin sections of gonadal tissue of red swamp crayfish according to an embodiment of the present invention is further described below with reference to the following examples.
Example 1:
a method for a probe suitable for in situ hybridization of red swamp crayfish gonad tissue mRNA paraffin section comprises the following steps:
s1, step: designing and constructing a primer sequence of a dsx probe vector according to the sequence of a dsx gene coding region of the culter ilishaeformis, wherein forward and reverse primers respectively have EcoRI and Xhol restriction sites, and the primer sequence is as follows:
the forward primer is: 5'-GAATTCTAATACGACTCACTATAGGGA-3' the number of the individual pieces of the plastic,
the reverse primer is as follows: 5'-CCTATAGTGAGTCGTATTAAAGCTT-3';
s2, step: using the cDNA of the gonad tissue of the red swamp crayfish obtained by reverse transcription as a template, and using a primer to amplify to obtain a partial ORF sequence of the dsx gene by a PCR reaction, wherein the sequence is used as a dsx protein specific probe sequence, and the PCR method is carried out for 4min at 94 ℃;94℃for 30s, 55℃for 30s, 72℃for 1min,32 cycles; extending at 72 ℃ for 7min, and purifying the target fragment by using an Axygen gel recovery kit;
s3, step: double enzyme digestion treatment is carried out on the target fragment and the pBluescriptII SK vector by EcoRI and Xhol, the reaction system is shown in table 1, the mixture is fully mixed after the reagent is added, the digestion is carried out for 4 hours in water bath at 37 ℃, and the purification is carried out after the completion;
table 1 double cleavage reaction System
| Component (A) | Volume of |
| Fragments or vectors of interest | 3μg |
| 10×H Buffer | 7μl |
| EcoRI | 3.5μl |
| Xhol | 3.5μl |
| ddH 2 O | Supplement to 70. Mu.l |
S4, step: connecting the digested vector and the target fragment in a water bath at 16 ℃, then converting, sequencing and identifying the correct recombinant plasmid through sequence analysis;
s5, step: the plasmid was subjected to single cleavage with EcoRI or Xhol, digested with 37℃in a water bath for 4 hours to give a linearized recombinant plasmid, which was purified using the Cleannip kit from Axygen. Then, taking the template as a transcription antisense RNA probe with digoxin marks, and the specific system is shown in the table 2, mixing uniformly after adding reagents, carrying out water bath reaction for 2.5 hours at 37 ℃, then adding 2 mu l DNaseI digestion plasmid template of RNase free, incubating for 20 minutes at 37 ℃, then adding 2 mu l 0.2M EDTA, and standing on ice until the digestion reaction is stopped;
TABLE 2 transcription RNA probe reactant
S6, step: after digestion, the RNA probe is purified by using Mini Quick Spin RNA Columns (Roche), electrophoresis and concentration detection are carried out on the purified product, and finally, the rest sample is added with deionized formamide with equal volume, and after light mixing and split charging, the mixture is preserved at-80 ℃ to prepare the mRNA in situ hybridization probe of dsx genes. The sex-determining double genes of the red swamp crayfish are all homologous genes of dsx, the double genes dsx are positioned at the most downstream of the sex-determining signal path, and are quite conservative relative to the important genes for the sex-determining at the upstream, and the splicing forms of the double genes are changed; and the splice enhancement or inhibition factors for regulating and controlling the splice and the trans-acting elements combined with the splice are hardly changed in the same subgrade, and the splice enhancement or inhibition factors are regularly circulated among different subgrades.
The dsx gene is expressed in the testis but not in the ovary.
The probe for in situ hybridization of the mRNA paraffin section of the gonad tissue of the red swamp crayfish is prepared by adopting the method.
The probe sequence is shown as SEQ ID NO.3, and concretely comprises the following steps:
GAATTCTAATACGACTCACTATAGGGATGGCTATGGCGCCTAAGAAGAAATTCGATTTTTTCAATGGTGGTCGGGGAATTGTCCTGACCGACACAACTGATGTGGAAGAATCGTCTGTAGAGCAGCAGCCACCAGAGCCCAAGGTAGTGCAGGAGTCTTATGGTTATGGAGGCGACTATGGGCGCTACTCTGGTTCTTCCGAGGGCTACGTAACCCCCAGCTCTCCTGGGGGCTATATGCCTCCAGGGGGCTATGTCCCCCCCAGATCTCCAGGGGGCTATGTCGTCCCCAGATCTCCAGGGGGCTGTGTGTCCTCTAGATCTCCAGGGGAGTATGTGCCCCCTGACAGCCCGAATGTTCATCAGTACCACGGTGACCCTATAGTGAGTCGTATTAAAGCTT。
example 2:
a method suitable for in situ hybridization of red swamp crayfish gonad tissue mRNA paraffin section comprises gonad tissue embedding, paraffin section, in situ hybridization and photographing record, wherein the in situ hybridization adopts the probe.
The in situ hybridization comprises the following steps:
1) Tissue fixation: tissue is taken out and washed, and then is immediately put into a fixing solution (prepared by DEPC water) for fixing for 2-12h;
2) Dehydrating: after tissue fixation is completed, the tissue is dehydrated by gradient alcohol and then is immersed in wax for embedding;
3) Slicing: slicing paraffin by a slicing machine, fishing out slices by a slice spreading machine, and baking slices by a 62 ℃ oven for 2h;
4) Paraffin sections dewaxed to water: sequentially placing the slices into xylene I15 min-xylene II 15 min-absolute ethanol I5 min-absolute ethanol II 5min-85% alcohol 5min-75% alcohol 5min-DEPC water washing;
5) Digestion: slicing according to the fixed time of tissue, boiling in repairing liquid for 10-15 min, naturally cooling, drawing circles with a gene pen, dripping proteinase K (20 ug/ml) according to different index characteristics of different tissues, digesting for 30min at 37 ℃, washing with PBS for 3 times×5min after washing with pure water;
6) Prehybridization: dropwise adding a prehybridization solution, and incubating for 1h at 37 ℃;
7) Hybridization: pouring out the prehybridization solution, dropwise adding the hybridization solution containing the probe, wherein the concentration is 50ng/ul, and hybridizing overnight at 37 ℃ in an incubator;
8) Washing after hybridization: washing off the hybridization solution, 2 XSSC, 10min at 37 ℃,1 XSSC, 2 XSSC in at 37 ℃,0.5 XSSC for 10min at room temperature, and if the number of nonspecific hybrids is large, the formamide washing can be increased;
9) And (3) dripping a sealing liquid: dripping and sealing serum BSA for 30min at room temperature;
10 Dripping mouse digoxin marked peroxidase (anti-DIG-HRP): the blocking solution was decanted and anti-DIG-HRP was added dropwise. Incubation at 37 ℃ for 40min, and then PBS washing for 4 times multiplied by 5min;
11 DAB color development): after the slices are slightly dried, dripping freshly prepared DAB color development liquid into the circles, controlling the color development time under a microscope, and washing the slices with pure water to terminate color development, wherein the positive color is brown;
12 Counterstaining the nuclei): the hematoxylin counterstain is carried out for about 3min, the washing is carried out by running water, 1% hydrochloric acid alcohol is differentiated for a plurality of seconds, the running water is carried out, the ammonia water returns to blue, and the running water is washed;
13 Sealing plate): sealing the neutral resin;
14 Microscopic examination, image acquisition and analysis, the result is shown in fig. 1, the left graph in fig. 1 is the testis, the right graph is the ovary, and the signal is in the testis, so that the dsx gene is strong in the testis, the signal cannot be detected in the ovary, and the dsx gene is expressed in the testis but not in the ovary.
Example 3:
in order to improve the dewaxing effect of paraffin sections, a further optimization scheme is as follows:
paraffin sections are dewaxed to a water step in which the xylene contains 0.03-0.06% chloral and 2.8-3.0% benzofluorenol. On one hand, the existence of chloral and lumefantrine promotes toluene to rapidly dissolve paraffin for supporting tissues and cells, so that the tissues are prevented from shrinking and deforming, the tissues are still kept in situ, and the tissue morphology is kept complete; on the other hand, the permeability of the antibody to cell membranes can be increased, the cells are well colored, the nucleoplasm is clear, the structure is clear, the tissues and the cells are more transparent and clear, the observation is convenient, and the in situ hybridization accuracy is improved in the DAB color development and hematoxylin dye solution counterstain process; in addition, the toughness of the DNA chain can be increased, the complex formed by the DNA chain and protein is avoided to prevent protease from being digested, the enzymolysis effect is improved, and finally the accuracy of in-situ hybridization is improved.
The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.
The above embodiments are merely for illustrating the present invention and not for limiting the same, and various changes and modifications may be made by one of ordinary skill in the art without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions are also within the scope of the present invention, which is defined by the claims.
Sequence listing
<110> Zhejiang province fresh water aquatic institute
<120> method for in situ hybridization of mRNA paraffin sections of gonadal tissue of red swamp crayfish
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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gaattctaat acgactcact ataggga 27
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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cctatagtga gtcgtattaa agctt 25
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gaattctaat acgactcact atagggatgg ctatggcgcc taagaagaaa ttcgattttt 60
tcaatggtgg tcggggaatt gtcctgaccg acacaactga tgtggaagaa tcgtctgtag 120
agcagcagcc accagagccc aaggtagtgc aggagtctta tggttatgga ggcgactatg 180
ggcgctactc tggttcttcc gagggctacg taacccccag ctctcctggg ggctatatgc 240
ctccaggggg ctatgtcccc cccagatctc cagggggcta tgtcgtcccc agatctccag 300
ggggctgtgt gtcctctaga tctccagggg agtatgtgcc ccctgacagc ccgaatgttc 360
atcagtacca cggtgaccct atagtgagtc gtattaaagc tt 402
Claims (3)
1. The probe for in situ hybridization of the red swamp crayfish gonad tissue mRNA paraffin section has a sequence shown as SEQ ID NO.3, and the preparation method of the probe comprises the following steps:
s1, step: using the cDNA of the gonad tissue of the red swamp crayfish obtained by reverse transcription as a template, and using a primer to amplify the cDNA by a PCR reactiondsxPartial ORF sequence of the gene, which is used as a dsx gene mRNA specific probe sequence, and the PCR method is 94 ℃ for 4min;94℃for 30s, 55℃for 30s, 72℃for 1min,32 cycles; extending at 72 ℃ for 7min, and purifying the target fragment by using an Axygen gel recovery kit;
s2, step: constructiondsxThe probe plasmid hybridized with the gene mRNA in situ is used for sequencing after bacterial culture after bacterial transformation;
s3, step: preparing dsx gene mRNA in situ hybridization probes;
the forward primer of the primer is as follows: 5'-GAATTCTAATACGACTCACTATAGGGA-3', the reverse primer is: 5'-CCTATAGTGAGTCGTATTAAAGCTT-3' the forward and reverse primers bear EcoRI and Xhol cleavage sites, respectively.
2. Use of the probe of claim 1 in the in situ hybridization of red swamp crayfish gonadal tissue mRNA paraffin sections.
3. A method for in situ hybridization of red swamp crayfish gonad tissue mRNA paraffin sections, comprising the following steps:
taking out and cleaning the tissue, immediately placing the tissue into a fixing solution for fixing for 2-12h, dehydrating the tissue by using gradient alcohol after the fixing is finished, immersing the tissue in wax, and embedding the tissue;
paraffin wax slicing dewaxing, namely slicing paraffin wax by a slicing machine, sequentially putting the slice into xylene I for 15min, xylene II for 15min, absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, 85% alcohol for 5min, 75% alcohol for 5min and DEPC water washing for dewaxing;
digestion, namely slicing the washed tissue into repairing liquid, boiling, naturally cooling, circling the gene pen, and dripping proteinase K for digestion and flushing according to different index characteristics of different tissues;
prehybridization: dripping prehybridization solution on the digested slices, and incubating;
hybridization: pouring out the prehybridization solution, dropwise adding the probe hybridization solution containing the probe hybridization solution of claim 1, wherein the concentration of the probe hybridization solution is 50ng/ul, and washing after hybridization overnight;
closing: dripping and sealing serum BSA, pouring off sealing liquid, dripping anti-DIG-HRP, and incubating;
DAB color development: after the slices are slightly dried, dripping freshly prepared DAB color development liquid into the circles, controlling the color development time under a microscope, and washing the slices with pure water to terminate color development, wherein the positive color is brown;
counterstaining the nuclei: counterstaining with hematoxylin dye, washing with water, differentiating with 1% hydrochloric acid alcohol, washing with water, returning to blue with ammonia water, and washing;
sealing piece: sealing the neutral resin;
microscopic examination, image acquisition and analysis,dsxthe gene signals stronger in the testis, but no signal was detected in the ovary, indicatingdsxGenes are expressed in the testis but not in the ovary;
the dimethylbenzene contains 0.03-0.06% of chloral and 2.8-3.0% of lumefantrine.
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